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Sample records for exons 13-15 comparison

  1. 40 CFR 13.15 - Taxpayer information.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Taxpayer information. 13.15 Section 13.15 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GENERAL CLAIMS COLLECTION STANDARDS Collection § 13.15 Taxpayer information. (a) The Administrator may obtain a debtor's current mailing address...

  2. Detailed comparison of two popular variant calling packages for exome and targeted exon studies

    Directory of Open Access Journals (Sweden)

    Charles D. Warden

    2014-09-01

    Full Text Available The Genome Analysis Toolkit (GATK is commonly used for variant calling of single nucleotide polymorphisms (SNPs and small insertions and deletions (indels from short-read sequencing data aligned against a reference genome. There have been a number of variant calling comparisons against GATK, but an equally comprehensive comparison for VarScan not yet been performed. More specifically, we compare (1 the effects of different pre-processing steps prior to variant calling with both GATK and VarScan, (2 VarScan variants called with increasingly conservative parameters, and (3 filtered and unfiltered GATK variant calls (for both the UnifiedGenotyper and the HaplotypeCaller. Variant calling was performed on three datasets (1 targeted exon dataset and 2 exome datasets, each with approximately a dozen subjects. In most cases, pre-processing steps (e.g., indel realignment and quality score base recalibration using GATK had only a modest impact on the variant calls, but the importance of the pre-processing steps varied between datasets and variant callers. Based upon concordance statistics presented in this study, we recommend GATK users focus on “high-quality” GATK variants by filtering out variants flagged as low-quality. We also found that running VarScan with a conservative set of parameters (referred to as “VarScan-Cons” resulted in a reproducible list of variants, with high concordance (>97% to high-quality variants called by the GATK UnifiedGenotyper and HaplotypeCaller. These conservative parameters result in decreased sensitivity, but the VarScan-Cons variant list could still recover 84–88% of the high-quality GATK SNPs in the exome datasets. This study also provides limited evidence that VarScan-Cons has a decreased false positive rate among novel variants (relative to high-quality GATK SNPs and that the GATK HaplotypeCaller has an increased false positive rate for indels (relative to VarScan-Cons and high-quality GATK Unified

  3. Detailed comparison of two popular variant calling packages for exome and targeted exon studies

    Science.gov (United States)

    Adamson, Aaron W.; Neuhausen, Susan L.

    2014-01-01

    The Genome Analysis Toolkit (GATK) is commonly used for variant calling of single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) from short-read sequencing data aligned against a reference genome. There have been a number of variant calling comparisons against GATK, but an equally comprehensive comparison for VarScan not yet been performed. More specifically, we compare (1) the effects of different pre-processing steps prior to variant calling with both GATK and VarScan, (2) VarScan variants called with increasingly conservative parameters, and (3) filtered and unfiltered GATK variant calls (for both the UnifiedGenotyper and the HaplotypeCaller). Variant calling was performed on three datasets (1 targeted exon dataset and 2 exome datasets), each with approximately a dozen subjects. In most cases, pre-processing steps (e.g., indel realignment and quality score base recalibration using GATK) had only a modest impact on the variant calls, but the importance of the pre-processing steps varied between datasets and variant callers. Based upon concordance statistics presented in this study, we recommend GATK users focus on “high-quality” GATK variants by filtering out variants flagged as low-quality. We also found that running VarScan with a conservative set of parameters (referred to as “VarScan-Cons”) resulted in a reproducible list of variants, with high concordance (>97%) to high-quality variants called by the GATK UnifiedGenotyper and HaplotypeCaller. These conservative parameters result in decreased sensitivity, but the VarScan-Cons variant list could still recover 84–88% of the high-quality GATK SNPs in the exome datasets. This study also provides limited evidence that VarScan-Cons has a decreased false positive rate among novel variants (relative to high-quality GATK SNPs) and that the GATK HaplotypeCaller has an increased false positive rate for indels (relative to VarScan-Cons and high-quality GATK UnifiedGenotyper indels

  4. A cross-country comparison of the prevalence of exposure to tobacco advertisements among adolescents aged 13-15 years in 20 low and middle income countries.

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    Agaku, Israel T; Adisa, Akinyele O; Akinyamoju, Akindayo O; Agboola, Samuel O

    2013-01-01

    This study assessed the prevalence and influence of exposure to pro-tobacco advertisements among adolescents in 20 low and middle income countries (LMICs). The 2007-2008 Global Youth Tobacco Survey was analyzed for students aged 13-15 years in 20 LMICs. Overall and sex-specific prevalence of exposure to tobacco advertisements in several media, as well as the prevalence of smoking susceptibility (i.e., the lack of a firm commitment among never smokers not to smoke in the future or if offered a cigarette by a friend) were assessed. The variability of the point estimates was assessed using 95% confidence intervals (CI). Logistic regression was used to assess the effect of exposure to multiple (i.e., ≥2) pro-tobacco advertisements on current smoking, adjusting for age and sex (P advertisement sources ranged as follows: movies/videos (78.4% in Lesotho to 97.8% in Belize); television programs (48.7% in Togo to 91.7% in the Philippines); newspapers/magazines (29.5% in Togo to 89.7% in the Philippines); and outdoor community events (30.6% in Rwanda to 79.4% in the Philippines). The overall proportion of never smokers who were susceptible to cigarette smoking ranged from 3.7% in Sri Lanka to 70.1% in Kyrgyzstan. Exposure to ≥2 sources of pro-tobacco advertisements was associated with significantly increased odds of cigarette smoking among adolescents in several countries including South Africa (adjusted odds ratio, aOR = 4.11; 95% CI:2.26-7.47), Togo (aOR = 3.77; 95% CI:1.27-11.21), the Former Yugoslav Republic of Macedonia (aOR = 1.42; 95% CI:1.01-1.99), Republic of Moldova (aOR = 1.53; 95% CI:1.11-2.12), Belize (aOR = 13.95; 95% CI:1.91-102.02), Panama (aOR = 5.14; 95% CI: 2.37-11.14) and Mongolia (aOR = 1.52; 95% CI:1.19-1.94). Prevalence of exposure to various pro-tobacco advertisements was high among adolescents in the LMICs surveyed. Enhanced and sustained national efforts are needed to reduce exposure to all forms of tobacco advertising and promotional activities.

  5. Comparison of uncommon EGFR exon 21 L858R compound mutations with single mutation.

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    Peng, Liang; Song, Zhigang; Jiao, Shunchang

    2015-01-01

    Non-small-cell lung cancer with epidermal growth factor receptor (EGFR) mutation is sensitive to EGFR tyrosine kinase inhibitors (TKIs). But little is known about the response to EGFR TKIs and the prognostic role of compound mutations. This study compared the uncommon EGFR exon 21 L858R compound mutations with single mutation to characterize EGFR compound mutations and investigated their response to EGFR TKI treatment. We retrospectively screened 799 non-small-cell lung cancer patients from August 1, 2009 to June 1, 2012 by EGFR mutation testing. EGFR mutations were detected in 443 patients, with 22 (4.97%) compound mutations. Subsequently, six patients with EGFR exon 21 L858R compound mutations and 18 paired patients with single L858R mutation were well characterized. Finally, we also analyzed the EGFR TKI treatment response and patients' outcomes of compound or single L858R mutations. There was no differential treatment effect on the disease control rate and objective response rate between the L858R compound mutations and single mutation groups. No significant difference in overall survival or progression-free survival of these two groups was found by log-rank test. In conclusion, we demonstrated that no significant difference was detected in the response to EGFR TKIs and patients' outcomes in the compound and single mutation groups.

  6. POEM, A 3-dimensional exon taxonomy and patterns in untranslated exons.

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    Knapp, Keith; Chonka, Ashley; Chen, Yi-Ping Phoebe

    2008-09-20

    The existence of exons and introns has been known for thirty years. Despite this knowledge, there is a lack of formal research into the categorization of exons. Exon taxonomies used by researchers tend to be selected ad hoc or based on an information poor de-facto standard. Exons have been shown to have specific properties and functions based on among other things their location and order. These factors should play a role in the naming to increase specificity about which exon type(s) are in question. POEM (Protein Oriented Exon Monikers) is a new taxonomy focused on protein proximal exons. It integrates three dimensions of information (Global Position, Regional Position and Region), thus its exon categories are based on known statistical exon features. POEM is applied to two congruent untranslated exon datasets resulting in the following statistical properties. Using the POEM taxonomy previous wide ranging estimates of initial 5' untranslated region exons are resolved. According to our datasets, 29-36% of genes have wholly untranslated first exons. Untranslated exon containing sequences are shown to have consistently up to 6 times more 5' untranslated exons than 3' untranslated exons. Finally, three exon patterns are determined which account for 70% of untranslated exon genes. We describe a thorough three-dimensional exon taxonomy called POEM, which is biologically and statistically relevant. No previous taxonomy provides such fine grained information and yet still includes all valid information dimensions. The use of POEM will improve the accuracy of genefinder comparisons and analysis by means of a common taxonomy. It will also facilitate unambiguous communication due to its fine granularity.

  7. Sequence comparison of the dopamine receptor D4 exon III repetitive region in several species of the order Carnivora.

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    Inoue-Murayama, Miho; Matsuura, Naoto; Murayama, Yuichi; Tsubota, Toshio; Iwasaki, Toshiroh; Kitagawa, Hitoshi; Ito, Shin'ichi

    2002-08-01

    It was previously demonstrated that the dog dopamine receptor D4 (DRD4) gene is polymorphic in terms of the repeat number and/or order of 39- and 12-bp sequences located in the third exon. To examine whether or not the repetitive region is present in other species of the order Carnivora, the homologous region of DRD4 genes were sequenced in the gray wolf, raccoon dog, Asiatic black bear, common raccoon and domestic cat. In the family Canidae, the wolf had an identical sequence to that of the dog 447b allele, and a repetitive sequence similar to the dog DRD4 was also recognized in the raccoon dog. On the other hand, no obvious repeated structure was observed in the sequences of the bear, raccoon and cat.

  8. Comparison between gentamycin and exon skipping treatments to restore ryanodine receptor subtype 2 functions in mdx mouse duodenum myocytes.

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    Dabertrand, Fabrice; Mironneau, Jean; Henaff, Morgana; Macrez, Nathalie; Morel, Jean-Luc

    2010-02-25

    In Duchenne muscular dystrophy, a stop-codon mutation in the dystrophin gene induces an impairment of skeletal and smooth muscles contraction. In duodenum from mdx mouse, the disease model, the decrease of contractility was linked with the decrease of calcium signals encoded by ryanodine receptor subtype 2. Aminoglycoside and antisense oligonucleotide strategies were investigated to restore calcium signalling in the mdx mouse. Mdx mice were treated by intraperitoneal injection of gentamycin or 2-O-methyl antisense ribonucleotide directed against exon 23 of dystrophin for 2 weeks. The efficiency of both therapeutic strategies was determined by the level of dystrophin protein expression. The physiological effects of both treatments on ryanodine receptor expression and function were followed by RT-PCR, western blot and calcium measurements. Fourteen days after injection of gentamycin or anti-dystrophin antisense, the expression of dystrophin was recovered in skeletal muscle from treated mdx mice. In duodenum cells, RT-PCR and western blot indicated that the expression of ryanodine receptor subtype 2 was similar in treated mice than in control mice in association with the recovery of caffeine-induced Ca(2+) response. No significant difference was observed in the ryanodine subtype 3-dependent spontaneous Ca(2+) oscillations in untreated and treated mice. Conclusions - these results may help to explain the efficiency of aminoglycoside and anti-dystrophin antisense treatments in smooth muscle. Both treatments could be an interesting therapeutic option to restore smooth muscle contraction in patients with Duchenne muscular dystrophy.

  9. The comparison of EGFR-TKI failure modes and subsequent management between exon 19 deletion and exon 21 L858R mutation in advanced non-small-cell lung cancer.

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    Zhang, Yaxiong; Chen, Gang; Chen, Xi; Fang, Wenfeng; Gao, Fei; Yang, Yunpeng; Zhao, Yuanyuan; Ma, Yuxiang; Hong, Shaodong; Zhang, Zhonghan; Miao, Siyu; Wu, Manli; Huang, Xiaodan; Luo, Youli; Zhou, Cong; Gong, Run; Huang, Yan; Chen, Likun; Zhou, Ningning; Zhao, Hongyun; Zhang, Li

    2017-01-01

    Background: Advanced non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 19 deletion (19 Del) and exon 21 L858R mutation (L858R) might be distinct diseases. Therefore, it is necessary to take EGFR mutation subgroups into consideration for making choices of subsequent treatment after tyrosine kinase inhibitors (TKIs) failure. Patients and methods : 174 patients who developed to EGFR-TKI failure were categorized into three cohorts of dramatic progression, gradual progression and local progression. Chi-square was used to compare the distribution of failure modes between 19 Del and L858R. Kaplan-Meier method and Cox Regression were performed for analyses of survival in different subsequent treatments. Results: The distribution of EGFR-TKI failure modes showed no significant difference between 19 Del and L858R. Patients in gradual progression had a longer progression-free survival (PFS) and overall survival (OS) compared with other failure modes in whole population, 19 Del cohort and L858R cohort. 19 Del patients with dramatic progression would obtain survival benefit from chemotherapy, while those with gradual progression got no survival benefit neither from chemotherapy nor previous TKI continuation. However, patients with dramatic or gradual progression would benefit from previous TKI continuation in L858R cohort. Conclusion: For advanced EGFR-positive NSCLC patients with acquired resistance to EGFR-TKI, subsequent treatment should be personalized according to EGFR-TKI failure modes & EGFR mutation subtypes.

  10. Periodicity of DNA in exons

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    Kinghorn Brian

    2004-08-01

    Full Text Available Abstract Background The periodic pattern of DNA in exons is a known phenomenon. It was suggested that one of the initial causes of periodicity could be the universal (RNYnpattern (R = A or G, Y = C or U, N = any base of ancient RNA. Two major questions were addressed in this paper. Firstly, the cause of DNA periodicity, which was investigated by comparisons between real and simulated coding sequences. Secondly, quantification of DNA periodicity was made using an evolutionary algorithm, which was not previously used for such purposes. Results We have shown that simulated coding sequences, which were composed using codon usage frequencies only, demonstrate DNA periodicity very similar to the observed in real exons. It was also found that DNA periodicity disappears in the simulated sequences, when the frequencies of codons become equal. Frequencies of the nucleotides (and the dinucleotide AG at each location along phase 0 exons were calculated for C. elegans, D. melanogaster and H. sapiens. Two models were used to fit these data, with the key objective of describing periodicity. Both of the models showed that the best-fit curves closely matched the actual data points. The first dynamic period determination model consistently generated a value, which was very close to the period equal to 3 nucleotides. The second fixed period model, as expected, kept the period exactly equal to 3 and did not detract from its goodness of fit. Conclusions Conclusion can be drawn that DNA periodicity in exons is determined by codon usage frequencies. It is essential to differentiate between DNA periodicity itself, and the length of the period equal to 3. Periodicity itself is a result of certain combinations of codons with different frequencies typical for a species. The length of period equal to 3, instead, is caused by the triplet nature of genetic code. The models and evolutionary algorithm used for characterising DNA periodicity are proven to be an effective tool

  11. The first PTPN1 1 mutations in hotspot exons reported in Moroccan children with Noonan syndrome and comparison of mutation rate to previous studies.

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    El Bouchikhi, Ihssane; Samri, Imane; Iraqui Houssaini, Mohammed; Trhanint, Saaid; Bouguenouch, Laila; Sayel, Hanane; Hida, Moustapha; Atmani, Samir; Ouldim, Karim

    2015-01-01

    Noonan syndrome is an autosomal dominant disorder with an incidence of 1/1000-2500. It results from protein-tyrosine phosphatase, nonreceptor type 11 (PTPN11) mutations in roughly 50% of cases. Mutational screening of PTPN11 has been carried out in different populations. Thus, the aim of this study was to screen, for the first time, PTPN11 mutations in a series of Moroccan Noonan syndrome patients. We used bidirectional sequencing of exons 3 and 8, considered as PTPN11 mutation hot spots, and then compared the rate of mutational events of these exons between different populations using chi-square and Fisher's exact tests. We detected 3 heterozygous mutations (Asp6lGly, Tyr63Cys, and Asn308Ser) in 4 individuals of 16 sporadic patients (25%). The rate of mutation in our cohort did not differ from that of other populations. However, we found significant differences in the mutation rate of exon 8 between one Japanese cohort and some populations, which requires more investigations to be explained. The present study allowed identification of mutations clustered in exons 3 and 8 of the PTPN11 gene in a Moroccan Noonan syndrome cohort and enabled us to give appropriate genetic counseling to the mutation-positive patients.

  12. Comparison of Nasal Epithelial Smoking-Induced Gene Expression on Affymetrix Exon 1.0 and Gene 1.0 ST Arrays

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    Xiaoling Zhang

    2013-01-01

    Full Text Available We have previously defined the impact of tobacco smoking on nasal epithelium gene expression using Affymetrix Exon 1.0 ST arrays. In this paper, we compared the performance of the Affymetrix GeneChip Human Gene 1.0 ST array with the Human Exon 1.0 ST array for detecting nasal smoking-related gene expression changes. RNA collected from the nasal epithelium of five current smokers and five never smokers was hybridized to both arrays. While the intersample correlation within each array platform was relatively higher in the Gene array than that in the Exon array, the majority of the genes most changed by smoking were tightly correlated between platforms. Although neither array dataset was powered to detect differentially expressed genes (DEGs at a false discovery rate (FDR <0.05, we identified more DEGs than expected by chance using the Gene ST array. These findings suggest that while both platforms show a high degree of correlation for detecting smoking-induced differential gene expression changes, the Gene ST array may be a more cost-effective platform in a clinical setting for gene-level genomewide expression profiling and an effective tool for exploring the host response to cigarette smoking and other inhaled toxins.

  13. Comparison of gefitinib as first- and second-line therapy for advanced lung adenocarcinoma patients with positive exon 21 or 19 del epidermal growth factor receptor mutation

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    Patel N

    2017-06-01

    Full Text Available Nishant Patel,1 Pingping Wu,2 Haijun Zhang1 1Department of Oncology, Zhongda Hospital, Medical School, Southeast University, 2Department of Medical Oncology, Jiangsu Cancer Hospital, Nanjing, People’s Republic of China Objectives: Gefitinib, a tyrosine kinase inhibitor (TKI targeting epidermal growth factor receptor (EGFR, shows excellent clinical benefit in treating advanced non-small-cell lung cancer (NSCLC. The aim of this study was to compare the efficacy and toxicity of gefitinib as first-line therapy and second-line therapy for advanced lung adenocarcinoma patients with positive exon 21 (L858R or exon 19 deletion of EGFR mutation. Methods: We retrospectively analyzed the clinical data of 60 EGFR-mutated advanced lung adenocarcinoma patients from July 2011 to November 2015 who have received oral gefitinib 250 mg once daily. Gefitinib was taken until disease progression, intolerable toxicity or death. Results: After a median follow-up of 792 days, one death had occurred. Among the 59 patients who survived, 17 patients progressed. Overall, the median progression-free survival (mPFS was 10 months (95% confidence interval [CI]: 7.53–12.46 months, p<0.05. The response rate (RR and disease control rate (DCR were 33.33% and 71.66%, respectively. However, there was longer mPFS in the first line-therapy than that in the second-line therapy: in the first-line gefitinib therapy, mPFS was 12 months among 41 patients (95% CI: 9.58–14.41 months, p<0.05, and in the second-line therapy, mPFS was 7 months among 19 patients (95% CI: 1.31–12.68 months, p<0.05. Furthermore, in subgroup analyses examining different EGFR mutation types, we noted that mPFS was significantly longer for patients with exon 19 deletion than for those with positive exon 21in both the first-line therapy and second-line therapy. Conclusion: Patients with advance lung adenocarcinoma who were selected by positive exon 21 or 19 deletion mutations had significantly longer m

  14. THE EFFECT OF 8-WEEK CORE TRAINING ON 100 M BACKSTROKE SWIMMING PERFORMANCE ON 13-15 AGE MALE SWIMMERS

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    Ahmet Gönener

    2017-12-01

    Full Text Available In this study, It was aimed to investigate the effect of 8-week core training on backstroke swimming style 100 m performance in male swimming group of 13-15 age group. 24 athletes from the Gebze Gençlerbirliği Swimming Club participated in this study. The groups are divided into experiment group and control group randomly as 12 persons per group. During the study, the experimental group participated in core training in addition to swimming for 3 days a week. Control group just participated in swimming training during the survey. The exercises were designed as strength training for core muscles for 8 weeks. 100 m swimming test was used as a test measurement tool. The obtained data from pre and post test comparison was analyzed with Wilcoxon Signed Ranks test in SPSS 21.0 statistical package program. When the pretest and posttest results were compared as intragroup, a statistically significant difference was found in the experimental group (p.05. As a result, it was seen that the core area trained by 8-week core training on 13-15 age male swimmers have a positive effect on 100 m backstroke style performance.

  15. The Relationship between Bible Reading and Attitude toward Substance Use among 13-15 Year Olds

    Science.gov (United States)

    Francis, Leslie J.

    2002-01-01

    The relationship between Bible reading and attitude toward drug use is examined among a sample of 25,888 teenagers, 13-15 year olds, throughout England and Wales. Information about sex, age, personality, belief in God, and church attendance was also considered. The conclusion is that Bible reading makes a small but significant contribution to…

  16. Third Forest Vegetation Simulator Conference; 2007 February 13-15; Fort Collins, CO

    Science.gov (United States)

    Robert N. Havis; Nicholas L. Crookston

    2008-01-01

    The Forest Vegetation Simulator (FVS) is a suite of computer modeling tools for predicting the long-term effects of alternative forest management actions. FVS was developed in the early 1980s and is used throughout the United Sates and British Columbia. The Third FVS conference, held February 13-15, 2007, in Fort Collins Colorado, contains 20 papers. They describe the...

  17. Structural insights into the exon junction complex.

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    Le Hir, Hervé; Andersen, Gregers Rom

    2008-02-01

    In higher eukaryotes, the exon junction complex is loaded onto spliced mRNAs at a precise position upstream of exon junctions, where it remains during nuclear export and cytoplasmic localisation until it is removed during the first translation round. The exon junction core complex consists of four proteins that form a dynamic binding platform for a variety of peripheral factors involved in mRNA metabolism. In the complex, mRNA binding is mediated by the DEAD-box protein eIF4AIII, and inhibition of its ATPase activity forms the mechanistic basis for the long-term stability of the complex. Recent crystal structures of the exon junction complex and eIF4AIII have provided the structural framework for investigating the function of the eIF4AIII ATPase and for localisation of surface patches involved in binding peripheral factors. Additionally, by comparison with the structure of a second DEAD-box protein also bound to RNA and ATP, general principles for the ATPase and unwinding/mRNP remodelling activities for this important group of enzymes can be proposed on the basis of atomic structures.

  18. [Photobiological properties of 13,15-N-(carboxymethyl)- and 13,15-N-(2-carboxyethyl)cycloimide derivatives of chlorin p6].

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    Feofanov, A V; Nazarova, A I; Karmakova, T A; Pliutinskaia, A D; Grishin, A I; Iakubovskaia, R I; Lebedeva, V S; Ruziev, R D; Mironov, A F; Maurizot, J C; Vigny, P

    2004-01-01

    Lipophilic derivatives of chlorin p6, 13,15-N-(carboxymethyl)cycloimide methyl ester (CIC1) and 13,15-N-(2-carboxyethyl)cycloimide methyl ester (CIC2), were shown to absorb light in 710 nm region and to be efficient IR photosensitizers. They exhibit similar phototoxicities on the cells of A549 human lung adenocarcinoma, which are 40- and 100-fold higher than those of chlorin p6 and the clinically used Photogem, respectively, and are not toxic in the absence of light irradiation. The confocal spectral imaging technique allowed us to demonstrate that the high phototoxicity of CIC1 and CIC2 is due to their ability to readily penetrate to cells and to be bound to the cell membranes and lipid-containing structures in the monomeric photoactive form. Under the irradiation, the membrane-bound CIC1 and CIC2 are characterized by high quantum yields of singlet oxygen generation (0.6 and 0.65, respectively) and the inability to produce hydroxyl radicals. A 1.5-microM content of CIC1 and CIC2 in the incubation medium provides for their average cytoplasmic concentrations of 21 and 16.5 microM, respectively. The incubation times to achieve 50% level of maximum accumulation for CIC1 and CIC2 in A549 cells are 30 +/- 6 and 24 +/- 12 min, and the times for 50% release of the dyes from the cells are 17 +/- 4 and 50 +/- 10 min, respectively. A diffuse distribution with the predominant accumulation in the membranes of the Golgi apparatus and mitochondria is characteristic of both CIC2 and CIC1, whereas, in addition, CIC1 is considerably accumulated in lipid droplets (cellular organelles responsible for the storage and metabolism of neutral lipids and steryl esters). Our results demonstrate that changes in the structure of the imide substituent could affect the intracellular localization and the rate of release of chlorin p6 cycloimide derivatives from cells while preserving their high photodynamic activity.

  19. Nano structures of group 13-15 mixed heptamer clusters: a computational study.

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    Mohajeri, Afshan; Ebadi, Mahsa

    2012-05-10

    The structural and thermodynamic characteristics of lowest-energy structures of group 13-15 mixed heptamers in two distinct series [(HM)(k)(HM')(l)(NH)(7)] (M, M' = B, Al, Ga and k + l = 7) and [(HGa)(7)(YH)(m)(Y'H)(n)] (Y,Y' = N, P, As and m + n = 7) have been systematically investigated using the density functional approach. Our main goal is to get knowledge of the preferential bonding patterns of the first three rows of group 13-15 elements for the construction of mixed heptameric clusters. Structural parameters, thermodynamic properties of oligomerization reaction, band gaps, and dipole moments of the 18 lowest-energy structures of the studied heptamers in each series are compared to their corresponding binary parents, that is, [(HM)(7)(NH)(7)] and [(HGa)(7)(YH)(7)]. The stability of different isomer structures is discussed to reveal the competitiveness of group 13 and 15 bonding. Mixed heptamers are predicted to be thermodynamically more stable compared to a mixture of monomers. However, the favorability for the generation of mixed heptamers strongly depends on the nature of inserted metal and nonmetal pairs of group 13-15. Moreover, it is found that among all studied heptamers the smaller band gaps correspond to arsenic containing species which are close to the semiconducting regime, around 4.62-4.98 eV.

  20. Committee Report of the BEPC-II Project Design Review May 13-15, 2002, SLAC

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    Kawakami, Traci M.

    2002-08-26

    As part of the US-China Cooperative Program in High Energy Physics for the year 2002, a BEPC-II Upgrade Review meeting was held at SLAC, May 13-15, 2002. The upgrade is aimed at improving the luminosity and performance of the BEPC facility at IHEP in Beijing, China with major upgrades to the injector linac, storage ring, and detector. This review addresses mainly the accelerator related issues. Prior to the review, an updated Draft Design Report was made available to the review team. Most important technical change since April 2001 has been a change from a single-ring configuration to a doublering. The goal of the review is to determine whether BEPC-II, if built as described, will meet the operations and physics goals. The charge to the review team is attached as Appendix A.

  1. Flood of June 13-15, 1981, in the Blanchard River basin, northwestern Ohio

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    Webber, E.E.

    1982-01-01

    The flood of June 13-15, 1981, in the Blanchard River basin in northwestern Ohio caused major damage in Findlay, Ottawa, and adjacent rural areas. Approximately 25 percent of Findlay and 55 percent of Ottawa were flooded. Estimated crop damage was $12 million in Hancock Country, $7 million in Wyandot Country, and $3 million in Putnam Country. During the flood, the maximum gage height of the Blanchard River at the U.S. Geological Survey gaging station near Findlay was 17.43 feet, gage datum. This was 0.67 foot higher than the previous peak of 16.76 feet, February 11, 1959. The corresponding peak discharge, 13,000 cubic feet per second, was estimated to have a recurrence interval of 50 years. Recurrence intervals of peak flows at other locations on the Blanchard River and on tributary streams were estimated to range from 25 to more than 100 years.

  2. PRO-HEALTH BEHAVIOURS AND BELIEF IN ONESELF AMONG 13-15-YEAR OLD TEENAGERS LIVING IN BIALA PODLASKA

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    Grad Rafal

    2013-04-01

    Full Text Available The aim of the research was to get information about pro-health behaviours and belief in oneself among 13-15-year old teenagers living in Biała Podlaska. The research included 229 pupils from three Grammar Schools (115 girls and 114 boys. The range of respondents’ age varied from 13 to 15 years old. The research tool was two anonymous questionnaires. The first questionnaire contained questions concerning teenagers’ belief in themselves and the efficacy of their activities (10 statements. The second questionnaire referred to the pro-health behaviours and it consisted of 14 situations described in the form of multiple choice questions. It was shown that Grammar School goers higly evaluated their own sense of efficacy (31,2 points. Girls (30,39 points in comparison to boys (32,05 evaluated their efficacy lower by 1,66 points. Grammar School goers in 85,7% of cases choose healthy behaviours and girls are the group which reach for such behaviours more often.

  3. [Body mass, self-esteem and life satisfaction in adolescents aged 13-15 years].

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    Tabak, Izabela; Mazur, Joanna; Oblacińska, Anna; Jodkowska, Maria

    2007-01-01

    The aim of the study was to analyse the relationships between objective body mass index and subjective body image, life satisfaction and self-esteem of adolescents. the study was carried in 5 regions in Poland, on the sample of over 8,000 pupils aged 13-15 yrs, from randomly chosen 112 lower secondary schools. School nurses measured the height and weight of pupils, calculated the BMI and qualified overweight pupils (BMI> or =85 percentile) to the obese group (n = 953). Matching gender and age, from the rest of pupils, they found the non-obese group with BMI between 15 and 75 percentile (n = 953). Pupils from both groups participated in a questionnaire study containing the Rosenberg Self-Esteem Scale, Cantril ladder and Stunkard Body Figure Perception Questionnaire. Hierarchic regression analyses and structural equation models were calculated. in the obese group the percentage of pupils satisfied with their life was lower (76% vs 82%, pesteem higher than in the non-obese (37% vs 23%, pesteem was subjective body image, and not the objective body mass index. Objective body mass (BMI) determined the body image and relationship between BMI and life satisfaction or self-esteem of adolescents was only indirect. change of subjective body image in obese adolescents is a chance for improving their quality of life and in consequence undertaking effective struggle with obesity.

  4. Operations managers conference: summary of proceedings. [Carmel, California, Feb. 13-15, 1979

    Energy Technology Data Exchange (ETDEWEB)

    None

    1980-02-01

    The fifth AESOP Operations Managers Conference was held in Carmel, California on February 13-15, 1979. The keynote address dealt with the changing role of the computer operations manager. Representatives presented contrasting approaches to managing the procurement and installation of new computer systems. The advantages and disadvantages of maintaining strong internal management support and good communication with the vendor and of increasing the involvement of broad-based planning committees and operations personnel in the computer procurement process were discussed. Two promising management programs, the PEER and 10/40 systems proved effective in improving employee morale by reducing job fragmentation, increasing internal mobility and modifying the work week. The topic of unionization of operations personnel was also discussed. The uneasy relationship between computers and the energy shortage was also considered. Techniques for avoiding computer problems caused by blackouts and brownouts were presented. The difficulty of conserving power at a computer facility was also discussed. Presentations concerning the President's committee on ADP reorganizaion and methods for predicting trends in computer innovation were made. Abstracts and two-page summaries of the papers given are included in this report.

  5. Proc. of the sixteenth symposium on energy engineering sciences, May 13-15, 1998, Argonne, IL.

    Energy Technology Data Exchange (ETDEWEB)

    None

    1998-05-13

    This Proceedings Volume includes the technical papers that were presented during the Sixteenth Symposium on Energy Engineering Sciences on May 13--15, 1998, at Argonne National Laboratory, Argonne, Illinois. The Symposium was structured into eight technical sessions, which included 30 individual presentations followed by discussion and interaction with the audience. A list of participants is appended to this volume. The DOE Office of Basic Energy Sciences (BES), of which Engineering Research is a component program, is responsible for the long-term, mission-oriented research in the Department. The Office has prime responsibility for establishing the basic scientific foundation upon which the Nation's future energy options will be identified, developed, and built. BES is committed to the generation of new knowledge necessary to solve present and future problems regarding energy exploration, production, conversion, and utilization, while maintaining respect for the environment. Consistent with the DOE/BES mission, the Engineering Research Program is charged with the identification, initiation, and management of fundamental research on broad, generic topics addressing energy-related engineering problems. Its stated goals are to improve and extend the body of knowledge underlying current engineering practice so as to create new options for enhancing energy savings and production, prolonging the useful life of energy-related structures and equipment, and developing advanced manufacturing technologies and materials processing. The program emphasis is on reducing costs through improved industrial production and performance and expanding the nation's store of fundamental knowledge for solving anticipated and unforeseen engineering problems in energy technologies. To achieve these goals, the Engineering Research Program supports approximately 130 research projects covering a broad spectrum of topics that cut across traditional engineering disciplines. The program

  6. Proficiency Assessment of Male Volleyball Teams of the 13-15-Year Age Group at Estonian Championships

    Science.gov (United States)

    Stamm, Meelis; Stamm, Raini; Koskel, Sade

    2008-01-01

    Study aim: Assessment of feasibility of using own computer software "Game" at competitions. Material and methods: The data were collected during Estonian championships in 2006 for male volleyball teams of the 13-15-years age group (n = 8). In all games, the performance of both teams was recorded in parallel with two computers. A total of…

  7. Splicing of designer exons informs a biophysical model for exon definition.

    Science.gov (United States)

    Arias, Mauricio A; Lubkin, Ashira; Chasin, Lawrence A

    2015-02-01

    Pre-mRNA molecules in humans contain mostly short internal exons flanked by longer introns. To explain the removal of such introns, exon recognition instead of intron recognition has been proposed. We studied this exon definition using designer exons (DEs) made up of three prototype modules of our own design: an exonic splicing enhancer (ESE), an exonic splicing silencer (ESS), and a Reference Sequence (R) predicted to be neither. Each DE was examined as the central exon in a three-exon minigene. DEs made of R modules showed a sharp size dependence, with exons shorter than 14 nt and longer than 174 nt splicing poorly. Changing the strengths of the splice sites improved longer exon splicing but worsened shorter exon splicing, effectively displacing the curve to the right. For the ESE we found, unexpectedly, that its enhancement efficiency was independent of its position within the exon. For the ESS we found a step-wise positional increase in its effects; it was most effective at the 3' end of the exon. To apply these results quantitatively, we developed a biophysical model for exon definition of internal exons undergoing cotranscriptional splicing. This model features commitment to inclusion before the downstream exon is synthesized and competition between skipping and inclusion fates afterward. Collision of both exon ends to form an exon definition complex was incorporated to account for the effect of size; ESE/ESS effects were modeled on the basis of stabilization/destabilization. This model accurately predicted the outcome of independent experiments on more complex DEs that combined ESEs and ESSs. © 2015 Arias et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  8. Unusual intron conservation near tissue-regulated exons found by splicing microarrays.

    Directory of Open Access Journals (Sweden)

    Charles W Sugnet

    2006-01-01

    Full Text Available Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5' splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families.

  9. Exon prediction in eucaryotic genomes.

    Science.gov (United States)

    Vignal, L; d'Aubenton-Carafa, Y; Lisacek, F; Mephu Ngüifo, E; Rouzé, P; Quinqueton, J; Thermes, C

    1996-01-01

    Two independent computer systems, NetPlantGene and AMELIE, dedicated to the identification of splice sites in plant and human genomes, respectively, are introduced here. Both methods were designed in relation to experimental work; they rely on automatically generated rules involving the nucleotide content of sequences regardless of the coding properties of exons. The specificity of plant sequences as considered in NetPlantGene is shown to enhance the quality of detection as opposed to general methods such as GRAIL. A scanning model of the acceptor site recognition is being simulated by AMELIE leading to a relatively accurate selection process of sites.

  10. ExonMiner: Web service for analysis of GeneChip Exon array data

    Directory of Open Access Journals (Sweden)

    Imoto Seiya

    2008-11-01

    Full Text Available Abstract Background Some splicing isoform-specific transcriptional regulations are related to disease. Therefore, detection of disease specific splice variations is the first step for finding disease specific transcriptional regulations. Affymetrix Human Exon 1.0 ST Array can measure exon-level expression profiles that are suitable to find differentially expressed exons in genome-wide scale. However, exon array produces massive datasets that are more than we can handle and analyze on personal computer. Results We have developed ExonMiner that is the first all-in-one web service for analysis of exon array data to detect transcripts that have significantly different splicing patterns in two cells, e.g. normal and cancer cells. ExonMiner can perform the following analyses: (1 data normalization, (2 statistical analysis based on two-way ANOVA, (3 finding transcripts with significantly different splice patterns, (4 efficient visualization based on heatmaps and barplots, and (5 meta-analysis to detect exon level biomarkers. We implemented ExonMiner on a supercomputer system in order to perform genome-wide analysis for more than 300,000 transcripts in exon array data, which has the potential to reveal the aberrant splice variations in cancer cells as exon level biomarkers. Conclusion ExonMiner is well suited for analysis of exon array data and does not require any installation of software except for internet browsers. What all users need to do is to access the ExonMiner URL http://ae.hgc.jp/exonminer. Users can analyze full dataset of exon array data within hours by high-level statistical analysis with sound theoretical basis that finds aberrant splice variants as biomarkers.

  11. Solid-State Precursor Routes to Iii-V Type Electronic (13-15) and Magnetic (3-15) Materials

    Science.gov (United States)

    Treece, Randolph Edward

    An interest in electronic materials has led me to investigate new synthetic approaches to "III-V" type semiconducting (13-15, current IUPAC designation for B and N groups in the Periodic Table) and magnetic (3-15) compounds. It is now possible to prepare binary (GaAs and GdP) and ternary mixed-metal (Al_ {x}Ga_{1-x}As) and mixed -pnictide (rm GaP_{x}As_ {1-x}) compounds in seconds from rapid, low-temperature-initiated metathesis reactions between a metal (III) trihalide and a trisodium pnictide, exemplified by MX_3 + Na_3Pn to MPn + 3 NaX, where M is Al, Ga, In, (Al,Ga), or a lanthanide; X is F, Cl, or I; and Pn is P, As, Sb, or (P,As). The precursors are mixed together in a dry box and ignited by light grinding with a mortar and pestle, or by brief, local heating from a hot filament. These reactions are very exothermic (calculated Delta H_{rxn} (GaAs) = -138 kcal/mol) and typically reach temperatures in excess of 700^circ C within seconds of initiation, and cool to room temperature in less than 15 seconds. The byproduct sodium halide is simply removed by washing with water or methanol, leaving the polycrystalline "III-V" products. In spite of the brief and explosive nature of these reactions, there are some clues as to the initiation and reaction processes. The initial steps in the reactions appear to be the decomposition of the precursors and the formation of the sodium halides. Since initiation occurs when one of the precursors goes through a phase change, such as melting or decomposing, the melting points of the GaX_3 precursors are good indicators of how easily the reactions can be initiated. Physical methods have been employed to characterize the bulk products. The 13-15 materials have been investigated using MAS-NMR spectroscopy. The 3-15 compounds have been extensively studied using magnetometry, and the effectiveness of the metathesis processes was examined by comparing the ignition reaction products to those generated by direct combination of the elements

  12. Differential transcription of exon 1 of the human c-fms gene in placental trophoblasts and monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Visvader, J.; Verma, I.M. (Salk Inst. for Biological Studies, San Diego, CA (USA))

    1989-03-01

    Structural analysis of the 5' end of the human c-fms gene revealed that a large intron of about 25 kilobases separates an upstream noncoding exon (exon 1) from the signal peptide-containing exon (exon 2). Northern (RNA) blot analysis, S1 nuclease mapping, and primer extensions showed that exon 1 is transcribed in placenta but not in cells of the monocytic lineage. This is due to the differential usage or promoters, separated by approximately 25 kilobases, in cell-specific manner. One major c-fms transcript was observed in U-937 cells, whereas multiple initiation sites for transcription appeared to be utilized in placental cells. Nucleotide sequence comparisons showed that the 3' end of the human platelet-derived growth factor receptor gene lies approximately 350 base pairs upstream of the major initiation sites for c-fms transcription in placental trophoblasts.

  13. Differential transcription of exon 1 of the human c-fms gene in placental trophoblasts and monocytes.

    Science.gov (United States)

    Visvader, J; Verma, I M

    1989-01-01

    Structural analysis of the 5' end of the human c-fms gene revealed that a large intron of about 25 kilobases separates an upstream noncoding exon (exon 1) from the signal peptide-containing exon (exon 2). Northern (RNA) blot analysis, S1 nuclease mapping, and primer extensions showed that exon 1 is transcribed in placenta but not in cells of the monocytic lineage. This is due to the differential usage of promoters, separated by approximately 25 kilobases, in a cell-specific manner. One major c-fms transcript was observed in U-937 cells, whereas multiple initiation sites for transcription appeared to be utilized in placental cells. Nucleotide sequence comparisons showed that the 3' end of the human platelet-derived growth factor receptor gene lies approximately 350 base pairs upstream of the major initiation sites for c-fms transcription in placental trophoblasts. Images PMID:2524648

  14. Overweight and lifestyle among 13-15 year olds: a cross-sectional study in northern Sweden.

    Science.gov (United States)

    Vaezghasemi, Masoud; Lindkvist, Marie; Ivarsson, Anneli; Eurenius, Eva

    2012-05-01

    To increase knowledge of self-rated health and lifestyle in relation to overweight/obesity among 13-15 year olds in northern Sweden. All 6768 13-15 year olds in nine out of 15 municipalities in Västerbotten County were asked to complete a cross-sectional school-based on-line survey in 2007. Eighty-two per cent participated in the study. Responses were considered reliable for 74% of the participants (2517 boys/2470 girls). The survey addressed demography, self-rated health, self-reported weight, height, and lifestyle characteristics. Simple and multiple logistic regression analyses were used. Overweight/obesity (ISO body mass index ≥25 kg/m(2)) was more prevalent among boys (20%) than girls (11%), but more girls (19%) than boys (9%) reported fair or bad health. Overweight/obese boys and girls were more often physically inactive. For the boys, overweight/obesity was also associated with skipping breakfast, insufficient tooth brushing, and using snuff. For the girls, overweight/obesity was also associated with living with one parent and more television watching. Boys reported healthier habits concerning sleep duration, physical activity, eating breakfast, and smoking compared to the girls. On the other hand, girls reported better dietary and tooth brushing habits. This study uncovered two alarming findings: a fifth of the boys were overweight/obese and a fifth of the girls reported fair or bad health. Girls living with a single parent and boys and girls with unhealthy lifestyles were more likely to be overweight. Our findings emphasise the need for developing and implementing effective health promotion strategies for school-aged children to prevent an overweight and obesity epidemic that could continue into adulthood.

  15. Exposure to Tobacco Advertising and Promotion among School Children Aged 13-15 in Vietnam - an Overview from GYTS 2014.

    Science.gov (United States)

    Long, Tran Khanh; Son, Phung Xuan; Giang, Kim Bao; Hai, Phan Thi; Huyen, Doan Thi Thu; Khue, Luong Ngoc; Nga, Pham Thi Quynh; Lam, Nguyen Tuan; Minh, Hoang Van; Huong, Le Thi Thanh

    2016-01-01

    Evidence shows that tobacco advertising and promotion activities may increase tobacco consumption and usage, especially in youth. Despite the regulation on prohibiting advertisement of any tobacco product, tobacco advertisement and promotion activities are still common in Vietnam. This article presents current exposure to tobacco advertising and promotion (TAP) among school children aged 13 to 15 years in Vietnam in 2014 and potential influencing factors. Data from the Global Youth Tobacco Survey 2014 in Vietnam covering 3,430 school aged children were used. Both descriptive and analytical statistics were carried out with Stata 13 statistical software. Binary logistic regression was applied to explain the exposure to TAP among youth and examine relationships with individual factors. A significance level of pexposure to at least 1 type of tobacco advertising or promotion. Wearing or otherwise using products related to tobacco was the most exposure TAP type reported by students (22.3%). The internet (22.1), points of sales (19.2) and social events (11.5) were three places that students aged 13-15 frequently were exposed to TAP. Binary logistic results showed that gender (female vs male) (OR = 0.61, 95%CI: 0.52 - 0.71), susceptibility to smoking (OR = 2.12, 95%CI: 1.53 - 2.92), closest friends' smoked (OR = 1.43, 95%CI: 1.2 - 1.7) and parents smoking status (OR = 2.83, 95%CI: 1.6 - 5.01) were significantly associated with TAP exposure among school-aged children. The research findings should contribute to effective implementation of measures for preventing and controlling tobacco use among students aged 13-15 in Viet Nam.

  16. Tobacco Use Among Students Aged 13-15 Years in South Korea: The 2013 Global Youth Tobacco Survey.

    Science.gov (United States)

    Choi, Sunhye; Kim, Yoonjung; Lee, Jihye; Kashiwabara, Mina; Oh, Kyungwon

    2017-01-01

    We examined the prevalence of tobacco use and exposure to secondhand smoke among middle-school students in Korea using the Global Youth Tobacco Survey (GYTS) in 2013. The GYTS in Korea was conducted between July and August 2013 by the Korea Centers for Disease Control and Prevention. Data were collected using a self-administered anonymous questionnaire from a nationally representative sample of middle-school students aged 13-15 years in sampled classrooms. The GYTS in Korea was completed by 4235 students aged 13-15 years in 43 middle schools. Approximately one in five of the students (17.8%) reported that they had tried cigarettes in the past, while 5.2% reported currently being cigarette smokers. Current cigarette smoking was higher in boys (7.5%) than in girls (2.6%). Of the students, 29.7% had been exposed to secondhand smoke at home, 47.4% inside enclosed public places, and 53.9% in outdoor public places. Of the current cigarette smokers, 25.7% bought their cigarettes from a store despite a law prohibiting this. Additionally, 58.0% of students noticed point-of-sale tobacco advertisements or promotions, 66.8% of current cigarette smokers wanted to stop smoking, and 70.9% of students had been taught about the dangers of tobacco use in school. These findings provide an opportunity to develop, implement, and evaluate a comprehensive tobacco control policy. The results suggest that youth have relatively easy access to cigarettes and are regularly exposed to secondhand smoke in public places, as well as to point-of-sale tobacco advertisements and promotions. Strict enforcement of the ban on tobacco sales to youth, expanding smoke-free areas, and advertising bans are needed to reduce tobacco use among youth.

  17. SR proteins induce alternative exon skipping through their activities on the flanking constitutive exons.

    Science.gov (United States)

    Han, Joonhee; Ding, Jian-Hua; Byeon, Cheol W; Kim, Jee H; Hertel, Klemens J; Jeong, Sunjoo; Fu, Xiang-Dong

    2011-02-01

    SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKIIδ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKIIδ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKIIδ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.

  18. Maturity-associated variation in sport-specific skills of youth soccer players aged 13-15 years.

    Science.gov (United States)

    Malina, Robert M; Cumming, Sean P; Kontos, Anthony P; Eisenmann, Joey C; Ribeiro, Basil; Aroso, João

    2005-05-01

    The aim of this study was to estimate the contribution of experience, body size and maturity status to variation in sport-specific skills of adolescent soccer players. The participants were 69 players aged 13.2-15.1 years from three clubs that competed in the highest division for their age group. Height and body mass were measured and stage of pubic hair development was assessed at clinical examination. Years of experience in football was obtained at interview. Six football skill tests were administered: ball control with the body, ball control with the head, dribbling with a pass, dribbling speed, shooting accuracy and passing accuracy. Multiple linear regression analysis was used to estimate the relative contributions of age, stage of sexual maturity, height, body mass and years of formal training in soccer to the six skill tests. Age, experience, body size and stage of puberty contributed significantly but in different combinations to the variance in four of the six skill tests: dribbling with a pass (21%; age, stage of maturity), ball control with the head (14%; stage of maturity, height, body height x body mass interaction), ball control with the body (13%; stage of maturity, years of training) and shooting accuracy (8%; stage of maturity, height; borderline significance, P = 0.06). There were no significant predictors for the tests of dribbling speed and passing accuracy. In conclusion, age, experience, body size and stage of puberty contributed relatively little to variation in performance in four of the six soccer-specific skill tests in adolescent footballers aged 13-15 years.

  19. A Brassica exon array for whole-transcript gene expression profiling.

    Directory of Open Access Journals (Sweden)

    Christopher G Love

    Full Text Available Affymetrix GeneChip® arrays are used widely to study transcriptional changes in response to developmental and environmental stimuli. GeneChip® arrays comprise multiple 25-mer oligonucleotide probes per gene and retain certain advantages over direct sequencing. For plants, there are several public GeneChip® arrays whose probes are localised primarily in 3' exons. Plant whole-transcript (WT GeneChip® arrays are not yet publicly available, although WT resolution is needed to study complex crop genomes such as Brassica, which are typified by segmental duplications containing paralogous genes and/or allopolyploidy. Available sequence data were sampled from the Brassica A and C genomes, and 142,997 gene models identified. The assembled gene models were then used to establish a comprehensive public WT exon array for transcriptomics studies. The Affymetrix GeneChip® Brassica Exon 1.0 ST Array is a 5 µM feature size array, containing 2.4 million 25-base oligonucleotide probes representing 135,201 gene models, with 15 probes per gene distributed among exons. Discrimination of the gene models was based on an E-value cut-off of 1E(-5, with ≤98% sequence identity. The 135 k Brassica Exon Array was validated by quantifying transcriptome differences between leaf and root tissue from a reference Brassica rapa line (R-o-18, and categorisation by Gene Ontologies (GO based on gene orthology with Arabidopsis thaliana. Technical validation involved comparison of the exon array with a 60-mer array platform using the same starting RNA samples. The 135 k Brassica Exon Array is a robust platform. All data relating to the array design and probe identities are available in the public domain and are curated within the BrassEnsembl genome viewer at http://www.brassica.info/BrassEnsembl/index.html.

  20. Diverse splicing patterns of exonized Alu elements in human tissues.

    Directory of Open Access Journals (Sweden)

    Lan Lin

    2008-10-01

    Full Text Available Exonization of Alu elements is a major mechanism for birth of new exons in primate genomes. Prior analyses of expressed sequence tags show that almost all Alu-derived exons are alternatively spliced, and the vast majority of these exons have low transcript inclusion levels. In this work, we provide genomic and experimental evidence for diverse splicing patterns of exonized Alu elements in human tissues. Using Exon array data of 330 Alu-derived exons in 11 human tissues and detailed RT-PCR analyses of 38 exons, we show that some Alu-derived exons are constitutively spliced in a broad range of human tissues, and some display strong tissue-specific switch in their transcript inclusion levels. Most of such exons are derived from ancient Alu elements in the genome. In SEPN1, mutations of which are linked to a form of congenital muscular dystrophy, the muscle-specific inclusion of an Alu-derived exon may be important for regulating SEPN1 activity in muscle. Realtime qPCR analysis of this SEPN1 exon in macaque and chimpanzee tissues indicates human-specific increase in its transcript inclusion level and muscle specificity after the divergence of humans and chimpanzees. Our results imply that some Alu exonization events may have acquired adaptive benefits during the evolution of primate transcriptomes.

  1. Limited HLA sequence variation outside of antigen recognition domain exons of 360 10 of 10 matched unrelated hematopoietic stem cell transplant donor-recipient pairs.

    Science.gov (United States)

    Hou, L; Vierra-Green, C; Lazaro, A; Brady, C; Haagenson, M; Spellman, S; Hurley, C K

    2017-01-01

    Traditional DNA-based typing focuses primarily on interrogating the exons of human leukocyte antigen (HLA) genes that form the antigen recognition domain (ARD). The relevance of mismatching donor and recipient for HLA variation outside the ARD on hematopoietic stem cell transplantation (HSCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in 10 of 10 (HLA-A, -B, -C, -DRB1, -DQB1) matched unrelated donor transplant pairs (n = 360). Next-generation DNA sequencing was used to characterize both HLA exons and introns for HLA-A, -B, -C alleles; exons 2, 3 and the intervening intron for HLA-DRB1 and exons only for HLA-DQA1 and -DQB1. Over 97% of alleles at each locus were matched for their nucleotide sequence outside of the ARD exons. Of the 4320 allele comparisons overall, only 17 allele pairs were mismatched for non-ARD exons, 41 for noncoding regions and 9 for ARD exons. The observed variation between donor and recipient usually involved a single nucleotide difference (88% of mismatches); 88% of the non-ARD exon variants impacted the amino acid sequence. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D-R pairs will be difficult to assess in HSCT outcome studies because these mismatches do not occur very frequently. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Exonic remnants of whole-genome duplication reveal cis-regulatory function of coding exons.

    Science.gov (United States)

    Dong, Xianjun; Navratilova, Pavla; Fredman, David; Drivenes, Øyvind; Becker, Thomas S; Lenhard, Boris

    2010-03-01

    Using a comparative genomics approach to reconstruct the fate of genomic regulatory blocks (GRBs) and identify exonic remnants that have survived the disappearance of their host genes after whole-genome duplication (WGD) in teleosts, we discover a set of 38 candidate cis-regulatory coding exons (RCEs) with predicted target genes. These elements demonstrate evolutionary separation of overlapping protein-coding and regulatory information after WGD in teleosts. We present evidence that the corresponding mammalian exons are still under both coding and non-coding selection pressure, are more conserved than other protein coding exons in the host gene and several control sets, and share key characteristics with highly conserved non-coding elements in the same regions. Their dual function is corroborated by existing experimental data. Additionally, we show examples of human exon remnants stemming from the vertebrate 2R WGD. Our findings suggest that long-range cis-regulatory inputs for developmental genes are not limited to non-coding regions, but can also overlap the coding sequence of unrelated genes. Thus, exonic regulatory elements in GRBs might be functionally equivalent to those in non-coding regions, calling for a re-evaluation of the sequence space in which to look for long-range regulatory elements and experimentally test their activity.

  3. Characterization of TTN Novex Splicing Variants across Species and the Role of RBM20 in Novex-Specific Exon Splicing

    Directory of Open Access Journals (Sweden)

    Zhilong Chen

    2018-02-01

    Full Text Available Titin (TTN is a major disease-causing gene in cardiac muscle. Titin (TTN contains 363 exons in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by RNA binding motif 20 (RBM20. Three isoforms of TTN protein are produced by mutually exclusive exons 45 (Novex 1, 46 (Novex 2, and 48 (Novex 3. Alternatively splicing in Novex isoforms across species and whether Novex isoforms are associated with heart disease remains completely unknown. Cross-species exon comparison with the mVISTA online tool revealed that exon 45 is more highly conserved across all species than exons 46 and 48. Importantly, a conserved region between exons 47 and 48 across species was revealed for the first time. Reverse transcript polymerase chain reaction (RT-PCR and DNA sequencing confirmed a new exon named as 48′ in Novex 3. In addition, with primer pairs for Novex 1, a new truncated form preserving introns 44 and 45 was discovered. We discovered that Novex 2 is not expressed in the pig, mouse, and rat with Novex 2 primer pairs. Unexpectedly, three truncated forms were identified. One TTN variant with intron 46 retention is mainly expressed in the human and frog heart, another variant with co-expression of exons 45 and 46 exists predominantly in chicken and frog heart, and a third with retention of introns 45 and 46 is mainly expressed in pig, mouse, rat, and chicken. Using Rbm20 knockout rat heart, we revealed that RBM20 is not a splicing regulator of Novex variants. Furthermore, the expression levels of Novex variants in human hearts with cardiomyopathies suggested that Novexes 2 and 3 could be associated with dilated cardiomyopathy (DCM and/or arrhythmogenic right ventricular cardiomyopathy (ARVC. Taken together, our study reveals that splicing diversity of Novex exons across species and Novex variants might play a role in cardiomyopathy.

  4. Exon - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...ontents Exons in variants Data file File name: astra_exon.zip File URL: ftp://ftp.biosciencedbc.jp/archive/a... About This Database Database Description Download License Update History of This Database Site Policy | Contact Us Exon - ASTRA | LSDB Archive ...

  5. Association between polymorphisms of exon 12 and exon 24 of JHDM2A gene and male infertility

    Directory of Open Access Journals (Sweden)

    Zohreh Hojati

    2016-06-01

    Full Text Available Background: Some dynamic changes occurs during spermatogenesis such as histone removal and its replacement with transition nuclear protein and protamine. These proteins are required for packing and condensation of sperm chromatin. JHDM2A is a histone demethylase that directly binds to promoter regions of Tnp1 and Prm1 genes and controls their expression by removing H3K9 at their promoters. Objective: The association between polymorphisms of exon 12 and exon 24 in JHDM2A gene and male infertility were evaluated for the first time. Materials and Methods: In this experimental study, 400 infertile men (oligospermia and azoospermia and normal healthy fathers were evaluated (n=200. Single Strand Conformation Polymorphism (SSCP-PCR and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP methods were used for screening any polymorphisms that are exist in exon 12 and exon 24. Results: Exon 24 PCR products were analyzed by RFLP but no polymorphism was found in this exon at the restriction site of EcoRV enzyme. Our monitoring along the whole nucleotides of exon 12 and exon 24 were continued using SSCP method, but we found no change along these exons. Conclusion: Generally, this study evaluated the association between polymorphisms in exon 12 and exon 24 of JHDM2A gene and male infertility which suggests that polymorphisms of these exons may not be associated with the risk of male infertility.

  6. Optimized Exon-Exon Junction Library and its Application on Rodents' Brain Transcriptome Analysis

    Directory of Open Access Journals (Sweden)

    Tong-Hai Dou

    2017-05-01

    Full Text Available ABSTRACT Background: Alternative splicing (AS, which plays an important role in gene expression and functional regulation, has been analyzed on genome-scale by various bioinformatic approaches based on RNA-seq data. Compared with the huge number of studies on mouse, the AS researches approaching the rat, whose genome is intermedia between mouse and human, were still limited. To enrich the knowledge on AS events in rodents' brain, we perfomed a comprehensive analysis on four transcriptome libraries (mouse cerebrum, mouse cerebellum, rat cerebrum, and rat cerebellum, recruiting high-throughput sequencing technology. An optimized exon-exon junction library approach was introduced to adapt the longer RNA-seq reads and to improve mapping efficiency. Results: In total, 7,106 mouse genes and 2,734 rat genes were differentially expressed between cerebrum and cerebellum, while 7,125 mouse genes and 1,795 rat genes exhibited varieties on transcript variant level. Only half of the differentially expressed exon-exon junctions could be reflected at gene expression level. Functional cluster analysis showed that 32 pathways in mouse and 9 pathways in rat were significantly enriched, and 6 of them were in both. Interestingly, some differentially expressed transcript variants did not show difference on gene expression level, such as PLCβ1 and Kcnma1. Conclusion: Our work provided a case study of a novel exon-exon junction strategy to analyze the expression of genes and isoforms, helping us understand which transcript contributes to the overall expression and further functional change.

  7. PENINGKATAN KEPOLARAN ASAM LINOLEAT DALAM BENTUK AMIDA MENJADI N-etanol-9,10,12,13,15,16 HEKSAHIDROKSI OLEIL-AMIDA

    Directory of Open Access Journals (Sweden)

    Daniel Tarigan

    2016-07-01

    Full Text Available Asam linolenat dari minyak nabati diesterifikasi dengan metanol menggunakan katalis asam dan dilanjutkan dengan amidasi dengan etanolamina menghasilkan alkanolamida selanjutnya dilakukan epoksidasi dengan tert-butyl hidroperoksida dan bantuan katalis peroksigenase dipanaskan pada suhu 40oC selama ± 12 jam sambil dialirkan gas Nitrogen UHP 99% untuk menghasilkan senyawa epoksi alkanolamida oktadekanoat campuran yang selanjutnya dihidrolisis dengan HCl 0,1 N untuk menghasilkan senyawa poliol alkanolamida heksahidroksi oktadekanoat. Uji HLB dari surfaktan alkanolamida heksahidroksi oktadekanoat (N-etanol-9,10,12,13,15,16 heksahidroksi oleil amida sebesar 14,13 Oleh karena itu senyawa poliol ini dapat digunakan sebagai pengemulsi sistem o/w. Seluruh hasil reaksi dikonfirmasikan secara analisis spektroskopi FT-IR, 1H-NMR, GC-MS dan analisis KLT sesuai dengan keperluan.    Kata kunci: Asam linolenat, N-etanol-9,10,12,13,15,16 heksahidroksi oleil amida dan esterifikasi

  8. Fuzzy-adaptive-thresholding-based exon prediction.

    Science.gov (United States)

    Agrawal, Ankit; Mittal, Ankush; Jain, Rahul; Takkar, Raghav

    2010-01-01

    Thresholding is always critical and decisive in many bioinformatics problems. In this paper, we propose and apply a fuzzy-logic-based adaptive thresholding approach to a well-known solution for the exon prediction problem, which uses a threshold on the frequency component at f = 1/3 in the nucleotide sequence. The proposed approach allows the thresholds to vary along the data set based on the local statistical properties. Experiments and results on the nucleotide data of Saccharomyces cerevisiae (Bakers yeast) illustrate the advantage of our approach. A user-friendly GUI in MATLAB is freely available for academic use at www.cs.iastate.edu/˜ankitag/FATBEP.html.

  9. The effect of a 12 week plyometric and soccer training on the cardiac structure and function among the 13-15 years old boys

    Directory of Open Access Journals (Sweden)

    Bakhtiar Tartibian

    2017-04-01

    Full Text Available Background: Exercise plays an important role in improving cardiovascular function. Due to the increasing tendency of children to the soccer the purpose of this investigation was to examine the effects of a 12 week plyometric and soccer training on the cardiac structure and function of healthy 13-15 years old boys. Materials and Methods: This quasi- experimental study was conducted with a pre-test and post-test design. The participants (n=20 were selected among the non-athletic healthy boys (13-15 years from Miyaneh (East Azerbaijan, Iran. After taking informed consent the participants were voluntarily participated in two equal Training and Control groups. The participants in the Exercise group performed a 12 week plyometric and soccer training. Under basic conditions and after a 12 week follow-up using the echocardiography both groups were examined for left ventricular end-diastolic dimension (LVEDD, left ventricular end-systolic dimension (LVESD, left ventricular end diastolic posterior wall dimension¬ (LVPWd, inter-ventricular septal end diastolic dimension (IVSd, stroke volume (SV, ejection fraction (EF and cardiac output (CO. Results: After a 12 week training program the LVEDD, LVPWd and SV were significantly increased (P<0.05. The EF in the Exercise group had no increase. Moreover, LVESD and IVSd were similar in both groups. Conclusion: A twelve week plyometric and soccer training in 13-15 years old boys result in significant changes in some cardiac structural and functional indexes.

  10. A rare missense variant in RET exon 8 in a Portuguese family with atypical multiple endocrine neoplasia type 2A.

    Science.gov (United States)

    Martins, Ana Filipa; Martins, João Martin; do Vale, Sónia; Dias, Teresa; Silveira, Catarina; da Silva, Inês Rodrigues; Carmo-Fonseca, Maria

    2016-07-01

    Multiple Endocrine Neoplasia type 2 (MEN2) is a rare genetic disorder characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and primary hyperparathyroidism. MEN2 is an autosomal dominant syndrome caused by mutations in the RET proto-oncogene. In the vast majority of patients, the mutations are localized in exons 10, 11 and 13-15 of the RET gene. Rare variants located in exon 8 were recently identified but their clinical significance remains unclear. We studied two sisters presenting with pheochromocytoma as the first tumor. One of the sisters was diagnosed with a right pheochromocytoma at the age of 44 and at age 53 she developed an invasive left pheochromocytoma with no other endocrine neoplasia. The other sister was diagnosed with a left pheochromocytoma at age 50 and at age 64 she had a right phemochromocytoma and MTC. Neither of the two sisters presented evidence of primary hyperparathyroidism. Mutations of the RET proto-oncogene were investigated by DNA sequencing. We detected a germline missense variant in RET exon 8 (p.Cys531Arg) in both sisters. The p.Cys531Arg variant was not present in a third 50-year-old sister who has remained to date clinically unaffected. This is the first case showing the p.Cys531Arg variant in RET exon 8 co-segregating with family members affected by a syndrome reminiscent of MEN2A. Our results suggest that this variant has a specific genotype-phenotype correlation as it is associated with the development of pheochromocytoma before the onset of MTC.

  11. Characteristics of transposable element exonization within human and mouse.

    Directory of Open Access Journals (Sweden)

    Noa Sela

    Full Text Available Insertion of transposed elements within mammalian genes is thought to be an important contributor to mammalian evolution and speciation. Insertion of transposed elements into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization. Elucidation of the evolutionary constraints that have shaped fixation of transposed elements within human and mouse protein coding genes and subsequent exonization is important for understanding of how the exonization process has affected transcriptome and proteome complexities. Here we show that exonization of transposed elements is biased towards the beginning of the coding sequence in both human and mouse genes. Analysis of single nucleotide polymorphisms (SNPs revealed that exonization of transposed elements can be population-specific, implying that exonizations may enhance divergence and lead to speciation. SNP density analysis revealed differences between Alu and other transposed elements. Finally, we identified cases of primate-specific Alu elements that depend on RNA editing for their exonization. These results shed light on TE fixation and the exonization process within human and mouse genes.

  12. Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable - Molecular pathology of mutations in PAH exon 11

    DEFF Research Database (Denmark)

    Heintz, Caroline; Dobrowolski, Steven F.; Andersen, Henriette Skovgaard

    2012-01-01

    distributed throughout the exon. Finally, we identified a pseudoexon in intron 11, which would have pathogenic consequences if activated by mutations or improved splicing conditions. Exonic mutations that disrupt splicing are unlikely to facilitate response to BH(4) and may lead to inconsistent genotype......In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation...... molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11...

  13. The silent mutation nucleotide 744 G --> A, Lys172Lys, in exon 6 of BRCA2 results in exon skipping

    DEFF Research Database (Denmark)

    Hansen, Thomas V O; Steffensen, Ane Y; Jønson, Lars

    2009-01-01

    and intron variants are of unknown significance. Here, we describe the functional characterization of a silent mutation (nucleotide 744 G --> A/c.516 G --> A, Lys172Lys) in exon 6 of BRCA2 in a Danish family with breast and ovarian cancer. Exon trapping analysis showed that the mutation results in skipping...... of exon 6 and/or both exon 5 and 6, which was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. We therefore conclude that the BRCA2 silent mutation Lys172Lys is a disease-causing mutation....

  14. Exon exchange approach to repair Duchenne dystrophin transcripts.

    Directory of Open Access Journals (Sweden)

    Stéphanie Lorain

    Full Text Available BACKGROUND: Trans-splicing strategies for mRNA repair involve engineered transcripts designed to anneal target mRNAs in order to interfere with their natural splicing, giving rise to mRNA chimeras where endogenous mutated exons have been replaced by exogenous replacement sequences. A number of trans-splicing molecules have already been proposed for replacing either the 5' or the 3' part of transcripts to be repaired. Here, we show the feasibility of RNA surgery by using a double trans-splicing approach allowing the specific substitution of a given mutated exon. METHODOLOGY/PRINCIPAL FINDINGS: As a target we used a minigene encoding a fragment of the mdx dystrophin gene enclosing the mutated exon (exon 23. This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains. We obtained accurate and efficient replacement of exon 23 in the mRNA target. Adding up a downstream intronic splice enhancer DISE in the exon exchange molecule enhanced drastically its efficiency up to 25-45% of repair depending on the construction in use. CONCLUSIONS/SIGNIFICANCE: These results demonstrate the possibility to fix up mutated exons, refurbish deleted exons and introduce protein motifs, while keeping natural untranslated sequences, which are essential for mRNA stability and translation regulation. Conversely to the well-known exon skipping, exon exchange has the advantage to be compatible with almost any type of mutations and more generally to a wide range of genetic conditions. In particular, it allows addressing disorders caused by dominant mutations.

  15. Exon Exchange Approach to Repair Duchenne Dystrophin Transcripts

    Science.gov (United States)

    Lorain, Stéphanie; Peccate, Cécile; Le Hir, Maëva; Garcia, Luis

    2010-01-01

    Background Trans-splicing strategies for mRNA repair involve engineered transcripts designed to anneal target mRNAs in order to interfere with their natural splicing, giving rise to mRNA chimeras where endogenous mutated exons have been replaced by exogenous replacement sequences. A number of trans-splicing molecules have already been proposed for replacing either the 5′ or the 3′ part of transcripts to be repaired. Here, we show the feasibility of RNA surgery by using a double trans-splicing approach allowing the specific substitution of a given mutated exon. Methodology/Principal Findings As a target we used a minigene encoding a fragment of the mdx dystrophin gene enclosing the mutated exon (exon 23). This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains. We obtained accurate and efficient replacement of exon 23 in the mRNA target. Adding up a downstream intronic splice enhancer DISE in the exon exchange molecule enhanced drastically its efficiency up to 25–45% of repair depending on the construction in use. Conclusions/Significance These results demonstrate the possibility to fix up mutated exons, refurbish deleted exons and introduce protein motifs, while keeping natural untranslated sequences, which are essential for mRNA stability and translation regulation. Conversely to the well-known exon skipping, exon exchange has the advantage to be compatible with almost any type of mutations and more generally to a wide range of genetic conditions. In particular, it allows addressing disorders caused by dominant mutations. PMID:20531943

  16. CD44 isoforms with exon v6 and metastasis of primary N0M0 breast carcinomas.

    Science.gov (United States)

    Guriec, N; Gairard, B; Marcellin, L; Wilk, A; Caldéroli, H; Renaud, R; Bergerat, J P; Oberling, F

    1997-07-01

    New isoforms of CD44 with alternatively spliced exons have recently been described. Expression of exon v6 seems to be of particular interest. It has indeed been associated with poorer outcome of breast cancer patients with node invasion at diagnosis. However, no data were available for patients N0M0 (with neither metastasis nor node invasion at diagnosis). Moreover, previous statistical analyses were realized using immunohistochemical methods to detect CD44v6 expression although several variants with exon v6 have been described. We investigated expression of isoforms containing CD44v6 using an RT-PCR approach and a panel of 25 normal breast specimens, 10 mammary fibroadenomas, 8 cystic samples and 52 primary breast tumors (38 invasive N0M0). Normal breasts, fibroadenomas, and cysts all express the same variant, A (with exon v6 only), while several transcripts are amplified in tumors. Expression of variants other than A correlates with acquisition of a malignant phenotype. Invasive cancers also express additional variants in comparison with in situ carcinomas. Metastasis capacities seem to be associated with transcription of variants other than A but also with no transcription of some of them, variants D (with exons v6 and v10) and L (with exons v6 to v10). Expression of variants D and L correlates with higher percentages of disease-free survival and better outcome. Expression of CD44 splice variants with exon v6, as detected by RT-PCR, might be a useful prognostic factor for breast cancer. However, since the series size is small, our results need to be confirmed by later studies on a larger number of patients.

  17. PTESFinder: a computational method to identify post-transcriptional exon shuffling (PTES) events.

    Science.gov (United States)

    Izuogu, Osagie G; Alhasan, Abd A; Alafghani, Hani M; Santibanez-Koref, Mauro; Elliott, David J; Elliot, David J; Jackson, Michael S

    2016-01-13

    Transcripts, which have been subject to Post-transcriptional exon shuffling (PTES), have an exon order inconsistent with the underlying genomic sequence. These have been identified in a wide variety of tissues and cell types from many eukaryotes, and are now known to be mostly circular, cytoplasmic, and non-coding. Although there is no uniformly ascribed function, several have been shown to be involved in gene regulation. Accurate identification of these transcripts can, however, be difficult due to artefacts from a wide variety of sources. Here, we present a computational method, PTESFinder, to identify these transcripts from high throughput RNAseq data. Uniquely, it systematically excludes potential artefacts emanating from pseudogenes, segmental duplications, and template switching, and outputs both PTES and canonical exon junction counts to facilitate comparative analyses. In comparison with four existing methods, PTESFinder achieves highest specificity and comparable sensitivity at a variety of read depths. PTESFinder also identifies between 13 % and 41.6 % more structures, compared to publicly available methods recently used to identify human circular RNAs. With high sensitivity and specificity, user-adjustable filters that target known sources of false positives, and tailored output to facilitate comparison of transcript levels, PTESFinder will facilitate the discovery and analysis of these poorly understood transcripts.

  18. Widespread evolutionary conservation of alternatively spliced exons in caenorhabditis

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob L; Penny, David

    2007-01-01

    Alternative splicing (AS) contributes to increased transcriptome and proteome diversity in various eukaryotic lineages. Previous studies showed low levels of conservation of alternatively spliced (cassette) exons within mammals and within dipterans. We report a strikingly different pattern...... in Caenorhabditis nematodes-more than 92% of cassette exons from Caenorhabditis elegans are conserved in Caenorhabditis briggsae and/or Caenorhabditis remanei. High levels of conservation extend to minor-form exons (present in a minority of transcripts) and are particularly pronounced for exons showing complex...... patterns of splicing. The functionality of the vast majority of cassette exons is underscored by various other features. We suggest that differences in conservation between lineages reflect differences in levels of functionality and further suggest that these differences are due to differences in intron...

  19. Current Tobacco Smoking and Desire to Quit Smoking Among Students Aged 13-15 Years - Global Youth Tobacco Survey, 61 Countries, 2012-2015.

    Science.gov (United States)

    Arrazola, René A; Ahluwalia, Indu B; Pun, Eugene; Garcia de Quevedo, Isabel; Babb, Stephen; Armour, Brian S

    2017-05-26

    Tobacco use is the world's leading cause of preventable morbidity and mortality, resulting in nearly 6 million deaths each year (1). Smoked tobacco products, such as cigarettes and cigars, are the most common form of tobacco consumed worldwide (2), and most tobacco smokers begin smoking during adolescence (3). The health benefits of quitting are greater for persons who stop smoking at earlier ages; however, quitting smoking at any age has health benefits (4). CDC used the Global Youth Tobacco Survey (GYTS) data from 61 countries across the six World Health Organization (WHO) regions from 2012 to 2015 to examine the prevalence of current tobacco smoking and desire to quit smoking among students aged 13-15 years. Across all 61 countries, the median current tobacco smoking prevalence among students aged 13-15 years was 10.7% (range = 1.7%, Sri Lanka to 35.0%, Timor-Leste). By sex, the median current tobacco smoking prevalence was 14.6% among males (range = 2.9%, Tajikistan to 61.4%, Timor-Leste) and 7.5% among females (range = 1.6%, Tajikistan to 29.0%, Bulgaria). In the majority of countries assessed, the proportion of current tobacco smokers who desired to quit smoking exceeded 50%. These findings could be used by country level tobacco control programs to inform strategies to prevent and reduce youth tobacco use (1,4).

  20. Skipping Multiple Exons to Treat DMD-Promises and Challenges.

    Science.gov (United States)

    Aslesh, Tejal; Maruyama, Rika; Yokota, Toshifumi

    2018-01-02

    Duchenne muscular dystrophy (DMD) is a lethal disorder caused by mutations in the DMD gene. Antisense-mediated exon-skipping is a promising therapeutic strategy that makes use of synthetic nucleic acids to skip frame-disrupting exon(s) and allows for short but functional protein expression by restoring the reading frame. In 2016, the U.S. Food and Drug Administration (FDA) approved eteplirsen, which skips DMD exon 51 and is applicable to approximately 13% of DMD patients. Multiple exon skipping, which is theoretically applicable to 80-90% of DMD patients in total, have been demonstrated in animal models, including dystrophic mice and dogs, using cocktail antisense oligonucleotides (AOs). Although promising, current drug approval systems pose challenges for the use of a cocktail AO. For example, both exons 6 and 8 need to be skipped to restore the reading frame in dystrophic dogs. Therefore, the cocktail of AOs targeting these exons has a combined therapeutic effect and each AO does not have a therapeutic effect by itself. The current drug approval system is not designed to evaluate such circumstances, which are completely different from cocktail drug approaches in other fields. Significant changes are needed in the drug approval process to promote the cocktail AO approach.

  1. Alternative splicing regulation of APP exon 7 by RBFox proteins.

    Science.gov (United States)

    Alam, Shafiul; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2014-12-01

    RBFox proteins are well-known alternative splicing regulators. We have shown previously that during neuronal differentiation of P19 cells induced by all-trans retinoic acid and cell aggregation, RBFox1 shows markedly increased temporal expression. To find its key splicing regulation, we examined the effect of RBFox1 on 33 previously reported and validated neuronal splicing events of P19 cells. We observed that alternative splicing of three genes, specifically, amyloid precursor protein (APP), disks large homolog 3 (DLG3), and G protein, alpha activating activity polypeptide O (GNAO1), was altered by transient RBFox1 expression in HEK293 and HeLa cells. Moreover, an RBFox1 mutant (RBFox1FA) that was unable to bind the target RNA sequence ((U)GCAUG) did not induce these splicing events. APP generates amyloid beta peptides that are involved in the pathology of Alzheimer's disease, and therefore we examined APP alternative splicing regulation by RBFox1 and other splicing regulators. Our results indicated that RBFox proteins promote the skipping of APP exon 7, but not the inclusion of exon 8. We made APP6789 minigenes and observed that two (U)GCAUG sequences, located upstream of exon 7 and in exon 7, functioned to induce skipping of exon 7 by RBFox proteins. Overall, RBFox proteins may shift APP from exon 7 containing isoforms, APP770 and APP751, toward the exon 7 lacking isoform, APP695, which is predominant in neural tissues. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. A novel splicing silencer generated by DMD exon 45 deletion junction could explain upstream exon 44 skipping that modifies dystrophinopathy.

    Science.gov (United States)

    Dwianingsih, Ery Kus; Malueka, Rusdy Ghazali; Nishida, Atsushi; Itoh, Kyoko; Lee, Tomoko; Yagi, Mariko; Iijima, Kazumoto; Takeshima, Yasuhiro; Matsuo, Masafumi

    2014-08-01

    Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease, is mostly caused by exon deletion mutations in the DMD gene. The reading frame rule explains that out-of-frame deletions lead to muscle dystrophin deficiency in DMD. In outliers to this rule, deletion junction sequences have never previously been explored as splicing modulators. In a Japanese case, we identified a single exon 45 deletion in the patient's DMD gene, indicating out-of-frame mutation. However, immunohistochemical examination disclosed weak dystrophin signals in his muscle. Reverse transcription-PCR amplification of DMD exons 42 to 47 revealed a major normally spliced product with exon 45 deletion and an additional in-frame product with deletion of both exons 44 and 45, indicating upstream exon 44 skipping. We considered the latter to underlie the observed dystrophin expression. Remarkably, the junction sequence cloned by PCR walking abolished the splicing enhancer activity of the upstream intron in a chimeric doublesex gene pre-mRNA in vitro splicing. Furthermore, antisense oligonucleotides directed against the junction site counteracted this effect. These indicated that the junction sequence was a splicing silencer that induced upstream exon 44 skipping. It was strongly suggested that creation of splicing regulator is a modifier of dystrophinopathy.

  3. Cigarette Smoking among Adolescents aged 13-15 in Viet Nam and Correlates of Current Cigarette Smoking: Results from GYTS 2014 Data.

    Science.gov (United States)

    Huong, Le Thi; Vu, Nga Thi Thu; Dung, Nguyen Ngoc; Xuan, Le Thi Thanh; Giang, Kim Bao; Hai, Phan Thi; Huyen, Doan Thu; Khue, Luong Ngoc; Lam, Nguyen Tuan; Minh, Hoang Van; Nga, Pham Thi Quynh

    2016-01-01

    The aim of this paper is to report the rate of current and ever cigarette smoking and explore correlates of current cigarette smoking among adolescents aged 13-15 in Viet Nam. This analysis was derived from GYTS survey, which comprised of 3,430 adolescents aged 13-15, conducted in 2014 in 13 cities and provinces of Viet Nam. We calculated the weighted rates of current and ever cigarette smoking and reported patterns of smoking behavior. We also performed logistic regression to explore correlates of current cigarette smoking behavior. The weighted rate of ever cigarette smoking was 9.5% (95% confidence interval (CI): 8.5 %-10.5%), in which the weighted rate among males (15.4%; 95% CI: 13.6%-17.0%) was higher than that among females (4.2%; 95% CI: 3.3%-5.1%). The weighted rate of current cigarette smoking was relatively low at 2.5% (95%CI: 2.0%- 3.0%) with higher weighted rate among males (4.9%; 95% CI: 3.8%-5.9%) compared to the corresponding figure among females (0.2%; 95% CI: 0.0 %-0.5%). Current cigarette smoking was significantly higher among males than females, in students aged 15 versus 13 years old, and in students who had several or all close friends smoking and students with daily observation of smoking at school. For greater smoking reduction outcomes, we recommend that tobacco interventions for adolescents should consider targeting more male students at older ages, establish stricter adherence to school-based banning of cigarette smoking, engage both smoking and nonsmoking adolescents and empower adolescents to resist peer smoking influence as well as changing their norms or beliefs towards smoking benefits.

  4. Origin of introns by 'intronization' of exonic sequences

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Penny, David

    2008-01-01

    The mechanisms of spliceosomal intron creation have proved elusive. Here we describe a new mechanism: the recruitment of internal exonic sequences ('intronization') in Caenorhabditis species. The numbers of intronization events and introns gained by other mechanisms are similar, suggesting...

  5. Human glucose phosphate isomerase: Exon mapping and gene structure

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Weiming; Lee, Pauline; Beutler, E. [Scripps Research Inst., La Jolla, CA (United States)

    1995-10-10

    The structure of the gene for human glucose phosphate isomerase (GPI) has been determined. Three GPI clones were isolated from a human genomic library by using a full-length GPI cDNA probe and were characterized. Oligonucleotides based on the known cDNA sequence were used as primers in amplification and sequence analyses. This led to the identification of the exon-intron junctions. By this approach, 18 exons and 17 introns have been identified. The exons range in size from 44 to 431 nucleotides. The intronic sequences surrounding the exons provide useful information for the identification of mutations that give rise to human GPI deficiency associated with chronic hemolytic anemia. 13 refs., 4 figs., 1 tab.

  6. Exon silencing by UAGG motifs in response to neuronal excitation.

    Directory of Open Access Journals (Sweden)

    Ping An

    2007-02-01

    Full Text Available Alternative pre-mRNA splicing plays fundamental roles in neurons by generating functional diversity in proteins associated with the communication and connectivity of the synapse. The CI cassette of the NMDA R1 receptor is one of a variety of exons that show an increase in exon skipping in response to cell excitation, but the molecular nature of this splicing responsiveness is not yet understood. Here we investigate the molecular basis for the induced changes in splicing of the CI cassette exon in primary rat cortical cultures in response to KCl-induced depolarization using an expression assay with a tight neuron-specific readout. In this system, exon silencing in response to neuronal excitation was mediated by multiple UAGG-type silencing motifs, and transfer of the motifs to a constitutive exon conferred a similar responsiveness by gain of function. Biochemical analysis of protein binding to UAGG motifs in extracts prepared from treated and mock-treated cortical cultures showed an increase in nuclear hnRNP A1-RNA binding activity in parallel with excitation. Evidence for the role of the NMDA receptor and calcium signaling in the induced splicing response was shown by the use of specific antagonists, as well as cell-permeable inhibitors of signaling pathways. Finally, a wider role for exon-skipping responsiveness is shown to involve additional exons with UAGG-related silencing motifs, and transcripts involved in synaptic functions. These results suggest that, at the post-transcriptional level, excitable exons such as the CI cassette may be involved in strategies by which neurons mount adaptive responses to hyperstimulation.

  7. Short Exon Detection via Wavelet Transform Modulus Maxima.

    Science.gov (United States)

    Zhang, Xiaolei; Shen, Zhiwei; Zhang, Guishan; Shen, Yuanyu; Chen, Miaomiao; Zhao, Jiaxiang; Wu, Renhua

    2016-01-01

    The detection of short exons is a challenging open problem in the field of bioinformatics. Due to the fact that the weakness of existing model-independent methods lies in their inability to reliably detect small exons, a model-independent method based on the singularity detection with wavelet transform modulus maxima has been developed for detecting short coding sequences (exons) in eukaryotic DNA sequences. In the analysis of our method, the local maxima can capture and characterize singularities of short exons, which helps to yield significant patterns that are rarely observed with the traditional methods. In order to get some information about singularities on the differences between the exon signal and the background noise, the noise level is estimated by filtering the genomic sequence through a notch filter. Meanwhile, a fast method based on a piecewise cubic Hermite interpolating polynomial is applied to reconstruct the wavelet coefficients for improving the computational efficiency. In addition, the output measure of a paired-numerical representation calculated in both forward and reverse directions is used to incorporate a useful DNA structural property. The performances of our approach and other techniques are evaluated on two benchmark data sets. Experimental results demonstrate that the proposed method outperforms all assessed model-independent methods for detecting short exons in terms of evaluation metrics.

  8. Short Exon Detection via Wavelet Transform Modulus Maxima.

    Directory of Open Access Journals (Sweden)

    Xiaolei Zhang

    Full Text Available The detection of short exons is a challenging open problem in the field of bioinformatics. Due to the fact that the weakness of existing model-independent methods lies in their inability to reliably detect small exons, a model-independent method based on the singularity detection with wavelet transform modulus maxima has been developed for detecting short coding sequences (exons in eukaryotic DNA sequences. In the analysis of our method, the local maxima can capture and characterize singularities of short exons, which helps to yield significant patterns that are rarely observed with the traditional methods. In order to get some information about singularities on the differences between the exon signal and the background noise, the noise level is estimated by filtering the genomic sequence through a notch filter. Meanwhile, a fast method based on a piecewise cubic Hermite interpolating polynomial is applied to reconstruct the wavelet coefficients for improving the computational efficiency. In addition, the output measure of a paired-numerical representation calculated in both forward and reverse directions is used to incorporate a useful DNA structural property. The performances of our approach and other techniques are evaluated on two benchmark data sets. Experimental results demonstrate that the proposed method outperforms all assessed model-independent methods for detecting short exons in terms of evaluation metrics.

  9. Exonization of the LTR transposable elements in human genome

    Directory of Open Access Journals (Sweden)

    Borodovsky Mark

    2007-08-01

    Full Text Available Abstract Background Retrotransposons have been shown to contribute to evolution of both structure and regulation of protein coding genes. It has been postulated that the primary mechanism by which retrotransposons contribute to structural gene evolution is through insertion into an intron or a gene flanking region, and subsequent incorporation into an exon. Results We found that Long Terminal Repeat (LTR retrotransposons are associated with 1,057 human genes (5.8%. In 256 cases LTR retrotransposons were observed in protein-coding regions, while 50 distinct protein coding exons in 45 genes were comprised exclusively of LTR RetroTransposon Sequence (LRTS. We go on to reconstruct the evolutionary history of an alternatively spliced exon of the Interleukin 22 receptor, alpha 2 gene (IL22RA2 derived from a sequence of retrotransposon of the Mammalian apparent LTR retrotransposons (MaLR family. Sequencing and analysis of the homologous regions of genomes of several primates indicate that the LTR retrotransposon was inserted into the IL22RA2 gene at least prior to the divergence of Apes and Old World monkeys from a common ancestor (~25 MYA. We hypothesize that the recruitment of the part of LTR as a novel exon in great ape species occurred prior to the divergence of orangutans and humans from a common ancestor (~14 MYA as a result of a single mutation in the proto-splice site. Conclusion Our analysis of LRTS exonization events has shown that the patterns of LRTS distribution in human exons support the hypothesis that LRTS played a significant role in human gene evolution by providing cis-regulatory sequences; direct incorporation of LTR sequences into protein coding regions was observed less frequently. Combination of computational and experimental approaches used for tracing the history of the LTR exonization process of IL22RA2 gene presents a promising strategy that could facilitate further studies of transposon initiated gene evolution.

  10. Linking data to tobacco control program action among students aged 13-15 in Association of Southeast Asian Nations (ASEAN) member states, 2000-2006.

    Science.gov (United States)

    Sirichotiratana, N; Sovann, S; Aditama, T Y; Krishnan, M; Kyaing, N N; Miguel-Baquilod, M; Hai, P T; Sinha, D N; Warren, C W; Jones, N R

    2008-12-01

    The Association of Southeast Asian Nations (ASEAN) has made tobacco use prevention a primary health issue. All ASEAN countries except Indonesia have ratified the World Health Organization Framework Convention on Tobacco Control (WHO FCTC), the world's first public health treaty on tobacco control. Global Youth Tobacco Survey (GYTS) data were collected from representative samples of students in school grades associated with ages 13-15 in Cambodia, Indonesia, Laos (Vientiane), Malaysia, Myanmar, Philippines, Singapore, Thailand and Vietnam (Hanoi). Current cigarette smoking ranged from less than 5% (Vietnam and Cambodia) to 20.2% in Malaysia. Current use of tobacco products other than cigarettes was less than 10% in all countries. Boys were significantly more likely than girls to smoke cigarettes or use other tobacco products. Exposure to second-hand smoke in public places was greater than 50%, direct pro-tobacco advertising exposure was greater than 75% and over 10% of students were exposed to indirect advertising. Over 60% of students who currently smoked cigarettes wanted to stop, but 80% who tried to quit in the year prior to the survey failed. Efforts to reduce the current and projected harm caused by tobacco use in the ASEAN countries are urgently needed. ASEAN countries need to expand their national comprehensive tobacco prevention and control programs and enforce those laws already passed. Without this effort little reduction can be expected in the burden of chronic diseases and tobacco-related mortality.

  11. Prevalence, demographic and psychosocial correlates for school truancy among students aged 13-15 in the Association of Southeast Asian Nations (ASEAN) member states.

    Science.gov (United States)

    Pengpid, Supa; Peltzer, Karl

    2017-11-01

    Truancy among adolescents may negatively affect the achievement of academic goals. The aim of the study was to investigate the prevalence and correlates of school truancy in the Association of Southeast Asian Nations (ASEAN) member states. The analysis included 28 419 school children aged 13-15 years from seven ASEAN member states that participated in the Global School-based Student Health Survey (GSHS) between 2007 and 2013. The overall prevalence of past 30 day truancy across six ASEAN countries (excluding Brunei) was 24.8%; ranging from below 20% in Myanmar, Thailand, and Vietnam to more than 30% in Brunei Darussalam, Indonesia, and the Philippines. In multivariable logistic regression analysis, socio-demographic factors (older age, being male, the experience of hunger), externalising behaviour (tobacco use, alcohol use, having been in a physical fight, being bullied, having sustained an injury), and lack of protective social-familial factors (lack of peer support and lack of parental or guardian support) were found to be associated with truancy. High rates of truancy were found in ASEAN member states calling for interventions aimed to reduce truancy considering identified associated factors.

  12. Prevalence and sociodemographic correlates of stunting, underweight, and overweight among Palestinian school adolescents (13-15 years in two major governorates in the West Bank

    Directory of Open Access Journals (Sweden)

    Awartani Faisal

    2009-12-01

    Full Text Available Abstract Background There is little information about height and weight status of Palestinian adolescents. The objective of this paper was to assess the prevalence of stunting, underweight, and overweight/obesity among Palestinian school adolescents (13-15 years and associated sociodemographic factors in 2 major governorates in the West Bank. Methods A Cross-sectional survey was conducted in 2005 comprising 1942 students in 65 schools in Ramallah and Hebron governorates. Data was collected through self-administered questionnaires from students and parents. Weights and heights were measured. Overweight and obesity were assessed using the 2000 Centers for Disease Control and Prevention (CDC reference and the International Obesity Task Force (IOTF criteria. Stunting and underweight were assessed using the 2000 CDC reference. Results Overweight/obesity was more prevalent in Ramallah than in Hebron and affected more girls than boys. Using the 2000 CDC reference, the prevalence of overweight and obesity in Ramallah among boys was 9.6% and 8.2%, respectively versus 15.6% and 6.0% among girls (P Conclusion Under- and overnutrition co-exist among Palestinian adolescents, with differences between sexes. Region, residence, STL, and onset of puberty were associated factors.

  13. Stress Analysis on Single Cobalt/Chrome Prosthesis With a 15-mm Cantilever Placed Over 10/13/15-mm-length Implants: A Simulated Photoelastic Model Study.

    Science.gov (United States)

    Gastaldo, José Fábio Guastelli; Pimentel, Angélica Castro; Gomes, Maria Helena; Sendyk, Wilson Roberto; Laganá, Dalva Cruz

    2015-12-01

    The aim of study was to assess the stress around 10/13/15-mm implants in the mandibular area with a 15-mm cantilevered acrylic-resin-coated prostheses following the application force, using the photoelasticity method. Three photoelastic mandibular models were created containing 10-, 13-, and 15-mm implants in length and 3.75 mm in diameter. The implants had bore internal hex connections and were placed parallel to the intermental region. Abutments with 1-mm high cuffs were placed over the implants, and a single cobalt/chrome metallic prosthesis with a 15-mm cantilever, coated with thermoplastic acrylic resin, was placed on top. Loads of 1.0 and 3.0 bars were applied, and the images were photographed and assessed by photoelasticity method. The greatest stress levels were observed for the 10-mm implants. The stress pattern was the same regardless of implant length; only the magnitude of the stress along the implant body revealed changes. Increased implant length played a role in reducing stress on the investigated area of the model, and the 15-mm implants exhibited the best performance in regard to stress distribution. The highest stress levels were found in the implants closest to the cantilever and the central implant. The longest implants were more favorable in regard to the stress distribution on the peri-implant support structures in the 15-mm cantilevered prosthesis under loads.

  14. Self-aggregation behavior of synthetic zinc 3-hydroxymethyl-13/15-carbonyl-chlorins as models of main light-harvesting components in photosynthetic green bacteria.

    Science.gov (United States)

    Tamiaki, Hitoshi; Yoshimura, Hideaki; Shimamura, Yasuhide; Kunieda, Michio

    2008-01-01

    Zinc complexes of 3-hydroxymethyl-13/15-carbonyl-chlorins having a six-membered lactone as the E-ring were prepared by modifying purpurin-18 as models of bacteriochlorophyll-d, one of the chlorophyllous pigments in the main light-harvesting antenna systems (chlorosomes) of green photosynthetic bacteria. The synthetic 13-carbonylated compound self-aggregated in 1%(v/v) tetrahydrofuran and hexane to give large oligomers possessing red-shifted and broadened electronic absorption bands and intense circular dichroism bands at the shifted Q ( y ) region, indicating that the supramolecular structure of the resulting self-aggregate was similar to those of natural and artificial chlorosomal aggregates. The red-shift value observed here was smaller than the reported values in chlorosomal pigments having a five-membered keto-ring, which was ascribable to a weaker intermolecular hydrogen-bonding of 13-C=O with 3(1)-OH in a supramolecule of the former self-aggregate and suppression of the pi-pi interaction among the composite chlorins. On the other hand, the isomeric 15-carbonylated molecule was monomeric even in the nonpolar organic solvent, confirming the reported proposal that the linear orientation of three interactive moieties, OH, C=O and Zn, in a molecule is requisite for its chlorosomal self-aggregation.

  15. Trends in Second-Hand Tobacco Smoke Exposure Levels at Home among Viet Nam School Children Aged 13-15 and Associated Factors.

    Science.gov (United States)

    Lam, Nguyen Tuan; Nga, Pham Thi Quynh; Minh, Hoang Van; Giang, Kim Bao; Hai, Phan Thi; Huyen, Doan Thu; Linh, Nguyen Thuy; Van, Duong Khanh; Khue, Luong Ngoc

    2016-01-01

    Second-hand tobacco smoke (SHS) exposure at home, especially among children, is a serious issue in Viet Nam. During the past decade, much effort has been taken for tobacco control in the country, including various prgorammes aiming to reduce SHS exposure among adults and children. This article analysed trends and factors associated with SHS exposure at home among school children aged 13-15 in Viet Nam, using the Global Youth Tobacco Surveys conducted in 2007 and 2014. Descriptive and inferential statistical methods with logistic regression were applied. Overall, there was a significant reduction in the level of exposure, from 58.5% (95%CI: 57.6-59.3) in 2007 to 47.1% (95%CI: 45.4-48.8) in 2014. Of the associated factors, having one or both parents smoking was significantly associated with the highest odds of SHS exposure at home (OR=5.0; 95%CI: 4.2-6.1). Conversely, having a mother with a college or higher education level was found to be a protective factor (OR=0.5; 95%CI: 0.3-0.8).

  16. Suicidal ideation and associated factors among students aged 13-15 years in Association of Southeast Asian Nations (ASEAN) member states, 2007-2013.

    Science.gov (United States)

    Peltzer, Karl; Pengpid, Supa

    2017-09-01

    The aim of this study was to assess suicidal ideation and associated factors in school-going adolescents in the Association of Southeast Asian Nations (ASEAN) member states. The analysis included 30,284 school children aged 13-15 years from seven ASEAN countries that participated in the cross-sectional global school-based student health survey (GSHS) between 2007 and 2013. The overall prevalence of suicidal ideation in the past 12 months across seven ASEAN countries was 12.3%, significantly higher in girls (15.1%) than boys (9.3%). Among seven ASEAN countries with the highest prevalence of suicidal ideation was in the Philippines (17.0%) and Vietnam (16.9%) and the lowest in Myanmar (1.1%) and Indonesia (4.2%). In multivariable logistic regression analysis, female gender, older age (14 or 15 years), living in a lower middle income country, having no friends, loneliness, bullying victimisation, having been in a physical fight in the past 12 months, lack of parental or guardian support, tobacco use and having a history of ever got drunk were associated with suicidal ideation. Different rates of suicidal ideation were observed in ASEAN member states. Several risk factors for suicidal ideation were identified which can help guide preventive efforts.

  17. OTEC thermal resource report for Caribbean Sea Plant Ship 13--15/sup 0/N 75--80/sup 0/N

    Energy Technology Data Exchange (ETDEWEB)

    Wolff, W. A.

    1979-05-01

    A large and consistent thermal resource exists in the area between 13--15/sup 0/ North latitude and 75--80/sup 0/ West longitude, which was studied for possible use by a plant ship. The area studied is primarily in the Colombian Basin of the Caribbean Sea. The average annual ..delta..T (surface temperature minus temperature at depth) for the area at 1000 meters is 22.4/sup 0/C. At 650 meters, an adequate ..delta..T exists, with no month of the year having a mean ..delta..T less than 19/sup 0/C. The total variability of temperature at all depths in this Caribbean area is small. Below 1000 meters, the total variation is extremely small. The area has a good mixed layer at all times of the year. It is occasionally exposed to strong winds from hurricanes. Currents generally show the same general pattern throughout the year with a predominant flow toward the west or northwest at average speeds of 40--50 cm/sec.

  18. Change in tobacco use among 13-15 year olds between 1999 and 2008: findings from the Global Youth Tobacco Survey.

    Science.gov (United States)

    Warren, Charles W; Lea, Veronica; Lee, Juliette; Jones, Nathan R; Asma, Samira; McKenna, Matthew

    2009-09-01

    Tobacco use is the leading preventable cause of death and disease in the world; yet little is known about the levels or patterns of youth tobacco use on a global basis. The purpose of this paper is to focus on change in youth tobacco use using data from 100 sites that have conducted repeat Global Youth Tobacco Surveys (GYTS). The GYTS is a school-based survey that collects data from students aged 13-15 years using a standardized methodology for constructing the sample frame, selecting schools and classes, and processing data. GYTS is conducted in school classes using self-administered anonymous data collection. The GYTS sample produces representative, independent, cross-sectional estimates for each sampling frame. Of the 100 sites surveyed, 61 reported no change over time in prevalence of cigarette smoking, likewise in 50 of the 97 sites with data on use of other tobacco products there was no change. However, 34 sites reported an increase in other tobacco use. This appears to be attributed to waterpipe, an emerging trend in tobacco use. Evidence was found supporting the idea that tobacco use among adolescent girls is likely increasing. The global tobacco control effort continues to face many challenges in reversing the tobacco epidemic. Few countries have implemented comprehensive tobacco control legislation laid out by the World Health Organization. The few countries that have adopted some of these proven policies can serve as examples in achieving positive results in curbing the tobacco epidemic.

  19. Intron Retention and TE Exonization Events in ZRANB2

    Directory of Open Access Journals (Sweden)

    Sang-Je Park

    2012-01-01

    Full Text Available The Zinc finger, RAN-binding domain-containing protein 2 (ZRANB2, contains arginine/serine-rich (RS domains that mediate its function in the regulation of alternative splicing. The ZRANB2 gene contains 2 LINE elements (L3b, Plat_L3 between the 9th and 10th exons. We identified the exonization event of a LINE element (Plat_L3. Using genomic PCR, RT-PCR amplification, and sequencing of primate DNA and RNA samples, we analyzed the evolutionary features of ZRANB2 transcripts. The results indicated that 2 of the LINE elements were integrated in human and all of the tested primate samples (hominoids: 3 species; Old World monkey: 8 species; New World monkey: 6 species; prosimian: 1 species. Human, rhesus monkey, crab-eating monkey, African-green monkey, and marmoset harbor the exon derived from LINE element (Plat_L3. RT-PCR amplification revealed the long transcripts and their differential expression patterns. Intriguingly, these long transcripts were abundantly expressed in Old World monkey lineages (rhesus, crab-eating, and African-green monkeys and were expressed via intron retention (IR. Thus, the ZRANB2 gene produces 3 transcript variants in which the Cterminus varies by transposable elements (TEs exonization and IR mechanisms. Therefore, ZRANB2 is valuable for investigating the evolutionary mechanisms of TE exonization and IR during primate evolution.

  20. First Exon Length Controls Active Chromatin Signatures and Transcription

    Directory of Open Access Journals (Sweden)

    Nicole I. Bieberstein

    2012-07-01

    Full Text Available Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

  1. Evaluation of dietary intake of vitamins and minerals in 13-15-years-old boys from a sport school in Warsaw.

    Science.gov (United States)

    Szczepańska, Beata; Malczewska-Lenczowska, Jadwiga; Wajszczyk, Bożena

    2016-01-01

    Insufficient intake of vitamins and minerals, in teenagers engaged in physical activity increases the risk of health disorders. The aim of this study was to evaluate selected vitamins and minerals intake in 13-15-year-old boys from sport school. The study of dietary intake was conducted among 44 boys from the School of Sport Championship (SSC). Nutritional data was collected using 24-hour recall for 3 days of week. Daily intake of minerals: sodium, potassium, calcium, phosphorus, magnesium, iron, zinc, copper, iodine and vitamins: A, E, D, B1, B2, B6, B12, C, folate and niacin was estimated. The probability of insufficient intake of nutrients in relation to the standard levels: Estimated Average Requirement (EAR) or Adequate Intake (AI) as well as excessive intake of them in relation to the Tolerable Upper Intake Level (UL) were assessed. The highest percentage of insufficient intake concerned vitamin D (100%), potassium (69%), folate (53%), and calcium (50%), slightly lower of magnesium (27%), vitamins C (24%) and E (15%). The risk of inadequate intake of other minerals: sodium, copper, iron, zinc, phosphorus, iodine and vitamins: B6, B1, B2, A, B12, niacin, was relatively lower and amounted from 0.3% to 5.4%. The disturbingly high probability of exceeding the UL for sodium (99.5%) was observed. A significant disproportion between the mean intake and the percentage of inadequate diets indicates a large diversity in the intake of vitamins and minerals in the group of studied boys, what was the reason of unbalanced diet. The insufficient intake concerns especially vitamin D, potassium, folate, calcium and a lesser extent magnesium, vitamins C and E. Sodium intake was disturbingly high. In order to avoid nutritional mistakes in the future education on the rational nutrition among students, their parents, and teachers is necessary.

  2. Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    van Ommen Gert-Jan B

    2007-07-01

    Full Text Available Abstract Background Antisense-mediated exon skipping is currently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD. Using antisense oligonucleotides (AONs targeting specific exons the DMD reading frame is restored and partially functional dystrophins are produced. Following proof of concept in cultured muscle cells from patients with various deletions and point mutations, we now focus on single and multiple exon duplications. These mutations are in principle ideal targets for this approach since the specific skipping of duplicated exons would generate original, full-length transcripts. Methods Cultured muscle cells from DMD patients carrying duplications were transfected with AONs targeting the duplicated exons, and the dystrophin RNA and protein were analyzed. Results For two brothers with an exon 44 duplication, skipping was, even at suboptimal transfection conditions, so efficient that both exons 44 were skipped, thus generating, once more, an out-of-frame transcript. In such cases, one may resort to multi-exon skipping to restore the reading frame, as is shown here by inducing skipping of exon 43 and both exons 44. By contrast, in cells from a patient with an exon 45 duplication we were able to induce single exon 45 skipping, which allowed restoration of wild type dystrophin. The correction of a larger duplication (involving exons 52 to 62, by combinations of AONs targeting the outer exons, appeared problematic due to inefficient skipping and mistargeting of original instead of duplicated exons. Conclusion The correction of DMD duplications by exon skipping depends on the specific exons targeted. Its options vary from the ideal one, restoring for the first time the true, wild type dystrophin, to requiring more 'classical' skipping strategies, while the correction of multi-exon deletions may need the design of tailored approaches.

  3. Reversible optic neuropathy with OPA1 exon 5b mutation

    DEFF Research Database (Denmark)

    Cornille, K.; Milea, D.; Amati-Bonneau, P.

    2008-01-01

    A new c.740G>A (R247H) mutation in OPA1 alternate spliced exon 5b was found in a patient presenting with bilateral optic neuropathy followed by partial, spontaneous visual recovery. R247H fibroblasts from the patient and his unaffected father presented unusual highly tubular mitochondrial network......, significant increased susceptibility to apoptosis, oxidative phosphorylation uncoupling, and altered OPA1 protein profile, supporting the pathogenicity of this mutation. These results suggest that the clinical spectrum of the OPA1-associated optic neuropathies may be larger than previously described......, and that spontaneous recovery may occur in cases harboring an exon 5b mutation Udgivelsesdato: 2008/5...

  4. Naturally occuring nucleosome positioning signals in human exons and introns

    DEFF Research Database (Denmark)

    Baldi, Pierre; Brunak, Søren; Chauvin, Yves

    1996-01-01

    We describe the structural implications of a periodic pattern found in human exons and introns by hidden Markov models. We show that exons (besides the reading frame) have a specific sequential structure in the form of a pattern with triplet consensus non-T(A/T)G, and a minimal periodicity...... faces inward and is compressed. The in-phase triplets are located adjacent to GCC/GGC triplets known to have the strongest bias in their positioning on the nucleosome. Analysis of mRNA sequences encoding proteins with known tertiary structure exclude the possibility that the pattern is a consequence...

  5. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency

    Directory of Open Access Journals (Sweden)

    HaiFang Yin

    2013-01-01

    Full Text Available We have recently reported that cell-penetrating peptides (CPPs and novel chimeric peptides containing CPP (referred as B peptide and muscle-targeting peptide (referred as MSP motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO and control peptide 3 (B-3-PMO and 3-B-PMO were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO, further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG, indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.

  6. Prevalence and sociodemographic correlates of stunting, underweight, and overweight among Palestinian school adolescents (13-15 years) in two major governorates in the West Bank

    Science.gov (United States)

    2009-01-01

    Background There is little information about height and weight status of Palestinian adolescents. The objective of this paper was to assess the prevalence of stunting, underweight, and overweight/obesity among Palestinian school adolescents (13-15 years) and associated sociodemographic factors in 2 major governorates in the West Bank. Methods A Cross-sectional survey was conducted in 2005 comprising 1942 students in 65 schools in Ramallah and Hebron governorates. Data was collected through self-administered questionnaires from students and parents. Weights and heights were measured. Overweight and obesity were assessed using the 2000 Centers for Disease Control and Prevention (CDC) reference and the International Obesity Task Force (IOTF) criteria. Stunting and underweight were assessed using the 2000 CDC reference. Results Overweight/obesity was more prevalent in Ramallah than in Hebron and affected more girls than boys. Using the 2000 CDC reference, the prevalence of overweight and obesity in Ramallah among boys was 9.6% and 8.2%, respectively versus 15.6% and 6.0% among girls (P < 0.01). In Hebron, the corresponding figures were 8.5% and 4.9% for boys and 13.5% and 3.4% for girls (P < 0.01). Using the IOTF criteria, the prevalence of overweight and obesity among boys in Ramallah was 13.3% and 5.2%, respectively versus 18.9% and 3.3% for girls. The prevalence of overweight and obesity among boys in Hebron was 10.9% and 2.2%, respectively versus 14.9% and 2.0% for girls. Overweight/obesity was associated with high standard of living (STL) among boys and with the onset of puberty among girls. More boys were underweight than girls, and the prevalence was higher in Hebron (12.9% and 6.0% in boys and girls, respectively (P < 0.01)) than in Ramallah (9.7% and 3.1% in boys and girls, respectively (p < 0.01)). The prevalence of stunting was similar in both governorates, and was higher among boys (9.2% and 9.4% in Ramallah and Hebron, respectively) than among girls (5

  7. Comparative analysis of antisense oligonucleotide sequences targeting exon 53 of the human DMD gene: Implications for future clinical trials.

    Science.gov (United States)

    Popplewell, Linda J; Adkin, Carl; Arechavala-Gomeza, Virginia; Aartsma-Rus, Annemieke; de Winter, Christa L; Wilton, Steve D; Morgan, Jennifer E; Muntoni, Francesco; Graham, Ian R; Dickson, George

    2010-02-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein, most commonly as a result of a range of out-of-frame mutations in the DMD gene. Modulation of pre-mRNA splicing with antisense oligonucleotides (AOs) to restore the reading frame has been demonstrated in vitro and in vivo, such that truncated but functional dystrophin is expressed. AO-induced skipping of exon 51 of the DMD gene, which could treat 13% of DMD patients, has now progressed to clinical trials. We describe here the methodical, cooperative comparison, in vitro (in DMD cells) and in vivo (in a transgenic mouse expressing human dystrophin), of 24 AOs of the phosphorodiamidate morpholino oligomer (PMO) chemistry designed to target exon 53 of the DMD gene, skipping of which could be potentially applicable to 8% of patients. A number of the PMOs tested should be considered worthy of development for clinical trial. Copyright 2009 Elsevier B.V. All rights reserved.

  8. Molecular characterization of exon 28 of von Willebrand's factor ...

    African Journals Online (AJOL)

    Background: Polymorphisms in von Willebrand factor (VWF) gene are an important contributor to the expression of VWF gene and differences in ethnic distribution of these single nucleotide polymorphisms (SNPs) exists. Aims: Our objective was to molecularly characterize the exon 28 of the VWF gene in the three major ...

  9. Designing exons for human olfactory receptor gene subfamilies ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences; Volume 35; Issue 3. Designing exons for human olfactory receptor gene subfamilies using a mathematical paradigm. Sk Sarif Hassan Pabitra Pal Choudhury Amita Pal R L Brahmachary Arunava Goswami. Articles Volume 35 Issue 3 September 2010 pp 389-393 ...

  10. Exon duplications in the ATP7A gene

    DEFF Research Database (Denmark)

    Mogensen, Mie; Skjørringe, Tina; Kodama, Hiroko

    2011-01-01

    BACKGROUND: Menkes disease (MD) is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene. Thirty-three Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in the ATP7A gene. ME...

  11. Investigation of ANGPTL3 expression, exon sequence and promotor ...

    African Journals Online (AJOL)

    like proteins, has been demonstrated to affect lipid metabolism by inhibiting the activity of lipoprotein lipase (LPL). Objective: To compare the ANGPTL3 mRNA and protein expression, exon mutation and promoter district CpG island methylation ...

  12. Exon-trapping mediated by the human retrotransposon SVA.

    Science.gov (United States)

    Hancks, Dustin C; Ewing, Adam D; Chen, Jesse E; Tokunaga, Katsushi; Kazazian, Haig H

    2009-11-01

    Although most human retrotransposons are inactive, both inactive and active retrotransposons drive genome evolution and may influence transcription through various mechanisms. In humans, three retrotransposon families are still active, but one of these, SVA, remains mysterious. Here we report the identification of a new subfamily of SVA, which apparently formed after an alternative splicing event where the first exon of the MAST2 gene spliced into an intronic SVA and subsequently retrotransposed. Additional examples of SVA retrotransposing upstream exons due to splicing into SVA were also identified in other primate genomes. After molecular and computational experiments, we found a number of functional 3' splice sites within many different transcribed SVAs across the human and chimpanzee genomes. Using a minigene splicing construct containing an SVA, we observed splicing in cell culture, along with SVA exonization events that introduced premature termination codons (PTCs). These data imply that an SVA residing within an intron in the same orientation as the gene may alter normal gene transcription either by gene-trapping or by introducing PTCs through exonization, possibly creating differences within and across species.

  13. Molecular characterization of exon 28 of von Willebrand's factor ...

    African Journals Online (AJOL)

    2016-05-12

    May 12, 2016 ... two probable cases among 95 patients with hemophilia A and 11 with hemophilia B between 1980 and 1986, but full investigation and family studies were not performed. In. Nigeria, we have been unable to find documented cases of. Molecular characterization of exon 28 of von Willebrand's factor gene in ...

  14. Characteristics of binding sites of intergenic, intronic and exonic ...

    African Journals Online (AJOL)

    user

    2013-03-06

    Mar 6, 2013 ... miRNAFinder 2.2 (https://sites.google.com/site/malaheenee/home) was used to find miRNA origins (intergenic, exonic or intronic). A literature review of genes coding intronic miRNAs led to 51 oncogenes (Supplementary Table 1) that encode proteins participating in gastrointestinal and breast cancer.

  15. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)

    DEFF Research Database (Denmark)

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most...

  16. Translational and Regulatory Challenges for Exon Skipping Therapies

    NARCIS (Netherlands)

    Aartsma-Rus, Annemieke; Ferlini, Alessandra; Goemans, Nathalie; Pasmooij, Anna M. G.; Wells, Dominic J.; Bushby, Katerine; Vroom, Elizabeth; Balabanov, Pavel

    2014-01-01

    Several translational challenges are currently impeding the therapeutic development of antisense-mediated exon skipping approaches for rare diseases. Some of these are inherent to developing therapies for rare diseases, such as small patient numbers and limited information on natural history and

  17. A fast algorithm for exonic regions prediction in DNA sequences.

    Science.gov (United States)

    Saberkari, Hamidreza; Shamsi, Mousa; Heravi, Hamed; Sedaaghi, Mohammad Hossein

    2013-07-01

    The main purpose of this paper is to introduce a fast method for gene prediction in DNA sequences based on the period-3 property in exons. First, the symbolic DNA sequences were converted to digital signal using the electron ion interaction potential method. Then, to reduce the effect of background noise in the period-3 spectrum, we used the discrete wavelet transform at three levels and applied it on the input digital signal. Finally, the Goertzel algorithm was used to extract period-3 components in the filtered DNA sequence. The proposed algorithm leads to decrease the computational complexity and hence, increases the speed of the process. Detection of small size exons in DNA sequences, exactly, is another advantage of the algorithm. The proposed algorithm ability in exon prediction was compared with several existing methods at the nucleotide level using: (i) specificity - sensitivity values; (ii) receiver operating curves (ROC); and (iii) area under ROC curve. Simulation results confirmed that the proposed method can be used as a promising tool for exon prediction in DNA sequences.

  18. Polymorphism of exon 3 of the HLA-G gene

    DEFF Research Database (Denmark)

    Hviid, T V; Meldgaard, Michael; Sørensen, S

    1997-01-01

    rate of embryos. HLA-G seems to play an important role in the feto-maternal relationship. The polymorphism of the HLA-G locus is not fully clarified. One study has shown extensive nucleotide sequence variation in the exon 3 (alpha-2 domain) in healthy African Americans. A few studies in other...... populations have only revealed a limited polymorphism. We investigated the polymorphism of the exon 3 of HLA-G by means of Polymerase Chain Reaction (PCR)-Single Strand Conformation Polymorphism (SSCP)- and DNA sequencing analysis in a Danish population. We detected four single-base substitutions in exon 3...... compared to the sequence of HLA-6.0 (G*01011); one of these has not been reported before. We also found a deletion of the first base of codon 130 or the third of codon 129 in a heterozygous individual. This study, together with previous results, suggests that the polymorphism of exon 3 of the HLA-G gene...

  19. Loss of exon identity is a common mechanism of human inherited disease

    National Research Council Canada - National Science Library

    Sterne-Weiler, Timothy; Howard, Jonathan; Mort, Matthew; Cooper, David N; Sanford, Jeremy R

    2011-01-01

    ...) and exonic splicing silencers (ESS) in human inherited disease is still poorly understood. Here we use a top-down approach to determine rates of loss or gain of known human exonic splicing regulatory (ESR...

  20. Deep RNA sequencing reveals the smallest known mitochondrial micro exon in animals: The placozoan cox1 single base pair exon.

    Science.gov (United States)

    Osigus, Hans-Jürgen; Eitel, Michael; Schierwater, Bernd

    2017-01-01

    The phylum Placozoa holds a key position for our understanding of the evolution of mitochondrial genomes in Metazoa. Placozoans possess large mitochondrial genomes which harbor several remarkable characteristics such as a fragmented cox1 gene and trans-splicing cox1 introns. A previous study also suggested the existence of cox1 mRNA editing in Trichoplax adhaerens, yet the only formally described species in the phylum Placozoa. We have analyzed RNA-seq data of the undescribed sister species, Placozoa sp. H2 ("Panama" clone), with special focus on the mitochondrial mRNA. While we did not find support for a previously postulated cox1 mRNA editing mechanism, we surprisingly found two independent transcripts representing intermediate cox1 mRNA splicing stages. Both transcripts consist of partial cox1 exon as well as overlapping intron fragments. The data suggest that the cox1 gene harbors a single base pair (cytosine) micro exon. Furthermore, conserved group I intron structures flank this unique micro exon also in other placozoans. We discuss the evolutionary origin of this micro exon in the context of a self-splicing intron gain in the cox1 gene of the last common ancestor of extant placozoans.

  1. Polymorphysims of Gene in the Exons Were Associated with the Reproductive Endocrine of Japanese Flounder (

    Directory of Open Access Journals (Sweden)

    R. Q. Ma

    2012-06-01

    Full Text Available The cytochrome P450c17-I (CYP17-I is one of the enzymes critical to gonadal development and the synthesis of androgens. Two single nucleotide polymorphisms (SNPs were detected within the coding region of the CYP17-I gene in a population of 75 male Japanese flounder (Paralichthys olivaceus. They were SNP1 (c.C445T located in exon2 and SNP2 (c.T980C (p.Phe307Leu located in exon5. Four physiological indices, which were serum testosterone (T, serum 17β-estradiol (E2, Hepatosomatic index (HSI, and Gonadosomatic index (GSI, were studied to examine the effect of the two SNPs on the reproductive endocrines of Japanese flounder. Multiple comparisons revealed that CT genotype of SNP1 had a much lower T level than CC genotype (p<0.05 and the GSI of individuals with CC genotype of SNP2 was higher than those with TT genotype (p<0.05. Four diplotypes were constructed based on the two SNPs and the diplotype D3 had a significantly lower T level and GSI. In conclusion, the two SNPs were significantly associated with reproductive traits of Japanese flounder.

  2. Determinants of Tobacco Use among Students Aged 13-15 Years in Nepal and Sri Lanka: Results from the Global Youth Tobacco Survey, 2007

    Science.gov (United States)

    Kabir, M. A.; Goh, Kim-Leng

    2014-01-01

    Objectives: This study aimed to investigate tobacco use behaviours and their correlates among secondary school students in Nepal and Sri Lanka together with cross-country comparisons. Design: Cross-sectional survey. Methods and Settings: The data were obtained from the Global Youth Tobacco Survey (GYTS), 2007. Current tobacco use was considered as…

  3. Proceedings of the Worldwide Air Force Pricing Conference ’Enhancing Air Force Pricing’ Held at Oklahoma City, OK on 13-15 March 1984.

    Science.gov (United States)

    1984-03-01

    rate of sustention for 101 defective pricing reports closed during the period was 18 percent. I consider that un-cceptable. By comparison, the overall... sustention rate for contract audit reports was 53 percent. As many of you know, I believe that the Department can do better on resolving defective

  4. Evaluating the protein coding potential of exonized transposable element sequences

    Directory of Open Access Journals (Sweden)

    Borodovsky Mark

    2007-11-01

    Full Text Available Abstract Background Transposable element (TE sequences, once thought to be merely selfish or parasitic members of the genomic community, have been shown to contribute a wide variety of functional sequences to their host genomes. Analysis of complete genome sequences have turned up numerous cases where TE sequences have been incorporated as exons into mRNAs, and it is widely assumed that such 'exonized' TEs encode protein sequences. However, the extent to which TE-derived sequences actually encode proteins is unknown and a matter of some controversy. We have tried to address this outstanding issue from two perspectives: i-by evaluating ascertainment biases related to the search methods used to uncover TE-derived protein coding sequences (CDS and ii-through a probabilistic codon-frequency based analysis of the protein coding potential of TE-derived exons. Results We compared the ability of three classes of sequence similarity search methods to detect TE-derived sequences among data sets of experimentally characterized proteins: 1-a profile-based hidden Markov model (HMM approach, 2-BLAST methods and 3-RepeatMasker. Profile based methods are more sensitive and more selective than the other methods evaluated. However, the application of profile-based search methods to the detection of TE-derived sequences among well-curated experimentally characterized protein data sets did not turn up many more cases than had been previously detected and nowhere near as many cases as recent genome-wide searches have. We observed that the different search methods used were complementary in the sense that they yielded largely non-overlapping sets of hits and differed in their ability to recover known cases of TE-derived CDS. The probabilistic analysis of TE-derived exon sequences indicates that these sequences have low protein coding potential on average. In particular, non-autonomous TEs that do not encode protein sequences, such as Alu elements, are frequently

  5. Efeito da idade de desmame no desempenho reprodutivo de novilhas de corte expostas à reprodução aos 13/15 meses de idade Effects of the weaning age on the reproductive performance of beef heifers exposed to reproduction at 13/15 months old

    Directory of Open Access Journals (Sweden)

    Ricardo Zambarda Vaz

    2010-01-01

    Full Text Available Este trabalho foi realizado com o objetivo de avaliar, por dois anos consecutivos, o efeito da idade de desmame no desempenho reprodutivo de 121 novilhas Braford acasaladas aos 13/15 meses de idade. Avaliaram-se duas idades de desmame - aos 77 dias pós-parto (desmame precoce; e aos 147 dias pós-parto (desmame convencional, utilizando-se 121 bezerras Braford nascidas nos anos de 2004 (66 e 2005 (55. Após o desmame, as bezerras foram mantidas, inicialmente, em pastagens cultivadas de verão (Pennisetum americanum, L. e de inverno/primavera (Avena strigosa Schreb + Lolium multiflorum Lam. Foram avaliados o peso vivo (PV, a condição corporal (CC e o ganho de peso médio diário (GMD durante o período reprodutivo. O desmame precoce não ocasionou diferença significativa no peso ao início e ao final do período reprodutivo. As novilhas nascidas em 2005 apresentaram menor peso durante o período reprodutivo em relação às nascidas em 2004. O ano influenciou a condição corporal ao início e ao final do período reprodutivo, que foi, respectivamente, 4,16 e 4,29 pontos para as novilhas nascidas em 2004; e 3,57 e 3,38 pontos para as nascidas em 2005. A porcentagem de prenhez não foi influenciada pela idade de desmame (55,5 e 70,7% para novilhas do desmame precoce e as do desmame convencional, respectivamente nem pelo ano (68,6% para novilhas nascidas em 2004 e 57,6% para as novilhas nascidas em 2005. As novilhas com diagnóstico de prenhez positivo tiveram maior desempenho reprodutivo durante a recria e o período de acasalamento em comparação àquelas com diagnóstico negativo. O desempenho reprodutivo das novilhas correlacionou-se positivamente com peso vivo e condição corporal ao início e ao final do período reprodutivo. O desmame precoce não afeta o desempenho reprodutivo de novilhas Braford aos 13/15 meses de idade.The objective of the study was to evaluate, for two consecutive years, the weaning age effect on the reproductive

  6. Detection of an exon 53 polymorphism in the dystrophin gene.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Sedra, M S

    1993-10-01

    We utilized a heteroduplex method to screen for small mutations in Duchenne muscular dystrophy patients who did not have deletions or duplications. A dystrophin exon 53 heteroduplex band was identified in 14.4% of the affected patients. Direct sequencing of the amplified product from DNA producing the heteroduplex revealed the presence of a polymorphism in the coding region. The codon for asparagine was converted from AAT to AAC.

  7. Characteristics of binding sites of intergenic, intronic and exonic ...

    African Journals Online (AJOL)

    The interaction of 784 intergenic (ig-miRNA), 686 intronic (in-miRNA) and 49 exonic miRNAs (ex-miRNA) with mRNAs of 51 oncogenes coding in-miRNAs was investigated. Out of the studied genes, 44 were targets for 94 ig-miRNAs, 29 were targets for 44 in-miRNAs and 7 were targets for 7 ex-miRNAs. The density of ...

  8. A simple physical model predicts small exon length variations.

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available One of the most common splice variations are small exon length variations caused by the use of alternative donor or acceptor splice sites that are in very close proximity on the pre-mRNA. Among these, three-nucleotide variations at so-called NAGNAG tandem acceptor sites have recently attracted considerable attention, and it has been suggested that these variations are regulated and serve to fine-tune protein forms by the addition or removal of a single amino acid. In this paper we first show that in-frame exon length variations are generally overrepresented and that this overrepresentation can be quantitatively explained by the effect of nonsense-mediated decay. Our analysis allows us to estimate that about 50% of frame-shifted coding transcripts are targeted by nonsense-mediated decay. Second, we show that a simple physical model that assumes that the splicing machinery stochastically binds to nearby splice sites in proportion to the affinities of the sites correctly predicts the relative abundances of different small length variations at both boundaries. Finally, using the same simple physical model, we show that for NAGNAG sites, the difference in affinities of the neighboring sites for the splicing machinery accurately predicts whether splicing will occur only at the first site, splicing will occur only at the second site, or three-nucleotide splice variants are likely to occur. Our analysis thus suggests that small exon length variations are the result of stochastic binding of the spliceosome at neighboring splice sites. Small exon length variations occur when there are nearby alternative splice sites that have similar affinity for the splicing machinery.

  9. The Exon-Florio National Security Test for Foreign Investment

    Science.gov (United States)

    2010-02-04

    Asia, Latin America, the Carribean , and North America. 24 Peninsular and Oriental Steam Company is a leading ports operator and transport company...CRS Report for Congress Prepared for Members and Committees of Congress The Exon-Florio National Security Test for Foreign Investment...c11173008 Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour

  10. Rodent-specific alternative exons are more frequent in rapidly evolving genes and in paralogs

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    Mironov Andrey A

    2009-06-01

    Full Text Available Abstract Background Alternative splicing is an important mechanism for generating functional and evolutionary diversity of proteins in eukaryotes. Here, we studied the frequency and functionality of recently gained, rodent-specific alternative exons. Results We projected the data about alternative splicing of mouse genes to the rat, human, and dog genomes, and identified exons conserved in the rat genome, but missing in more distant genomes. We estimated the frequency of rodent-specific exons while controlling for possible residual conservation of spurious exons. The frequency of rodent-specific exons is higher among predominantly skipped exons and exons disrupting the reading frame. Separation of all genes by the rate of sequence evolution and by gene families has demonstrated that rodent-specific cassette exons are more frequent in rapidly evolving genes and in rodent-specific paralogs. Conclusion Thus we demonstrated that recently gained exons tend to occur in fast-evolving genes, and their inclusion rate tends to be lower than that of older exons. This agrees with the theory that gain of alternative exons is one of the major mechanisms of gene evolution.

  11. Efficient use of a translation start codon in BDNF exon I.

    Science.gov (United States)

    Koppel, Indrek; Tuvikene, Jürgen; Lekk, Ingrid; Timmusk, Tõnis

    2015-09-01

    The brain-derived neurotrophic factor (BDNF) gene contains a number of 5' exons alternatively spliced with a common 3' exon. BDNF protein is synthesized from alternative transcripts as a prepro-precursor encoded by the common 3' exon IX, which has a translation start site 21 bp downstream of the splicing site. BDNF mRNAs containing exon I are an exception to this arrangement as the last three nucleotides of this exon constitute an in-frame AUG. Here, we show that this AUG is efficiently used for translation initiation in PC12 cells and cultured cortical neurons. Use of exon I-specific AUG produces higher levels of BDNF protein than use of the common translation start site, resulting from a higher translation rate. No differences in protein degradation, constitutive or regulated secretion were detected between BDNF isoforms with alternative 5' termini. As the BDNF promoter preceding exon I is known to be highly regulated by neuronal activity, our results suggest that the function of this translation start site may be efficient stimulus-dependent synthesis of BDNF protein. The brain-derived neurotrophic factor (BDNF) gene contains multiple untranslated 5' exons alternatively spliced to one common protein-coding 3' exon. However, exon I contains an in-frame ATG in a favorable translation context. Here, we show that use of this ATG is associated with more efficient protein synthesis than the commonly used ATG in exon IX. © 2015 International Society for Neurochemistry.

  12. Variants affecting exon skipping contribute to complex traits.

    Directory of Open Access Journals (Sweden)

    Younghee Lee

    Full Text Available DNA variants that affect alternative splicing and the relative quantities of different gene transcripts have been shown to be risk alleles for some Mendelian diseases. However, for complex traits characterized by a low odds ratio for any single contributing variant, very few studies have investigated the contribution of splicing variants. The overarching goal of this study is to discover and characterize the role that variants affecting alternative splicing may play in the genetic etiology of complex traits, which include a significant number of the common human diseases. Specifically, we hypothesize that single nucleotide polymorphisms (SNPs in splicing regulatory elements can be characterized in silico to identify variants affecting splicing, and that these variants may contribute to the etiology of complex diseases as well as the inter-individual variability in the ratios of alternative transcripts. We leverage high-throughput expression profiling to 1 experimentally validate our in silico predictions of skipped exons and 2 characterize the molecular role of intronic genetic variations in alternative splicing events in the context of complex human traits and diseases. We propose that intronic SNPs play a role as genetic regulators within splicing regulatory elements and show that their associated exon skipping events can affect protein domains and structure. We find that SNPs we would predict to affect exon skipping are enriched among the set of SNPs reported to be associated with complex human traits.

  13. Translational and regulatory challenges for exon skipping therapies.

    Science.gov (United States)

    Aartsma-Rus, Annemieke; Ferlini, Alessandra; Goemans, Nathalie; Pasmooij, Anna M G; Wells, Dominic J; Bushby, Katerine; Vroom, Elizabeth; Balabanov, Pavel

    2014-10-01

    Several translational challenges are currently impeding the therapeutic development of antisense-mediated exon skipping approaches for rare diseases. Some of these are inherent to developing therapies for rare diseases, such as small patient numbers and limited information on natural history and interpretation of appropriate clinical outcome measures. Others are inherent to the antisense oligonucleotide (AON)-mediated exon skipping approach, which employs small modified DNA or RNA molecules to manipulate the splicing process. This is a new approach and only limited information is available on long-term safety and toxicity for most AON chemistries. Furthermore, AONs often act in a mutation-specific manner, in which case multiple AONs have to be developed for a single disease. A workshop focusing on preclinical development, trial design, outcome measures, and different forms of marketing authorization was organized by the regulatory models and biochemical outcome measures working groups of Cooperation of Science and Technology Action: "Networking towards clinical application of antisense-mediated exon skipping for rare diseases." The workshop included participants from patient organizations, academia, and members of staff from the European Medicine Agency and Medicine Evaluation Board (the Netherlands). This statement article contains the key outcomes of this meeting.

  14. Suplementação energética pré-acasalamento aos 13/15 meses de idade para novilhas de corte: desenvolvimento e desempenho reprodutivo Pre-mating energetic supplementation at 13/15 months of age for beef heifers: development and reproductive performance

    Directory of Open Access Journals (Sweden)

    Alcides Pilau

    2009-12-01

    Full Text Available Avaliou-se o efeito da suplementação pré-acasalamento no desempenho reprodutivo de novilhas de corte aos 13/15 meses de idade. Foram utilizadas 89 novilhas com média de 12 meses de idade e peso corporal (PC médio inicial de 242 kg, provenientes de três rebanhos (novilhas Aberdeen Angus das Fazendas Santa Cecília e Capitão Rodrigo e novilhas mestiças Aberdeen Angus submetidas a regime alimentar exclusivamente em pastejo ou com suplementação a pasto com grão de milho moído. As novilhas foram agrupadas dentro dos regimes alimentares considerando o peso inicial e o rebanho. O suplemento foi fornecido diariamente na proporção de 0,7% do peso corporal por 29 dias em pastagem de aveia (Avena strigosa Schreb e azevém (Lolium multiflorum Lam e 19 dias em pastagem natural. O pastejo foi contínuo com taxa de lotação variável. As variáveis analisadas foram: peso corporal, condição corporal, ganho de peso médio diário, ganho de condição corporal, porcentagem de novilhas púberes, taxa de prenhez e ordem de concepção. Não houve interação regimes alimentares ×rebanhos. Durante o pré-acasalamento, o maior ganho médio diário das novilhas sob suplementação foi maior (0,800 kg que o daquelas sem suplementação (0,658 kg. No período reprodutivo, as novilhas perderam peso, média de -0,104 kg/dia. O peso corporal e a condição corporal não diferiram ao início do período reprodutivo, média de 277 kg e 3,6 pontos, respectivamente. As novilhas sob suplementação apresentaram maior PNP (por extenso (78% e maior taxa de prenhez (47%. Houve interação rebanho ×período para peso corporal. A variação no peso vivo foi quadrática entre os três rebanhos. O peso corporal das novilhas mestiças foi sempre superior ao das Aberdeen Angus do rebanho Santa Cecília. As novilhas Aberdeen Angus do rebanho Capitão Rodrigo tiveram maior percentagem de novilhas púberes (80% e taxa de prenhez (48%. A suplementação energética por 48

  15. A founder synonymous COL7A1 mutation in three Danish families with dominant dystrophic epidermolysis bullosa pruriginosa identifies exonic regulatory sequences required for exon 87 splicing

    DEFF Research Database (Denmark)

    Covaciu, C; Grosso, F; Pisaneschi, E

    2011-01-01

    shoulders. DEB-Pr is caused by either dominant (DDEB-Pr) or recessive mutations in the COL7A1 gene encoding type VII collagen (COLVII). The full spectrum of COL7A1 mutations in DEB-Pr remains elusive and the genotype-phenotype correlation is largely incomplete. Here, we report and functionally characterize...... a previously unrecognized translationally silent exonic COL7A1 mutation that results in skipping of exon 87 and is associated with DDEB-Pr phenotypes in several members of three apparently unrelated Danish families. A haplotype segregation study suggested a common ancestor in these kindred. Functional splicing...... analysis of the mutant exon by a COL7A1 minigene construct and computational prediction for splicing regulatory cis-sequences prove that the mutation alters the activity of an exonic splicing enhancer (ESE) critical for exon inclusion. These findings substantiate for the first time the involvement...

  16. Exon and junction microarrays detect widespread mouse strain- and sex-bias expression differences

    Directory of Open Access Journals (Sweden)

    Schadt Eric E

    2008-06-01

    Full Text Available Abstract Background Studies have shown that genetic and sex differences strongly influence gene expression in mice. Given the diversity and complexity of transcripts produced by alternative splicing, we sought to use microarrays to establish the extent of variation found in mouse strains and genders. Here, we surveyed the effect of strain and sex on liver gene and exon expression using male and female mice from three different inbred strains. Results 71 liver RNA samples from three mouse strains – DBA/2J, C57BL/6J and C3H/HeJ – were profiled using a custom-designed microarray monitoring exon and exon-junction expression of 1,020 genes representing 9,406 exons. Gene expression was calculated via two different methods, using the 3'-most exon probe ("3' gene expression profiling" and using all probes associated with the gene ("whole-transcript gene expression profiling", while exon expression was determined using exon probes and flanking junction probes that spanned across the neighboring exons ("exon expression profiling". Widespread strain and sex influences were detected using a two-way Analysis of Variance (ANOVA regardless of the profiling method used. However, over 90% of the genes identified in 3' gene expression profiling or whole transcript profiling were identified in exon profiling, along with 75% and 38% more genes, respectively, showing evidence of differential isoform expression. Overall, 55% and 32% of genes, respectively, exhibited strain- and sex-bias differential gene or exon expression. Conclusion Exon expression profiling identifies significantly more variation than both 3' gene expression profiling and whole-transcript gene expression profiling. A large percentage of genes that are not differentially expressed at the gene level demonstrate exon expression variation suggesting an influence of strain and sex on alternative splicing and a need to profile expression changes at sub-gene resolution.

  17. Mutations in Exons 9 and 13 of KIT Gene Are Rare Events in Gastrointestinal Stromal Tumors

    Science.gov (United States)

    Lasota, Jerzy; Wozniak, Agnieszka; Sarlomo-Rikala, Maarit; Rys, Janusz; Kordek, Radzislaw; Nassar, Aziza; Sobin, Leslie H.; Miettinen, Markku

    2000-01-01

    Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the gastrointestinal tract, typically express the KIT protein. Activating mutations in the juxtamembrane domain (exon 11) of the c-kit gene have been shown in a subset of GISTs. These mutations lead into ligand-independent activation of the tyrosine kinase of c-kit, and have a transforming effect in vitro. Several groups have studied the clinical implication of the c-kit mutation status of exon 11 in GISTs and a possible relationship between c-kit mutations and malignant behavior has been established. Recently, a 1530ins6 mutation in exon 9 and missense mutations, 1945A>G in exon 13 of the c-kit gene were reported. The frequency and clinical importance of these findings are unknown. In this study we evaluated 200 GISTs for the presence of mutations in exons 9 and 13 of c-kit. Six cases revealed 1530ins6 mutation in exon 9 and two cases 1945A>G mutation in exon 13. All tumors with mutations in exon 9 and 13 lacked mutations in exon 11 of c-kit. None of the analyzed tumors had more than one type of c-kit mutation. All but one of the eight tumors with mutations in exon 9 or 13 of the c-kit gene were histologically and clinically malignant. All four of six cases with exon 9 mutation of which location of primary tumor was known, were small intestinal, suggesting that this type of mutation could preferentially occur in small intestinal tumors. Exon 9 and 13 mutations seem to be rare, and they cover only a small portion (8%) of the balance of GISTs that do not have mutations in exon 11 of c-kit. This finding indicates that other genetic alterations may activate c-kit in GISTs, or that KIT is not activated by mutations in all cases. PMID:11021812

  18. Targeted exon sequencing in Usher syndrome type I.

    Science.gov (United States)

    Bujakowska, Kinga M; Consugar, Mark; Place, Emily; Harper, Shyana; Lena, Jaclyn; Taub, Daniel G; White, Joseph; Navarro-Gomez, Daniel; Weigel DiFranco, Carol; Farkas, Michael H; Gai, Xiaowu; Berson, Eliot L; Pierce, Eric A

    2014-12-02

    Patients with Usher syndrome type I (USH1) have retinitis pigmentosa, profound congenital hearing loss, and vestibular ataxia. This syndrome is currently thought to be associated with at least six genes, which are encoded by over 180 exons. Here, we present the use of state-of-the-art techniques in the molecular diagnosis of a cohort of 47 USH1 probands. The cohort was studied with selective exon capture and next-generation sequencing of currently known inherited retinal degeneration genes, comparative genomic hybridization, and Sanger sequencing of new USH1 exons identified by human retinal transcriptome analysis. With this approach, we were able to genetically solve 14 of the 47 probands by confirming the biallelic inheritance of mutations. We detected two likely pathogenic variants in an additional 19 patients, for whom family members were not available for cosegregation analysis to confirm biallelic inheritance. Ten patients, in addition to primary disease-causing mutations, carried rare likely pathogenic USH1 alleles or variants in other genes associated with deaf-blindness, which may influence disease phenotype. Twenty-one of the identified mutations were novel among the 33 definite or likely solved patients. Here, we also present a clinical description of the studied cohort at their initial visits. We found a remarkable genetic heterogeneity in the studied USH1 cohort with multiplicity of mutations, of which many were novel. No obvious influence of genotype on phenotype was found, possibly due to small sample sizes of the genotypes under study. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  19. DNA-methylation effect on cotranscriptional splicing is dependent on GC architecture of the exon-intron structure.

    Science.gov (United States)

    Gelfman, Sahar; Cohen, Noa; Yearim, Ahuvi; Ast, Gil

    2013-05-01

    DNA methylation is known to regulate transcription and was recently found to be involved in exon recognition via cotranscriptional splicing. We recently observed that exon-intron architectures can be grouped into two classes: one with higher GC content in exons compared to the flanking introns, and the other with similar GC content in exons and introns. The first group has higher nucleosome occupancy on exons than introns, whereas the second group exhibits weak nucleosome marking of exons, suggesting another type of epigenetic marker distinguishes exons from introns when GC content is similar. We find different and specific patterns of DNA methylation in each of the GC architectures; yet in both groups, DNA methylation clearly marks the exons. Exons of the leveled GC architecture exhibit a significantly stronger DNA methylation signal in relation to their flanking introns compared to exons of the differential GC architecture. This is accentuated by a reduction of the DNA methylation level in the intronic sequences in proximity to the splice sites and shows that different epigenetic modifications mark the location of exons already at the DNA level. Also, lower levels of methylated CpGs on alternative exons can successfully distinguish alternative exons from constitutive ones. Three positions at the splice sites show high CpG abundance and accompany elevated nucleosome occupancy in a leveled GC architecture. Overall, these results suggest that DNA methylation affects exon recognition and is influenced by the GC architecture of the exon and flanking introns.

  20. The exon junction complex differentially marks spliced junctions.

    Science.gov (United States)

    Saulière, Jérôme; Haque, Nazmul; Harms, Scot; Barbosa, Isabelle; Blanchette, Marco; Le Hir, Hervé

    2010-10-01

    The exon junction complex (EJC), which is deposited onto mRNAs as a consequence of splicing, is involved in multiple post-transcriptional events in metazoa. Here, using Drosophila melanogaster cells, we show that only some introns trigger EJC-dependent nonsense-mediated mRNA decay and that EJC association with particular spliced junctions depends on RNA cis-acting sequences. This study provides the first evidence to our knowledge that EJC deposition is not constitutive but instead is a regulated process.

  1. Evolution of hydra, a recently evolved testis-expressed gene with nine alternative first exons in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Shou-Tao Chen

    2007-07-01

    Full Text Available We describe here the Drosophila gene hydra that appears to have originated de novo in the melanogaster subgroup and subsequently evolved in both structure and expression level in Drosophila melanogaster and its sibling species. D. melanogaster hydra encodes a predicted protein of approximately 300 amino acids with no apparent similarity to any previously known proteins. The syntenic region flanking hydra on both sides is found in both D. ananassae and D. pseudoobscura, but hydra is found only in melanogaster subgroup species, suggesting that it originated less than approximately 13 million y ago. Exon 1 of hydra has undergone recurrent duplications, leading to the formation of nine tandem alternative exon 1s in D. melanogaster. Seven of these alternative exons are flanked on their 3' side by the transposon DINE-1 (Drosophila interspersed element-1. We demonstrate that at least four of the nine duplicated exon 1s can function as alternative transcription start sites. The entire hydra locus has also duplicated in D. simulans and D. sechellia. D. melanogaster hydra is expressed most intensely in the proximal testis, suggesting a role in late-stage spermatogenesis. The coding region of hydra has a relatively high Ka/Ks ratio between species, but the ratio is less than 1 in all comparisons, suggesting that hydra is subject to functional constraint. Analysis of sequence polymorphism and divergence of hydra shows that it has evolved under positive selection in the lineage leading to D. melanogaster. The dramatic structural changes surrounding the first exons do not affect the tissue specificity of gene expression: hydra is expressed predominantly in the testes in D. melanogaster, D. simulans, and D. yakuba. However, we have found that expression level changed dramatically (approximately >20-fold between D. melanogaster and D. simulans. While hydra initially evolved in the absence of nearby transposable element insertions, we suggest that the subsequent

  2. Clinical phenotypes as predictors of the outcome of skipping around DMD exon 45.

    Science.gov (United States)

    Findlay, Andrew R; Wein, Nicolas; Kaminoh, Yuuki; Taylor, Laura E; Dunn, Diane M; Mendell, Jerry R; King, Wendy M; Pestronk, Alan; Florence, Julaine M; Mathews, Katherine D; Finkel, Richard S; Swoboda, Kathryn J; Howard, Michael T; Day, John W; McDonald, Craig; Nicolas, Aurélie; Le Rumeur, Elisabeth; Weiss, Robert B; Flanigan, Kevin M

    2015-04-01

    Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion-exon 45 (Δ45)-may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients. Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy were analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions. As expected, deletions of either exons 45 to 47 (Δ45-47) or exons 45 to 48 (Δ45-48) result in BMD in 97% (36 of 37) of subjects. Unexpectedly, deletion of exons 45 to 46 (Δ45-46) is associated with the more severe DMD phenotype in 4 of 4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44 to 45 (Δ44-45) were found within the United Dystrophinopathy Project database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype. The observation that Δ45-46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of exon 44 and multiexon skipping of exons 46 and 47 (or exons 46-48) are better potential therapies than skipping of exon 46 alone. © 2015 American Neurological Association.

  3. Conserved usage of alternative 5' untranslated exons of the GATA4 gene.

    Directory of Open Access Journals (Sweden)

    Séverine Mazaud Guittot

    2009-12-01

    Full Text Available GATA4 is an essential transcription factor required for the development and function of multiple organs. Despite this important role, our knowledge of how the GATA4 gene is regulated remains limited. To better understand this regulation, we characterized the 5' region of the mouse, rat, and human GATA4 genes.Using 5' RACE, we identified novel transcription start sites in all three species. GATA4 is expressed as multiple transcripts with varying 5' ends encoded by alternative untranslated first exons. Two of these non-coding first exons are conserved between species: exon 1a located 3.5 kb upstream of the GATA4 ATG site in exon 2, and a second first exon (exon 1b located 28 kb further upstream. Expression of both mRNA variants was found in all GATA4-expressing organs but with a preference for the exon 1a-containing transcript. The exception was the testis where exon 1a- and 1b-containing transcripts were similarly expressed. In some tissues such as the intestine, alternative transcript expression appears to be regionally regulated. Polysome analysis suggests that both mRNA variants contribute to GATA4 protein synthesis.Taken together, our results indicate that the GATA4 gene closely resembles the other GATA family members in terms of gene structure where alternative first exon usage appears to be an important mechanism for regulating its tissue- and cell-specific expression.

  4. Amelogenin Exon4 Forms a Novel miRNA That Directs Ameloblast and Osteoblast Differentiation.

    Science.gov (United States)

    Le, M H; Warotayanont, R; Stahl, J; Den Besten, P K; Nakano, Y

    2016-04-01

    Amelogenins constitute the major portion of secretory enamel matrix proteins and are known to be highly alternative spliced. Of all the alternatively spliced forms of amelogenins, exon4 is most commonly spliced out. Our analyses of the exon4 sequence led us to hypothesize that when spliced out, exon4 may generate a novel mature miRNA. To explore this possibility, we used in vivo mouse models (wild-type and Amel knockout mice) and in vitro cell culture to investigate the presence and function of a mature miRNA derived from exon4 (miR-exon4). When ameloblast-like cells (LS8) were transfected with an amelogenin minigene to increase amelogenin synthesis, the transfected cells synthesized miR-exon4. Introduction of a mutation in the conserved CNNC sequence required for primary miRNA recognition, downstream of the mature miR-exon4 sequence, resulted in a significantly reduced production of miR-exon4 in the transfected cells. In vivo, miR-exon4 was most highly amplified from wild-type mouse enamel organs at the secretory stage. In Amel knockout mice, an in vivo model for reduced amelogenin synthesis, we found reduced miR-exon4, with no changes in expression of enamel matrix-related genes. However, expression of Runx2 and its downstream genes Odam and Amtn were significantly downregulated. Transfection of miR-exon4 mimic to the LS8 cells also significantly upregulated Runx2. The mature miR-exon4 as well as Runx2 was also present in mouse osteoblasts with no apparent change in expression level between wild-type and Amel knockout mice. However, transfecting miR-exon4 inhibitor to the MC3T3-E1 osteoblastic cells resulted in a significant downregulation of Runx2 expression. These data indicate that when exon4 is spliced out, as occurs most of the time during alternative splicing of amelogenin pre-mRNA, a novel mature miRNA is generated from exon4. This miR-exon4 may contribute to the differentiation of ameloblasts and osteoblasts through regulation of Runx2 expression.

  5. Genomic V exons from whole genome shotgun data in reptiles.

    Science.gov (United States)

    Olivieri, D N; von Haeften, B; Sánchez-Espinel, C; Faro, J; Gambón-Deza, F

    2014-08-01

    Reptiles and mammals diverged over 300 million years ago, creating two parallel evolutionary lineages amongst terrestrial vertebrates. In reptiles, two main evolutionary lines emerged: one gave rise to Squamata, while the other gave rise to Testudines, Crocodylia, and Aves. In this study, we determined the genomic variable (V) exons from whole genome shotgun sequencing (WGS) data in reptiles corresponding to the three main immunoglobulin (IG) loci and the four main T cell receptor (TR) loci. We show that Squamata lack the TRG and TRD genes, and snakes lack the IGKV genes. In representative species of Testudines and Crocodylia, the seven major IG and TR loci are maintained. As in mammals, genes of the IG loci can be grouped into well-defined IMGT clans through a multi-species phylogenetic analysis. We show that the reptilian IGHV and IGLV genes are distributed amongst the established mammalian clans, while their IGKV genes are found within a single clan, nearly exclusive from the mammalian sequences. The reptilian and mammalian TRAV genes cluster into six common evolutionary clades (since IMGT clans have not been defined for TR). In contrast, the reptilian TRBV genes cluster into three clades, which have few mammalian members. In this locus, the V exon sequences from mammals appear to have undergone different evolutionary diversification processes that occurred outside these shared reptilian clans. These sequences can be obtained in a freely available public repository (http://vgenerepertoire.org).

  6. Exon Shuffling and Origin of Scorpion Venom Biodiversity

    Directory of Open Access Journals (Sweden)

    Xueli Wang

    2016-12-01

    Full Text Available Scorpion venom is a complex combinatorial library of peptides and proteins with multiple biological functions. A combination of transcriptomic and proteomic techniques has revealed its enormous molecular diversity, as identified by the presence of a large number of ion channel-targeted neurotoxins with different folds, membrane-active antimicrobial peptides, proteases, and protease inhibitors. Although the biodiversity of scorpion venom has long been known, how it arises remains unsolved. In this work, we analyzed the exon-intron structures of an array of scorpion venom protein-encoding genes and unexpectedly found that nearly all of these genes possess a phase-1 intron (one intron located between the first and second nucleotides of a codon near the cleavage site of a signal sequence despite their mature peptides remarkably differ. This observation matches a theory of exon shuffling in the origin of new genes and suggests that recruitment of different folds into scorpion venom might be achieved via shuffling between body protein-coding genes and ancestral venom gland-specific genes that presumably contributed tissue-specific regulatory elements and secretory signal sequences.

  7. Polymorphism of exon 3 of the HLA-G gene

    DEFF Research Database (Denmark)

    Hviid, T V; Meldgaard, M; Sørensen, Steen

    1997-01-01

    HLA-G is a non-classical MHC class I gene with a limited tissue distribution. The most pronounced expression is detected in the cytotrophoblast of first trimester placenta. It is possible to detect mRNA for HLA-G in preimplantation blastocysts where expression is correlated with a high cleavage...... compared to the sequence of HLA-6.0 (G*01011); one of these has not been reported before. We also found a deletion of the first base of codon 130 or the third of codon 129 in a heterozygous individual. This study, together with previous results, suggests that the polymorphism of exon 3 of the HLA-G gene...... rate of embryos. HLA-G seems to play an important role in the feto-maternal relationship. The polymorphism of the HLA-G locus is not fully clarified. One study has shown extensive nucleotide sequence variation in the exon 3 (alpha-2 domain) in healthy African Americans. A few studies in other...

  8. The effects of multiple features of alternatively spliced exons on the KA/KS ratio test

    Directory of Open Access Journals (Sweden)

    Chen Feng-Chi

    2006-05-01

    Full Text Available Abstract Background The evolution of alternatively spliced exons (ASEs is of primary interest because these exons are suggested to be a major source of functional diversity of proteins. Many exon features have been suggested to affect the evolution of ASEs. However, previous studies have relied on the KA/KS ratio test without taking into consideration information sufficiency (i.e., exon length > 75 bp, cross-species divergence > 5% of the studied exons, leading to potentially biased interpretations. Furthermore, which exon feature dominates the results of the KA/KS ratio test and whether multiple exon features have additive effects have remained unexplored. Results In this study, we collect two different datasets for analysis – the ASE dataset (which includes lineage-specific ASEs and conserved ASEs and the ACE dataset (which includes only conserved ASEs. We first show that information sufficiency can significantly affect the interpretation of relationship between exons features and the KA/KS ratio test results. After discarding exons with insufficient information, we use a Boolean method to analyze the relationship between test results and four exon features (namely length, protein domain overlapping, inclusion level, and exonic splicing enhancer (ESE frequency for the ASE dataset. We demonstrate that length and protein domain overlapping are dominant factors, and they have similar impacts on test results of ASEs. In addition, despite the weak impacts of inclusion level and ESE motif frequency when considered individually, combination of these two factors still have minor additive effects on test results. However, the ACE dataset shows a slightly different result in that inclusion level has a marginally significant effect on test results. Lineage-specific ASEs may have contributed to the difference. Overall, in both ASEs and ACEs, protein domain overlapping is the most dominant exon feature while ESE frequency is the weakest one in affecting

  9. The role of exon shuffling in shaping protein-protein interaction networks

    Directory of Open Access Journals (Sweden)

    França Gustavo S

    2010-12-01

    Full Text Available Abstract Background Physical protein-protein interaction (PPI is a critical phenomenon for the function of most proteins in living organisms and a significant fraction of PPIs are the result of domain-domain interactions. Exon shuffling, intron-mediated recombination of exons from existing genes, is known to have been a major mechanism of domain shuffling in metazoans. Thus, we hypothesized that exon shuffling could have a significant influence in shaping the topology of PPI networks. Results We tested our hypothesis by compiling exon shuffling and PPI data from six eukaryotic species: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Cryptococcus neoformans and Arabidopsis thaliana. For all four metazoan species, genes enriched in exon shuffling events presented on average higher vertex degree (number of interacting partners in PPI networks. Furthermore, we verified that a set of protein domains that are simultaneously promiscuous (known to interact to multiple types of other domains, self-interacting (able to interact with another copy of themselves and abundant in the genomes presents a stronger signal for exon shuffling. Conclusions Exon shuffling appears to have been a recurrent mechanism for the emergence of new PPIs along metazoan evolution. In metazoan genomes, exon shuffling also promoted the expansion of some protein domains. We speculate that their promiscuous and self-interacting properties may have been decisive for that expansion.

  10. Lack of exon 10 in the murine tau gene results in mild sensorimotor defects with aging

    OpenAIRE

    Gumucio, Astrid; Lannfelt, Lars; Nilsson, Lars N

    2013-01-01

    Background Complex species-specific, developmental- and tissue-dependent mechanisms regulate alternative splicing of tau, thereby diversifying tau protein synthesis. The functional role of alternative splicing of tau e.g. exon 10 has never been examined in vivo, although genetic studies suggest that it is important to neurodegenerative disease. Results Gene-targeting was used to delete exon 10 in muri...

  11. Mutation in an exonic splicing enhancer site causing chronic granulomatous disease

    NARCIS (Netherlands)

    de Boer, Martin; van Leeuwen, Karin; Geissler, Judy; Belohradsky, Bernd H.; Kuijpers, Taco W.; Roos, Dirk

    2017-01-01

    In a male patient suffering from X-linked chronic granulomatous disease (CGD) we found a c. 389G > T mutation in exon 5 of the CYBB gene. We have analyzed why 95% of the transcripts of this gene lacked exon 5, leading to a frameshift and premature termination codon. The mutation was located in a

  12. Association between A59V polymorphism in exon 3 of leptin gene ...

    African Journals Online (AJOL)

    We used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique to screen for DNA polymorphisms of the leptin gene in 255 cows of Iranian Holstein. Amplified region is located in exon 3 of leptin gene. The genomic bovine leptin sequences, which consist of three exons, were ...

  13. Exonic variants associated with development of aspirin exacerbated respiratory diseases.

    Directory of Open Access Journals (Sweden)

    Seung-Woo Shin

    Full Text Available Aspirin-exacerbated respiratory disease (AERD is one phenotype of asthma, often occurring in the form of a severe and sudden attack. Due to the time-consuming nature and difficulty of oral aspirin challenge (OAC for AERD diagnosis, non-invasive biomarkers have been sought. The aim of this study was to identify AERD-associated exonic SNPs and examine the diagnostic potential of a combination of these candidate SNPs to predict AERD. DNA from 165 AERD patients, 397 subjects with aspirin-tolerant asthma (ATA, and 398 normal controls were subjected to an Exome BeadChip assay containing 240K SNPs. 1,023 models (210-1 were generated from combinations of the top 10 SNPs, selected by the p-values in association with AERD. The area under the curve (AUC of the receiver operating characteristic (ROC curves was calculated for each model. SNP Function Portal and PolyPhen-2 were used to validate the functional significance of candidate SNPs. An exonic SNP, exm537513 in HLA-DPB1, showed the lowest p-value (p = 3.40×10-8 in its association with AERD risk. From the top 10 SNPs, a combination model of 7 SNPs (exm537513, exm83523, exm1884673, exm538564, exm2264237, exm396794, and exm791954 showed the best AUC of 0.75 (asymptotic p-value of 7.94×10-21, with 34% sensitivity and 93% specificity to discriminate AERD from ATA. Amino acid changes due to exm83523 in CHIA were predicted to be "probably damaging" to the structure and function of the protein, with a high score of '1'. A combination model of seven SNPs may provide a useful, non-invasive genetic marker combination for predicting AERD.

  14. Exon microarray analysis of human dorsolateral prefrontal cortex in alcoholism.

    Science.gov (United States)

    Manzardo, Ann M; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G

    2014-06-01

    Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function, and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC; Brodmann area 9) of 7 adult alcoholic (6 males, 1 female, mean age 49 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using quantitative reverse transcription polymerase chain reaction, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN), and signaling (e.g., RASGRP3, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease and development including cellular assembly and organization impacting on psychological disorders. Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation, and signaling that targets white matter of the brain. Copyright © 2014 by the Research Society on Alcoholism.

  15. Exon 44 nonsense mutation in two-Duchenne muscular dystrophy brothers detected by heteroduplex analysis.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartolo, C; Mendell, J R

    1993-01-01

    Utilizing a heteroduplex method, we screened the dystrophin exon 43-45 region for point mutations, including small deletions and insertions. The method depends upon the formation of a heteroduplex between wild-type and mutant DNA PCR products. DNA specimens from one hundred and four DMD patients without detected deletions or duplications were multiplexed amplified for exons 43, 44, and 45. The PCR products were mixed with the PCR products from nonaffected controls, electrophoresed, and examined for the presence of altered mobility heteroduplex bands. An exon 44 nonsense mutation in two DMD brothers and a common intron 44 polymorphism were identified using this approach. Although the exon 44-45 region is a hotspot for deletion breakpoints, it does not appear to be prone to point mutations. The technique is extremely useful for screening several exons simultaneously and it allowed us to screen a large number of patients.

  16. Kapitel 1-7, 13, 15

    DEFF Research Database (Denmark)

    Heiberg, Morten Rievers

    2009-01-01

    Den 30. september 1968 udsendte VKR-regeringen en erklæring, hvis hovedsynspunkt var "...at registrering af danske statsborgere ikke længere må finde sted alene på grundlag af lovlig politisk virksomhed." Det fremgik ydermere af erklæringen, at såfremt tjenesten lå inde med sådanne registreringer...

  17. BEAT: Bioinformatics Exon Array Tool to store, analyze and visualize Affymetrix GeneChip Human Exon Array data from disease experiments

    Directory of Open Access Journals (Sweden)

    Consiglio Arianna

    2012-03-01

    Full Text Available Abstract Background It is known from recent studies that more than 90% of human multi-exon genes are subject to Alternative Splicing (AS, a key molecular mechanism in which multiple transcripts may be generated from a single gene. It is widely recognized that a breakdown in AS mechanisms plays an important role in cellular differentiation and pathologies. Polymerase Chain Reactions, microarrays and sequencing technologies have been applied to the study of transcript diversity arising from alternative expression. Last generation Affymetrix GeneChip Human Exon 1.0 ST Arrays offer a more detailed view of the gene expression profile providing information on the AS patterns. The exon array technology, with more than five million data points, can detect approximately one million exons, and it allows performing analyses at both gene and exon level. In this paper we describe BEAT, an integrated user-friendly bioinformatics framework to store, analyze and visualize exon arrays datasets. It combines a data warehouse approach with some rigorous statistical methods for assessing the AS of genes involved in diseases. Meta statistics are proposed as a novel approach to explore the analysis results. BEAT is available at http://beat.ba.itb.cnr.it. Results BEAT is a web tool which allows uploading and analyzing exon array datasets using standard statistical methods and an easy-to-use graphical web front-end. BEAT has been tested on a dataset with 173 samples and tuned using new datasets of exon array experiments from 28 colorectal cancer and 26 renal cell cancer samples produced at the Medical Genetics Unit of IRCCS Casa Sollievo della Sofferenza. To highlight all possible AS events, alternative names, accession Ids, Gene Ontology terms and biochemical pathways annotations are integrated with exon and gene level expression plots. The user can customize the results choosing custom thresholds for the statistical parameters and exploiting the available clinical

  18. Alternative splicing of exon 17 and a missense mutation in exon 20 of the insulin receptor gene in two brothers with a novel syndrome of insulin resistance (congenital fiber-type disproportion myopathy)

    DEFF Research Database (Denmark)

    Vorwerk, P; Christoffersen, C T; Müller, J

    1999-01-01

    to be compound heterozygotes for mutations in the IR gene. The maternal allele was alternatively spliced in exon 17 due to a point mutation in the -1 donor splice site of the exon. The abnormal skipping of exon 17 shifts the amino acid reading frame and leads to a truncated IR, missing the entire tyrosine kinase...

  19. Detection of imprinting mutations in Angelman syndrome using a probe for exon {alpha} of SNRPN

    Energy Technology Data Exchange (ETDEWEB)

    Beuten, J.; Sutcliffe, J.S.; Casey, B.M. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1996-05-17

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct clinical disorders resulting from deficiency of paternal (PWS) or maternal (AS) expression of imprinted genes within chromosome 15q11-q13. 15 refs., 1 fig.

  20. The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

    Directory of Open Access Journals (Sweden)

    Seim Inge

    2009-05-01

    Full Text Available Abstract Background The murine ghrelin gene (Ghrl, originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0. We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. Findings 5' RACE revealed two transcription start sites (TSSs in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR, we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. Conclusion We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1 raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.

  1. Identification, characterization and expression of novel Sex Hormone Binding Globulin alternative first exons in the human prostate

    Directory of Open Access Journals (Sweden)

    de Torres Inés

    2009-06-01

    Full Text Available Abstract Background The human Sex Hormone Binding Globulin (SHBG gene, located at 17p13.1, comprises, at least, two different transcription units regulated by two different promoters. The first transcription unit begins with the exon 1 sequence and is responsible for the production of plasma SHBG by the hepatocytes, while the second begins with an alternative exon 1 sequence, which replaces the exon 1 present in liver transcripts. Alternative exon 1 transcription and translation has only been demonstrated in the testis of transgenic mice containing an 11-kb human SHBG transgene and in the human testis. Our goal has been to further characterize the 5' end of the SHBG gene and analyze the presence of the SHBG alternative transcripts in human prostate tissue and derived cell lines. Results Using a combination of in silico and in vitro studies, we have demonstrated that the SHBG gene, along with exon 1 and alternative exon 1 (renamed here exon 1A, contains four additional alternative first exons: the novel exons 1B, 1C, and 1E, and a previously identified exon 1N, which has been further characterized and renamed as exon 1D. We have shown that these four alternative first exons are all spliced to the same 3' splice site of SHBG exon 2, and that exon 1A and the novel exon 1B can be spliced to exon 1. We have also demonstrated the presence of SHBG transcripts beginning with exons 1B, 1C and 1D in prostate tissues and cell lines, as well as in several non-prostatic cell lines. Finally, the alignment of the SHBG mammalian sequences revealed that, while exons 1C, 1D and 1E are very well conserved phylogenetically through non-primate mammal species, exon 1B probably aroused in apes due to a single nucleotide change that generated a new 5' splice site in exon 1B. Conclusion The identification of multiple transcription start sites (TSS upstream of the annotated first exon of human SHBG, and the detection of the alternative transcripts in human prostate

  2. The identification of exons from the MED/PSACH region of human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Li, Quan-Yi; Brook, J.D. [Univ. of Nottingham (United Kingdom); Lennon, G.G. [Lawrence Livermore National Lab., Livermore, CA (United States)

    1996-03-01

    We have used exon amplification to identify putative transcribed sequences from an 823-kb contig consisting of 28 cosmids that form a minimum tiling path from the interval 19p12-p13.1. This region contains the genes responsible for multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). We have trapped 66 exons (an average of 2.4 exons per cosmid) from pools of 2 or 3 cosmids. The majority of exons (51.5%) show only weak similarity or no similarity (36.3%) to sequences in current databases. Six of 8 exons examined from these groups, however, show cross-species sequence conservation, indicating that many of them probably represent authentic exons. Eight exons show identity or significant similarity to ESTs or known genes, including the human TNF receptor 3{prime}-flanking region gene, human epoxide hydrolase (EPHX), human growth/differentiation factor (GOF-1), human myocyte-specific enhancer factor 2, the rat neurocan gene, and the human cartilage oligomeric matrix protein gene (COMP). Mutations in this latter gene have recently been shown to be responsible for MED and PSACH. 33 refs., 4 figs., 2 tabs.

  3. 11p15-subband specific search for transcribed sequences using exon trapping

    Energy Technology Data Exchange (ETDEWEB)

    Loebbert, R.; Prawitt, D. [Univ. of Mainz (Germany); Monroe, D. [MIT, Cambridge, MA (United States)] [and others

    1994-09-01

    Evidence from cytogenetic and molecular data suggest that the region 11p15 contains genes involved in different disorders, like Beckwith-Wiedemann syndrome (BWS), long QT syndrome (LQT), Usher syndrome type I and tumor development. Focusing on the subregion 11p15.1, we are isolating and characterizing new transcribed sequences. The applied strategy includes exon amplification and subsequent PCR screening of cDNA libraries. So far 100 YACs and 38 cosmid clones from 11p15.1-15.3 have been collected and are currently arrayed. 16 cosmids have been analyzed for transcribed sequences using the exon amplification scheme developed by Buckler et al. (1991). We were able to identify 18 exons that contain correct open reading frames and map back to the cosmid clones. A data base search revealed that two exons represent parts of known genes from this region (ST5 and AMPD3). Moreover, we identified one exon that represents an EGF-like repeat with homologies to various proteins. Using PCR and primers from the exon sequences, a fetal brain library, which has been arranged in the form of hierarchic arrayed phage pools, was screened. Up to now, two cDNA clones corresponding to different exons were isolated and are currently sequenced.

  4. Patterns of exon-intron architecture variation of genes in eukaryotic genomes

    Directory of Open Access Journals (Sweden)

    Chen Jian-Qun

    2009-01-01

    Full Text Available Abstract Background The origin and importance of exon-intron architecture comprises one of the remaining mysteries of gene evolution. Several studies have investigated the variations of intron length, GC content, ordinal position in a gene and divergence. However, there is little study about the structural variation of exons and introns. Results We investigated the length, GC content, ordinal position and divergence in both exons and introns of 13 eukaryotic genomes, representing plant and animal. Our analyses revealed that three basic patterns of exon-intron variation were present in nearly all analyzed genomes (P Conclusion Although the factors contributing to these patterns have not been identified, our results provide three important clues: common factor(s exist and may shape both exons and introns; the ordinal reduction patterns may reflect a time-orderly evolution; and the larger first and last exons may be splicing-required. These clues provide a framework for elucidating mechanisms involved in the organization of eukaryotic genomes and particularly in building exon-intron structures.

  5. Antisense Oligonucleotide-mediated Exon Skipping as a Systemic Therapeutic Approach for Recessive Dystrophic Epidermolysis Bullosa

    Directory of Open Access Journals (Sweden)

    Jeroen Bremer

    2016-01-01

    Full Text Available The “generalized severe” form of recessive dystrophic epidermolysis bullosa (RDEB-gen sev is caused by bi-allelic null mutations in COL7A1, encoding type VII collagen. The absence of type VII collagen leads to blistering of the skin and mucous membranes upon the slightest trauma. Because most patients carry exonic point mutations or small insertions/deletions, most exons of COL7A1 are in-frame, and low levels of type VII collagen already drastically improve the disease phenotype, this gene seems a perfect candidate for antisense oligonucleotide (AON-mediated exon skipping. In this study, we examined the feasibility of AON-mediated exon skipping in vitro in primary cultured keratinocytes and fibroblasts, and systemically in vivo using a human skin-graft mouse model. We show that treatment with AONs designed against exon 105 leads to in-frame exon 105 skipping at the RNA level and restores type VII collagen protein production in vitro. Moreover, we demonstrate that systemic delivery in vivo induces de novo expression of type VII collagen in skin grafts generated from patient cells. Our data demonstrate strong proof-of-concept for AON-mediated exon skipping as a systemic therapeutic strategy for RDEB.

  6. SR Proteins Induce Alternative Exon Skipping through Their Activities on the Flanking Constitutive Exons▿

    Science.gov (United States)

    Han, Joonhee; Ding, Jian-Hua; Byeon, Cheol W.; Kim, Jee H.; Hertel, Klemens J.; Jeong, Sunjoo; Fu, Xiang-Dong

    2011-01-01

    SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKIIδ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKIIδ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKIIδ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs. PMID:21135118

  7. A new explanation for recessive myotonia congenita: exon deletions and duplications in CLCN1.

    Science.gov (United States)

    Raja Rayan, D L; Haworth, A; Sud, R; Matthews, E; Fialho, D; Burge, J; Portaro, S; Schorge, S; Tuin, K; Lunt, P; McEntagart, M; Toscano, A; Davis, M B; Hanna, M G

    2012-06-12

    To assess whether exon deletions or duplications in CLCN1 are associated with recessive myotonia congenita (MC). We performed detailed clinical and electrophysiologic characterization in 60 patients with phenotypes consistent with MC. DNA sequencing of CLCN1 followed by multiplex ligation-dependent probe amplification to screen for exon copy number variation was undertaken in all patients. Exon deletions or duplications in CLCN1 were identified in 6% of patients with MC. Half had heterozygous exonic rearrangements. The other 2 patients (50%), with severe disabling infantile onset myotonia, were identified with both a homozygous mutation, Pro744Thr, which functional electrophysiology studies suggested was nonpathogenic, and a triplication/homozygous duplication involving exons 8-14, suggesting an explanation for the severe phenotype. These data indicate that copy number variation in CLCN1 may be an important cause of recessive MC. Our observations suggest that it is important to check for exon deletions and duplications as part of the genetic analysis of patients with recessive MC, especially in patients in whom sequencing identifies no mutations or only a single recessive mutation. These results also indicate that additional, as yet unidentified, genetic mechanisms account for cases not currently explained by either CLCN1 point mutations or exonic deletions or duplications.

  8. Tracking the evolution of alternatively spliced exons within the Dscam family

    Directory of Open Access Journals (Sweden)

    Vision Todd J

    2006-02-01

    Full Text Available Abstract Background The Dscam gene in the fruit fly, Drosophila melanogaster, contains twenty-four exons, four of which are composed of tandem arrays that each undergo mutually exclusive alternative splicing (4, 6, 9 and 17, potentially generating 38,016 protein isoforms. This degree of transcript diversity has not been found in mammalian homologs of Dscam. We examined the molecular evolution of exons within this gene family to locate the point of divergence for this alternative splicing pattern. Results Using the fruit fly Dscam exons 4, 6, 9 and 17 as seed sequences, we iteratively searched sixteen genomes for homologs, and then performed phylogenetic analyses of the resulting sequences to examine their evolutionary history. We found homologs in the nematode, arthropod and vertebrate genomes, including homologs in several vertebrates where Dscam had not been previously annotated. Among these, only the arthropods contain homologs arranged in tandem arrays indicative of mutually exclusive splicing. We found no homologs to these exons within the Arabidopsis, yeast, tunicate or sea urchin genomes but homologs to several constitutive exons from fly Dscam were present within tunicate and sea urchin. Comparing the rate of turnover within the tandem arrays of the insect taxa (fruit fly, mosquito and honeybee, we found the variants within exons 4 and 17 are well conserved in number and spatial arrangement despite 248–283 million years of divergence. In contrast, the variants within exons 6 and 9 have undergone considerable turnover since these taxa diverged, as indicated by deeply branching taxon-specific lineages. Conclusion Our results suggest that at least one Dscam exon array may be an ancient duplication that predates the divergence of deuterostomes from protostomes but that there is no evidence for the presence of arrays in the common ancestor of vertebrates. The different patterns of conservation and turnover among the Dscam exon arrays

  9. Genetic associations of nonsynonymous exonic variants with psychophysiological endophenotypes

    Science.gov (United States)

    Vrieze, Scott I.; Malone, Stephen M.; Pankratz, Nathan; Vaidyanathan, Uma; Miller, Michael B.; Kang, Hyun Min; McGue, Matt; Abecasis, Gonçalo; Iacono, William G.

    2014-01-01

    We mapped ~85,000 rare nonsynonymous exonic single nucleotide polymorphisms (SNPs) to 17 psychophysiological endophenotypes in 4,905 individuals, including antisaccade eye movements, resting EEG, P300 amplitude, electrodermal activity, affect-modulated startle eye blink. Nonsynonymous SNPs are predicted to directly change or disrupt proteins encoded by genes and are expected to have significant biological consequences. Most such variants are rare, and new technologies can efficiently assay them on a large scale. We assayed 247,870 mostly rare SNPs on an Illumina exome array. Approximately 85,000 of the SNPs were polymorphic, rare (MAF < .05), and nonsynonymous. Single variant association tests identified a SNP in the PARD3 gene associated with theta resting EEG power. The sequence kernel association test, a gene-based test, identified a gene PNPLA7 associated with pleasant difference startle, the difference in startle magnitude between pleasant and neutral images. No other single nonsynonymous variant, or gene-based group of variants, was strongly associated with any endophenotype. PMID:25387709

  10. Conservation of the Exon-Intron Structure of Long Intergenic Non-Coding RNA Genes in Eutherian Mammals

    Directory of Open Access Journals (Sweden)

    Diana Chernikova

    2016-07-01

    Full Text Available The abundance of mammalian long intergenic non-coding RNA (lincRNA genes is high, yet their functions remain largely unknown. One possible way to study this important question is to use large-scale comparisons of various characteristics of lincRNA with those of protein-coding genes for which a large body of functional information is available. A prominent feature of mammalian protein-coding genes is the high evolutionary conservation of the exon-intron structure. Comparative analysis of putative intron positions in lincRNA genes from various mammalian genomes suggests that some lincRNA introns have been conserved for over 100 million years, thus the primary and/or secondary structure of these molecules is likely to be functionally important.

  11. A Large Inversion Involving GNAS Exon A/B and All Exons Encoding Gsα Is Associated With Autosomal Dominant Pseudohypoparathyroidism Type Ib (PHP1B).

    Science.gov (United States)

    Grigelioniene, Giedre; Nevalainen, Pasi I; Reyes, Monica; Thiele, Susanne; Tafaj, Olta; Molinaro, Angelo; Takatani, Rieko; Ala-Houhala, Marja; Nilsson, Daniel; Eisfeldt, Jesper; Lindstrand, Anna; Kottler, Marie-Laure; Mäkitie, Outi; Jüppner, Harald

    2017-04-01

    Pseudohypoparathyroidism type Ib (PHP1B) is characterized primarily by resistance to parathyroid hormone (PTH) and thus hypocalcemia and hyperphosphatemia, in most cases without evidence for Albright hereditary osteodystrophy (AHO). PHP1B is associated with epigenetic changes at one or several differentially-methylated regions (DMRs) within GNAS, which encodes the α-subunit of the stimulatory G protein (Gsα) and splice variants thereof. Heterozygous, maternally inherited STX16 or GNAS deletions leading to isolated loss-of-methylation (LOM) at exon A/B alone or at all maternal DMRs are the cause of autosomal dominant PHP1B (AD-PHP1B). In this study, we analyzed three affected individuals, the female proband and her two sons. All three revealed isolated LOM at GNAS exon A/B, whereas the proband's healthy maternal grandmother and uncle showed normal methylation at this locus. Haplotype analysis was consistent with linkage to the STX16/GNAS region, yet no deletion could be identified. Whole-genome sequencing of one of the patients revealed a large heterozygous inversion (1,882,433 bp). The centromeric breakpoint of the inversion is located 7,225 bp downstream of GNAS exon XL, but its DMR showed no methylation abnormality, raising the possibility that the inversion disrupts a regulatory element required only for establishing or maintaining exon A/B methylation. Because our three patients presented phenotypes consistent with PHP1B, and not with PHP1A, the Gsα promoter is probably unaffected by the inversion. Our findings expand the spectrum of genetic mutations that lead to LOM at exon A/B alone and thus biallelic expression of the transcript derived from this alternative first GNAS exon. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

  12. Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene.

    Science.gov (United States)

    Flanigan, Kevin M; Dunn, Diane M; von Niederhausern, Andrew; Soltanzadeh, Payam; Howard, Michael T; Sampson, Jacinda B; Swoboda, Kathryn J; Bromberg, Mark B; Mendell, Jerry R; Taylor, Laura E; Anderson, Christine B; Pestronk, Alan; Florence, Julaine M; Connolly, Anne M; Mathews, Katherine D; Wong, Brenda; Finkel, Richard S; Bonnemann, Carsten G; Day, John W; McDonald, Craig; Weiss, Robert B

    2011-03-01

    Nonsense mutations are usually predicted to function as null alleles due to premature termination of protein translation. However, nonsense mutations in the DMD gene, encoding the dystrophin protein, have been associated with both the severe Duchenne Muscular Dystrophy (DMD) and milder Becker Muscular Dystrophy (BMD) phenotypes. In a large survey, we identified 243 unique nonsense mutations in the DMD gene, and for 210 of these we could establish definitive phenotypes. We analyzed the reading frame predicted by exons flanking those in which nonsense mutations were found, and present evidence that nonsense mutations resulting in BMD likely do so by inducing exon skipping, confirming that exonic point mutations affecting exon definition have played a significant role in determining phenotype. We present a new model based on the combination of exon definition and intronic splicing regulatory elements for the selective association of BMD nonsense mutations with a subset of DMD exons prone to mutation-induced exon skipping. © 2011 Wiley-Liss, Inc.

  13. Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene

    Science.gov (United States)

    Flanigan, Kevin M.; Dunn, Diane M.; von Niederhausern, Andrew; Soltanzadeh, Payam; Howard, Michael T.; Sampson, Jacinda B.; Swoboda, Kathryn J.; Bromberg, Mark B.; Mendell, Jerry R.; Taylor, Laura; Anderson, Christine B.; Pestronk, Alan; Florence, Julaine; Connolly, Anne M.; Mathews, Katherine D.; Wong, Brenda; Finkel, Richard S.; Bonnemann, Carsten G.; Day, John W.; McDonald, Craig; Weiss, Robert B.

    2013-01-01

    Nonsense mutations are usually predicted to function as null alleles due to premature termination of protein translation. However, nonsense mutations in the DMD gene, encoding the dystrophin protein, have been associated with both the severe Duchenne Muscular Dystrophy (DMD) and milder Becker Muscular Dystrophy (BMD) phenotypes. In a large survey, we identified 243 unique nonsense mutations in the DMD gene, and for 210 of these we could establish definitive phenotypes. We analyzed the reading frame predicted by exons flanking those in which nonsense mutations were found, and present evidence that nonsense mutations resulting in BMD likely do so by inducing exon skipping, confirming that exonic point mutations affecting exon definition have played a significant role in determining phenotype. We present a new model based on the combination of exon definition and intronic splicing regulatory elements for the selective association of BMD nonsense mutations with a subset of DMD exons prone to mutation-induced exon skipping. PMID:21972111

  14. Polymorphism in exon 1 of adiponectin gene and its association with ...

    African Journals Online (AJOL)

    hope&shola

    2010-11-08

    -. Weinberg equilibrium (P > 0.05). Simultaneously, the locus belonged to medium polymorphism in three populations. (0.25 < PIC < 0.5). Allelic effect of the adiponectin gene exon 1 on meat quality, serum total cholesterol and ...

  15. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy

    National Research Council Canada - National Science Library

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-01-01

    .... Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin...

  16. Polymorphism in exon 1 of adiponectin gene and its association with ...

    African Journals Online (AJOL)

    hope&shola

    2010-11-08

    . Up to now, the research about association of polymorphisms in duck adiponectin gene with production traits has not been reported. The objectives of the present study were to identify SNPs in the duck adiponectin gene exon ...

  17. Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

    Science.gov (United States)

    Grinev, Vasily V; Migas, Alexandr A; Kirsanava, Aksana D; Mishkova, Olga A; Siomava, Natalia; Ramanouskaya, Tatiana V; Vaitsiankova, Alina V; Ilyushonak, Ilia M; Nazarov, Petr V; Vallar, Laurent; Aleinikova, Olga V

    2015-11-01

    The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process. Copyright © 2015 Elsevier Ltd. All rights

  18. Plant Proteins Are Smaller Because They Are Encoded by Fewer Exons than Animal Proteins.

    Science.gov (United States)

    Ramírez-Sánchez, Obed; Pérez-Rodríguez, Paulino; Delaye, Luis; Tiessen, Axel

    2016-12-01

    Protein size is an important biochemical feature since longer proteins can harbor more domains and therefore can display more biological functionalities than shorter proteins. We found remarkable differences in protein length, exon structure, and domain count among different phylogenetic lineages. While eukaryotic proteins have an average size of 472 amino acid residues (aa), average protein sizes in plant genomes are smaller than those of animals and fungi. Proteins unique to plants are ∼81aa shorter than plant proteins conserved among other eukaryotic lineages. The smaller average size of plant proteins could neither be explained by endosymbiosis nor subcellular compartmentation nor exon size, but rather due to exon number. Metazoan proteins are encoded on average by ∼10 exons of small size [∼176 nucleotides (nt)]. Streptophyta have on average only ∼5.7 exons of medium size (∼230nt). Multicellular species code for large proteins by increasing the exon number, while most unicellular organisms employ rather larger exons (>400nt). Among subcellular compartments, membrane proteins are the largest (∼520aa), whereas the smallest proteins correspond to the gene ontology group of ribosome (∼240aa). Plant genes are encoded by half the number of exons and also contain fewer domains than animal proteins on average. Interestingly, endosymbiotic proteins that migrated to the plant nucleus became larger than their cyanobacterial orthologs. We thus conclude that plants have proteins larger than bacteria but smaller than animals or fungi. Compared to the average of eukaryotic species, plants have ∼34% more but ∼20% smaller proteins. This suggests that photosynthetic organisms are unique and deserve therefore special attention with regard to the evolutionary forces acting on their genomes and proteomes. Copyright © 2016 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  19. Post-transcriptional exon shuffling events in humans can be evolutionarily conserved and abundant

    OpenAIRE

    Al-Balool, Haya H.; Weber, David; Liu, Yilei; Wade, Mark; Guleria, Kamlesh; Nam, Pitsien Lang Ping; Clayton, Jake; Rowe, William; Coxhead, Jonathan; Irving, Julie; Elliott, David J.; Hall, Andrew G.; Santibanez-Koref, Mauro; Jackson, Michael S.

    2011-01-01

    In silico analyses have established that transcripts from some genes can be processed into RNAs with rearranged exon order relative to genomic structure (post-transcriptional exon shuffling, or PTES). Although known to contribute to transcriptome diversity in some species, to date the structure, distribution, abundance, and functional significance of human PTES transcripts remains largely unknown. Here, using high-throughput transcriptome sequencing, we identify 205 putative human PTES produc...

  20. Exon size affects competition between splicing and cleavage-polyadenylation in the immunoglobulin mu gene.

    Science.gov (United States)

    Peterson, M L; Bryman, M B; Peiter, M; Cowan, C

    1994-01-01

    The alternative RNA processing of microseconds and microns mRNAs from a single primary transcript depends on competition between a cleavage-polyadenylation reaction to produce microseconds mRNA and a splicing reaction to produce microns mRNA. The ratio of microseconds to microns mRNA is regulated during B-cell maturation; relatively more spliced microns mRNA is made in B cells than in plasma cells. The balance between the efficiencies of splicing and cleavage-polyadenylation is critical to the regulation. The mu gene can be modified to either reduce or improve the efficiency of each reaction and thus alter the ratio of the two RNAs produced. However, as long as neither reaction is so strong that it totally dominates, expression of the modified mu genes is regulated in B cells and plasma cells. The current experiments reveal a relationship between the C mu 4 exon size and the microseconds/microns expression ratio. The shorter the distance between the C mu 4 5' splice site and the nearest upstream 3' splice site, the more spliced microns mRNA was produced. Conversely, when this exon was expanded, more microseconds mRNA was produced. Expression from these mu genes with altered exon sizes were regulated between B cells and plasma cells. Since RNA processing in the mu gene can be considered a competition between defining the C mu 4 exon as an internal exon (in microns mRNA) versus a terminal exon (in microseconds mRNA), exon size may affect the competition among factors interacting with this exon.

  1. Plant Proteins Are Smaller Because They Are Encoded by Fewer Exons than Animal Proteins

    Directory of Open Access Journals (Sweden)

    Obed Ramírez-Sánchez

    2016-12-01

    Full Text Available Protein size is an important biochemical feature since longer proteins can harbor more domains and therefore can display more biological functionalities than shorter proteins. We found remarkable differences in protein length, exon structure, and domain count among different phylogenetic lineages. While eukaryotic proteins have an average size of 472 amino acid residues (aa, average protein sizes in plant genomes are smaller than those of animals and fungi. Proteins unique to plants are ∼81 aa shorter than plant proteins conserved among other eukaryotic lineages. The smaller average size of plant proteins could neither be explained by endosymbiosis nor subcellular compartmentation nor exon size, but rather due to exon number. Metazoan proteins are encoded on average by ∼10 exons of small size [∼176 nucleotides (nt]. Streptophyta have on average only ∼5.7 exons of medium size (∼230 nt. Multicellular species code for large proteins by increasing the exon number, while most unicellular organisms employ rather larger exons (>400 nt. Among subcellular compartments, membrane proteins are the largest (∼520 aa, whereas the smallest proteins correspond to the gene ontology group of ribosome (∼240 aa. Plant genes are encoded by half the number of exons and also contain fewer domains than animal proteins on average. Interestingly, endosymbiotic proteins that migrated to the plant nucleus became larger than their cyanobacterial orthologs. We thus conclude that plants have proteins larger than bacteria but smaller than animals or fungi. Compared to the average of eukaryotic species, plants have ∼34% more but ∼20% smaller proteins. This suggests that photosynthetic organisms are unique and deserve therefore special attention with regard to the evolutionary forces acting on their genomes and proteomes.

  2. Crystal Structure of the CLOCK Transactivation Domain Exon19 in Complex with a Repressor

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Zhiqiang; Su, Lijing; Pei, Jimin; Grishin, Nick V.; Zhang, Hong (UTSMC)

    2017-08-01

    In the canonical clock model, CLOCK:BMAL1-mediated transcriptional activation is feedback regulated by its repressors CRY and PER and, in association with other coregulators, ultimately generates oscillatory gene expression patterns. How CLOCK:BMAL1 interacts with coregulator(s) is not well understood. Here we report the crystal structures of the mouse CLOCK transactivating domain Exon19 in complex with CIPC, a potent circadian repressor that functions independently of CRY and PER. The Exon19:CIPC complex adopts a three-helical coiled-coil bundle conformation containing two Exon19 helices and one CIPC. Unique to Exon19:CIPC, three highly conserved polar residues, Asn341 of CIPC and Gln544 of the two Exon19 helices, are located at the mid-section of the coiled-coil bundle interior and form hydrogen bonds with each other. Combining results from protein database search, sequence analysis, and mutagenesis studies, we discovered for the first time that CLOCK Exon19:CIPC interaction is a conserved transcription regulatory mechanism among mammals, fish, flies, and other invertebrates.

  3. Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression

    Science.gov (United States)

    Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Dombrowski, Stephen M.; Miller, Tyler E.; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; von Elverfeldt, Dominik; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.; Bredel, Markus

    2014-01-01

    Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones. PMID:24865424

  4. Molecular characterization of exon 3 of caprine myostatin gene in Marwari goat

    Directory of Open Access Journals (Sweden)

    Jai Prakash Khichar

    2016-06-01

    Full Text Available Aim: To estimate genetic variability in exon 3 of caprine myostatin gene in Marwari goats. Materials and Methods: A total of 120 blood samples from unrelated Marwari goats were randomly collected from different villages of Bikaner (Rajasthan, India. Genomic DNA was extracted from whole blood using blood DNA isolation kit (Himedia Ltd. as per manufacturer’s protocol. The quality of extracted genomic DNA was checked on 0.8% agarose gel. Specifically designed a primer set for caprine myostatin (MSTN gene (Genebank accession no. DQ167575 was used to amplify the exon 3 region of MSTN gene in Marwari goat. The genetic variability in exon 3 of MSTN gene in Marwari goat was assessed on 8% polyacrylamide gel electrophoresis to detect single strand conformation polymorphism (SSCP pattern. Results: The exon 3 of MSTN gene in Marwari goat showed two types of conformation patterns on 8% polyacrylamide gel. One of the patterns showed only two bands and was considered as genotype AA, whereas another pattern having an extra band was designated as genotype AB. The frequencies of AA and AB genotype for exon 3 region of MSTN gene were calculated as 0.90 and 0.10, respectively. Conclusion: Low level of polymorphism was observed at exon 3 region of MSTN gene in Marwari goat through SSCP analysis. This information could be utilized in future breeding plan to exploit the unique characteristics of Marwari goat of Rajasthan.

  5. Molecular characterization of exon 3 of caprine myostatin gene in Marwari goat

    Science.gov (United States)

    Khichar, Jai Prakash; Gahlot, Gyan Chand; Agrawal, Vijay Kumar; Kiran; Dewna, Ajay Singh; Prakash; Ashraf, Mohammad

    2016-01-01

    Aim: To estimate genetic variability in exon 3 of caprine myostatin gene in Marwari goats. Materials and Methods: A total of 120 blood samples from unrelated Marwari goats were randomly collected from different villages of Bikaner (Rajasthan), India. Genomic DNA was extracted from whole blood using blood DNA isolation kit (Himedia Ltd.) as per manufacturer’s protocol. The quality of extracted genomic DNA was checked on 0.8% agarose gel. Specifically designed a primer set for caprine myostatin (MSTN) gene (Genebank accession no. DQ167575) was used to amplify the exon 3 region of MSTN gene in Marwari goat. The genetic variability in exon 3 of MSTN gene in Marwari goat was assessed on 8% polyacrylamide gel electrophoresis to detect single strand conformation polymorphism (SSCP) pattern. Results: The exon 3 of MSTN gene in Marwari goat showed two types of conformation patterns on 8% polyacrylamide gel. One of the patterns showed only two bands and was considered as genotype AA, whereas another pattern having an extra band was designated as genotype AB. The frequencies of AA and AB genotype for exon 3 region of MSTN gene were calculated as 0.90 and 0.10, respectively. Conclusion: Low level of polymorphism was observed at exon 3 region of MSTN gene in Marwari goat through SSCP analysis. This information could be utilized in future breeding plan to exploit the unique characteristics of Marwari goat of Rajasthan. PMID:27397994

  6. Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot.

    Science.gov (United States)

    Suzuki, Hitoshi; Aoki, Yoshitsugu; Kameyama, Toshiki; Saito, Takashi; Masuda, Satoru; Tanihata, Jun; Nagata, Tetsuya; Mayeda, Akila; Takeda, Shin'ichi; Tsukahara, Toshifumi

    2016-10-13

    Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45-55 of the DMD gene, might improve patients' symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45-55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44-56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5' splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.

  7. Loss of exon identity is a common mechanism of human inherited disease.

    Science.gov (United States)

    Sterne-Weiler, Timothy; Howard, Jonathan; Mort, Matthew; Cooper, David N; Sanford, Jeremy R

    2011-10-01

    It is widely accepted that at least 10% of all mutations causing human inherited disease disrupt splice-site consensus sequences. In contrast to splice-site mutations, the role of auxiliary cis-acting elements such as exonic splicing enhancers (ESE) and exonic splicing silencers (ESS) in human inherited disease is still poorly understood. Here we use a top-down approach to determine rates of loss or gain of known human exonic splicing regulatory (ESR) sequences associated with either disease-causing mutations or putatively neutral single nucleotide polymorphisms (SNPs). We observe significant enrichment toward loss of ESEs and gain of ESSs among inherited disease-causing variants relative to neutral polymorphisms, indicating that exon skipping may play a prominent role in aberrant gene regulation. Both computational and biochemical approaches underscore the relevance of exonic splicing enhancer loss and silencer gain in inherited disease. Additionally, we provide direct evidence that both SRp20 (SRSF3) and possibly PTB (PTBP1) are involved in the function of a splicing silencer that is created de novo by a total of 83 different inherited disease mutations in 67 different disease genes. Taken together, we find that ~25% (7154/27,681) of known mis-sense and nonsense disease-causing mutations alter functional splicing signals within exons, suggesting a much more widespread role for aberrant mRNA processing in causing human inherited disease than has hitherto been appreciated.

  8. Crystal Structure of the CLOCK Transactivation Domain Exon19 in Complex with a Repressor.

    Science.gov (United States)

    Hou, Zhiqiang; Su, Lijing; Pei, Jimin; Grishin, Nick V; Zhang, Hong

    2017-08-01

    In the canonical clock model, CLOCK:BMAL1-mediated transcriptional activation is feedback regulated by its repressors CRY and PER and, in association with other coregulators, ultimately generates oscillatory gene expression patterns. How CLOCK:BMAL1 interacts with coregulator(s) is not well understood. Here we report the crystal structures of the mouse CLOCK transactivating domain Exon19 in complex with CIPC, a potent circadian repressor that functions independently of CRY and PER. The Exon19:CIPC complex adopts a three-helical coiled-coil bundle conformation containing two Exon19 helices and one CIPC. Unique to Exon19:CIPC, three highly conserved polar residues, Asn341 of CIPC and Gln544 of the two Exon19 helices, are located at the mid-section of the coiled-coil bundle interior and form hydrogen bonds with each other. Combining results from protein database search, sequence analysis, and mutagenesis studies, we discovered for the first time that CLOCK Exon19:CIPC interaction is a conserved transcription regulatory mechanism among mammals, fish, flies, and other invertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Conservation of CD44 exon v3 functional elements in mammals

    Directory of Open Access Journals (Sweden)

    Fernández-Bellon Hugo

    2008-07-01

    Full Text Available Abstract Background The human CD44 gene contains 10 variable exons (v1 to v10 that can be alternatively spliced to generate hundreds of different CD44 protein isoforms. Human CD44 variable exon v3 inclusion in the final mRNA depends on a multisite bipartite splicing enhancer located within the exon itself, which we have recently described, and provides the protein domain responsible for growth factor binding to CD44. Findings We have analyzed the sequence of CD44v3 in 95 mammalian species to report high conservation levels for both its splicing regulatory elements (the 3' splice site and the exonic splicing enhancer, and the functional glycosaminglycan binding site coded by v3. We also report the functional expression of CD44v3 isoforms in peripheral blood cells of different mammalian taxa with both consensus and variant v3 sequences. Conclusion CD44v3 mammalian sequences maintain all functional splicing regulatory elements as well as the GAG binding site with the same relative positions and sequence identity previously described during alternative splicing of human CD44. The sequence within the GAG attachment site, which in turn contains the Y motif of the exonic splicing enhancer, is more conserved relative to the rest of exon. Amplification of CD44v3 sequence from mammalian species but not from birds, fish or reptiles, may lead to classify CD44v3 as an exclusive mammalian gene trait.

  10. Inactivating Mutation screening of Exon 6 and Exon 10E of FSHR gene in women with Polycystic Ovarian Syndrome in Vellore population

    Science.gov (United States)

    Sekar, Nishu; Sapre, Madhura; Kale, Vaikhari; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

    2017-11-01

    Polycystic Ovarian syndrome (PCOS) is a major cause of infertility in females of reproducing age and is typified by oligo-anovulation, hyperandrogenism, hirsutism and polycystic ovaries. FSHR gene located on chromosome 2 p21 is responsible for the normal follicular development and any deletion or mutation in the gene affects the interaction of FSH with its receptor. Thus, it becomes the candidate gene for PCOS study. Inactivating mutation in FSHR gene limits the receptor’s function by creating a complete block, changing the receptor-ligand complex or the basic hormone signal transduction.To screen the inactivating mutations in Exon 6 and Exon 10E of FSHR gene in women diagnosed with PCOS.PCR-RFLP analysis indicated that there were no inactivating mutations found in Exon 6 and Exon 10E. Variations in hormone levels were seen amongst the PCOS patients. There were no inactivating mutations found in FSHR gene of the women diagnosed with PCOS according to the Rotterdam criteria in Vellore population.

  11. RBFOX and PTBP1 proteins regulate the alternative splicing of micro-exons in human brain transcripts.

    Science.gov (United States)

    Li, Yang I; Sanchez-Pulido, Luis; Haerty, Wilfried; Ponting, Chris P

    2015-01-01

    Ninety-four percent of mammalian protein-coding exons exceed 51 nucleotides (nt) in length. The paucity of micro-exons (≤ 51 nt) suggests that their recognition and correct processing by the splicing machinery present greater challenges than for longer exons. Yet, because thousands of human genes harbor processed micro-exons, specialized mechanisms may be in place to promote their splicing. Here, we survey deep genomic data sets to define 13,085 micro-exons and to study their splicing mechanisms and molecular functions. More than 60% of annotated human micro-exons exhibit a high level of sequence conservation, an indicator of functionality. While most human micro-exons require splicing-enhancing genomic features to be processed, the splicing of hundreds of micro-exons is enhanced by the adjacent binding of splice factors in the introns of pre-messenger RNAs. Notably, splicing of a significant number of micro-exons was found to be facilitated by the binding of RBFOX proteins, which promote their inclusion in the brain, muscle, and heart. Our analyses suggest that accurate regulation of micro-exon inclusion by RBFOX proteins and PTBP1 plays an important role in the maintenance of tissue-specific protein-protein interactions. © 2015 Li et al.; Published by Cold Spring Harbor Laboratory Press.

  12. Population genetics of duplicated alternatively spliced exons of the Dscam gene in Daphnia and Drosophila.

    Directory of Open Access Journals (Sweden)

    Daniela Brites

    Full Text Available In insects and crustaceans, the Down syndrome cell adhesion molecule (Dscam occurs in many different isoforms. These are produced by mutually exclusive alternative splicing of dozens of tandem duplicated exons coding for parts or whole immunoglobulin (Ig domains of the Dscam protein. This diversity plays a role in the development of the nervous system and also in the immune system. Structural analysis of the protein suggested candidate epitopes where binding to pathogens could occur. These epitopes are coded by regions of the duplicated exons and are therefore diverse within individuals. Here we apply molecular population genetics and molecular evolution analyses using Daphnia magna and several Drosophila species to investigate the potential role of natural selection in the divergence between orthologs of these duplicated exons among species, as well as between paralogous exons within species. We found no evidence for a role of positive selection in the divergence of these paralogous exons. However, the power of this test was low, and the fact that no signs of gene conversion between paralogous exons were found suggests that paralog diversity may nonetheless be maintained by selection. The analysis of orthologous exons in Drosophila and in Daphnia revealed an excess of non-synonymous polymorphisms in the epitopes putatively involved in pathogen binding. This may be a sign of balancing selection. Indeed, in Dr. melanogaster the same derived non-synonymous alleles segregate in several populations around the world. Yet other hallmarks of balancing selection were not found. Hence, we cannot rule out that the excess of non-synonymous polymorphisms is caused by segregating slightly deleterious alleles, thus potentially indicating reduced selective constraints in the putative pathogen binding epitopes of Dscam.

  13. Allelic combinations of promoter and exon 2 in DQB1 in dogs and wolves.

    Science.gov (United States)

    Berggren, Karin T; Seddon, Jennifer M

    2008-07-01

    Polymorphism of PBRs of the major histocompatibility complex (MHC) genes is well recognized, but the polymorphism also extends to proximal promoter regions. Examining DQB1 variability in dogs and wolves, we identified 7 promoter variants and 13 exon 2 alleles among 89 dogs, including a previously unknown DQB1 exon 2 allele, and 8 promoter variants and 9 exon 2 alleles among 85 wolves. As expected from previous studies and from a close chromosomal location, strong linkage disequilibrium was demonstrated in both wolves and dogs by having significantly fewer promoter/exon 2 combinations than expected from simulations of randomized data sets. Interestingly, we noticed weaker haplotypic associations in dogs than in wolves. Dogs had twice as many promoter/exon 2 combinations as wolves and an almost 2-fold difference in the number of exon 2 alleles per promoter variant. This difference was not caused by an admixture of breeds in our group of dogs because the high ratio of observed to expected number of haplotypes persisted within a single dog breed, the German Shepherd. Ewens-Watterson tests indicated that both the promoter and exon 2 are under the balancing selection, and both regions appear to be more recently derived in the dog than in the wolf. Hence, although reasons for the differences are unknown, they may relate to altered selection pressure on patterns of expression. Deviations from normal MHC expression patterns have been associated with autoimmune diseases, which occur frequently in several dog breeds. Further knowledge about these deviations may help us understand the source of such diseases.

  14. Deletion of exon 26 of the dystrophin gene is associated with a mild Becker muscular dystrophy phenotype

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, Morten; Vissing, John

    2011-01-01

    With the possible introduction of exon skipping therapy in Duchenne muscular dystrophy, it has become increasingly important to know the role of each exon of the dystrophin gene to protein expression, and thus the phenotype. In this report, we present two related men with an unusually mild BMD...... calf hypertrophy was noted. Creatine kinase was normal or raised maximally to 500 U/l. The muscle biopsy was myopathic with increased fiber size variation and many internal nuclei, but no dystrophy. No comorbidity was found. In both cases, western blot showed a reduced dystrophin band. Genetic...... evaluation revealed a deletion of exon 26 of the dystrophin gene in both. This is the first description of patients with a exon 26 deletion of the dystrophin gene. Assuming the proband's comorbidity is unrelated, exon 26 deletion results in a very mild phenotype. This might be of interest in planning exon...

  15. Clinical implications of cytosine deletion of exon 5 of P53 gene in non small cell lung cancer patients

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    Rashid Mir

    2016-01-01

    Full Text Available Aim: Lung cancer is considered to be the most common cancer in the world. In humans, about 50% or more cancers have a mutated tumor suppressor p53 gene thereby resulting in accumulation of p53 protein and losing its function to activate the target genes that regulate the cell cycle and apoptosis. Extensive research conducted in murine cancer models with activated p53, loss of p53, or p53 missense mutations have facilitated researchers to understand the role of this key protein. Our study was aimed to evaluate the frequency of cytosine deletion in nonsmall cell lung cancer (NSCLC patients. Methods: One hundred NSCLC patients were genotyped for P53 (exon5, codon168 cytosine deletion leading to loss of its function and activate the target genes by allele-specific polymerase chain reaction. The P53 cytosine deletion was correlated with all the clinicopathological parameters of the patients. Results and Analysis: 59% cases were carrying P53 cytosine deletion. Similarly, the significantly higher incidence of cytosine deletion was reported in current smokers (75% in comparison to exsmoker and nonsmoker. Significantly higher frequency of cytosine deletion was reported in adenocarcinoma (68.08% than squamous cell carcinoma (52.83%. Also, a significant difference was reported between p53 cytosine deletion and metastasis (64.28%. Further, the majority of the cases assessed for response carrying P53 cytosine deletion were found to show faster disease progression. Conclusion: The data suggests that there is a significant association of the P53 exon 5 deletion of cytosine in codon 168 with metastasis and staging of the disease.

  16. Familial hypertrophic cardiomyopathy owing to double heterozygosity for a 403Arg--> Trp mutation in exon 13 of the MYH7 gene and a novel mutation, 453Arg--> His, in exon 14 of the MYH7 gene: A case report.

    Science.gov (United States)

    Haluza, R; Halouzková, S; Buncek, M; Smíd, O; Kvasnicka, J

    2001-01-01

    An unusual clinical history of a 23-year-old male proband with obstructive hypertrophic cardiomyopathy associated with a rare genotype is presented. Genetic analysis of the proband found evidence for two distinct mutations of the MYH7 gene (the gene coding for the beta-myosin heavy chain): 403Arg--> Trp in exon 13 and a novel mutation, 453Arg--> His, in exon 14. A heterozygous site mutation was identified in exon 13 in the proband's father but no mutation site was found in his mother. Thus, the novel mutation in exon 14 is a de novo mutation.

  17. Familial hypertrophic cardiomyopathy owing to double heterozygosity for a 403Arg→ Trp mutation in exon 13 of the MYH7 gene and a novel mutation, 453Arg→ His, in exon 14 of the MYH7 gene: A case report

    Science.gov (United States)

    Haluza, Radovan; Halouzková, Štěpánka; Bunček, Martin; Šmíd, Ondřej; Kvasnička, Jiří

    2001-01-01

    An unusual clinical history of a 23-year-old male proband with obstructive hypertrophic cardiomyopathy associated with a rare genotype is presented. Genetic analysis of the proband found evidence for two distinct mutations of the MYH7 gene (the gene coding for the beta-myosin heavy chain): 403Arg→ Trp in exon 13 and a novel mutation, 453Arg→ His, in exon 14. A heterozygous site mutation was identified in exon 13 in the proband’s father but no mutation site was found in his mother. Thus, the novel mutation in exon 14 is a de novo mutation. PMID:20428263

  18. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    Energy Technology Data Exchange (ETDEWEB)

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H. [Kobe Univ. School of Medicine and Science (Japan)

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  19. Categorization of 77 dystrophin exons into 5 groups by a decision tree using indexes of splicing regulatory factors as decision markers.

    Science.gov (United States)

    Malueka, Rusdy Ghazali; Takaoka, Yutaka; Yagi, Mariko; Awano, Hiroyuki; Lee, Tomoko; Dwianingsih, Ery Kus; Nishida, Atsushi; Takeshima, Yasuhiro; Matsuo, Masafumi

    2012-03-31

    Duchenne muscular dystrophy, a fatal muscle-wasting disease, is characterized by dystrophin deficiency caused by mutations in the dystrophin gene. Skipping of a target dystrophin exon during splicing with antisense oligonucleotides is attracting much attention as the most plausible way to express dystrophin in DMD. Antisense oligonucleotides have been designed against splicing regulatory sequences such as splicing enhancer sequences of target exons. Recently, we reported that a chemical kinase inhibitor specifically enhances the skipping of mutated dystrophin exon 31, indicating the existence of exon-specific splicing regulatory systems. However, the basis for such individual regulatory systems is largely unknown. Here, we categorized the dystrophin exons in terms of their splicing regulatory factors. Using a computer-based machine learning system, we first constructed a decision tree separating 77 authentic from 14 known cryptic exons using 25 indexes of splicing regulatory factors as decision markers. We evaluated the classification accuracy of a novel cryptic exon (exon 11a) identified in this study. However, the tree mislabeled exon 11a as a true exon. Therefore, we re-constructed the decision tree to separate all 15 cryptic exons. The revised decision tree categorized the 77 authentic exons into five groups. Furthermore, all nine disease-associated novel exons were successfully categorized as exons, validating the decision tree. One group, consisting of 30 exons, was characterized by a high density of exonic splicing enhancer sequences. This suggests that AOs targeting splicing enhancer sequences would efficiently induce skipping of exons belonging to this group. The decision tree categorized the 77 authentic exons into five groups. Our classification may help to establish the strategy for exon skipping therapy for Duchenne muscular dystrophy.

  20. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    Energy Technology Data Exchange (ETDEWEB)

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  1. Noncoder: a web interface for exon array-based detection of long non-coding RNAs.

    Science.gov (United States)

    Gellert, Pascal; Ponomareva, Yuliya; Braun, Thomas; Uchida, Shizuka

    2013-01-07

    Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called 'noncoder'. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de.

  2. Assessment of the feasibility of exon 45–55 multiexon skipping for duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    van Ommen Gert-Jan B

    2008-12-01

    Full Text Available Abstract Background The specific skipping of an exon, induced by antisense oligonucleotides (AON during splicing, has shown to be a promising therapeutic approach for Duchenne muscular dystrophy (DMD patients. As different mutations require skipping of different exons, this approach is mutation dependent. The skipping of an entire stretch of exons (e.g. exons 45 to 55 has recently been suggested as an approach applicable to larger groups of patients. However, this multiexon skipping approach is technically challenging. The levels of intended multiexon skips are typically low and highly variable, and may be dependent on the order of intron removal. We hypothesized that the splicing order might favor the induction of multiexon 45–55 skipping. Methods We here tested the feasibility of inducing multiexon 45–55 in control and patient muscle cell cultures using various AON cocktails. Results In all experiments, the exon 45–55 skip frequencies were minimal and comparable to those observed in untreated cells. Conclusion We conclude that current state of the art does not sufficiently support clinical development of multiexon skipping for DMD.

  3. Prognostic and Predictive Value of RAS Gene Mutations in Colorectal Cancer: Moving Beyond KRAS Exon 2.

    Science.gov (United States)

    Boeckx, Nele; Peeters, Marc; Van Camp, Guy; Pauwels, Patrick; Op de Beeck, Ken; Deschoolmeester, Vanessa

    2015-10-01

    The advent of anti-EGFR (epidermal growth factor receptor) therapy resulted in significant progress in the treatment of metastatic colorectal cancer patients. However, many patients do not respond to this therapy or develop acquired resistance within a few months after the start of treatment. Since 2008, anti-EGFR therapy is restricted to KRAS wild-type patients as it has been shown that KRAS exon 2-mutated patients do not respond to this therapy. Still, up to 60 % of KRAS exon 2 wild-type patients show primary resistance to this treatment. Recently, several studies investigating the predictive and prognostic role of RAS mutations other than in KRAS exon 2 demonstrated that patients with these mutations are not responding to therapy. However, the role of these mutations has long been questioned as The National Comprehensive Cancer Network Guidelines in Oncology and the European Medicines Agency indications had already been changed in order to restrict anti-EGFR therapy to all RAS wild-type colorectal cancer patients, while the Food and Drug Administration guidelines remained unchanged. Recently, the Food and Drug Administration guidelines have also been changed, which implies the importance of RAS mutations beyond KRAS exon 2 in colorectal cancer. In this review, we discuss the most important studies regarding the predictive and prognostic role of RAS mutations other than in KRAS exon 2 in order to demonstrate the importance of these RAS mutations in patients with metastatic colorectal cancer treated with anti-EGFR therapy.

  4. Novel Rbfox2 isoforms associated with alternative exon usage in rat cortex and suprachiasmatic nucleus.

    Science.gov (United States)

    Partridge, L M M; Carter, D A

    2017-08-30

    Transcriptome diversity in adult neurons is partly mediated by RNA binding proteins (RBPs), including the RBFOX factors. RBFOX3/NeuN, a neuronal maturity marker, is strangely depleted in suprachiasmatic nucleus (SCN) neurons, and may be compensated by a change in Rbfox2 expression. In this study, we found no superficial changes in Rbfox2 expression in the SCN, but mRNA population analysis revealed a distinct SCN transcript profile that includes multiple novel Rbfox2 isoforms. Of eleven isoforms in SCN and cerebral cortex that exhibit exon variation across two protein domains, we found a 3-fold higher abundance of a novel ('-12-40') C-terminal domain (CTD)-variant in the SCN. This isoform embraces an alternative reading frame that imparts a 50% change in CTD protein sequence, and functional impairment of exon 7 exclusion activity in a RBFOX2-target, the L-type calcium channel gene, Cacna1c. We have also demonstrated functional correlates in SCN gene transcripts; inclusion of Cacna1c exon 7, and also exclusion of both NMDA receptor gene Grin1 exon 4, and Enah exon 12, all consistent with a change in SCN RBFOX activity. The demonstrated regional diversity of Rbfox2 in adult brain highlights the functional adaptability of this RBP, enabling neuronal specialization, and potentially responding to disease-related neuronal dysfunction.

  5. Leisure Time Physical Inactivity and Sedentary Behaviour and Lifestyle Correlates among Students Aged 13-15 in the Association of Southeast Asian Nations (ASEAN) Member States, 2007-2013.

    Science.gov (United States)

    Peltzer, Karl; Pengpid, Supa

    2016-02-15

    The aim of this study was to examine the relationship between self-reported leisure time physical inactivity frequency and sedentary behaviour and lifestyle correlates among school children in the Association of Southeast Asian Nations (ASEAN) region. The analysis included 30,284 school children aged 13-15 years from seven ASEAN countries that participated in the Global School-based Student Health Survey (GSHS) between 2007 and 2013. The measure asked about overall physical activity, walking or biking to school, and on time spent sitting. Overall, the prevalence of physical inactivity was 80.4%, ranging from 74.8% in Myanmar to 90.7% in Cambodia and sedentary behaviour 33.0%, ranging from 10.5% in Cambodia and Myanmar to 42.7% in Malaysia. In multivariate logistic regression, not walking or biking to school, not attending physical education classes, inadequate vegetable consumption and lack of protective factors (peer and parental or guardian support) were associated with physical inactivity, and older age (14 and 15 years old), coming from an upper middle income country, being overweight or obese, attending physical education classes, alcohol use, loneliness, peer support and lack of parental or guardian supervision were associated with sedentary behaviour. In boys, lower socioeconomic status (in the form of having experienced hunger) and coming from a low income or lower middle income country were additionally associated with physical inactivity, and in girls, higher socioeconomic status, not walking or biking to school and being bullied were additionally associated with sedentary behaviour. In conclusion, a very high prevalence of leisure physical inactivity and sedentary behaviour among school going adolescents in ASEAN was found and several factors identified that may inform physical activity promotion programmes in school-going adolescents in ASEAN.

  6. Molecular evolution of the leptin exon 3 in some species of the family Canidae

    Directory of Open Access Journals (Sweden)

    Switonski Marek

    2003-09-01

    Full Text Available Abstract The structure of the leptin gene seems to be well conserved. The polymorphism of this gene in four species belonging to the Canidae family (the dog (Canis familiaris – 16 different breeds, the Chinese racoon dog (Nyctereutes procyonoides procyonoides, the red fox (Vulpes vulpes and the arctic fox (Alopex lagopus were studied with the use of single strand conformation polymorphism (SSCP, restriction fragment length polymorphism (RFLP and DNA sequencing techniques. For exon 2, all species presented the same SSCP pattern, while in exon 3 some differences were found. DNA sequencing of exon 3 revealed the presence of six nucleotide substitutions, differentiating the studied species. Three of them cause amino acid substitutions as well. For all dog breeds studied, SSCP patterns were identical.

  7. DISC1 exon 11 rare variants found more commonly in schizoaffective spectrum cases than controls.

    Science.gov (United States)

    Green, E K; Grozeva, D; Sims, R; Raybould, R; Forty, L; Gordon-Smith, K; Russell, E; St Clair, D; Young, A H; Ferrier, I N; Kirov, G; Jones, I; Jones, L; Owen, M J; O'Donovan, M C; Craddock, N

    2011-06-01

    We previously performed a linkage study using families identified through probands meeting criteria for DSM-IV schizoaffective disorder, bipolar type (SABP) and observed a genome-wide significant signal (LOD = 3.54) at chromosome 1q42 close to DISC1. An initial sequencing study of DISC1 using 14 unrelated DSM-IV SABP samples from the linkage study identified 2 non-synonymous coding SNPs in exon 11 in 2 separate individuals. Here we provide evidence of additional rare coding SNPs within exon 11. In sequencing exon 11 in 506 cases and 1,211 controls for variants that occurred only once, 4 additional rare variants were found in cases (P-value = 0.008, Fisher's exact trend test). Copyright © 2011 Wiley-Liss, Inc.

  8. Multi-exon deletions of the FBN1 gene in Marfan syndrome

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    Schrijver Iris

    2001-10-01

    Full Text Available Abstract Background Mutations in the fibrillin -1 gene (FBN1 cause Marfan syndrome (MFS, an autosomal dominant multi-system connective tissue disorder. The 200 different mutations reported in the 235 kb, 65 exon-containing gene include only one family with a genomic multi-exon deletion. Methods We used long-range RT-PCR for mutation detection and long-range genomic PCR and DNA sequencing for identification of deletion breakpoints, allele-specific transcript analyses to determine stability of the mutant RNA, and pulse-chase studies to quantitate fibrillin synthesis and extracellular matrix deposition in cultured fibroblasts. Southern blots of genomic DNA were probed with three overlapping fragments covering the FBN1 coding exons Results Two novel multi-exon FBN1 deletions were discovered. Identical nucleotide pentamers were found at or near the intronic breakpoints. In a Case with classic MFS, an in-frame deletion of exons 42 and 43 removed the C-terminal 24 amino acids of the 5th LTBP (8-cysteine domain and the adjacent 25th calcium-binding EGF-like (6-cysteine domain. The mutant mRNA was stable, but fibrillin synthesis and matrix deposition were significantly reduced. A Case with severe childhood-onset MFS has a de novo deletion of exons 44–46 that removed three EGF-like domains. Fibrillin protein synthesis was normal, but matrix deposition was strikingly reduced. No genomic rearrangements were detected by Southern analysis of 18 unrelated MFS samples negative for FBN1 mutation screening. Conclusions Two novel deletion cases expand knowledge of mutational mechanisms and genotype/phenotype correlations of fibrillinopathies. Deletions or mutations affecting an LTBP domain may result in unstable mutant protein cleavage products that interfere with microfibril assembly.

  9. Characterization of Major Histocompatibility Complex (MHC) DRB Exon 2 and DRA Exon 3 Fragments in a Primary Terrestrial Rabies Vector (Procyon lotor)

    Science.gov (United States)

    Castillo, Sarrah; Srithayakumar, Vythegi; Meunier, Vanessa; Kyle, Christopher J.

    2010-01-01

    The major histocompatibility complex (MHC) presents a unique system to explore links between genetic diversity and pathogens, as diversity within MHC is maintained in part by pathogen driven selection. While the majority of wildlife MHC studies have investigated species that are of conservation concern, here we characterize MHC variation in a common and broadly distributed species, the North American raccoon (Procyon lotor). Raccoons host an array of broadly distributed wildlife diseases (e.g., canine distemper, parvovirus and raccoon rabies virus) and present important human health risks as they persist in high densities and in close proximity to humans and livestock. To further explore how genetic variation influences the spread and maintenance of disease in raccoons we characterized a fragment of MHC class II DRA exon 3 (250bp) and DRB exon 2 (228 bp). MHC DRA was found to be functionally monomorphic in the 32 individuals screened; whereas DRB exon 2 revealed 66 unique alleles among the 246 individuals screened. Between two and four alleles were observed in each individual suggesting we were amplifying a duplicated DRB locus. Nucleotide differences between DRB alleles ranged from 1 to 36 bp (0.4–15.8% divergence) and translated into 1 to 21 (1.3–27.6% divergence) amino acid differences. We detected a significant excess of nonsynonymous substitutions at the peptide binding region (P = 0.005), indicating that DRB exon 2 in raccoons has been influenced by positive selection. These data will form the basis of continued analyses into the spatial and temporal relationship of the raccoon rabies virus and the immunogenetic response in its primary host. PMID:20706587

  10. Characterization of major histocompatibility complex (MHC DRB exon 2 and DRA exon 3 fragments in a primary terrestrial rabies vector (Procyon lotor.

    Directory of Open Access Journals (Sweden)

    Sarrah Castillo

    Full Text Available The major histocompatibility complex (MHC presents a unique system to explore links between genetic diversity and pathogens, as diversity within MHC is maintained in part by pathogen driven selection. While the majority of wildlife MHC studies have investigated species that are of conservation concern, here we characterize MHC variation in a common and broadly distributed species, the North American raccoon (Procyon lotor. Raccoons host an array of broadly distributed wildlife diseases (e.g., canine distemper, parvovirus and raccoon rabies virus and present important human health risks as they persist in high densities and in close proximity to humans and livestock. To further explore how genetic variation influences the spread and maintenance of disease in raccoons we characterized a fragment of MHC class II DRA exon 3 (250 bp and DRB exon 2 (228 bp. MHC DRA was found to be functionally monomorphic in the 32 individuals screened; whereas DRB exon 2 revealed 66 unique alleles among the 246 individuals screened. Between two and four alleles were observed in each individual suggesting we were amplifying a duplicated DRB locus. Nucleotide differences between DRB alleles ranged from 1 to 36 bp (0.4-15.8% divergence and translated into 1 to 21 (1.3-27.6% divergence amino acid differences. We detected a significant excess of nonsynonymous substitutions at the peptide binding region (P = 0.005, indicating that DRB exon 2 in raccoons has been influenced by positive selection. These data will form the basis of continued analyses into the spatial and temporal relationship of the raccoon rabies virus and the immunogenetic response in its primary host.

  11. Histone hyperacetylation and exon skipping: a calcium-mediated dynamic regulation in cardiomyocytes

    Science.gov (United States)

    Sharma, Alok; Nguyen, Hieu; Cai, Lu; Lou, Hua

    2015-01-01

    In contrast to cell type-specific pre-mRNA alternative splicing, mechanisms controlling activity-dependent alternative splicing is under-studied and not well understood. In a recent study, we conducted a comprehensive analysis of calcium-mediated mechanism that regulates alternative exon skipping in mouse cardiomyocytes. Our results reveal a strong link between histone hyperacetylation and skipping of cassette exons, and provide support to the kinetic coupling model of the epigenetic regulation of alternative splicing at the chromatin level. PMID:26325491

  12. De novo exonic mutation in MYH7 gene leading to exon skipping in a patient with early onset muscular weakness and fiber-type disproportion.

    Science.gov (United States)

    Pajusalu, Sander; Talvik, Inga; Noormets, Klari; Talvik, Tiina; Põder, Haide; Joost, Kairit; Puusepp, Sanna; Piirsoo, Andres; Stenzel, Werner; Goebel, Hans H; Nikopensius, Tiit; Annilo, Tarmo; Nõukas, Margit; Metspalu, Andres; Õunap, Katrin; Reimand, Tiia

    2016-03-01

    Here we report on a case of MYH7-related myopathy in a boy with early onset of muscular weakness and delayed motor development in infancy. His most affected muscles were neck extensors showing a dropped head sign, proximal muscles of lower limbs with positive Gower's sign, and trunk muscles. Brain and spinal cord MRI scans, echocardiography, and laboratory analyses including creatine kinase and lactate did not reveal any abnormalities. Muscle histopathology showed fiber-type disproportion. Whole exome sequencing of the parents-offspring trio revealed a novel de novo c.5655G>A p.(Ala1885=) synonymous substitution of the last nucleotide in exon 38 of the MYH7 gene. Further RNA investigations proved the skipping of exon 38 (p.1854_1885del). This is a first report of an exon-skipping mutation in the MYH7 gene causing myopathy. This report broadens both the phenotypic and genotypic spectra of MYH7-related myopathies. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

    Science.gov (United States)

    Saito, Takashi; Nakamura, Akinori; Aoki, Yoshitsugu; Yokota, Toshifumi; Okada, Takashi; Osawa, Makiko; Takeda, Shin'ichi

    2010-08-18

    Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J)) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. We converted fibroblasts of CXMD(J) and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J) and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.

  14. Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

    Directory of Open Access Journals (Sweden)

    Takashi Saito

    Full Text Available BACKGROUND: Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD. We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. METHODOLOGY/PRINCIPAL FINDINGS: We converted fibroblasts of CXMD(J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. CONCLUSION/SIGNIFICANCE: Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.

  15. Epidermal growth factor receptor exon 20 p.S768I mutation in non-small cell lung carcinoma

    DEFF Research Database (Denmark)

    Improta, Giuseppina; Pettinato, Angela; Gieri, Stefania

    2016-01-01

    to the comparative rarity of EGFR exon 20 mutations, clinical information concerning the association between EGFR exon 20 mutations and responsiveness to TKIs has been limited within the relevant literature, particularly for certain rare mutations, including p.S768I. The current study reports the case of a patient...

  16. Efficient Skipping of Single Exon Duplications in DMD Patient-Derived Cell Lines Using an Antisense Oligonucleotide Approach.

    Science.gov (United States)

    Wein, Nicolas; Vulin, Adeline; Findlay, Andrew R; Gumienny, Felecia; Huang, Nianyuan; Wilton, Steve D; Flanigan, Kevin M

    2017-01-01

    Exon skipping strategies in Duchenne muscular dystrophy (DMD) have largely been directed toward altering splicing of exons flanking out-of-frame deletions, with the goal of restoring an open mRNA reading frame that leads to production of an internally deleted but partially functional dystrophin protein. We sought to apply exon skipping to duplication mutations, assuming that the inherently limited efficiency of antisense oligonucleotide-induced exon skipping would more frequently skip a single copy of a duplicated exon, rather than both and result in significant amounts of wild-type DMD mRNA. We tested this hypothesis in fibroblast cell lines derived from patients with a variety of single or multiple exon duplications that have been modified to allow transdifferentiation into a myogenic lineage. Using a variety of 2'O-methyl antisense oligonucleotides, significant skipping was induced for each duplication leading to a wild-type transcript as a major mRNA product. This study provides another proof of concept for the feasibility of therapeutic skipping in patients carrying exon duplications in order to express wild-type, full-length mRNA, although careful evaluation of the skipping efficiency should be performed as some exons are easier to skip than others. Such a personalized strategy is expected to be highly beneficial for this subset of DMD patients, compared to inducing expression of an internally-deleted dystrophin.

  17. Genetic variation at Exon2 of TLR4 gene and its association with ...

    African Journals Online (AJOL)

    Jane

    2011-08-08

    Aug 8, 2011 ... role as a key linkage between innate immunity and specific immunity. But to date, no mutation has been detected in exon 2 in chicken. The correlation between polymorphism of TLR4 gene and its resistance to disease have rarely been reported in china. This study analyzed the polymorphism of chicken ...

  18. Comprehensive survey of SNPs in the Affymetrix exon array using the 1000 Genomes dataset.

    Directory of Open Access Journals (Sweden)

    Eric R Gamazon

    Full Text Available Microarray gene expression data has been used in genome-wide association studies to allow researchers to study gene regulation as well as other complex phenotypes including disease risks and drug response. To reach scientifically sound conclusions from these studies, however, it is necessary to get reliable summarization of gene expression intensities. Among various factors that could affect expression profiling using a microarray platform, single nucleotide polymorphisms (SNPs in target mRNA may lead to reduced signal intensity measurements and result in spurious results. The recently released 1000 Genomes Project dataset provides an opportunity to evaluate the distribution of both known and novel SNPs in the International HapMap Project lymphoblastoid cell lines (LCLs. We mapped the 1000 Genomes Project genotypic data to the Affymetrix GeneChip Human Exon 1.0ST array (exon array, which had been used in our previous studies and for which gene expression data had been made publicly available. We also evaluated the potential impact of these SNPs on the differentially spliced probesets we had identified previously. Though the 1000 Genomes Project data allowed a comprehensive survey of the SNPs in this particular array, the same approach can certainly be applied to other microarray platforms. Furthermore, we present a detailed catalogue of SNP-containing probesets (exon-level and transcript clusters (gene-level, which can be considered in evaluating findings using the exon array as well as benefit the design of follow-up experiments and data re-analysis.

  19. Longitudinal changes in glucocorticoid receptor exon 1(F) methylation and psychopathology after military deployment

    NARCIS (Netherlands)

    Schur, R. R.; Boks, M. P.; Rutten, B. P. F.; Daskalakis, N. P.; de Nijs, L.; van Zuiden, M.; Kavelaars, A.; Heijnen, C. J.; Joels, M.; Kahn, R. S.; Geuze, E.; Vermetten, E.; Vinkers, C. H.

    2017-01-01

    Several cross-sectional studies have demonstrated the relevance of DNA methylation of the glucocorticoid receptor exon 1(F) region (GR-1(F)) for trauma-related psychopathology. We conducted a longitudinal study to examine GR-1(F) methylation changes over time in relation to trauma exposure and the

  20. Deletion of SNURF/SNRPN U1B and U1B* upstream exons in a ...

    Indian Academy of Sciences (India)

    we report the case of a patient who was referred to us with Prader–Willi syndrome-like symptoms including obesity and developmental delay. Examination of this patient revealed that he was a carrier of a paternally inherited deletion that affected the U1B and U1B* upstream exons of the SNURF–SNRNP gene within the ...

  1. Genetic polymorphism of exon 9-11 of the leptin gene receptor in ...

    African Journals Online (AJOL)

    Genomic DNA was extracted using modified salting-out method and amplified polymerase chain reaction technique. Exon and intron 9-11 of the fowl leptin gene ... Further association analysis is required to clarify the effects of these marker genotypes on production traits in this breeder flock. Key words: Leptin gene receptor, ...

  2. Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Fiorella Guadagni

    2012-01-01

    Full Text Available The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine at codon 57. In addition, we found in the same patient’s sample a silent polymorphism at codon 11 (Ala11Ala of exon 1. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 729–733

  3. Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients

    NARCIS (Netherlands)

    Aartsma-Rus, Annemieke; Janson, Anneke A. M.; Kaman, Wendy E.; Bremmer-Bout, Mattie; den Dunnen, Johan T.; Baas, Frank; van Ommen, Gert-Jan B.; van Deutekom, Judith C. T.

    2003-01-01

    The dystrophin deficiency leading to the severely progressing muscle degeneration in Duchenne muscular dystrophy (DMD) patients is caused by frame-shifting mutations in the DMD gene. We are developing a reading frame correction therapy aimed at the antisense-induced skipping of targeted exons from

  4. Improvements to previous algorithms to predict gene structure and isoform concentrations using Affymetrix Exon arrays

    Directory of Open Access Journals (Sweden)

    Aramburu Ander

    2010-11-01

    Full Text Available Abstract Background Exon arrays provide a way to measure the expression of different isoforms of genes in an organism. Most of the procedures to deal with these arrays are focused on gene expression or on exon expression. Although the only biological analytes that can be properly assigned a concentration are transcripts, there are very few algorithms that focus on them. The reason is that previously developed summarization methods do not work well if applied to transcripts. In addition, gene structure prediction, i.e., the correspondence between probes and novel isoforms, is a field which is still unexplored. Results We have modified and adapted a previous algorithm to take advantage of the special characteristics of the Affymetrix exon arrays. The structure and concentration of transcripts -some of them possibly unknown- in microarray experiments were predicted using this algorithm. Simulations showed that the suggested modifications improved both specificity (SP and sensitivity (ST of the predictions. The algorithm was also applied to different real datasets showing its effectiveness and the concordance with PCR validated results. Conclusions The proposed algorithm shows a substantial improvement in the performance over the previous version. This improvement is mainly due to the exploitation of the redundancy of the Affymetrix exon arrays. An R-Package of SPACE with the updated algorithms have been developed and is freely available.

  5. Genetic variation at Exon2 of TLR4 gene and its association with ...

    African Journals Online (AJOL)

    This study was conducted to analyze the polymorphisms of chicken Toll-like receptors 4(TLR4) gene and aimed to provide a theoretical foundation for a further research on correlation between chicken TLR4 gene and disease resistance. Genetic variations at exon 2 of TLR4 gene in 14 chicken breeds and the red jungle ...

  6. Investigation of biochemical changes of the ovine calpain 3 exon-10 polymorphism.

    Science.gov (United States)

    Muto, Yukiyo; Morton, Jim; Palmer, David

    2015-12-01

    Calpain 3 (CAPN3) is a tissue specific calpain, and its mRNA is the most expressed calpain isoform in skeletal muscles. Many mutations and polymorphisms within the human CAPN3 gene have been reported and related to limb-girdle muscular dystrophy. Several reports link CAPN3 polymorphisms and meat quality. An association between three allele variants in exon-10 of ovine CAPN3 and the yield of fat trimmed meat cuts has been reported. This research investigated the biochemical significance of polymorphic variation in CAPN3. CAPN3 mRNA sequences were obtained from muscle samples collected from lambs which were homozygous for each of the three alleles. Four single base substitutions were found besides those in exon-10, but none of them, including the variations within exon-10, caused a change in amino acid sequence. The expression of CAPN3 mRNA and the amounts of CAPN3 protein were also compared among genotypes, and no significant differences were found. These results suggest that the reported association of specific allele variants within CAPN3 exon-10 to phenotype variations were not direct effects of CAPN3 polymorphisms. Interspecies analyses of the CAPN3 sequences indicated that the sequence reported here is more likely to be the correct common ovine CAPN3 sequence than the reference sequence. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Deletion of SNURF/SNRPN U1B and U1B* upstream exons in a ...

    Indian Academy of Sciences (India)

    Here, we report the case of a patient who was referred to us with Prader–Willi syndrome-like symptoms including obesity and developmental delay. Examination of this patient revealed that he was a carrier of a paternally inherited deletion that affected the U1B and U1B* upstream exons of the SNURF–SNRNP gene within ...

  8. Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    van Ommen Gert Jan B

    2011-04-01

    Full Text Available Abstract Background Myostatin is a potent muscle growth inhibitor that belongs to the Transforming Growth Factor-β (TGF-β family. Mutations leading to non functional myostatin have been associated with hypermuscularity in several organisms. By contrast, Duchenne muscular dystrophy (DMD is characterized by a loss of muscle fibers and impaired regeneration. In this study, we aim to knockdown myostatin by means of exon skipping, a technique which has been successfully applied to reframe the genetic defect of dystrophin gene in DMD patients. Methods We targeted myostatin exon 2 using antisense oligonucleotides (AON in healthy and DMD-derived myotubes cultures. We assessed the exon skipping level, transcriptional expression of myostatin and its target genes, and combined myostatin and several dystrophin AONs. These AONs were also applied in the mdx mice models via intramuscular injections. Results Myostatin AON induced exon 2 skipping in cell cultures and to a lower extent in the mdx mice. It was accompanied by decrease in myostatin mRNA and enhanced MYOG and MYF5 expression. Furthermore, combination of myostatin and dystrophin AONs induced simultaneous skipping of both genes. Conclusions We conclude that two AONs can be used to target two different genes, MSTN and DMD, in a straightforward manner. Targeting multiple ligands of TGF-beta family will be more promising as adjuvant therapies for DMD.

  9. Exon skipping: a first in class strategy for Duchenne muscular dystrophy.

    Science.gov (United States)

    Niks, Erik H; Aartsma-Rus, Annemieke

    2017-02-01

    Exon skipping is a therapeutic approach for Duchenne muscular dystrophy (DMD) that has been in development for close to two decades. This approach uses antisense oligonucleotides (AONs) to modulate pre-mRNA splicing of dystrophin transcripts to restore the disrupted DMD reading frame. The approach has moved from in vitro proof of concept studies to the clinical trial phase and marketing authorization applications with regulators. The first AON (eteplirsen) has recently received accelerated approval by the Food and Drug Administration in the US. Areas covered: In this review the authors explain the antisense-mediated exon skipping approach, outline how it needs be tailored for different DMD mutation types and describe the challenges and opportunities for each mutation type. The authors summarize the clinical development of antisense-mediated exon 51 skipping, and discuss methods to improve efficiency. Finally, the authors provide their opinion on current developments and identify topics for future prioritization. Expert opinion: Exon skipping development has been a learning experience for all those involved. Aside from an approved therapy, its development has yielded side benefits including the development of tools for clinical trials and has increased collaboration between academics, patients, industry and regulators.

  10. Mutations in exon 10 of the RET proto-oncogene in Hirschsprung`s disease

    Energy Technology Data Exchange (ETDEWEB)

    Attie, T.; Eng, C.; Mulligan, L.M. [Hospital des Enfants-Malades, Paris (France)] [and others

    1994-09-01

    Hirschsprung`s disease (HSCR) is a frequent congenital malformation ascribed to the absence of autonomic ganglion cells in the terminal hindgut. Recently, we have identified mutations in the RET proto-oncogene in HSCR families. Mutations of the RET gene have also been reported in multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). While RET mutations in HSCR are scattered on the whole coding sequence, MEN 2A and FMTC mutations are clustered in 5 cystein codons of exons 10 and 11. Here, we report on HSCR families carrying mutations in exon 10 of the RET gene, one of them involving a cystein codon. Germ-line mutations in exon 10 of the RET gene may contribute to either an early development defect (HSCR) or inherited predisposition to cancer (MEN 2A and FMTC), probable depending on the nature and location of the mutation. These data also suggest that HSCR patients with mutations in exon 10 might subsequently prove to be at risk for MEN 2A or FMTC since several MEN 2A/HSCR associations have been reported.

  11. [Polymorphism of exon 4 in the CANP-3 gene in patients with primary myopathies].

    Science.gov (United States)

    Lipatova, N A; Krakhmaleva, I N; Shishkin, S S; Shakhovskaia, N I; Podnikova, N I; Lunga, I N; Tarksh, M A; Gerasimova, N L

    1999-12-01

    The structures of the gene for calpain (CANP-3) and of the DMD gene were analyzed in patients with primary myopathies [limb-girdle muscular distrophy (LGMD) and Duchenne-Becker myodystrophy (DBM)] from various regions of Russia. Via amplification of DNA isolated from the peripheral blood lymphocytes of 74 patients, extended deletions were found in 18 out of 55 patients with DBM. In none of the 19 patients with LGMD, were extended deletions in the CANP-3 gene found. In most patients with LGMD, the amplification of the promoter region and exons 1, 2, 3, 4, 5, and 6 of the CANP-3 gene yielded a single product of corresponding length, but in six patients (three sib pairs), amplification of exon 4 of the CANP-3 gene yielded two products of different size. The following single-strand conformation polymorphism (SSCP) analysis revealed a pronounced polymorphism of exon 4 of the CANP-3 gene in 14 out of 19 patients with LGMD. This structure of exon 4 of the CANP-3 gene was found neither in 16 patients with DBM who had deletions in the DMD gene nor in 16 patients with DBM who had no deletions in the DMD gene.

  12. Folding Landscape of Mutant Huntingtin Exon1: Diffusible Multimers, Oligomers and Fibrils, and No Detectable Monomer.

    Directory of Open Access Journals (Sweden)

    Bankanidhi Sahoo

    Full Text Available Expansion of the polyglutamine (polyQ track of the Huntingtin (HTT protein above 36 is associated with a sharply enhanced risk of Huntington's disease (HD. Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6-9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the

  13. Identification of novel candidate disease genes from de novo exonic copy number variants.

    Science.gov (United States)

    Gambin, Tomasz; Yuan, Bo; Bi, Weimin; Liu, Pengfei; Rosenfeld, Jill A; Coban-Akdemir, Zeynep; Pursley, Amber N; Nagamani, Sandesh C S; Marom, Ronit; Golla, Sailaja; Dengle, Lauren; Petrie, Heather G; Matalon, Reuben; Emrick, Lisa; Proud, Monica B; Treadwell-Deering, Diane; Chao, Hsiao-Tuan; Koillinen, Hannele; Brown, Chester; Urraca, Nora; Mostafavi, Roya; Bernes, Saunder; Roeder, Elizabeth R; Nugent, Kimberly M; Bader, Patricia I; Bellus, Gary; Cummings, Michael; Northrup, Hope; Ashfaq, Myla; Westman, Rachel; Wildin, Robert; Beck, Anita E; Immken, LaDonna; Elton, Lindsay; Varghese, Shaun; Buchanan, Edward; Faivre, Laurence; Lefebvre, Mathilde; Schaaf, Christian P; Walkiewicz, Magdalena; Yang, Yaping; Kang, Sung-Hae L; Lalani, Seema R; Bacino, Carlos A; Beaudet, Arthur L; Breman, Amy M; Smith, Janice L; Cheung, Sau Wai; Lupski, James R; Patel, Ankita; Shaw, Chad A; Stankiewicz, Paweł

    2017-09-21

    Exon-targeted microarrays can detect small (<1000 bp) intragenic copy number variants (CNVs), including those that affect only a single exon. This genome-wide high-sensitivity approach increases the molecular diagnosis for conditions with known disease-associated genes, enables better genotype-phenotype correlations, and facilitates variant allele detection allowing novel disease gene discovery. We retrospectively analyzed data from 63,127 patients referred for clinical chromosomal microarray analysis (CMA) at Baylor Genetics laboratories, including 46,755 individuals tested using exon-targeted arrays, from 2007 to 2017. Small CNVs harboring a single gene or two to five non-disease-associated genes were identified; the genes involved were evaluated for a potential disease association. In this clinical population, among rare CNVs involving any single gene reported in 7200 patients (11%), we identified 145 de novo autosomal CNVs (117 losses and 28 intragenic gains), 257 X-linked deletion CNVs in males, and 1049 inherited autosomal CNVs (878 losses and 171 intragenic gains); 111 known disease genes were potentially disrupted by de novo autosomal or X-linked (in males) single-gene CNVs. Ninety-one genes, either recently proposed as candidate disease genes or not yet associated with diseases, were disrupted by 147 single-gene CNVs, including 37 de novo deletions and ten de novo intragenic duplications on autosomes and 100 X-linked CNVs in males. Clinical features in individuals with de novo or X-linked CNVs encompassing at most five genes (224 bp to 1.6 Mb in size) were compared to those in individuals with larger-sized deletions (up to 5 Mb in size) in the internal CMA database or loss-of-function single nucleotide variants (SNVs) detected by clinical or research whole-exome sequencing (WES). This enabled the identification of recently published genes (BPTF, NONO, PSMD12, TANGO2, and TRIP12), novel candidate disease genes (ARGLU1 and STK3), and further confirmation

  14. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen thi Man; Morris, G.E. (North East Wales Inst., Clwyd (United Kingdom))

    1993-06-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random [open quotes]libraries[close quotes] of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25--60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1--41, and the authors now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. 38 refs., 2 figs., 4 tabs.

  15. SNP discovery in candidate adaptive genes using exon capture in a free-ranging alpine ungulate

    Science.gov (United States)

    Roffler, Gretchen H.; Amish, Stephen J.; Smith, Seth; Cosart, Ted F.; Kardos, Marty; Schwartz, Michael K.; Luikart, Gordon

    2016-01-01

    Identification of genes underlying genomic signatures of natural selection is key to understanding adaptation to local conditions. We used targeted resequencing to identify SNP markers in 5321 candidate adaptive genes associated with known immunological, metabolic and growth functions in ovids and other ungulates. We selectively targeted 8161 exons in protein-coding and nearby 5′ and 3′ untranslated regions of chosen candidate genes. Targeted sequences were taken from bighorn sheep (Ovis canadensis) exon capture data and directly from the domestic sheep genome (Ovis aries v. 3; oviAri3). The bighorn sheep sequences used in the Dall's sheep (Ovis dalli dalli) exon capture aligned to 2350 genes on the oviAri3 genome with an average of 2 exons each. We developed a microfluidic qPCR-based SNP chip to genotype 476 Dall's sheep from locations across their range and test for patterns of selection. Using multiple corroborating approaches (lositan and bayescan), we detected 28 SNP loci potentially under selection. We additionally identified candidate loci significantly associated with latitude, longitude, precipitation and temperature, suggesting local environmental adaptation. The three methods demonstrated consistent support for natural selection on nine genes with immune and disease-regulating functions (e.g. Ovar-DRA, APC, BATF2, MAGEB18), cell regulation signalling pathways (e.g. KRIT1, PI3K, ORRC3), and respiratory health (CYSLTR1). Characterizing adaptive allele distributions from novel genetic techniques will facilitate investigation of the influence of environmental variation on local adaptation of a northern alpine ungulate throughout its range. This research demonstrated the utility of exon capture for gene-targeted SNP discovery and subsequent SNP chip genotyping using low-quality samples in a nonmodel species.

  16. JAK2 Exon 12 Mutations in Polycythemia Vera and Idiopathic Erythrocytosis

    Science.gov (United States)

    Scott, Linda M.; Tong, Wei; Levine, Ross L.; Scott, Mike A.; Beer, Philip A.; Stratton, Michael R.; Futreal, P. Andrew; Erber, Wendy N.; McMullin, Mary Frances; Harrison, Claire N.; Warren, Alan J.; Gilliland, D. Gary; Lodish, Harvey F.; Green, Anthony R.

    2010-01-01

    BACKGROUND The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. METHODS We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. RESULTS We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. CONCLUSIONS JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis

  17. Splicing Analysis of Exonic OCRL Mutations Causing Lowe Syndrome or Dent-2 Disease

    Directory of Open Access Journals (Sweden)

    Lorena Suarez-Artiles

    2018-01-01

    Full Text Available Mutations in the OCRL gene are associated with both Lowe syndrome and Dent-2 disease. Patients with Lowe syndrome present congenital cataracts, mental disabilities and a renal proximal tubulopathy, whereas patients with Dent-2 disease exhibit similar proximal tubule dysfunction but only mild, or no additional clinical defects. It is not yet understood why some OCRL mutations cause the phenotype of Lowe syndrome, while others develop the milder phenotype of Dent-2 disease. Our goal was to gain new insights into the consequences of OCRL exonic mutations on pre-mRNA splicing. Using predictive bioinformatics tools, we selected thirteen missense mutations and one synonymous mutation based on their potential effects on splicing regulatory elements or splice sites. These mutations were analyzed in a minigene splicing assay. Results of the RNA analysis showed that three presumed missense mutations caused alterations in pre-mRNA splicing. Mutation c.741G>T; p.(Trp247Cys generated splicing silencer sequences and disrupted splicing enhancer motifs that resulted in skipping of exon 9, while mutations c.2581G>A; p.(Ala861Thr and c.2581G>C; p.(Ala861Pro abolished a 5′ splice site leading to skipping of exon 23. Mutation c.741G>T represents the first OCRL exonic variant outside the conserved splice site dinucleotides that results in alteration of pre-mRNA splicing. Our results highlight the importance of evaluating the effects of OCRL exonic mutations at the mRNA level.

  18. Evolution, functional divergence and conserved exon-intron structure of bHLH/PAS gene family.

    Science.gov (United States)

    Yan, Jun; Ma, Zhaowu; Xu, Xiaopeng; Guo, An-Yuan

    2014-02-01

    bHLH/PAS genes encode a family of basic helix-loop-helix (bHLH) transcription factors with bHLH, PAS and PAS_3 domain. bHLH/PAS genes are involved in many essential physiological and developmental processes, such as hypoxic response neural development, the circadian clock, and learning ability. Despite their important functions, the origin and evolution of this bHLH/PAS gene family has yet to be elucidated. In this study, we aim to explore the origin, evolution, gene structure conservation of this gene family and provide a model to analyze the evolution of other gene families. Our results show that genes of the bHLH/PAS family only exist in metazoans. They may have originated from the common ancestor of metazoans and expanded into vertebrates. We identified bHLH/PAS genes in more than ten species representing the main lineages and constructed the phylogenetic trees (Beyasian, ML and NJ) to classify them into three groups. The exon-intron structure analysis revealed that a relatively conserved "1001-0210" eight-exon structure exists in most groups and lineages. In addition, we found the exon fusion pattern in several groups in this conserved eight-exon structure. Further analysis indicated that bHLH/PAS protein paralogs evolved from several gene duplication events followed by functional divergence and purifying selection. We presented a phylogenetic model to describe the evolutionary history of the exon structures of bHLH/PAS genes. Taken together, our study revealed the evolutionary model, functional divergence and gene structure conservation of bHLH/PAS genes. These findings provide clues for the functional and evolutionary mechanism of bHLH/PAS genes.

  19. Post-transcriptional exon shuffling events in humans can be evolutionarily conserved and abundant.

    Science.gov (United States)

    Al-Balool, Haya H; Weber, David; Liu, Yilei; Wade, Mark; Guleria, Kamlesh; Nam, Pitsien Lang Ping; Clayton, Jake; Rowe, William; Coxhead, Jonathan; Irving, Julie; Elliott, David J; Hall, Andrew G; Santibanez-Koref, Mauro; Jackson, Michael S

    2011-11-01

    In silico analyses have established that transcripts from some genes can be processed into RNAs with rearranged exon order relative to genomic structure (post-transcriptional exon shuffling, or PTES). Although known to contribute to transcriptome diversity in some species, to date the structure, distribution, abundance, and functional significance of human PTES transcripts remains largely unknown. Here, using high-throughput transcriptome sequencing, we identify 205 putative human PTES products from 176 genes. We validate 72 out of 112 products analyzed using RT-PCR, and identify additional PTES products structurally related to 61% of validated targets. Sequencing of these additional products reveals GT-AG dinucleotides at >95% of the splice junctions, confirming that they are processed by the spliceosome. We show that most PTES transcripts are expressed in a wide variety of human tissues, that they can be polyadenylated, and that some are conserved in mouse. We also show that they can extend into 5' and 3' UTRs, consistent with formation via trans-splicing of independent pre-mRNA molecules. Finally, we use real-time PCR to compare the abundance of PTES exon junctions relative to canonical exon junctions within the transcripts from seven genes. PTES exon junctions are present at 90% of the levels of canonical junctions, with transcripts from MAN1A2, PHC3, TLE4, and CDK13 exhibiting the highest levels. This is the first systematic experimental analysis of PTES in human, and it suggests both that the phenomenon is much more widespread than previously thought and that some PTES transcripts could be functional.

  20. Short Tandem Repeats in Human Exons: A Target for Disease Mutations

    Directory of Open Access Journals (Sweden)

    Villesen Palle

    2008-09-01

    Full Text Available Abstract Background In recent years it has been demonstrated that structural variations, such as indels (insertions and deletions, are common throughout the genome, but the implications of structural variations are still not clearly understood. Long tandem repeats (e.g. microsatellites or simple repeats are known to be hypermutable (indel-rich, but are rare in exons and only occasionally associated with diseases. Here we focus on short (imperfect tandem repeats (STRs which fall below the radar of conventional tandem repeat detection, and investigate whether STRs are targets for disease-related mutations in human exons. In particular, we test whether they share the hypermutability of the longer tandem repeats and whether disease-related genes have a higher STR content than non-disease-related genes. Results We show that validated human indels are extremely common in STR regions compared to non-STR regions. In contrast to longer tandem repeats, our definition of STRs found them to be present in exons of most known human genes (92%, 99% of all STR sequences in exons are shorter than 33 base pairs and 62% of all STR sequences are imperfect repeats. We also demonstrate that STRs are significantly overrepresented in disease-related genes in both human and mouse. These results are preserved when we limit the analysis to STRs outside known longer tandem repeats. Conclusion Based on our findings we conclude that STRs represent hypermutable regions in the human genome that are linked to human disease. In addition, STRs constitute an obvious target when screening for rare mutations, because of the relatively low amount of STRs in exons (1,973,844 bp and the limited length of STR regions.

  1. Splicing reporter mice revealed the evolutionally conserved switching mechanism of tissue-specific alternative exon selection.

    Directory of Open Access Journals (Sweden)

    Akihide Takeuchi

    Full Text Available Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of "splice code". So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this "balance" model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2 gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the "switch-like" mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved "splice code," in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions.

  2. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity.

    Science.gov (United States)

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-07-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a "splicing memory" hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. © 2015 Marquez et al.; Published by Cold Spring Harbor Laboratory Press.

  3. Model for a transcript map of human chromosome 21: isolation of new coding sequences from exon and enriched cDNA libraries.

    Science.gov (United States)

    Yaspo, M L; Gellen, L; Mott, R; Korn, B; Nizetic, D; Poustka, A M; Lehrach, H

    1995-08-01

    The construction of a transcriptional map for human chromosome 21 requires the generation of a specific catalogue of genes, together with corresponding mapping information. Towards this goal, we conducted a pilot study on a pool of random chromosome 21 cosmids representing 2 Mb of non-contiguous DNA. Exon-amplification and cDNA selection methods were used in combination to extract the coding content from these cosmids, and to derive expressed sequences libraries. These libraries and the source cosmid library were arrayed at high density for hybridisation screening. A strategy was used which related data obtained by multiple hybridisations of clones originating from one library, screened against the other libraries. In this way, it was possible to integrate the information with the physical map and to compare the gene recovery rate of each technique. cDNAs and exons were grouped into bins delineated by EcoRI cosmid fragments, and a subset of 91 cDNAs and 29 exons have been sequenced. These sequences defined 79 non-overlapping potential coding segments distributed in 24 transcriptional units, which were mapped along 21q. Northern blot analysis performed for a subset of cDNAs indicated the existence of a cognate transcript. Comparison to databases indicated three segments matching to known chromosome 21 genes: PFKL, COL6A1 and S100B and six segments matching to unmapped anonymous expressed sequence tags (ESTs). At the translated nucleotide level, strong homologies to known proteins were found with ATP-binding transporters of the ABC family and the dihydroorotase domain of pyrimidine synthetases. These data strongly suggest that bona fide partial genes have been isolated. Several of the newly isolated transcriptional units map to clinically important regions, in particular those involved in Down's syndrome, progressive myoclonus epilepsia and auto-immune polyglandular disease. The study presented here illustrates the complementarity of exon-amplification and c

  4. SMA carrier testing - validation of hemizygous SMN exon 7 deletion test for the identification of proximal spinal muscular atrophy carriers and patients with a single allele deletion

    NARCIS (Netherlands)

    Scheffer, H; Cobben, JM; Mensink, RGJ; Stulp, RP; van der Steege, G; Buys, CHCM

    To facilitate the detection of carriers of a hemizygous survival motor neuron (SMN) exon 7 deletion we have modified the quantitative SMN exon 7 assay described by McAndrew ct al (1997). The major changes include quantitative analysis of the amount of SMN exon 7-specific fluorescently-labelled PCR

  5. ExDom: an integrated database for comparative analysis of the exon-intron structures of protein domains in eukaryotes.

    Science.gov (United States)

    Bhasi, Ashwini; Philip, Philge; Manikandan, Vinu; Senapathy, Periannan

    2009-01-01

    We have developed ExDom, a unique database for the comparative analysis of the exon-intron structures of 96 680 protein domains from seven eukaryotic organisms (Homo sapiens, Mus musculus, Bos taurus, Rattus norvegicus, Danio rerio, Gallus gallus and Arabidopsis thaliana). ExDom provides integrated access to exon-domain data through a sophisticated web interface which has the following analytical capabilities: (i) intergenomic and intragenomic comparative analysis of exon-intron structure of domains; (ii) color-coded graphical display of the domain architecture of proteins correlated with their corresponding exon-intron structures; (iii) graphical analysis of multiple sequence alignments of amino acid and coding nucleotide sequences of homologous protein domains from seven organisms; (iv) comparative graphical display of exon distributions within the tertiary structures of protein domains; and (v) visualization of exon-intron structures of alternative transcripts of a gene correlated to variations in the domain architecture of corresponding protein isoforms. These novel analytical features are highly suited for detailed investigations on the exon-intron structure of domains and make ExDom a powerful tool for exploring several key questions concerning the function, origin and evolution of genes and proteins. ExDom database is freely accessible at: http://66.170.16.154/ExDom/.

  6. Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome.

    Directory of Open Access Journals (Sweden)

    Norishige Yamada

    2015-08-01

    Full Text Available To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

  7. A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation

    Science.gov (United States)

    Ke, Shengdong; Alemu, Endalkachew A.; Mertens, Claudia; Gantman, Emily Conn; Fak, John J.; Mele, Aldo; Haripal, Bhagwattie; Zucker-Scharff, Ilana; Moore, Michael J.; Park, Christopher Y.; Vågbø, Cathrine Broberg; Kusśnierczyk, Anna; Klungland, Arne; Darnell, James E.; Darnell, Robert B.

    2015-01-01

    We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m6A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3′-most (last) exons, with a very sharp rise (sixfold) within 150–400 nucleotides of the start of the last exon. Two-thirds of last exon m6A and >40% of all m6A in mRNA are present in 3′ untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m6A sites around stop codons. Moreover, m6A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m6A density peaks early in the 3′ UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m6A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m6A modification in regulating proximal alternative polyA choice. PMID:26404942

  8. Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy.

    Science.gov (United States)

    Taniguchi-Ikeda, Mariko; Kobayashi, Kazuhiro; Kanagawa, Motoi; Yu, Chih-chieh; Mori, Kouhei; Oda, Tetsuya; Kuga, Atsushi; Kurahashi, Hiroki; Akman, Hasan O; DiMauro, Salvatore; Kaji, Ryuji; Yokota, Toshifumi; Takeda, Shin'ichi; Toda, Tatsushi

    2011-10-05

    Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene. In FCMD, the SVA insertion occurs in the 3' untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant messenger RNA (mRNA) splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in patients with FCMD. This region spans part of the 3' end of the fukutin coding region, a proximal part of the 3' UTR and the SVA insertion. Correspondingly, fukutin mRNA transcripts in patients with FCMD and SVA knock-in model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin carboxy (C) terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in cells of patients with FCMD and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of α-dystroglycan (α-DG) and laminin binding by α-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia, neutral lipid storage disease) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical

  9. Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants.

    Science.gov (United States)

    Pagenstecher, Constanze; Wehner, Maria; Friedl, Waltraut; Rahner, Nils; Aretz, Stefan; Friedrichs, Nicolaus; Sengteller, Marlies; Henn, Wolfram; Buettner, Reinhard; Propping, Peter; Mangold, Elisabeth

    2006-03-01

    Single base substitutions in DNA mismatch repair genes which are predicted to lead either to missense or silent mutations, or to intronic variants outside the highly conserved splicing region are often found in hereditary nonpolyposis colorectal cancer (HNPCC) families. In order to use the variants for predictive testing in persons at risk, their pathogenicity has to be evaluated. There is growing evidence that some substitutions have a detrimental influence on splicing. We examined 19 unclassified variants (UVs) detected in MSH2 or MLH1 genes in patients suspected of HNPCC for expression at RNA level. We demonstrate that 10 of the 19 UVs analyzed affect splicing. For example, the substitution MLH1,c.2103G > C in the last position of exon 18 does not result in a missense mutation as theoretically predicted (p.Gln701His), but leads to a complete loss of exon 18. The substitution MLH1,c.1038G > C (predicted effect p.Gln346His) leads to complete inactivation of the mutant allele by skipping of exons 10 and 11, and by activation of a cryptic intronic splice site. Similarly, the intronic variant MLH1,c.306+2dupT results in loss of exon 3 and a frameshift mutation due to a new splice donor site 5 bp upstream. Furthermore, we confirmed complete exon skipping for the mutations MLH1,c.1731G > A and MLH1,c.677G > A. Partial exon skipping was demonstrated for the mutations MSH2,c.1275A > G, MLH1,c.588+5G > A, MLH1,c.790+4A > G and MLH1,c.1984A > C. In contrast, five missense mutations (MSH2,c.4G > A, MSH2,c.2123T > A, MLH1,c.464T > G, MLH1,c.875T > C and MLH1,c.2210A > T) were found in similar proportions in the mRNA as in the genomic DNA. We conclude that the mRNA examination should precede functional tests at protein level.

  10. Transcriptome instability in colorectal cancer identified by exon microarray analyses: Associations with splicing factor expression levels and patient survival.

    Science.gov (United States)

    Sveen, Anita; Agesen, Trude H; Nesbakken, Arild; Rognum, Torleiv O; Lothe, Ragnhild A; Skotheim, Rolf I

    2011-05-27

    Colorectal cancer (CRC) is a heterogeneous disease that, on the molecular level, can be characterized by inherent genomic instabilities; chromosome instability and microsatellite instability. In the present study we analyze genome-wide disruption of pre-mRNA splicing, and propose transcriptome instability as a characteristic that is analogous to genomic instability on the transcriptome level. Exon microarray profiles from two independent series including a total of 160 CRCs were investigated for their relative amounts of exon usage differences. Each exon in each sample was assigned an alternative splicing score calculated by the FIRMA algorithm. Amounts of deviating exon usage per sample were derived from exons with extreme splicing scores. There was great heterogeneity within both series in terms of sample-wise amounts of deviating exon usage. This was strongly associated with the expression levels of approximately half of 280 splicing factors (54% and 48% of splicing factors were significantly correlated to deviating exon usage amounts in the two series). Samples with high or low amounts of deviating exon usage, associated with overall transcriptome instability, were almost completely separated into their respective groups by hierarchical clustering analysis of splicing factor expression levels in both sample series. Samples showing a preferential tendency towards deviating exon skipping or inclusion were associated with skewed transcriptome instability. There were significant associations between transcriptome instability and reduced patient survival in both sample series. In the test series, patients with skewed transcriptome instability showed the strongest prognostic association (P = 0.001), while a combination of the two characteristics showed the strongest association with poor survival in the validation series (P = 0.03). We have described transcriptome instability as a characteristic of CRC. This transcriptome instability has associations with splicing

  11. Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing.

    Directory of Open Access Journals (Sweden)

    Elisabete Hernández-Imaz

    Full Text Available Neurofibromatosis type 1 (NF1 is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene.

  12. Exon organization of the mouse entactin gene corresponds to the structural domains of the polypeptide and has regional homology to the low-density lipoprotein receptor gene

    DEFF Research Database (Denmark)

    Durkin, M E; Wewer, U M; Chung, A E

    1995-01-01

    Entactin is a widespread basement membrane protein of 150 kDa that binds to type IV collagen and laminin. The complete exon-intron structure of the mouse entactin gene has been determined from lambda genomic DNA clones. The gene spans at least 65 kb and contains 20 exons. The exon organization...... of the mouse entactin gene closely corresponds to the organization of the polypeptide into distinct structural and functional domains. The two amino-terminal globular domains are encoded by three exons each. Single exons encode the two protease-sensitive, O-glycosylated linking regions. The six EGF...

  13. A 5' Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo.

    Science.gov (United States)

    Lu, Jiamiao; Williams, James A; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A

    2017-01-01

    We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5' UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo.

  14. Screening of BRCA1 sequence variants within exon 11 by heteroduplex analysis

    Directory of Open Access Journals (Sweden)

    Lucian Negura

    2013-03-01

    Full Text Available Germ-line mutations of either BRCA1 or BRCA2 represents the major hereditary risk to breast and ovariancancer. Screening for mutations in these genes is now standard practice in molecular diagnosis, opening the way tooncogenetic counselling and follow-up. Because mutations in both BRCA1 and BRCA2 are distributed throughout theloci, accepted clinical protocols involve screening their entire coding regions. Systematic Sanger sequencing is time andmoney consuming. Therefore, a lot of pre-screening techniques evolved over time in order to identify anomalousamplicons prior to sequencing. Because BRCA mutations are always heterozygous, heteroduplex analysis proved to be asuitable pre-screening step. We previously implemented mismatch specific endonuclease heteroduplex analysis forBRCA1 exon7. Here we show the utility of the same method for mutations and SNPs found in BRCA1 exon 11

  15. Exonization of active mouse L1s: a driver of transcriptome evolution?

    Directory of Open Access Journals (Sweden)

    Badge Richard

    2007-10-01

    Full Text Available Abstract Background Long interspersed nuclear elements (LINE-1s, L1s have been recently implicated in the regulation of mammalian transcriptomes. Results Here, we show that members of the three active mouse L1 subfamilies (A, GF and TF contain, in addition to those on their sense strands, conserved functional splice sites on their antisense strands, which trigger multiple exonization events. The latter is particularly intriguing in the light of the strong antisense orientation bias of intronic L1s, implying that the toleration of antisense insertions results in an increased potential for exonization. Conclusion In a genome-wide analysis, we have uncovered evidence suggesting that the mobility of the large number of retrotransposition-competent mouse L1s (~2400 potentially active L1s in NCBIm35 has significant potential to shape the mouse transcriptome by continuously generating insertions into transcriptional units.

  16. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity

    OpenAIRE

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-01-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and...

  17. Towards a transcription map of human chromosome 21: Identification of expressed sequences by exon trapping

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.M.; Chrast, R.; Rossier, C. [Geneva Univ. Medical School (Switzerland)] [and others

    1994-09-01

    Chromosome 21q contains about 1% of the human genome, and when triplicated is responsible for Down syndrome. The genetic and physical maps of this chromosome are amongst the most developed of all human chromosomes. A considerable international effort is now under way with the aims of cloning and mapping all chromosome 21 genes, assigning functions, and determining their involvement in disease phenotypes. We have used exon trapping/amplification methods to identify exons of genes that map on chromosome 21. EcoR1 or Bam HI-digested DNA from pools of 96 cosmids from the chromosome 21 library LL21NC02{open_quotes}Q{close_quotes} were used for cloning in vector pSLP3 (after elimination of cosmids positive for ribosomal RNR genes and mouse DNA); recombinant plasmids were transfected into cos7 cells and trapped sequences were subcloned. False positive clones, i.e. those containing vector self-spliced sequences (which represented between 8-30% of clones in different experiments), have been eliminated by hybridization of oligonucleotides corresponding to sequences of the vector self-spliced events. More than 100 different trapped {open_quotes}exons{close_quotes} have been identified to date after single or double pass sequencing. Two sequences matched exons of known genes on chromosome 21 (COL6A 1 and MX1). About 45% of the sequences were entirely new, i.e. there was no homology with entries in the nucleotide or protein databases (blastin and blastx searches). An additional 48% of the sequences were homologous but not identical to sequences in the databases. Only 4% were repetitive elements. Specific homologies will be presented. All of the trapped sequences that have been mapped by filter hybridization, PCR, or FISH, map back to cosmids or YACs of chromosome 21. This approach permits rapid identification of expressed sequences of this chromosome, the cloning of its genes, and the understanding of its disorders.

  18. Leishmania donovani complex: genotyping with the ribosomal internal transcribed spacer and the mini-exon

    OpenAIRE

    Mauricio, IL; Stothard, JR; Miles, MA

    2004-01-01

    Intergenic region typing by restriction analysis of the ribosomal internal transcribed spacer (ITS) and mini-exon provide diagnostic markers for some Leishmania. Here, we evaluate restriction analysis of these targets for genotyping and phylogenetic analysis within the Leishmania donovani complex (agents of visceral leishmaniasis). Each method was useful for genotyping of both L. donovani complex strains and Old World Leishmania species. The targets produced less robust groups than gp63 inter...

  19. GMDH-GA Hybrid Model Extracting Exon Region from DNA Sequences

    OpenAIRE

    Ohta, Kouji; Yoshihara, Ikuo; Yamamori, Kunihito; Yasunaga, Moritoshi

    2004-01-01

    Abstract ###A model building method based on Group Method of Data Handling (GMDH) optimized by ###GA is developed for extracting exon regions. GMDH, that is originally a method to construct ###higher order polynomial model, is extended to constructing higher order logical model. ###The model built by proposed method is compared with Genetic Programming (GP)-based ###model as to the extraction rate of best, worst and average. The proposed method is superior to GP ###as to extraction rate of al...

  20. Selective Blockade of Periostin Exon 17 Preserves Cardiac Performance in Acute Myocardial Infarction.

    Science.gov (United States)

    Taniyama, Yoshiaki; Katsuragi, Naruto; Sanada, Fumihiro; Azuma, Junya; Iekushi, Kazuma; Koibuchi, Nobutaka; Okayama, Keita; Ikeda-Iwabu, Yuka; Muratsu, Jun; Otsu, Rei; Rakugi, Hiromi; Morishita, Ryuichi

    2016-02-01

    We previously reported that overexpression of full-length periostin, Pn-1, resulted in ventricular dilation with enhanced interstitial collagen deposition in a rat model. However, other reports have documented that the short-form splice variants Pn-2 (lacking exon 17) and Pn-4 (lacking exons 17 and 21) promoted cardiac repair by angiogenesis and prevented cardiac rupture after acute myocardial infarction. The apparently differing findings from those reports prompted us to use a neutralizing antibody to selectively inhibit Pn-1 by blockade of exon 17 in a rat acute myocardial infarction model. Administration of Pn neutralizing antibody resulted in a significant decrease in the infarcted and fibrotic areas of the myocardium, which prevented ventricular wall thinning and dilatation. The inhibition of fibrosis by Pn neutralizing antibody was associated with a significant decrease in gene expression of fibrotic markers, including collagen I, collagen III, and transforming growth factor-β1. Importantly, the number of α-smooth muscle actin-positive myofibroblasts was significantly reduced in the hearts of animals treated with Pn neutralizing antibody, whereas cardiomyocyte proliferation and angiogenesis were comparable in the IgG and neutralizing antibody groups. Moreover, the level of Pn-1 expression was significantly correlated with the severity of myocardial infarction. In addition, Pn-1, but not Pn-2 or Pn-4, inhibited fibroblast and myocyte attachment, which might account for the cell slippage observed during cardiac remodeling. Collectively, these results indicate that therapeutics that specifically inhibit Pn exon-17, via a neutralizing antibody or drug, without suppressing other periostin variants might offer a new class of medication for the treatment of acute myocardial infarction patients. © 2015 American Heart Association, Inc.

  1. Exon deletions and intragenic insertions are not rare in ataxia with oculomotor apraxia 2

    Directory of Open Access Journals (Sweden)

    Kreuz Friedmar

    2009-09-01

    Full Text Available Abstract Background The autosomal recessively inherited ataxia with oculomotor apraxia 2 (AOA2 is a neurodegenerative disorder characterized by juvenile or adolescent age of onset, gait ataxia, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia, and elevated serum AFP levels. AOA2 is caused by mutations within the senataxin gene (SETX. The majority of known mutations are nonsense, missense, and splice site mutations, as well as small deletions and insertions. Methods To detect mutations in patients showing a clinical phenotype consistent with AOA2, the coding region including splice sites of the SETX gene was sequenced and dosage analyses for all exons were performed on genomic DNA. The sequence of cDNA fragments of alternative transcripts isolated after RT-PCR was determined. Results Sequence analyses of the SETX gene in four patients revealed a heterozygous nonsense mutation or a 4 bp deletion in three cases. In another patient, PCR amplification of exon 11 to 15 dropped out. Dosage analyses and breakpoint localisation yielded a 1.3 kb LINE1 insertion in exon 12 (patient P1 and a 6.1 kb deletion between intron 11 and intron 14 (patient P2 in addition to the heterozygous nonsense mutation R1606X. Patient P3 was compound heterozygous for a 4 bp deletion in exon 10 and a 20.7 kb deletion between intron 10 and 15. This deletion was present in a homozygous state in patient P4. Conclusion Our findings indicate that gross mutations seem to be a frequent cause of AOA2 and reveal the importance of additional copy number analysis for routine diagnostics.

  2. An erythroid differentiation-specific splicing switch in protein 4.1R mediated by the interaction of SF2/ASF with an exonic splicing enhancer.

    Science.gov (United States)

    Yang, Guang; Huang, Shu-Ching; Wu, Jane Y; Benz, Edward J

    2005-03-01

    Protein 4.1R is a vital component of the red blood cell membrane cytoskeleton. Promotion of cytoskeletal junctional complex stability requires an erythroid differentiation stage-specific splicing switch promoting inclusion of exon 16 within the spectrin/actin binding domain. We showed earlier that an intricate combination of positive and negative RNA elements controls exon 16 splicing. In this report, we further identified 3 putative exonic splicing enhancers within exon 16 and investigated the function of the sequence CAGACAT in the regulation of exon 16 splicing. Mutation of these sequences leads to increased exclusion of exon 16 in both in vivo and in vitro splicing assays, indicating that CAGACAT is a functional exonic splicing enhancer. UV cross-linking further detects an approximately 33-kDa protein that specifically binds to the CAGACAT-containing transcript. An anti-SF2/ASF antibody specifically immunoprecipitates the approximately 33-kDa protein. Furthermore, SF2/ASF stimulates exon 16 inclusion in both in vitro complementation assays and minigene-transfected mouse erythroleukemia cells (MELCs). Finally, SF2/ASF expression is up-regulated and correlates with exon 16 inclusion in differentiated MELCs. These results suggest that increased splicing factor 2/alternative splicing factor (SF2/ASF) expression in differentiated mouse erythroleukemia mediates a differentiation stage-specific exon 16 splicing switch through its interaction with the exonic splicing enhancer.

  3. 3′ Splice Site Sequences of Spinal Muscular Atrophy Related SMN2 Pre-mRNA Include Enhancers for Nearby Exons

    Directory of Open Access Journals (Sweden)

    Sunghee Cho

    2014-01-01

    Full Text Available Spinal muscular atrophy (SMA is a human genetic disease which occurs because of the deletion or mutation of SMN1 gene. SMN1 gene encodes the SMN protein which plays a key role in spliceosome assembly. Although human patients contain SMN2, a duplicate of SMN1, splicing of SMN2 produces predominantly exon 7 skipped isoform. In order to understand the functions of splice site sequences on exon 7 and 8, we analyzed the effects of conserved splice site sequences on exon 7 skipping of SMN2 and SMN1 pre-mRNA. We show here that conserved 5′ splice site sequence of exon 7 promoted splicing of nearby exons and subsequently reduced splicing of distant exons. However, to our surprise, conserved 3′ splice site sequence of exon 7 and 8 did not promote splicing of nearby exons. By contrast, the mutation inhibited splicing of nearby exons and subsequently promoted splicing of distant exons. Our study shows that 3′ splice sites of exon 7 and 8 contain enhancer for their splice site selection, in addition to providing cleavage sites.

  4. Dynamic ASXL1 Exon Skipping and Alternative Circular Splicing in Single Human Cells.

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    Winston Koh

    Full Text Available Circular RNAs comprise a poorly understood new class of noncoding RNA. In this study, we used a combination of targeted deletion, high-resolution splicing detection, and single-cell sequencing to deeply probe ASXL1 circular splicing. We found that efficient circular splicing required the canonical transcriptional start site and inverted AluSx elements. Sequencing-based interrogation of isoforms after ASXL1 overexpression identified promiscuous linear splicing between all exons, with the two most abundant non-canonical linear products skipping the exons that produced the circular isoforms. Single-cell sequencing revealed a strong preference for either the linear or circular ASXL1 isoforms in each cell, and found the predominant exon skipping product is frequently co-expressed with its reciprocal circular isoform. Finally, absolute quantification of ASXL1 isoforms confirmed our findings and suggests that standard methods overestimate circRNA abundance. Taken together, these data reveal a dynamic new view of circRNA genesis, providing additional framework for studying their roles in cellular biology.

  5. Whole exon 5 and intron 5 replaced by RHCE in DVa(Hus).

    Science.gov (United States)

    Shao, Chaopeng; Xiong, Wen; Wang, Wei

    2004-01-01

    The DVa(Hus) was previously investigated through cDNA analysis, which revealed an RHD-CE(5)-D hybrid allele. However, the 5' and 3' breakpoints remain unknown. In this article, gene recombinations between the RHD and RHCE alleles were investigated by a combination approach of a sequence-specific primer PCR (PCR-SSP) and an RHD full-length coding region sequencing method on two Chinese subjects with weak D phenotypes. The hybrid Rhesus box of each individual was also investigated through an established PCR-based method. As a result, two partial D phenotypes, DVa(Hus) and DVI type III, were identified, each carrying one hybrid RHD-CE-D allele. The two samples were also serotyped with Rh phontypes of DccEe and DCcee, respectively. Other sequencing analyses of the DVaHus sample showed that the sequence of intron 4 is identical with RHD, whereas the whole sequence of exon 5 and intron 5 is identical with RHCE except for seven polymorphisms in the intron 5. We may concluded that in the case of this Chinese DVa(Hus), the whole exon 5 and complete intron 5 of a total segment of 1801 nucleotides were replaced by RHCE suggesting that the breakpoints of the replaced region are the 5' end of the exon 5 and the 3' end of the intron 5.

  6. Performance of genotype imputation for rare variants identified in exons and flanking regions of genes.

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    Li Li

    Full Text Available Genotype imputation has the potential to assess human genetic variation at a lower cost than assaying the variants using laboratory techniques. The performance of imputation for rare variants has not been comprehensively studied. We utilized 8865 human samples with high depth resequencing data for the exons and flanking regions of 202 genes and Genome-Wide Association Study (GWAS data to characterize the performance of genotype imputation for rare variants. We evaluated reference sets ranging from 100 to 3713 subjects for imputing into samples typed for the Affymetrix (500K and 6.0 and Illumina 550K GWAS panels. The proportion of variants that could be well imputed (true r(2>0.7 with a reference panel of 3713 individuals was: 31% (Illumina 550K or 25% (Affymetrix 500K with MAF (Minor Allele Frequency less than or equal 0.001, 48% or 35% with 0.0010.05. The performance for common SNPs (MAF>0.05 within exons and flanking regions is comparable to imputation of more uniformly distributed SNPs. The performance for rare SNPs (0.01exon resequencing into additional samples with GWAS data, but imputation of very rare variants (MAF< = 0.005 will require reference panels with thousands of subjects.

  7. Two-exon skipping within MLPH is associated with coat color dilution in rabbits.

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    Stefanie Lehner

    Full Text Available Coat color dilution turns black coat color to blue and red color to cream and is a characteristic in many mammalian species. Matings among Netherland Dwarf, Loh, and Lionhead Dwarf rabbits over two generations gave evidence for a monogenic autosomal recessive inheritance of coat colour dilution. Histological analyses showed non-uniformly distributed, large, agglomerating melanin granules in the hair bulbs of coat color diluted rabbits. We sequenced the cDNA of MLPH in two dilute and one black rabbit for polymorphism detection. In both color diluted rabbits, skipping of exons 3 and 4 was present resulting in altered amino acids at p.QGL[37-39]QWA and a premature stop codon at p.K40*. Sequencing of genomic DNA revealed a c.111-5C>A splice acceptor mutation within the polypyrimidine tract of intron 2 within MLPH. This mutation presumably causes skipping of exons 3 and 4. In 14/15 dilute rabbits, the c.111-5C>A mutation was homozygous and in a further dilute rabbit, heterozygous and in combination with a homozygous frame shift mutation within exon 6 (c.585delG. In conclusion, our results demonstrated a colour dilution associated MLPH splice variant causing a strongly truncated protein (p.Q37QfsX4. An involvement of further MLPH-associated mutations needs further investigations.

  8. SVA retrotransposition in exon 6 of the coagulation factor IX gene causing severe hemophilia B.

    Science.gov (United States)

    Nakamura, Yuki; Murata, Moe; Takagi, Yuki; Kozuka, Toshihiro; Nakata, Yukiko; Hasebe, Ryo; Takagi, Akira; Kitazawa, Jun-ichi; Shima, Midori; Kojima, Tetsuhito

    2015-07-01

    Hemophilia B is an X-linked recessive bleeding disorder caused by abnormalities of the coagulation factor IX gene (F9). Insertion mutations in F9 ranging from a few to more than 100 base pairs account for only a few percent of all hemophilia B cases. We investigated F9 to elucidate genetic abnormalities causing severe hemophilia B in a Japanese subject. We performed PCR-mediated analysis of F9 and identified a large insertion in exon 6. Next, we carried out direct sequencing of a PCR clone of the whole insert using nested deletion by exonuclease III and S1 nuclease. We identified an approximately 2.5-kb SINE-VNTR-Alu (SVA)-F element flanked by 15-bp duplications in the antisense orientation in exon 6. Additionally, we carried out exontrap analysis to assess the effect of this retrotransposition on mRNA splicing. We observed that regular splicing at exons 5 and 6 of F9 was disturbed by the SVA retrotransposition, suggesting that abnormal FIX mRNA may be reduced by nonsense-mediated mRNA decay. In conclusion, this is the first report of SVA retrotransposition causing severe hemophilia B; only five cases of LINE-1 or Alu retrotranspositions in F9 have been reported previously.

  9. Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF.

    Science.gov (United States)

    Chen, Zhiqi; Ma, Xuezhong; Zhang, Jianhua; Hu, Jim; Gorczynski, Reginald M

    2010-10-01

    CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.

  10. SH3BP2 is rarely mutated in exon 9 in giant cell lesions outside cherubism.

    Science.gov (United States)

    Lietman, Steven A; Prescott, Nichole L; Hicks, David G; Westra, William H; Levine, Michael A

    2007-06-01

    Giant cell tumor of bone and giant cell reparative granuloma are benign lesions with prominent giant (multinucleated) cells, and an understanding of the molecular biology and genetics of these lesions will likely aid in more effective treatment. Cherubism is a benign lesion of the maxilla and mandible histologically similar to giant cell tumor of bone and giant cell reparative granuloma. Germline mutations in exon 9 of the gene encoding Src homology 3 binding protein 2 (SH3BP2) occur in most patients with cherubism. We therefore hypothesized SH3BP2 and its putative downstream effector nuclear factor of activated T cells c1 isoform (NFATc1) are highly expressed in sporadic nonsyndromic giant cell lesions and associated with somatic SH3BP2 mutations. We analyzed giant cell lesions for SH3BP2 and NFATc1 expression by RNA blot and/or immunohistochemistry and for exon 9 SH3BP2 mutations. We found the SH3BP2 transcripts and protein were abundantly expressed in giant cell tumors of bone, as well as NFATc1 protein. Sequencing of exon 9 of SH3BP2 was normal in all sporadic nonsyndromic giant cell lesions. Although many multinucleated giant cell lesions of bone share histologic features, the primary genetic defect in cherubism and these other giant cell lesions appears different.

  11. mRNA-Associated Processes and Their Influence on Exon-Intron Structure in Drosophila melanogaster

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    Gildas Lepennetier

    2016-06-01

    Full Text Available mRNA-associated processes and gene structure in eukaryotes are typically treated as separate research subjects. Here, we bridge this separation and leverage the extensive multidisciplinary work on Drosophila melanogaster to examine the roles that capping, splicing, cleavage/polyadenylation, and telescripting (i.e., the protection of nascent transcripts from premature cleavage/polyadenylation by the splicing factor U1 might play in shaping exon-intron architecture in protein-coding genes. Our findings suggest that the distance between subsequent internal 5′ splice sites (5′ss in Drosophila genes is constrained such that telescripting effects are maximized, in theory, and thus nascent transcripts are less vulnerable to premature termination. Exceptionally weak 5′ss and constraints on intron-exon size at the gene 5′ end also indicate that capping might enhance the recruitment of U1 and, in turn, promote telescripting at this location. Finally, a positive correlation between last exon length and last 5′ss strength suggests that optimal donor splice sites in the proximity of the pre-mRNA tail may inhibit the processing of downstream polyadenylation signals more than weak donor splice sites do. These findings corroborate and build upon previous experimental and computational studies on Drosophila genes. They support the possibility, hitherto scantly explored, that mRNA-associated processes impose significant constraints on the evolution of eukaryotic gene structure.

  12. Rat tenascin-R gene: structure, chromosome location and transcriptional activity of promoter and exon 1.

    Science.gov (United States)

    Leprini, A; Gherzi, R; Vecchi, E; Borsi, L; Zardi, L; Siri, A

    1998-01-01

    Tenascin-R is an extracellular matrix protein expressed exclusively in the central nervous system where it is thought to play a relevant role in regulating neurite outgrowth. We have i) cloned the cDNA of the rat tenascin-R 5' region; ii) defined its genomic organization, obtaining the sequence of two novel untranslated exons; iii) mapped the gene to rat chromosome 13q23 and suggested a previously unreported synteny between rat chromosome 13q23, human chromosome 1q24, and mouse chromosome 4E; and iv) sequenced and characterized the elements responsible for its neural cell-restricted transcription. We found that two discrete regions of the rat gene (the first in the proximal promoter, the second in the first exon) are independently able to activate to a high degree the transcription of a reporter gene in either human or rat neuroblastoma cell lines but not in other cell lines. Based on this observation, we re-evaluated the arrangement of transcriptionally active regions in the human tenascin-R gene we recently cloned and found that the human gene also contains an exon sequence able to initiate and sustain transcription independently of promoter sequences.

  13. Relationship between polymorphisms in exon 10 of FSHR gene and litter size in swine.

    Science.gov (United States)

    Zhang, X D; Zhu, H Y; Zhou, J; Wang, N; Zhou, N; Huang, L; Wu, T; Feng, Y F; Ding, Y Y; Yin, Z J

    2015-07-27

    Follicle-stimulating hormone (FSH), a glycoprotein secreted by the anterior pituitary, can regulate ovarian function through the FSH receptor (FSHR). To evaluate the effects of the FSHR gene on reproductive traits in pigs, polymorphisms in exon 10 of the FSHR gene were observed by polymerase chain reaction-single-strand conformation polymorphism, and 3 single nucleotide polymorphisms (C1491T, G1885A, and C1977T) in exon 10 of the porcine FSHR gene, and 3 genotypes (AA, AB, and BB) for C1491T and 2 haplotypes (D and E) for G1885A and C1977T were identified. Further analysis of single nucleotide polymorphism genotypes associated with reproductive traits including total number born (TNB) and number born alive (NBA) was carried out in 3 pig populations including Berkshire, Wannan Black (a Chinese indigenous pig breed), and BW pigs (two-way crossbred pigs produced from Berkshire ♂ and Wannan Black pig ♀). The results showed that the TNB and NBA of Wannan Black pigs with the AB genotype were significantly higher than in AA genotype sows (P pigs with the DE genotype were significantly higher than the DD and EE genotype sows (P DD > EE. The results showed that polymorphisms in exon 10 of the FSHR gene had a significant effect on litter size traits of Wannan Black and Berkshire pigs. These results can be applied for marker-assisted selection in the 2 swine breeds.

  14. MET exon 14 juxtamembrane splicing mutations: clinical and therapeutical perspectives for cancer therapy

    Science.gov (United States)

    Pilotto, Sara; Gkountakos, Anastasios; Carbognin, Luisa; Scarpa, Aldo; Tortora, Giampaolo

    2017-01-01

    The MET proto-oncogene plays crucial roles in cell growth and proliferation, survival and apoptosis, epithelial-mesenchymal transition (EMT) and invasion, potentially conditioning the development and progression of the carcinogenesis process. The MET-associated aberrant signaling could be triggered by a variety of mechanisms, such as mutations, gene amplification, increased gene copy number and Met/HGF protein expression. Among the various MET alterations, MET exon 14 splicing abnormalities, causing the loss of the Met juxtamembrane (JM) domain, recently emerged as a new potential oncogenic driver and have been identified and validated across different cancer and histology subtypes. Moreover, this aberration was found to be mutually exclusive with other recognized drivers, thus strongly nominating its potential oncogenic role. Recently, the clinical activity of anti-Met-targeted therapy was demonstrated particularly in patients harboring MET exon 14 skipping lung cancer, resulting in a renewed enthusiasm to further test MET precision therapy in prospective trials. In this review, the key preclinical and clinical data regarding MET exon 14 skipping splicing variants as an actionable genomic aberration in cancer are described, and the perspectives deriving from the validation of such alteration as a potential target, which may further allow driving the therapeutic approach in this molecularly selected patients’ subgroup, are explored. PMID:28164087

  15. Deletion of exons 1-5 of the STS gene causing X-linked ichthyosis.

    Science.gov (United States)

    Valdes-Flores, M; Kofman-Alfaro, S H; Vaca, A L; Cuevas-Covarrubias, S A

    2001-03-01

    X-linked ichthyosis is an inherited disorder due to steroid sulfatase deficiency. It is clinically characterized by dark, adhesive, and regular scales of the skin. Most X-linked ichthyosis patients present large deletions of the STS gene and flanking markers; a minority show a point mutation or partial deletion of the STS gene. In this study we analyzed the STS gene in a family with simultaneous occurrence of X-linked ichthyosis and ichthyosis vulgaris. X-linked ichthyosis diagnosis was confirmed through steroid sulfatase assay in leukocytes using 7-[3H]-dehydroepiandrosterone sulfate as a substrate. Exons 1, 2, 5, and 6-10, and the 5' flanking markers DXS1130, DXS1139, and DXS996 of the STS gene were analyzed by polymerase chain reaction. X-linked ichthyosis patients of the family (n = 4 males) had undetectable levels of STS activity (0.00 pmol per mg protein per h). The DNA analysis showed that only exons 6-10 and the 5' flanking markers of the STS gene were present. We report the first partial deletion of the STS gene spanning exons 1-5 in X-linked ichthyosis patients.

  16. Transcriptome-wide modulation of splicing by the exon junction complex.

    Science.gov (United States)

    Wang, Zhen; Murigneux, Valentine; Le Hir, Hervé

    2014-01-01

    The exon junction complex (EJC) is a dynamic multi-protein complex deposited onto nuclear spliced mRNAs upstream of exon-exon junctions. The four core proteins, eIF4A3, Magoh, Y14 and MLN51, are stably bound to mRNAs during their lifecycle, serving as a binding platform for other nuclear and cytoplasmic proteins. Recent evidence has shown that the EJC is involved in the splicing regulation of some specific events in both Drosophila and mammalian cells. Here, we show that knockdown of EJC core proteins causes widespread alternative splicing changes in mammalian cells. These splicing changes are specific to EJC core proteins, as knockdown of eIF4A3, Y14 and MLN51 shows similar splicing changes, and are different from knockdown of other splicing factors. The splicing changes can be rescued by a siRNA-resistant form of eIF4A3, indicating an involvement of EJC core proteins in regulating alternative splicing. Finally, we find that the splicing changes are linked with RNA polymerase II elongation rates. Taken together, this study reveals that the coupling between EJC proteins and splicing is broader than previously suspected, and that a possible link exists between mRNP assembly and splice site recognition.

  17. Representation of DNA sequences in genetic codon context with applications in exon and intron prediction.

    Science.gov (United States)

    Yin, Changchuan

    2015-04-01

    To apply digital signal processing (DSP) methods to analyze DNA sequences, the sequences first must be specially mapped into numerical sequences. Thus, effective numerical mappings of DNA sequences play key roles in the effectiveness of DSP-based methods such as exon prediction. Despite numerous mappings of symbolic DNA sequences to numerical series, the existing mapping methods do not include the genetic coding features of DNA sequences. We present a novel numerical representation of DNA sequences using genetic codon context (GCC) in which the numerical values are optimized by simulation annealing to maximize the 3-periodicity signal to noise ratio (SNR). The optimized GCC representation is then applied in exon and intron prediction by Short-Time Fourier Transform (STFT) approach. The results show the GCC method enhances the SNR values of exon sequences and thus increases the accuracy of predicting protein coding regions in genomes compared with the commonly used 4D binary representation. In addition, this study offers a novel way to reveal specific features of DNA sequences by optimizing numerical mappings of symbolic DNA sequences.

  18. Rare exonic deletions implicate the synaptic organizer Gephyrin (GPHN) in risk for autism, schizophrenia and seizures.

    Science.gov (United States)

    Lionel, Anath C; Vaags, Andrea K; Sato, Daisuke; Gazzellone, Matthew J; Mitchell, Elyse B; Chen, Hong Yang; Costain, Gregory; Walker, Susan; Egger, Gerald; Thiruvahindrapuram, Bhooma; Merico, Daniele; Prasad, Aparna; Anagnostou, Evdokia; Fombonne, Eric; Zwaigenbaum, Lonnie; Roberts, Wendy; Szatmari, Peter; Fernandez, Bridget A; Georgieva, Lyudmila; Brzustowicz, Linda M; Roetzer, Katharina; Kaschnitz, Wolfgang; Vincent, John B; Windpassinger, Christian; Marshall, Christian R; Trifiletti, Rosario R; Kirmani, Salman; Kirov, George; Petek, Erwin; Hodge, Jennelle C; Bassett, Anne S; Scherer, Stephen W

    2013-05-15

    The GPHN gene codes for gephyrin, a key scaffolding protein in the neuronal postsynaptic membrane, responsible for the clustering and localization of glycine and GABA receptors at inhibitory synapses. Gephyrin has well-established functional links with several synaptic proteins that have been implicated in genetic risk for neurodevelopmental disorders such as autism spectrum disorder (ASD), schizophrenia and epilepsy including the neuroligins (NLGN2, NLGN4), the neurexins (NRXN1, NRXN2, NRXN3) and collybistin (ARHGEF9). Moreover, temporal lobe epilepsy has been linked to abnormally spliced GPHN mRNA lacking exons encoding the G-domain of the gephyrin protein, potentially arising due to cellular stress associated with epileptogenesis such as temperature and alkalosis. Here, we present clinical and genomic characterization of six unrelated subjects, with a range of neurodevelopmental diagnoses including ASD, schizophrenia or seizures, who possess rare de novo or inherited hemizygous microdeletions overlapping exons of GPHN at chromosome 14q23.3. The region of common overlap across the deletions encompasses exons 3-5, corresponding to the G-domain of the gephyrin protein. These findings, together with previous reports of homozygous GPHN mutations in connection with autosomal recessive molybdenum cofactor deficiency, will aid in clinical genetic interpretation of the GPHN mutation spectrum. Our data also add to the accumulating evidence implicating neuronal synaptic gene products as key molecular factors underlying the etiologies of a diverse range of neurodevelopmental conditions.

  19. Un gene con intrones en vez de exones / Envejecimiento Prematuro de la Piel

    Directory of Open Access Journals (Sweden)

    Tobías Mojica

    1996-04-01

    Full Text Available Un gene con intrones en vez de exones. La noción de que los genes son discontinuos (compuestos de exones e intrones en forma alterna y en cuya organización los exones representan regiones presentes, por medio del código genético en las proteínas, y los intrones nadie sabe todavía que representan produjo una cierta cantidad de desasosiego entre los genetistas mayores de edad, pero hoy día es ampliamente aceptada, con poco o ningún dolor, y se ha convertido en parte del cánon científico. / Envejecimiento Prematuro de la Piel. La exposición a largo plazo de la piel a la luz ultravioleta proveniente del sol resulta en daño al colágeno de la piel y a la elastina de la matriz extracelular; se cree que este daño es responsable de la apariencia típicamente arrugadita de la piel expuesta al sol por mucho tiempo (como en los vaqueros de los comerciales de la televisión.

  20. Plug-and-Play Genetic Access to Drosophila Cell Types using Exchangeable Exon Cassettes

    Directory of Open Access Journals (Sweden)

    Fengqiu Diao

    2015-03-01

    Full Text Available Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here, we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes that leverages the untapped potential of “coding introns” (i.e., introns between coding exons. Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted “plug-and-play” cassettes (called “Trojan exons” that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC (Minos-mediated integration cassette transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system.

  1. Decreased Usage of Specific Scrib Exons Defines a More Malignant Phenotype of Breast Cancer With Worsened Survival

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    Gergana Metodieva

    2016-06-01

    Full Text Available SCRIB is a polarity regulator known to be abnormally expressed in cancer at the protein level. Here we report that, in breast cancer, an additional and hidden dimension of deregulations exists: an unexpected SCRIB exon usage pattern appears to mark a more malignant tumor phenotype and significantly correlates with survival. Conserved exons encoding the leucine-rich repeats tend to be overexpressed while others are underused. Mechanistic studies revealed that the underused exons encode part of the protein necessary for interaction with Vimentin and Numa1, a protein which is required for proper positioning of the mitotic spindle. Thus, the inclusion/exclusion of specific SCRIB exons is a mechanistic hallmark of breast cancer, which could potentially be exploited to develop more efficient diagnostics and therapies.

  2. Acetylcholinesterase (AChE) gene modification in transgenic animals: functional consequences of selected exon and regulatory region deletion.

    Science.gov (United States)

    Camp, Shelley; Zhang, Limin; Marquez, Michael; de la Torre, Brian; Long, Jeffery M; Bucht, Goran; Taylor, Palmer

    2005-12-15

    AChE is an alternatively spliced gene. Exons 2, 3 and 4 are invariantly spliced, and this sequence is responsible for catalytic function. The 3' alternatively spliced exons, 5 and 6, are responsible for AChE disposition in tissue [J. Massoulie, The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11 (3) (2002) 130-143; Y. Li, S. Camp, P. Taylor, Tissue-specific expression and alternative mRNA processing of the mammalian acetylcholinesterase gene. J. Biol. Chem. 268 (8) (1993) 5790-5797]. The splice to exon 5 produces the GPI anchored form of AChE found in the hematopoietic system, whereas the splice to exon 6 produces a sequence that binds to the structural subunits PRiMA and ColQ, producing AChE expression in brain and muscle. A third alternative RNA species is present that is not spliced at the 3' end; the intron 3' of exon 4 is used as coding sequence and produces the read-through, unanchored form of AChE. In order to further understand the role of alternative splicing in the expression of the AChE gene, we have used homologous recombination in stem cells to produce gene specific deletions in mice. Alternatively and together exon 5 and exon 6 were deleted. A cassette containing the neomycin gene flanked by loxP sites was used to replace the exon(s) of interest. Tissue analysis of mice with exon 5 deleted and the neomycin cassette retained showed very low levels of AChE expression, far less than would have been anticipated. Only the read-through species of the enzyme was produced; clearly the inclusion of the selection cassette disrupted splicing of exon 4 to exon 6. The selection cassette was then deleted in exon 5, exon 6 and exons 5 + 6 deleted mice by breeding to Ella-cre transgenic mice. AChE expression in serum, brain and muscle has been analyzed. Another AChE gene targeted mouse strain involving a region in the first intron, found to be critical for AChE expression in muscle cells [S. Camp, L. Zhang, M. Marquez, B

  3. Nucleobase-modified antisense oligonucleotides containing 5-(phenyltriazol)-2′-deoxyuridine nucleotides induce exon-skipping

    DEFF Research Database (Denmark)

    Le, Bao T.; Hornum, Mick; Sharma, Pawan K.

    2017-01-01

    Chemically-modified antisense oligonucleotide-mediated exon-skipping has been validated as a therapeutic strategy for tackling several disease pathologies, particularly duchenne muscular dystrophy. To date, only sugar-modified and internucleotide linkage-modified oligonucleotide chemistries have...

  4. Development of Exon Skipping Therapies for Duchenne Muscular Dystrophy: A Critical Review and a Perspective on the Outstanding Issues

    Science.gov (United States)

    Aartsma-Rus, Annemieke; Straub, Volker; Hemmings, Robert; Haas, Manuel; Schlosser-Weber, Gabriele; Stoyanova-Beninska, Violeta; Mercuri, Eugenio; Muntoni, Francesco; Sepodes, Bruno; Vroom, Elizabeth

    2017-01-01

    Duchenne muscular dystrophy (DMD) is a rare, severe, progressive muscle-wasting disease leading to disability and premature death. Patients lack the muscle membrane-stabilizing protein dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach that aims to induce production of partially functional dystrophins. Recently, an AON targeting exon 51 became the first of its class to be approved by the United States regulators [Food and Drug Administration (FDA)] for the treatment of DMD. A unique aspect of the exon-skipping approach for DMD is that, depending on the size and location of the mutation, different exons need to be skipped. This challenge raises a number of questions regarding the development and regulatory approval of those individual compounds. In this study, we present a perspective on those questions, following a European stakeholder meeting involving academics, regulators, and representatives from industry and patient organizations, and in the light of the most recent scientific and regulatory experience. PMID:28796573

  5. A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

    DEFF Research Database (Denmark)

    Kjær, L.; Riley, C.H.; Westman, M.

    2012-01-01

    Mutations in the Janus kinase 2 (JAK2) gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We...... present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel...... tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well....

  6. Triple-layer dissection of the lung adenocarcinoma transcriptome – regulation at the gene, transcript, and exon levels

    Science.gov (United States)

    Hsu, Min-Kung; Wu, I-Ching; Cheng, Ching-Chia; Su, Jen-Liang; Hsieh, Chang-Huain; Lin, Yeong-Shin; Chen, Feng-Chi

    2015-01-01

    Lung adenocarcinoma is one of the most deadly human diseases. However, the molecular mechanisms underlying this disease, particularly RNA splicing, have remained underexplored. Here, we report a triple-level (gene-, transcript-, and exon-level) analysis of lung adenocarcinoma transcriptomes from 77 paired tumor and normal tissues, as well as an analysis pipeline to overcome genetic variability for accurate differentiation between tumor and normal tissues. We report three major results. First, more than 5,000 differentially expressed transcripts/exonic regions occur repeatedly in lung adenocarcinoma patients. These transcripts/exonic regions are enriched in nicotine metabolism and ribosomal functions in addition to the pathways enriched for differentially expressed genes (cell cycle, extracellular matrix receptor interaction, and axon guidance). Second, classification models based on rationally selected transcripts or exonic regions can reach accuracies of 0.93 to 1.00 in differentiating tumor from normal tissues. Of the 28 selected exonic regions, 26 regions correspond to alternative exons located in such regulators as tumor suppressor (GDF10), signal receptor (LYVE1), vascular-specific regulator (RASIP1), ubiquitination mediator (RNF5), and transcriptional repressor (TRIM27). Third, classification systems based on 13 to 14 differentially expressed genes yield accuracies near 100%. Genes selected by both detection methods include C16orf59, DAP3, ETV4, GABARAPL1, PPAR, RADIL, RSPO1, SERTM1, SRPK1, ST6GALNAC6, and TNXB. Our findings imply a multilayered lung adenocarcinoma regulome in which transcript-/exon-level regulation may be dissociated from gene-level regulation. Our described method may be used to identify potentially important genes/transcripts/exonic regions for the tumorigenesis of lung adenocarcinoma and to construct accurate tumor vs. normal classification systems for this disease. PMID:26356813

  7. In silico screening based on predictive algorithms as a design tool for exon skipping oligonucleotides in Duchenne muscular dystrophy.

    Science.gov (United States)

    Echigoya, Yusuke; Mouly, Vincent; Garcia, Luis; Yokota, Toshifumi; Duddy, William

    2015-01-01

    The use of antisense 'splice-switching' oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD), for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1) the binding energetics of the oligonucleotide to the RNA, and (2) the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many) into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted) and/or 2'O Methyl RNA oligonucleotides (76% correctly predicted). Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R² 0.89) and 53 (R² 0.89), one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each position of

  8. De novo disruption of promoter and exon 1 of STAR gene reveals essential role for gonadal development.

    Science.gov (United States)

    Piya, Anil; Kaur, Jasmeet; Rice, Alan M; Bose, Himangshu S

    2017-01-01

    Cholesterol transport into the mitochondria is required for synthesis of the first steroid, pregnenolone. Cholesterol is transported by the steroidogenic acute regulatory protein (STAR), which acts at the outer mitochondrial membrane prior to its import. Mutations in the STAR protein result in lipoid congenital adrenal hyperplasia (CAH). Although the STAR protein consists of seven exons, biochemical analysis in nonsteroidogenic COS-1 cells showed that the first two were not essential for pregnenolone synthesis. Here, we present a patient with ambiguous genitalia, salt-lossing crisis within two weeks after birth and low cortisol levels. Sequence analysis of the STAR, including the exon-intron boundaries, showed the complete deletion of exon 1 as well as more than 50 nucleotides upstream of STAR promoter. Mitochondrial protein import with the translated protein through synthesis cassette of the mutant STAR lacking exon 1 showed protein translation, but it is less likely to have synthesized without a promoter in our patient. Thus, a full-length STAR gene is necessary for physiological mitochondrial cholesterol transport in vivo. STAR exon 1 deletion caused lipoid CAH.Exon 1 substitution does not affect biochemical activity.StAR promoter is responsible for gonadal development.

  9. The effect of 6-thioguanine on alternative splicing and antisense-mediated exon skipping treatment for duchenne muscular dystrophy.

    Science.gov (United States)

    Verhaart, Ingrid E C; Aartsma-Rus, Annemieke

    2012-12-12

    The severe muscle wasting disorder Duchenne muscular dystrophy (DMD) is caused by genetic defects in the DMD gene, leading to a complete absence of dystrophin protein. Of the therapeutic approaches addressing the underlying genetic defect, exon skipping through antisense oligonucleotides (AONs) is the closest to clinical application. Several strategies to improve the efficiency of this approach are currently being investigated, such as the use of small chemical compounds that improve AONmediated exon skipping levels. Recently, enhanced exon skipping in combination with a guanine analogue, 6-thioguanine (6TG) was reported for phosphorodiamidate morpholino oligomers (PMO). Here the effect of 6TG on the exon skipping efficacy of 2'-O-methyl phosphorothioate RNA (2OMePS) and PMO AONs in vitro and in vivo was further evaluated, as well as the effect of 6TG by itself. Results confirm an increase of exon skipping levels in vitro, however, in contrast to the previous report, no effect was observed in vivo. Importantly, 6TG treatment in vitro resulted in numerous additional DMD exon skipping events. This, in combination with the known cytotoxic effects of 6TG after incorporation in DNA, warrants reconsidering of the use of 6TG as enhancer of AON efficiency in DMD, were chronic treatment will be required.

  10. Mutations in exons 9 and 13 of KIT gene are rare events in gastrointestinal stromal tumors. A study of 200 cases.

    Science.gov (United States)

    Lasota, J; Wozniak, A; Sarlomo-Rikala, M; Rys, J; Kordek, R; Nassar, A; Sobin, L H; Miettinen, M

    2000-10-01

    Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the gastrointestinal tract, typically express the KIT protein. Activating mutations in the juxtamembrane domain (exon 11) of the c-kit gene have been shown in a subset of GISTs. These mutations lead into ligand-independent activation of the tyrosine kinase of c-kit, and have a transforming effect in vitro. Several groups have studied the clinical implication of the c-kit mutation status of exon 11 in GISTs and a possible relationship between c-kit mutations and malignant behavior has been established. Recently, a 1530ins6 mutation in exon 9 and missense mutations, 1945A>G in exon 13 of the c-kit gene were reported. The frequency and clinical importance of these findings are unknown. In this study we evaluated 200 GISTs for the presence of mutations in exons 9 and 13 of c-kit. Six cases revealed 1530ins6 mutation in exon 9 and two cases 1945A>G mutation in exon 13. All tumors with mutations in exon 9 and 13 lacked mutations in exon 11 of c-kit. None of the analyzed tumors had more than one type of c-kit mutation. All but one of the eight tumors with mutations in exon 9 or 13 of the c-kit gene were histologically and clinically malignant. All four of six cases with exon 9 mutation of which location of primary tumor was known, were small intestinal, suggesting that this type of mutation could preferentially occur in small intestinal tumors. Exon 9 and 13 mutations seem to be rare, and they cover only a small portion (8%) of the balance of GISTs that do not have mutations in exon 11 of c-kit. This finding indicates that other genetic alterations may activate c-kit in GISTs, or that KIT is not activated by mutations in all cases.

  11. The exon-level biomarker SLC39A14 has organ-confined cancer-specificity in colorectal cancer.

    Science.gov (United States)

    Sveen, Anita; Bakken, Anne Cathrine; Ågesen, Trude H; Lind, Guro E; Nesbakken, Arild; Nordgård, Oddmund; Brackmann, Stephan; Rognum, Torleiv O; Lothe, Ragnhild A; Skotheim, Rolf I

    2012-09-15

    An alternative transcript variant of SLC39A14, caused by pre-mRNA splicing, was recently suggested as a biomarker for colorectal cancer (CRC). In our study, we have validated the cancer-specific splicing pattern of the mutually exclusive exons 4A and 4B in altogether 244 colorectal tissue samples. Exon-specific quantitative RT-PCR analyses across 136 Stage I-IV CRC samples and 44 normal colonic mucosa samples showed complete cancer-specificity, as well as 94% sensitivity of SLC39A14-exon4B relative to SLC39A14-exon4A expression. However, across 20 samples from a range of healthy tissues, 18 expressed the CRC variant. This was true also for ten benign lymph nodes, demonstrating that the cancer-specificity is mainly confined to the colon and rectum. Hence, clinical use of SLC39A14-exon4B as a detection marker for CRC other than in samples taken from the bowel wall is diminished. Prognostic value by detection of metastasis to lymph nodes is also abated, elucidating an important pitfall to biomarker discovery. However, analyses of ten nondysplastic biopsies from patients with active inflammatory bowel disease showed negative results in seven samples and only weakly positive results in three samples, suggesting value of SLC39A14-exon4B as a marker to distinguish CRC from other pathologic conditions of the colon. In conclusion, the SLC39A14-exon4B transcript variant is a CRC biomarker with high sensitivity and organ-confined specificity. Further use of the transcript and its encoded protein isoform should be explored in an organ-confined context. Copyright © 2011 UICC.

  12. Deleting exon 55 from the nebulin gene induces severe muscle weakness in a mouse model for nemaline myopathy.

    Science.gov (United States)

    Ottenheijm, Coen A C; Buck, Danielle; de Winter, Josine M; Ferrara, Claudia; Piroddi, Nicoletta; Tesi, Chiara; Jasper, Jeffrey R; Malik, Fady I; Meng, Hui; Stienen, Ger J M; Beggs, Alan H; Labeit, Siegfried; Poggesi, Corrado; Lawlor, Michael W; Granzier, Henk

    2013-06-01

    Nebulin--a giant sarcomeric protein--plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy, the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which Neb exon 55 is deleted (Neb(ΔExon55)) to replicate a founder mutation seen frequently in patients with nemaline myopathy with Ashkenazi Jewish heritage. Neb(ΔExon55) mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies--a hallmark feature of nemaline myopathy--in muscle fibres from Neb(ΔExon55) mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from Neb(ΔExon55) mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibres and single myofibrils is reduced in Neb(ΔExon55) mice with a more pronounced reduction at longer sarcomere lengths. Finally, in Neb(ΔExon55) mice the regulation of contraction is impaired, as evidenced by marked changes in crossbridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based nemaline myopathy model, which recapitulates important features of the phenotype observed in patients harbouring this particular mutation, and which has severe muscle weakness caused by

  13. Antisense oligonucleotide-mediated exon skipping of CHRNA1 pre-mRNA as potential therapy for Congenital Myasthenic Syndromes.

    Science.gov (United States)

    Tei, Shoin; Ishii, Hiroshige T; Mitsuhashi, Hiroaki; Ishiura, Shoichi

    2015-06-05

    CHRNA1 encodes the α subunit of nicotinic acetylcholine receptors (nAChRs) and is expressed at the neuromuscular junction. Moreover, it is one of the causative genes of Congenital Myasthenic Syndromes (CMS). CHRNA1 undergoes alternative splicing to produce two splice variants: P3A(-), without exon P3A, and P3A(+), with the exon P3A. Only P3A(-) forms functional nAChR. Aberrant alternative splicing caused by intronic or exonic point mutations in patients leads to an extraordinary increase in P3A(+) and a concomitant decrease in P3A(-). Consequently this resulted in a shortage of functional receptors. Aiming to restore the imbalance between the two splice products, antisense oligonucleotides (AONs) were employed to induce exon P3A skipping. Three AON sequences were designed to sterically block the putative binding sequences for splicing factors necessary for exon recognition. Herein, we show that AON complementary to the 5' splice site of the exon was the most effective at exon skipping of the minigene with causative mutations, as well as endogenous wild-type CHRNA1. We conclude that single administration of the AON against the 5' splice site is a promising therapeutic approach for patients based on the dose-dependent effect of the AON and the additive effect of combined AONs. This conclusion is favorable to patients with inherited diseases of uncertain etiology that arise from aberrant splicing leading to a subsequent loss of functional translation products because our findings encourage the option of AON treatment as a therapeutic for these prospectively identified diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers.

    Science.gov (United States)

    Smith, Philip J; Zhang, Chaolin; Wang, Jinhua; Chew, Shern L; Zhang, Michael Q; Krainer, Adrian R

    2006-08-15

    Numerous disease-associated point mutations exert their effects by disrupting the activity of exonic splicing enhancers (ESEs). We previously derived position weight matrices to predict putative ESEs specific for four human SR proteins. The score matrices are part of ESEfinder, an online resource to identify ESEs in query sequences. We have now carried out a refined functional SELEX screen for motifs that can act as ESEs in response to the human SR protein SF2/ASF. The test BRCA1 exon under selection was internal, rather than the 3'-terminal IGHM exon used in our earlier studies. A naturally occurring heptameric ESE in BRCA1 exon 18 was replaced with two libraries of random sequences, one seven nucleotides in length, the other 14. Following three rounds of selection for in vitro splicing via internal exon inclusion, new consensus motifs and score matrices were derived. Many winner sequences were demonstrated to be functional ESEs in S100-extract-complementation assays with recombinant SF2/ASF. Motif-score threshold values were derived from both experimental and statistical analyses. Motif scores were shown to correlate with levels of exon inclusion, both in vitro and in vivo. Our results confirm and extend our earlier data, as many of the same motifs are recognized as ESEs by both the original and our new score matrix, despite the different context used for selection. Finally, we have derived an increased specificity score matrix that incorporates information from both of our SF2/ASF-specific matrices and that accurately predicts the exon-skipping phenotypes of deleterious point mutations.

  15. HER2 exon 27 mutations predict worse survival of breast cancer patients, especially in HER2-negative patients.

    Science.gov (United States)

    Si, Pilei; Chen, Tao; Fang, Bin; Yao, Jiabing; Liu, Gaoxiu; Chen, Haijun; Zhai, Baoping; Li, Wentao

    2017-12-01

    The aims of this study were to assess the prognostic value of the HER2 exon 27 mutations in breast cancer patients. Genomic DNA was isolated from peripheral blood leukocytes, and then HER2 exon 27 mutations were detected by direct sequencing. Survival curves were estimated by Kaplan-Meier curves and the differences between the curves were compared by log-rank tests. A total cohort of 892 female patients with operable primary breast cancer was included in this study. The median follow-up was 47 months. Of these 892 patients, 3.7% (33/892) had HER2 exon 27 mutations. Patients with the HER2 exon 27 mutations had a significant worse recurrence-free survival (RFS, unadjusted hazard ratio [HR] 2.42; 95% CI: 1.05-5.58; P = 0.032) and distant recurrence-free survival (DRFS, unadjusted HR 2.81; 95% CI: 1.21-6.50; P = 0.012) than the patients with the wild-type exon 27. Among the 673 patients with negative HER2 expression, 24 mutants were found. Patients with the HER2 mutations showed a worse RFS (unadjusted HR 5.08; 95% CI: 2.14-12.02; P HER2 exon 27 mutations have a worse survival, especially in HER2-negative patients. HER2-negative patients with HER2 exon 27 mutations are potential subgroup of breast cancer patients benefiting from HER2-targeted therapy in future. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  16. Exclusion of alternative exon 33 of CaV1.2 calcium channels in heart is proarrhythmogenic.

    Science.gov (United States)

    Li, Guang; Wang, Juejin; Liao, Ping; Bartels, Peter; Zhang, Hengyu; Yu, Dejie; Liang, Mui Cheng; Poh, Kian Keong; Yu, Chye Yun; Jiang, Fengli; Yong, Tan Fong; Wong, Yuk Peng; Hu, Zhenyu; Huang, Hua; Zhang, Guangqin; Galupo, Mary Joyce; Bian, Jin-Song; Ponniah, Sathivel; Trasti, Scott Lee; See, Kelvin; Foo, Roger; Hoppe, Uta C; Herzig, Stefan; Soong, Tuck Wah

    2017-05-23

    Alternative splicing changes the CaV1.2 calcium channel electrophysiological property, but the in vivo significance of such altered channel function is lacking. Structure-function studies of heterologously expressed CaV1.2 channels could not recapitulate channel function in the native milieu of the cardiomyocyte. To address this gap in knowledge, we investigated the role of alternative exon 33 of the CaV1.2 calcium channel in heart function. Exclusion of exon 33 in CaV1.2 channels has been reported to shift the activation potential -10.4 mV to the hyperpolarized direction, and increased expression of CaV1.2Δ33 channels was observed in rat myocardial infarcted hearts. However, how a change in CaV1.2 channel electrophysiological property, due to alternative splicing, might affect cardiac function in vivo is unknown. To address these questions, we generated mCacna1c exon 33(-/-)-null mice. These mice contained CaV1.2Δ33 channels with a gain-of-function that included conduction of larger currents that reflects a shift in voltage dependence and a modest increase in single-channel open probability. This altered channel property underscored the development of ventricular arrhythmia, which is reflected in significantly more deaths of exon 33(-/-) mice from β-adrenergic stimulation. In vivo telemetric recordings also confirmed increased frequencies in premature ventricular contractions, tachycardia, and lengthened QT interval. Taken together, the significant decrease or absence of exon 33-containing CaV1.2 channels is potentially proarrhythmic in the heart. Of clinical relevance, human ischemic and dilated cardiomyopathy hearts showed increased inclusion of exon 33. However, the possible role that inclusion of exon 33 in CaV1.2 channels may play in the pathogenesis of human heart failure remains unclear.

  17. A majority of m6A residues are in the last exons, allowing the potential for 3' UTR regulation.

    Science.gov (United States)

    Ke, Shengdong; Alemu, Endalkachew A; Mertens, Claudia; Gantman, Emily Conn; Fak, John J; Mele, Aldo; Haripal, Bhagwattie; Zucker-Scharff, Ilana; Moore, Michael J; Park, Christopher Y; Vågbø, Cathrine Broberg; Kusśnierczyk, Anna; Klungland, Arne; Darnell, James E; Darnell, Robert B

    2015-10-01

    We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m(6)A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3'-most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m(6)A and >40% of all m(6)A in mRNA are present in 3' untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m(6)A sites around stop codons. Moreover, m(6)A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m(6)A density peaks early in the 3' UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m(6)A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m(6)A modification in regulating proximal alternative polyA choice. © 2015 Ke et al.; Published by Cold Spring Harbor Laboratory Press.

  18. Clinical characterisation of Becker muscular dystrophy patients predicts favourable outcome in exon-skipping therapy.

    Science.gov (United States)

    van den Bergen, J C; Schade van Westrum, S M; Dekker, L; van der Kooi, A J; de Visser, M; Wokke, B H A; Straathof, C S; Hulsker, M A; Aartsma-Rus, A; Verschuuren, J J; Ginjaar, H B

    2014-01-01

    Duchenne and Becker muscular dystrophy (DMD/BMD) are both caused by mutations in the DMD gene. Out-of-frame mutations in DMD lead to absence of the dystrophin protein, while in-frame BMD mutations cause production of internally deleted dystrophin. Clinically, patients with DMD loose ambulance around the age of 12, need ventilatory support at their late teens and die in their third or fourth decade due to pulmonary or cardiac failure. BMD has a more variable disease course. The disease course of patients with BMD with specific mutations could be very informative to predict the outcome of the exon-skipping therapy, aiming to restore the reading-frame in patients with DMD. Patients with BMD with a mutation equalling a DMD mutation after successful exon skipping were selected from the Dutch Dystrophinopathy Database. Information about disease course was gathered through a standardised questionnaire. Cardiac data were collected from medical correspondence and a previous study on cardiac function in BMD. Forty-eight patients were included, representing 11 different mutations. Median age of patients was 43 years (range 6-67). Nine patients were wheelchair users (26-56 years). Dilated cardiomyopathy was present in 7/36 patients. Only one patient used ventilatory support. Three patients had died at the age of 45, 50 and 76 years, respectively. This study provides mutation specific data on the course of disease in patients with BMD. It shows that the disease course of patients with BMD, with a mutation equalling a 'skipped' DMD mutation is relatively mild. This finding strongly supports the potential benefit of exon skipping in patients with DMD.

  19. LRRK2 exon 41 mutations in sporadic Parkinson disease in Europeans.

    Science.gov (United States)

    Lesage, Suzanne; Janin, Sabine; Lohmann, Ebba; Leutenegger, Anne-Louise; Leclere, Laurence; Viallet, François; Pollak, Pierre; Durif, Franck; Thobois, Stéphane; Layet, Valérie; Vidailhet, Marie; Agid, Yves; Dürr, Alexandra; Brice, Alexis; Bonnet, Anne-Marie; Borg, Michel; Broussolle, Emmanuel; Damier, Philippe; Destée, Alain; Martinez, Maria; Penet, Christiane; Rasco, Olivier; Tison, François; Tranchan, Christine; Vérin, Marc

    2007-03-01

    Mutations in leucine-rich repeat kinase 2 gene (LRRK2), particularly the G2019S mutation in exon 41, have been detected in familial and sporadic Parkinson disease (PD) cases. To assess the frequency of LRRK2 exon 41 mutations in a series of sporadic PD cases from Europe and to determine the clinical features of LRRK2 mutation carriers. We analyzed European cases of sporadic PD for the presence of LRRK2 exon 41 mutations. These mutations were screened by denaturing high-performance liquid chromatography, and abnormal chromatograph traces were investigated by direct sequencing to determine the exact nature of the variants. Early-onset sporadic PD cases were also screened for parkin mutations. The haplotypes associated with the G2019S mutation were determined. The clinical characteristics of patients carrying LRRK2 mutations were detailed. French Network for the Study of Parkinson Disease Genetics. Patients Three hundred twenty patients with apparently sporadic PD from Europe. Results of genetic analyses. We found the G2019S mutation in 6 patients and identified 2 new variants (Y2006H and T2031S) in 1 patient each. Their clinical features were similar to those of typical PD. All G2019S mutation carriers shared a common haplotype. The G2019S mutation is almost as frequent in sporadic cases (1.9%) as in previously reported familial cases (2.9%) in Europe and occurs in the same common founder. We identified 2 novel variants. Although the phenotype of LRRK2 mutation carriers closely resembles that of typical PD, the age at onset was younger (29 years in 1 patient) than previously described, and 3 patients were improved by deep brain stimulation.

  20. Rules and tools to predict the splicing effects of exonic and intronic mutations.

    Science.gov (United States)

    Ohno, Kinji; Takeda, Jun-Ichi; Masuda, Akio

    2017-09-26

    Development of next generation sequencing technologies has enabled detection of extensive arrays of germline and somatic single nucleotide variations (SNVs) in human diseases. SNVs affecting intronic GT-AG dinucleotides invariably compromise pre-mRNA splicing. Most exonic SNVs introduce missense/nonsense codons, but some affect auxiliary splicing cis-elements or generate cryptic GT-AG dinucleotides. Similarly, most intronic SNVs are silent, but some affect canonical and auxiliary splicing cis-elements or generate cryptic GT-AG dinucleotides. However, prediction of the splicing effects of SNVs is challenging. The splicing effects of SNVs generating cryptic AG or disrupting canonical AG can be inferred from the AG-scanning model. Similarly, the splicing effects of SNVs affecting the first nucleotide G of an exon can be inferred from AG-dependence of the 3' splice site (ss). A variety of tools have been developed for predicting the splicing effects of SNVs affecting the 5' ss, as well as exonic and intronic splicing enhancers/silencers. In contrast, only two tools, the Human Splicing Finder and the SVM-BP finder, are available for predicting the position of the branch point sequence. Similarly, IntSplice and Splicing based Analysis of Variants (SPANR) are the only tools to predict the splicing effects of intronic SNVs. The rules and tools introduced in this review are mostly based on observations of a limited number of genes, and no rule or tool can ensure 100% accuracy. Experimental validation is always required before any clinically relevant conclusions are drawn. Development of efficient tools to predict aberrant splicing, however, will facilitate our understanding of splicing pathomechanisms in human diseases. For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

  1. Characterization of CaV1.2 exon 33 heterozygous knockout mice and negative correlation between Rbfox1 and CaV1.2 exon 33 expressions in human heart failure.

    Science.gov (United States)

    Wang, Juejin; Li, Guang; Yu, Dejie; Wong, Yuk Peng; Yong, Tan Fong; Liang, Mui Cheng; Liao, Ping; Foo, Roger; Hoppe, Uta C; Soong, Tuck Wah

    2017-09-26

    Recently, we reported that homozygous deletion of alternative exon 33 of CaV1.2 calcium channel in the mouse resulted in ventricular arrhythmias arising from increased CaV1.2Δ33 ICaL current density in the cardiomyocytes. We wondered whether heterozygous deletion of exon 33 might produce cardiac phenotype in a dose-dependent manner, and whether the expression levels of RNA splicing factors known to regulate alternative splicing of exon 33 might change in human heart failure. Unexpectedly, we found that exon 33+/- cardiomyocytes showed similar CaV1.2 channel properties as wild-type cardiomyocyte, even though CaV1.2Δ33 channels exhibit a gain-in-function. In human hearts, we found that the mRNA level of splicing factor Rbfox1, but not Rbfox2, was downregulated in dilated cardiomyopathy, and CACNA1C mRNA level was dramatically decreased in the both of dilated and ischemic cardiomyopathy. These data imply Rbfox1 may be involved in the development of cardiomyopathies via regulating the alternative splicing of CaV1.2 exon 33. (149 words).

  2. Becker muscular dystrophy with widespread muscle hypertrophy and a non-sense mutation of exon 2

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, M; Vissing, J

    2013-01-01

    , and Western blot showed a 95% reduction of dystrophin levels. Genetic analyses revealed a non-sense mutation in exon 2 of the dystrophin gene. This mutation is predicted to result in a Duchenne phenotype, but resulted in a mild Becker muscular dystrophy with widespread muscle hypertrophy. We suggest......Becker muscular dystrophy features progressive proximal weakness, wasting and often focal hypertrophy. We present a patient with pain and cramps from adolescence. Widespread muscle hypertrophy, preserved muscle strength and a 10-20-fold raised CPK were noted. Muscle biopsy was dystrophic...

  3. ABERRANT SPLICING OF A BRAIN-ENRICHED ALTERNATIVE EXON ELIMINATES TUMOR SUPPRESSOR FUNCTION AND PROMOTES ONCOGENE FUNCTION DURING BRAIN TUMORIGENESIS

    Science.gov (United States)

    Bredel, Markus; Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; Elverfeldt, Dominik v.; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.

    2014-01-01

    BACKGROUND: Tissue-specific alternative splicing is known to be critical to emergence of tissue identity during development, yet its role in malignant transformation is undefined. Tissue-specific splicing involves evolutionary-conserved, alternative exons, which represent only a minority of total alternative exons. Many, however, have functional features that influence activity in signaling pathways to profound biological effect. Given that tissue-specific splicing has a determinative role in brain development and the enrichment of genes containing tissue-specific exons for proteins with roles in signaling and development, it is thus plausible that changes in such exons could rewire normal neurogenesis towards malignant transformation. METHODS: We used integrated molecular genetic and cell biology analyses, computational biology, animal modeling, and clinical patient profiles to characterize the effect of aberrant splicing of a brain-enriched alternative exon in the membrane-binding tumor suppressor Annexin A7 (ANXA7) on oncogene regulation and brain tumorigenesis. RESULTS: We show that aberrant splicing of a tissue-specific cassette exon in ANXA7 diminishes endosomal targeting and consequent termination of the signal of the EGFR oncoprotein during brain tumorigenesis. Splicing of this exon is mediated by the ribonucleoprotein Polypyrimidine Tract-Binding Protein 1 (PTBP1), which is normally repressed during brain development but, we find, is excessively expressed in glioblastomas through either gene amplification or loss of a neuron-specific microRNA, miR-124. Silencing of PTBP1 attenuates both malignancy and angiogenesis in a stem cell-derived glioblastoma animal model characterized by a high native propensity to generate tumor endothelium or vascular pericytes to support tumor growth. We show that EGFR amplification and PTBP1 overexpression portend a similarly poor clinical outcome, further highlighting the importance of PTBP1-mediated activation of EGFR

  4. Clinical Characteristics and Outcomes of Lung Cancer Patients 
with EGFR Mutations in Exons 19 and 21

    Directory of Open Access Journals (Sweden)

    Renwang LIU

    2014-11-01

    Full Text Available Background and objective Studies on the epidermal growth factor receptor (EGFR signaling pathways and the therapeutic effects of EGFR-tyrosine kinase inhibitors (EGFR-TKIs have recently proven that targeted therapy has a major role in the treatment of lung cancer. However, the therapeutic effects of EGFR-TKIs on lung cancers with different EGFR mutation subtypes remain unclear. And if there is a significant difference in the effects of EGFR-TKIs, the mechanisms for the difference remain unclear. The aim of this study was to investigate the clinical importance of EGFR mutations in exons 19 and 21 of lung cancer patients and to compare the outcomes of these patients. Methods The study recruited 113 patients who had non-small cell lung cancer (NSCLC with EGFR mutations. EGFR mutations were detected for 47 patients using Real-time PCR or DNA sequencinag. The mutations of the remaining patients were determined using xTag-EGFR liquid chip technology. All stages I-III patients underwent radical resection followed by 4 cycles of postoperative chemotherapy. Patients with pleural metastases underwent pleural biopsy, pleurodesis, and chemotherapy only. Patients with distant metastases underwent biopsy and chemotherapy only. Collected clinical data were analyzed using SPSS 19.0 software. Results EGFR exon mutations 19 and 21 were found in 56 and 57 patients, respectively. The mean age of patients with exon 19 mutations was lower than the age of the patients with exon 21 mutations (57.02±11.31 years vs 62.25±7.76 years, respectively; P0.05 between the patients with exon 19 and 21 mutations; and survival analysis of 91 (80.5% patients with complete clinical data found no differences in overall survival. Stratification analysis found out that patients with exon 19 mutations had longer overall survival associated with age>61 years, male gender, ever smoking, and stage IV disease; although the differences were not significant. Conclusion Compared to the lung

  5. Design of a tobacco exon array with application to investigate the differential cadmium accumulation property in two tobacco varieties.

    Science.gov (United States)

    Martin, Florian; Bovet, Lucien; Cordier, Audrey; Stanke, Mario; Gunduz, Irfan; Peitsch, Manuel C; Ivanov, Nikolai V

    2012-11-28

    For decades the tobacco plant has served as a model organism in plant biology to answer fundamental biological questions in the areas of plant development, physiology, and genetics. Due to the lack of sufficient coverage of genomic sequences, however, none of the expressed sequence tag (EST)-based chips developed to date cover gene expression from the whole genome. The availability of Tobacco Genome Initiative (TGI) sequences provides a useful resource to build a whole genome exon array, even if the assembled sequences are highly fragmented. Here, the design of a Tobacco Exon Array is reported and an application to improve the understanding of genes regulated by cadmium (Cd) in tobacco is described. From the analysis and annotation of the 1,271,256 Nicotiana tabacum fasta and quality files from methyl filtered genomic survey sequences (GSS) obtained from the TGI and ~56,000 ESTs available in public databases, an exon array with 272,342 probesets was designed (four probes per exon) and tested on two selected tobacco varieties.Two tobacco varieties out of 45 accumulating low and high cadmium in leaf were identified based on the GGE biplot analysis, which is analysis of the genotype main effect (G) plus analysis of the genotype by environment interaction (GE) of eight field trials (four fields over two years) showing reproducibility across the trials. The selected varieties were grown under greenhouse conditions in two different soils and subjected to exon array analyses using root and leaf tissues to understand the genetic make-up of the Cd accumulation. An Affymetrix Exon Array was developed to cover a large (~90%) proportion of the tobacco gene space. The Tobacco Exon Array will be available for research use through Affymetrix array catalogue. As a proof of the exon array usability, we have demonstrated that the Tobacco Exon Array is a valuable tool for studying Cd accumulation in tobacco leaves. Data from field and greenhouse experiments supported by gene

  6. Transcriptome-based exon capture enables highly cost-effective comparative genomic data collection at moderate evolutionary scales

    Directory of Open Access Journals (Sweden)

    Bi Ke

    2012-08-01

    Full Text Available Abstract Background To date, exon capture has largely been restricted to species with fully sequenced genomes, which has precluded its application to lineages that lack high quality genomic resources. We developed a novel strategy for designing array-based exon capture in chipmunks (Tamias based on de novo transcriptome assemblies. We evaluated the performance of our approach across specimens from four chipmunk species. Results We selectively targeted 11,975 exons (~4 Mb on custom capture arrays, and enriched over 99% of the targets in all libraries. The percentage of aligned reads was highly consistent (24.4-29.1% across all specimens, including in multiplexing up to 20 barcoded individuals on a single array. Base coverage among specimens and within targets in each species library was uniform, and the performance of targets among independent exon captures was highly reproducible. There was no decrease in coverage among chipmunk species, which showed up to 1.5% sequence divergence in coding regions. We did observe a decline in capture performance of a subset of targets designed from a much more divergent ground squirrel genome (30 My, however, over 90% of the targets were also recovered. Final assemblies yielded over ten thousand orthologous loci (~3.6 Mb with thousands of fixed and polymorphic SNPs among species identified. Conclusions Our study demonstrates the potential of a transcriptome-enabled, multiplexed, exon capture method to create thousands of informative markers for population genomic and phylogenetic studies in non-model species across the tree of life.

  7. Bottom-up design of small molecules that stimulate exon 10 skipping in mutant MAPT pre-mRNA.

    Science.gov (United States)

    Luo, Yiling; Disney, Matthew D

    2014-09-22

    One challenge in chemical biology is to develop small molecules that control cellular protein content. The amount and identity of proteins are influenced by the RNAs that encode them; thus, protein content in a cell could be affected by targeting mRNA. However, RNA has been traditionally difficult to target with small molecules. In this report, we describe controlling the protein products of the mutated microtubule-associated protein tau (MAPT) mature mRNA with a small molecule. MAPT mutations in exon 10 are associated with inherited frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), an incurable disease that is directly caused by increased inclusion of exon 10 in MAPT mRNA. Recent studies have shown that mutations within a hairpin at the MAPT exon 10-intron junction decrease the thermodynamic stability of the RNA, increasing binding to U1 snRNP and thus exon 10 inclusion. Therefore, we designed small molecules that bind and stabilize a mutant MAPT by using Inforna, a computational approach based on information about RNA-small-molecule interactions. The optimal compound selectively bound the mutant MAPT hairpin and thermodynamically stabilized its folding, facilitating exon 10 exclusion. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Investigation of Mutations in Exons 12-15 MYH7 Gene in Hypertrophic Cardiomyopathie Patients Using PCR-SSCP Technique

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    Soraya Heydari

    2013-10-01

    Full Text Available Background: Hypertrophic cardiomyopathy (HCM is the most common kind of Mendelian inherited heart disease, affects 0.2% of the global population. HCM is also the most common cause of sudden cardiac death in individuals younger than 35 years old. To date more than 900 individual mutations has been identified in over 20 genes, such as MYH7, MYBPC3, and TNNT2. Interestingly, most of these genes encode sarcomeric proteins. In the present study, we investigated the possible presence of mutation in exons 12-15 MYH7 gene, which has already been reported to accommodate some mutations, in 30 patients with HCM in Chaharmahal va Bakhtiyari province. Materials and Methods: DNA was extracted using standard phenol-chloroform method and then was used for amplification and gel electroploresis by PCR-SSCP procedure. Finally, the suspected cases were selected for the direct sequencing and the results were analyzed using chromas software.Results: There is no mutation in these exons, but two polymorphisms including: 5811 C>T and 5845 G> were found in the exon 12 of 1 and 5 separate patients, respectively.Conclusion: In this study with respect to none amino acid codon changes arisen from these polymorphisms, we concluded that mutations in these exons of MYH7 gene have a very low contribution in patients in this province and this is necessary to study other exons for better assessment.

  9. Exon Skipping and Gene Transfer Restore Dystrophin Expression in Human Induced Pluripotent Stem Cells-Cardiomyocytes Harboring DMD Mutations

    Science.gov (United States)

    Dick, Emily; Kalra, Spandan; Anderson, David; George, Vinoj; Ritso, Morten; Laval, Steven H.; Barresi, Rita; Aartsma-Rus, Annemieke; Lochmüller, Hanns

    2013-01-01

    With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47–50 or 48–50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart. PMID:23829870

  10. A consensus CaMK IV-responsive RNA sequence mediates regulation of alternative exons in neurons.

    Science.gov (United States)

    Xie, Jiuyong; Jan, Calvin; Stoilov, Peter; Park, Jennifer; Black, Douglas L

    2005-12-01

    Neurons make extensive use of alternative pre-mRNA splicing to regulate gene expression and diversify physiological responses. We showed previously in a pituitary cell line that the Ca(++)/calmodulin-dependent protein kinase CaMK IV specifically repressed splicing of the BK channel STREX exon. This repression is dependent on a CaMK IV-responsive RNA element (CaRRE) within the STREX 3' splice site. Here, we report that similar Ca(++) regulation of splicing, mediated by L-type calcium channels and CaM kinase IV, occurs in cultured neurons and in the brain. We identify a critical CaRRE motif (CACATNRTTAT) that is essential for conferring CaMK IV repression on an otherwise constitutive exon. Additional Ca(++)-regulated exons that carry this consensus sequence are also identified in the human genome. Thus, the Ca(++)/CaMK IV pathway in neurons controls the alternative splicing of a group of exons through this short CaRRE consensus sequence. The functions of some of these exons imply that splicing control through the CaMK IV pathway will alter neuronal activity.

  11. Genotyping of Human Papillomavirus and TP53 Mutaions at Exons 5 to 7 in Lung Cancer Patients from Iran

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    Hossein Jafari

    2013-06-01

    Full Text Available Introduction: There is a powerful relationship between high-risk human papillomaviruses and lung cancer. In fact, inactivation of p53 is the most common genetic abnormality in lung cancer. Indeed, the frequency of HPV types and TP53 mutations in squamous cell carcinoma of lung, among patients from the northwest of Iran has been evaluated in this article. Methodes: Fifty Paraffin embedded blocks of lung SCC were selected for detection of HPV DNA by Nested PCR, and then DNA was sequenced for HPV typing. Equal numbers of positive and negative samples for the HPV DNA were examined for the presence of mutations in exons 5-7 of the TP53 gene by PCR and direct sequencing. Results: Overtly 9 (18% of 50 samples presented the HPV DNA: eight were HPV-18 and one was HPV-6. TP53 mutations were found in 5 samples (27.7%. Of these, 4 cases showed mutations in exon 5 and one case contained a mutation in exon 7.The most frequent mutation in exon 5 was the C to G transversion (c.409C>G, and also the T to A tansversion (c.770T>A in exon 7. Conclusion: This study showed that HPV-18 is more likely to conscequence in the development of lung cancer among some communities. Genetic alterations, alongside with environmental factors, all play a significant role in the pathogenesis of lung cancer.

  12. Human peptidylglycine alpha-amidating monooxygenase transcripts derived by alternative mRNA splicing of an unreported exon.

    Science.gov (United States)

    Vos, M D; Jones, J E; Treston, A M

    1995-10-03

    We are characterizing the alternatively spliced human peptidylglycine alpha-amidating monooxygenase (hPAM)-encoding mRNA transcripts expressed by human cells. Reverse transcription coupled to the polymerase chain reaction (RT-PCR) has been used to identify four alternatively spliced variants that differ in the region joining the two catalytic domains. Two of the transcripts represent previously reported splice variants differentiated by the presence (hPAM-A) or absence (hPAM-B) of a 321-nucleotide (nt) linker (optional exon A) which in the rat produce functionally distinct enzymes. Different mRNAs represent two splice variants, hPAM-C and hPAM-D, that show the presence of an exon unreported for PAM in any other species. This new exon, designated exon C, is 54 nt in length, encodes an 18-amino-acid (aa) peptide containing a conserved dibasic aa endoproteolytic processing motif, and is located 3' of exon A in human genomic DNA. We propose that cell-specific regulation of mRNA splicing would provide a mechanism for control of prohormone activation by these variants of the PAM enzyme.

  13. GAA Deficiency in Pompe Disease Is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells.

    Science.gov (United States)

    van der Wal, Erik; Bergsma, Atze J; van Gestel, Tom J M; In 't Groen, Stijn L M; Zaehres, Holm; Araúzo-Bravo, Marcos J; Schöler, Hans R; van der Ploeg, Ans T; Pijnappel, W W M Pim

    2017-06-16

    Pompe disease is a metabolic myopathy caused by deficiency of the acid α-glucosidase (GAA) enzyme and results in progressive wasting of skeletal muscle cells. The c.-32-13T>G (IVS1) GAA variant promotes exon 2 skipping during pre-mRNA splicing and is the most common variant for the childhood/adult disease form. We previously identified antisense oligonucleotides (AONs) that promoted GAA exon 2 inclusion in patient-derived fibroblasts. It was unknown how these AONs would affect GAA splicing in skeletal muscle cells. To test this, we expanded induced pluripotent stem cell (iPSC)-derived myogenic progenitors and differentiated these to multinucleated myotubes. AONs restored splicing in myotubes to a similar extent as in fibroblasts, suggesting that they act by modulating the action of shared splicing regulators. AONs targeted the putative polypyrimidine tract of a cryptic splice acceptor site that was part of a pseudo exon in GAA intron 1. Blocking of the cryptic splice donor of the pseudo exon with AONs likewise promoted GAA exon 2 inclusion. The simultaneous blocking of the cryptic acceptor and cryptic donor sites restored the majority of canonical splicing and alleviated GAA enzyme deficiency. These results highlight the relevance of cryptic splicing in human disease and its potential as therapeutic target for splicing modulation using AONs. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. Alternative splicing of exon 17 and a missense mutation in exon 20 of the insulin receptor gene in two brothers with a novel syndrome of insulin resistance (congenital fiber-type disproportion myopathy)

    DEFF Research Database (Denmark)

    Vorwerk, P; Christoffersen, C T; Müller, J

    1999-01-01

    The insulin receptor (IR) in two brothers with a rare syndrome of congenital muscle fiber type disproportion myopathy (CFTDM) associated with diabetes and severe insulin resistance was studied. By direct sequencing of Epstein-Barr virus-transformed lymphocytes both patients were found...... domain. In the correct spliced variant, the point mutation is silent and results in a normally translated IR. The paternal allele carries a missense mutation in the tyrosine kinase domain. All three cDNA variants were present in the lymphocytes of the patients. Purified IR from 293 cells overexpressing...... to be compound heterozygotes for mutations in the IR gene. The maternal allele was alternatively spliced in exon 17 due to a point mutation in the -1 donor splice site of the exon. The abnormal skipping of exon 17 shifts the amino acid reading frame and leads to a truncated IR, missing the entire tyrosine kinase...

  15. Accurate clinical detection of exon copy number variants in a targeted NGS panel using DECoN [version 1; referees: 2 approved

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    Anna Fowler

    2016-11-01

    Full Text Available Background: Targeted next generation sequencing (NGS panels are increasingly being used in clinical genomics to increase capacity, throughput and affordability of gene testing. Identifying whole exon deletions or duplications (termed exon copy number variants, ‘exon CNVs’ in exon-targeted NGS panels has proved challenging, particularly for single exon CNVs.  Methods: We developed a tool for the Detection of Exon Copy Number variants (DECoN, which is optimised for analysis of exon-targeted NGS panels in the clinical setting. We evaluated DECoN performance using 96 samples with independently validated exon CNV data. We performed simulations to evaluate DECoN detection performance of single exon CNVs and to evaluate performance using different coverage levels and sample numbers. Finally, we implemented DECoN in a clinical laboratory that tests BRCA1 and BRCA2 with the TruSight Cancer Panel (TSCP. We used DECoN to analyse 1,919 samples, validating exon CNV detections by multiplex ligation-dependent probe amplification (MLPA.  Results: In the evaluation set, DECoN achieved 100% sensitivity and 99% specificity for BRCA exon CNVs, including identification of 8 single exon CNVs. DECoN also identified 14/15 exon CNVs in 8 other genes. Simulations of all possible BRCA single exon CNVs gave a mean sensitivity of 98% for deletions and 95% for duplications. DECoN performance remained excellent with different levels of coverage and sample numbers; sensitivity and specificity was >98% with the typical NGS run parameters. In the clinical pipeline, DECoN automatically analyses pools of 48 samples at a time, taking 24 minutes per pool, on average. DECoN detected 24 BRCA exon CNVs, of which 23 were confirmed by MLPA, giving a false discovery rate of 4%. Specificity was 99.7%.  Conclusions: DECoN is a fast, accurate, exon CNV detection tool readily implementable in research and clinical NGS pipelines. It has high sensitivity and specificity and acceptable

  16. Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro

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    Caples Matt

    2004-07-01

    Full Text Available Abstract Background Incorporation of exon 11 of the insulin receptor gene is both developmentally and hormonally-regulated. Previously, we have shown the presence of enhancer and silencer elements that modulate the incorporation of the small 36-nucleotide exon. In this study, we investigated the role of inherent splice site strength in the alternative splicing decision and whether recognition of the splice sites is the major determinant of exon incorporation. Results We found that mutation of the flanking sub-optimal splice sites to consensus sequences caused the exon to be constitutively spliced in-vivo. These findings are consistent with the exon-definition model for splicing. In-vitro splicing of RNA templates containing exon 11 and portions of the upstream intron recapitulated the regulation seen in-vivo. Unexpectedly, we found that the splice sites are occupied and spliceosomal complex A was assembled on all templates in-vitro irrespective of splicing efficiency. Conclusion These findings demonstrate that the exon-definition model explains alternative splicing of exon 11 in the IR gene in-vivo but not in-vitro. The in-vitro results suggest that the regulation occurs at a later step in spliceosome assembly on this exon.

  17. FGFR2 exon IIIa and IIIc mutations in Crouzon, Jackson-Weiss, and Pfeiffer syndromes: Evidence for missense changes, insertions, and a deletion due to alternative RNA splicing

    Energy Technology Data Exchange (ETDEWEB)

    Meyers, G.A.; Przylepa, K.A.; Scott, A.F. [Johns Hopkins Hospital, Baltimore, MD (United States)] [and others

    1996-03-01

    Fibroblast growth factor receptor 2 (FGFR2) mutations have been associated with the craniosynostotic conditions Crouzon, Jackson-Weiss, and Pfeiffer syndromes. Previously, mutations were described in the exons IIIa and IIIc, which form the extracellular, third immunoglobulin-like domain (IgM) and adjacent linker regions, both of which are normally involved in ligand binding. For all three conditions, mutations were found in exon IIIc. Only in Crouzon syndrome were mutations identified in exon IIIa. In this study, 39 cases with one of these three conditions were screened for exon IIIa or IIIc mutations. Eleven mutations are reported in 17 unrelated cases. Mutations in exon IIIa are identified for not only Crouzon but also Jackson-Weiss and Pfeiffer syndromes. Four mutations in either exon IIIa or exon IIIc reported only in Crouzon syndrome are present also in one of the other two syndromes. Two insertions, one in exon IIIa in a Crouzon syndrome patient and the other in exon IIIc in a Pfeiffer syndrome patient, were observed. The latter mutation has the same alternative RNA splicing effect as a reported synonymous mutation for Crouzon syndrome. A missense mutation was detected in one Pfeiffer syndrome family in which two members had craniosynostosis without limb anomalies. The inter- and intrafamilial variability in expression of FGFR2 mutations suggests that these three syndromes, presumed to be clinically distinct, are instead representative of a spectrum of related craniosynostotic and digital disorders. 16 refs., 3 figs., 1 tab.

  18. Novel homozygous VHL mutation in exon 2 is associated with congenital polycythemia but not with cancer.

    Science.gov (United States)

    Lanikova, Lucie; Lorenzo, Felipe; Yang, Chunzhang; Vankayalapati, Hari; Drachtman, Richard; Divoky, Vladimir; Prchal, Josef T

    2013-05-09

    Germline von Hippel-Lindau (VHL) gene mutations underlie dominantly inherited familial VHL tumor syndrome comprising a predisposition for renal cell carcinoma, pheochromocytoma/paraganglioma, cerebral hemangioblastoma, and endolymphatic sac tumors. However, recessively inherited congenital polycythemia, exemplified by Chuvash polycythemia, has been associated with 2 separate 3' VHL gene mutations in exon 3. It was proposed that different positions of loss-of-function VHL mutations are associated with VHL syndrome cancer predisposition and only C-terminal domain-encoding VHL mutations would cause polycythemia. However, now we describe a new homozygous VHL exon 2 mutation of the VHL gene:(c.413C>T):P138L, which is associated in the affected homozygote with congenital polycythemia but not in her, or her-heterozygous relatives, with cancer or other VHL syndrome tumors. We show that VHL(P138L) has perturbed interaction with hypoxia-inducible transcription factor (HIF)1α. Further, VHL(P138L) protein has decreased stability in vitro. Similarly to what was reported in Chuvash polycythemia and some other instances of HIFs upregulation, VHL(P138L) erythroid progenitors are hypersensitive to erythropoietin. Interestingly, the level of RUNX1/AML1 and NF-E2 transcripts that are specifically upregulated in acquired polycythemia vera were also upregulated in VHL(P138L) granulocytes.

  19. Molecular characterization of exon 28 of von Willebrand's factor gene in Nigerian population.

    Science.gov (United States)

    Ezigbo, E D; Ukaejiofo, E O; Nwagha, T U

    2017-02-01

    Polymorphisms in von Willebrand factor (VWF) gene are an important contributor to the expression of VWF gene and differences in ethnic distribution of these single nucleotide polymorphisms (SNPs) exists. Our objective was to molecularly characterize the exon 28 of the VWF gene in the three major ethnic groups of Nigeria. We recruited 90 subjects, 45 had a history of bleeding. Questions included those used in the Zimmerman Program for the Molecular and Clinical Biology of von Willebrand disease (VWD), and the bleeding scores were calculated using the Molecular and Clinical Markers for the Diagnosis and Management of type 1 VWD scoring system. Full blood count, coagulation profile, VWF:antigen level and VWF:collagen-binding activities were carried out. Data were analyzed using GraphPad Prism (5.03). GraphPad Software, Inc USA. The BigDye terminator chemistry was used to determine the nucleotide sequences of VWF gene (exon 28). Eight SNPs were identified, rs 216310 (T1547), rs 1800385 (V1565L), rs1800384 (A1515), rs1800383 (D1472H), rs 1800386 (Y1584C), rs 216311 (T1381A), rs 216312 (intronic) and rs 1800381 (P1337). The SNPs rs 216311, rs 1800383 and rs 1800386 associated significantly with bleeding in study subjects. rs1800386 occurred in all with bleeding history, no ethnic variations were noted.

  20. Global analysis of aberrant pre-mRNA splicing in glioblastoma using exon expression arrays

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    Nixon Tamara J

    2008-05-01

    Full Text Available Abstract Background Tumor-predominant splice isoforms were identified during comparative in silico sequence analysis of EST clones, suggesting that global aberrant alternative pre-mRNA splicing may be an epigenetic phenomenon in cancer. We used an exon expression array to perform an objective, genome-wide survey of glioma-specific splicing in 24 GBM and 12 nontumor brain samples. Validation studies were performed using RT-PCR on glioma cell lines, patient tumor and nontumor brain samples. Results In total, we confirmed 14 genes with glioma-specific splicing; seven were novel events identified by the exon expression array (A2BP1, BCAS1, CACNA1G, CLTA, KCNC2, SNCB, and TPD52L2. Our data indicate that large changes (> 5-fold in alternative splicing are infrequent in gliomagenesis ( Conclusion While we observed some tumor-specific alternative splicing, the number of genes showing exclusive tumor-specific isoforms was on the order of tens, rather than the hundreds suggested previously by in silico mining. Given the important role of alternative splicing in neural differentiation, there may be selective pressure to maintain a majority of splicing events in order to retain glial-like characteristics of the tumor cells.

  1. Mannose-binding lectin exon 1 and promoter polymorphisms in tuberculosis disease in a Mediterranean area.

    Science.gov (United States)

    García-Gasalla, M; Milá Llambí, J; Losada-López, I; Cifuentes-Luna, C; Fernández-Baca, V; Pareja-Bezares, A; Mir-Villadrich, I; Payeras-Cifré, A

    2014-08-01

    Mannose-binding lectin (MBL) is a serum protein that activates the complement and mediates phagocytosis. MBL levels and MBL2 genotype may impact upon host susceptibility to tuberculosis (TB) disease but evidence to date has been conflicting. MBL2 exon 1 and promoter genotyping and serum MBL concentrations were determined in 79 patients with active tuberculosis (58 pulmonary TB and 21 extrapulmonary or miliary TB) and 120 household healthy contacts (HHC) from a Mediterranean area (Majorca Island, Spain). Significantly higher serum MBL levels were found in patients with active tuberculosis than in HHC [median MBL concentrations 3430 ng mL(-1) (10-28 415) and 2600 ng mL(-1) (5-20 000) respectively, P = 0.002]. These higher MBL levels were mainly related to the most prevalent YA/YA wild-type diplotype. There was a strong correlation between MBL2 exon 1 and promoter genotype and MBL levels. The diplotype LYQA/HYPA was present in 12 out of 57 of the pulmonary TB cases but in none of the extrapulmonary TB patients. Diplotype LXPA/HYPA, producer of high levels of MBL, was significantly more frequent in HHC than in patients (16.8% vs. 6.4%, P = 0.031) suggesting a protective role against the development of TB disease that has not been previously found. © 2014 John Wiley & Sons Ltd.

  2. Exon skipping and translation in patients with frameshift deletions in the dystrophin gene

    Energy Technology Data Exchange (ETDEWEB)

    Sherratt, T.G.; Dubowitz, V.; Sewry, C.A.; Strong, P.N. (Royal Postgraduate Medical School, London (United Kingdom)); Vulliamy, T. (Hammersmith Hospital, London (United Kingdom))

    1993-11-01

    Although many Duchenne muscular dystrophy patients have a deletion in the dystrophin gene which disrupts the translational reading frame, they express dystrophin in a small proportion of skeletal muscle fibers ([open quotes]revertant fibers[close quotes]). Antibody studies have shown, indirectly, that dystrophin synthesis in revertant fibers is facilitated by a frame-restoring mechanism; in the present study, the feasibility of mRNA splicing was investigated. Dystrophin transcripts were analyzed in skeletal muscle from individuals possessing revertant fibers and a frameshift deletion in the dystrophin gene. In each case a minor in-frame transcript was detected, in which exons adjacent to those deleted from the genome had been skipped. There appeared to be some correlation between the levels of in-frame transcripts and the predicted translation products. Low levels of alternatively spliced transcripts were also present in normal muscle. The results provide further evidence of exon skipping in the dystrophin gene and indicate that this may be involved in the synthesis of dystrophin by revertant fibers. 44 refs., 12 figs.

  3. An Inframe Trinucleotide Deletion in MTRR Exon 1 is Associated with the Risk of Spina Bifida.

    Science.gov (United States)

    Zhang, Jun; Dai, Xiao-Lu; Liu, Gui-Cen; Wang, Juan; Ren, Xue-Yi; Jin, Mu-Hua; Mi, Nan-Nan; Wang, Shu-Qin

    2017-09-01

    Maternal genetic variants of enzymes in folate-homocysteine metabolic network are significantly correlative with the risk of spina bifida. To survey the genetic causality, the genotypes of three women having spina bifida fetuses from two unrelated Chinese families were screened in candidate alleles. Polymerase chain reaction, capillary electrophoresis and Sanger sequencing were employed to recognize the allelic variation. A trinucleotide deletion (c.4_6delAGG) was identified in the first exon of MTRR. All the three women showed the novel clinical variation including one heterozygous and two homozygous. The siblings who had healthy babies from the same families did not harbor the variation. In the unaffected control individuals, the variant was also not observed. Eukaryotic expression and bioinformatics techniques were utilized to explore the molecular pathogenesis of the potential genetic risk of developing spina bifida. Exceptionally, the functional examination revealed that the Arg2del variant kept subcellular localization unaltered with catalytic activity intact, but failed to efficiently activate MTR compared with the wild type. Genetic disorder of folate and homocysteine metabolism during pregnancy is believed to be associated with folate-sensitive neural tube defects. The report highlights that the inframe deletion in MTRR exon 1 could be a high risk factor susceptibility to spina bifida.

  4. Random Splicing of Several Exons Caused by a Single Base Change in the Target Exon of CRISPR/Cas9 Mediated Gene Knockout.

    Science.gov (United States)

    Kapahnke, Marcel; Banning, Antje; Tikkanen, Ritva

    2016-12-14

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated sequence 9 (CRISPR/Cas9) system is widely used for genome editing purposes as it facilitates an efficient knockout of a specific gene in, e.g. cultured cells. Targeted double-strand breaks are introduced to the target sequence of the guide RNAs, which activates the cellular DNA repair mechanism for non-homologous-end-joining, resulting in unprecise repair and introduction of small deletions or insertions. Due to this, sequence alterations in the coding region of the target gene frequently cause frame-shift mutations, facilitating degradation of the mRNA. We here show that such CRISPR/Cas9-mediated alterations in the target exon may also result in altered splicing of the respective pre-mRNA, most likely due to mutations of splice-regulatory sequences. Using the human FLOT-1 gene as an example, we demonstrate that such altered splicing products also give rise to aberrant protein products. These may potentially function as dominant-negative proteins and thus interfere with the interpretation of the data generated with these cell lines. Since most researchers only control the consequences of CRISPR knockout at genomic and protein level, our data should encourage to also check the alterations at the mRNA level.

  5. Random Splicing of Several Exons Caused by a Single Base Change in the Target Exon of CRISPR/Cas9 Mediated Gene Knockout

    Directory of Open Access Journals (Sweden)

    Marcel Kapahnke

    2016-12-01

    Full Text Available The clustered regularly interspaced short palindromic repeats (CRISPR-associated sequence 9 (CRISPR/Cas9 system is widely used for genome editing purposes as it facilitates an efficient knockout of a specific gene in, e.g. cultured cells. Targeted double-strand breaks are introduced to the target sequence of the guide RNAs, which activates the cellular DNA repair mechanism for non-homologous-end-joining, resulting in unprecise repair and introduction of small deletions or insertions. Due to this, sequence alterations in the coding region of the target gene frequently cause frame-shift mutations, facilitating degradation of the mRNA. We here show that such CRISPR/Cas9-mediated alterations in the target exon may also result in altered splicing of the respective pre-mRNA, most likely due to mutations of splice-regulatory sequences. Using the human FLOT-1 gene as an example, we demonstrate that such altered splicing products also give rise to aberrant protein products. These may potentially function as dominant-negative proteins and thus interfere with the interpretation of the data generated with these cell lines. Since most researchers only control the consequences of CRISPR knockout at genomic and protein level, our data should encourage to also check the alterations at the mRNA level.

  6. Partial correlation analysis indicates causal relationships between GC-content, exon density and recombination rate in the human genome.

    Science.gov (United States)

    Freudenberg, Jan; Wang, Mingyi; Yang, Yaning; Li, Wentian

    2009-01-30

    Several features are known to correlate with the GC-content in the human genome, including recombination rate, gene density and distance to telomere. However, by testing for pairwise correlation only, it is impossible to distinguish direct associations from indirect ones and to distinguish between causes and effects. We use partial correlations to construct partially directed graphs for the following four variables: GC-content, recombination rate, exon density and distance-to-telomere. Recombination rate and exon density are unconditionally uncorrelated, but become inversely correlated by conditioning on GC-content. This pattern indicates a model where recombination rate and exon density are two independent causes of GC-content variation. Causal inference and graphical models are useful methods to understand genome evolution and the mechanisms of isochore evolution in the human genome.

  7. Identification of a mutation in exon 27 of the RB1 gene associated with incomplete penetrance retinoblastoma.

    Science.gov (United States)

    Mitter, Diana; Rushlow, Diane; Nowak, Inga; Ansperger-Rescher, Birgit; Gallie, Brenda L; Lohmann, Dietmar R

    2009-01-01

    Retinoblastoma (Rb) is initiated by germline mutations in the RB1 gene. Up to date, no mutation was identified in exons 26 and 27. We have identified a 2 bp frameshift insertion in exon 27 of the RB1 gene (RBg.177008_177009dup) in a boy with unilateral Rb and his healthy father that has occurred de novo on the allele transmitted by the father's father. RT-PCR showed that the mutant +2 bp transcript is present in RNA from peripheral leukocytes after short-term culture. The level of the mutant transcript was low compared to the normal transcript indicating abnormal expression of the variant allele. The mutant transcript was further reduced after puromycin treatment suggesting that NMD is not involved. Although oncogenic mutations in the terminal exons of the RB1 gene are rare molecular testing is important as those terminal mutations can be associated with incomplete penetrance and cause high recurrence risk in family members.

  8. AON-mediated Exon Skipping Restores Ciliation in Fibroblasts Harboring the Common Leber Congenital Amaurosis CEP290 Mutation

    Directory of Open Access Journals (Sweden)

    Xavier Gerard

    2012-01-01

    Full Text Available Leber congenital amaurosis (LCA is a severe hereditary retinal dystrophy responsible for congenital or early-onset blindness. The most common disease-causing mutation (>10% is located deep in intron 26 of the CEP290 gene (c.2991+1655A>G. It creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. In the present study, we show that the use of antisense oligonucleotides (AONs allow an efficient skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients. These data support the feasibility of an AON-mediated exon skipping strategy to correct the aberrant splicing.

  9. Novel Cationic Carotenoid Lipids as Delivery Vectors of Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Vassilia Partali

    2012-01-01

    Full Text Available Duchenne Muscular Dystrophy (DMD is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs. The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC.

  10. Deletion of exon 26 of the dystrophin gene is associated with a mild Becker muscular dystrophy phenotype

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, Morten; Vissing, John

    2011-01-01

    With the possible introduction of exon skipping therapy in Duchenne muscular dystrophy, it has become increasingly important to know the role of each exon of the dystrophin gene to protein expression, and thus the phenotype. In this report, we present two related men with an unusually mild BMD...... associated with an exon 26 deletion. The proband, a 23-year-old man, had slightly delayed motor milestones, walking 1 1/2 years old. He had no complaints of muscle weakness, but had muscle pain. Clinical examination revealed no muscle wasting or loss of power, but his CK was 1500-7000 U/l. Muscle biopsy...... skipping therapy for Duchenne muscular dystrophy. This report also shows that BMD may present with a normal CK....

  11. Molecular analyses of novel ASAH1 mutations causing Farber lipogranulomatosis: analyses of exonic splicing enhancer inactivating mutation.

    Science.gov (United States)

    Bashyam, M D; Chaudhary, A K; Kiran, M; Reddy, V; Nagarajaram, H A; Dalal, A; Bashyam, L; Suri, D; Gupta, A; Gupta, N; Kabra, M; Puri, R D; RamaDevi, R; Kapoor, S; Danda, S

    2014-12-01

    Farber lipogranulomatosis is a rare autosomal recessive lysosomal storage disorder caused by mutations in the ASAH1 gene. In the largest ever study, we identified and characterized ASAH1 mutations from 11 independent Farber disease (FD) families. A total of 13 different mutations were identified including 1 splice, 1 polypyrimidine tract (PPT) deletion and 11 missense mutations. Eleven mutations were exclusive to the Indian population. The IVS6+4A>G splice and IVS5-16delTTTTC PPT deletion mutations resulted in skipping of exon 6 precluding thereby the region responsible for cleavage of enzyme precursor. A missense mutation (p.V198A) resulted in skipping of exon 8 due to inactivation of an exonic splicing enhancer (ESE) element. This is the first report of mutations affecting PPT and ESE in the ASAH1 gene resulting in FD. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Deletion of exon 26 of the dystrophin gene is associated with a mild Becker muscular dystrophy phenotype

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, Morten; Vissing, John

    2011-01-01

    calf hypertrophy was noted. Creatine kinase was normal or raised maximally to 500 U/l. The muscle biopsy was myopathic with increased fiber size variation and many internal nuclei, but no dystrophy. No comorbidity was found. In both cases, western blot showed a reduced dystrophin band. Genetic......With the possible introduction of exon skipping therapy in Duchenne muscular dystrophy, it has become increasingly important to know the role of each exon of the dystrophin gene to protein expression, and thus the phenotype. In this report, we present two related men with an unusually mild BMD...... associated with an exon 26 deletion. The proband, a 23-year-old man, had slightly delayed motor milestones, walking 1 1/2 years old. He had no complaints of muscle weakness, but had muscle pain. Clinical examination revealed no muscle wasting or loss of power, but his CK was 1500-7000 U/l. Muscle biopsy...

  13. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools.

    Directory of Open Access Journals (Sweden)

    Omar Soukarieh

    2016-01-01

    Full Text Available The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient's RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants, including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs. We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.

  14. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools.

    Science.gov (United States)

    Soukarieh, Omar; Gaildrat, Pascaline; Hamieh, Mohamad; Drouet, Aurélie; Baert-Desurmont, Stéphanie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

    2016-01-01

    The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient's RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.

  15. Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development.

    Directory of Open Access Journals (Sweden)

    Sushma Grellscheid

    2011-12-01

    Full Text Available Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10 is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl; Nestin-Cre(tg/+. This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.

  16. Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts

    Directory of Open Access Journals (Sweden)

    Herington Adrian C

    2007-08-01

    Full Text Available Abstract Background Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0. The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus. Results We have demonstrated the presence of an additional novel exon (exon -1 and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons. Conclusion The sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin

  17. PCR detection of a C/T polymorphism in exon 1 of the porphobilinogen deaminase gene (PBGD)

    Energy Technology Data Exchange (ETDEWEB)

    Picat, C.; Bourgeois, F.; Grandchamp, B. (Faculte de Medicine, Paris (France))

    1991-09-25

    Sequencing of exon 1 revealed a C/T polymorphism in exon 1 of human porphobilinogen deaminase gene (PBGD) at position {minus}64 relatively to the initiation translational codon. Genetic defects of PBGD are responsible for acute intermittent porphyria. The use of a 5{prime} primer with a mutated sequence to amplify the region containing this polymorphism allows its restriction analysis. After a ApaI digest of the amplified fragment, two alleles can be identified: F1: 164 bp, F2: 145 bp + 19 bp. Codominant inheritance was demonstrated in two large families with AIP.

  18. DVL1 frameshift mutations clustering in the penultimate exon cause autosomal-dominant Robinow syndrome

    DEFF Research Database (Denmark)

    White, Janson; Mazzeu, Juliana F; Hoischen, Alexander

    2015-01-01

    mutations in three of them. Targeted Sanger sequencing in additional subjects with DRS uncovered DVL1 exon 14 mutations in five individuals, including a pair of monozygotic twins. In total, six distinct frameshift mutations were found in eight subjects, and all were heterozygous truncating variants within......-canonical signaling gene WNT5A underlie a subset of autosomal-dominant Robinow syndrome (DRS) cases, but most individuals with DRS remain without a molecular diagnosis. We performed whole-exome sequencing in four unrelated DRS-affected individuals without coding mutations in WNT5A and found heterozygous DVL1 exon 14...

  19. Screening the dystrophin gene suggests a high rate of polymorphism in general but no exonic deletions in schizophrenics

    Energy Technology Data Exchange (ETDEWEB)

    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N. [Mayo Clinic/Foundation, Rochester, MN (United States)] [and others

    1994-03-15

    The dystrophin gene, located at chromosome Xp21, was evaluated as a candidate gene in chronic schizophrenia in response to the report of a large family in which schizophrenia cosegregated with Becker muscular dystrophy. Genomic DNA from 94 men with chronic schizophrenia was evaluated by Southern blot analysis using cDNA probes that span exons 1-59. No exonic deletions were identified. An unexpectedly high rate of polymorphism was calculated in this study and two novel polymorphisms were found, demonstrating the usefulness of the candidate gene approach even when results of the original study are negative. 41 refs., 1 fig., 4 tabs.

  20. The JAK2V617F and CALR exon 9 mutations are shared immunogenic neoantigens in hematological malignancy

    DEFF Research Database (Denmark)

    Holmstrom, Morten Orebo; Hasselbalch, Hans Carl; Andersen, Mads Hald

    2017-01-01

    Approximately 90% of patients with the hematological malignancies termed the chronic myeloproliferative neoplasms harbor either the JAK2V617F-mutation or CALR exon 9 mutation. Both of these are recognized by T-cells, which make the mutations ideal targets for cancer immune therapy as they are sha......Approximately 90% of patients with the hematological malignancies termed the chronic myeloproliferative neoplasms harbor either the JAK2V617F-mutation or CALR exon 9 mutation. Both of these are recognized by T-cells, which make the mutations ideal targets for cancer immune therapy...

  1. Identification of POMC exonic variants associated with substance dependence and body mass index.

    Directory of Open Access Journals (Sweden)

    Fan Wang

    Full Text Available Risk of substance dependence (SD and obesity has been linked to the function of melanocortin peptides encoded by the proopiomelanocortin gene (POMC.POMC exons were Sanger sequenced in 280 African Americans (AAs and 308 European Americans (EAs. Among them, 311 (167 AAs and 114 EAs were affected with substance (alcohol, cocaine, opioid and/or marijuana dependence and 277 (113 AAs and164 EAs were screened controls. We identified 23 variants, including two common polymorphisms (rs10654394 and rs1042571 and 21 rare variants; 12 of which were novel. We used logistic regression to analyze the association between the two common variants and SD or body mass index (BMI, with sex, age, and ancestry proportion as covariates. The common variant rs1042571 in the 3'UTR was significantly associated with BMI in EAs (Overweight: P(adj = 0.005; Obese: P(adj = 0.018; Overweight+Obese: P(adj = 0.002 but not in AAs. The common variant, rs10654394, was not associated with BMI and neither common variant was associated with SD in either population. To evaluate the association between the rare variants and SD or BMI, we collapsed rare variants and tested their prevalence using Fisher's exact test. In AAs, rare variants were nominally associated with SD overall and with specific SD traits (SD: P(FET,1df = 0.026; alcohol dependence: P(FET,1df = 0.027; cocaine dependence: P(FET,1df = 0.007; marijuana dependence: P(FET,1df = 0.050 (the P-value from cocaine dependence analysis survived Bonferroni correction. There was no such effect in EAs. Although the frequency of the rare variants did not differ significantly between the normal-weight group and the overweight or obese group in either population, certain rare exonic variants occurred only in overweight or obese subjects without SD.These findings suggest that POMC exonic variants may influence risk for both SD and elevated BMI, in a population-specific manner. However, common and rare variants in this gene may exert

  2. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Δ48-50 DMD cells

    Science.gov (United States)

    De Angelis, Fernanda Gabriella; Sthandier, Olga; Berarducci, Barbara; Toso, Silvia; Galluzzi, Giuliana; Ricci, Enzo; Cossu, Giulio; Bozzoni, Irene

    2002-01-01

    Deletions and point mutations in the dystrophin gene cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy, depending on whether the translational reading frame is lost or maintained. Because internal in-frame deletions in the protein produce only mild myopathic symptoms, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore a partially corrected phenotype. Such control has been previously accomplished by the use of synthetic oligonucleotides; nevertheless, a significant drawback to this approach is caused by the fact that oligonucleotides would require periodic administrations. To circumvent this problem, we have produced several constructs able to express in vivo, in a stable fashion, large amounts of chimeric RNAs containing antisense sequences. In this paper we show that antisense molecules against exon 51 splice junctions are able to direct skipping of this exon in the human DMD deletion 48–50 and to rescue dystrophin synthesis. We also show that the highest skipping activity was found when antisense constructs against the 5′ and 3′ splice sites are coexpressed in the same cell. PMID:12077324

  3. Mutation in exon 1a of PLEC, leading to disruption of plectin isoform 1a, causes autosomal-recessive skin-only epidermolysis bullosa simplex

    NARCIS (Netherlands)

    Gostynska, Katarzyna B.; Nijenhuis, Miranda; Lemmink, Henny; Pas, Hendrikus; Pasmooij, Anna M. G.; Lang, Kristin Kernland; Castanon, Maria J.; Wiche, Gerhard; Jonkman, Marcel F.

    2015-01-01

    PLEC, the gene encoding the cytolinker protein plectin, has eight tissue-specific isoforms in humans, arising by alternate splicing of the first exon. To date, all PLEC mutations that cause epidermolysis bullosa simplex (EBS) were found in exons common to all isoforms. Due to the ubiquitous presence

  4. Species-Dependent Splice Recognition of a Cryptic Exon Resulting from a Recurrent Intronic CEP290 Mutation that Causes Congenital Blindness

    NARCIS (Netherlands)

    Garanto, A.; Duijkers, L.E.M.; Collin, R.W.J.

    2015-01-01

    A mutation in intron 26 of CEP290 (c.2991+1655A>G) is the most common genetic cause of Leber congenital amaurosis (LCA), a severe type of inherited retinal degeneration. This mutation creates a cryptic splice donor site, resulting in the insertion of an aberrant exon (exon X) into ~50% of all

  5. The Transposon Provides Messages That Yield Unique Profiles of Protein Isoforms and Acts Synergistically With to Enrich Proteome Complexity via Exonization

    Directory of Open Access Journals (Sweden)

    Yuh-Chyang Charng

    2017-02-01

    Full Text Available In exonization events, Ds1 may provide donor and/or acceptor sites for splicing after inserting into genes and be incorporated into new transcripts with new exon(s. In this study, the protein variants of Ds1 exonization yielding additional functional profile(s were studied. Unlike Ds exonization, which creates new profiles mostly by incorporating flanking intron sequences with the Ds message, Ds1 exonization additionally creates new profiles through the presence or absence of Ds1 messages. The number of unique functional profiles harboring Ds1 messages is 1.3-fold more than that of functional profiles without Ds1 messages. The highly similar 11 protein isoforms at a single insertion site also contribute to proteome complexity enrichment by exclusively creating new profiles. Particularly, Ds1 exonization produces 459 unique profiles, of which 129 cannot be built by Ds . We thus conclude that Ds and Ds1 are independent but synergistic in their capacity to enrich proteome complexity through exonization.

  6. Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease

    NARCIS (Netherlands)

    E. van der Wal (Erik); A.J. Bergsma (Atze); J. Pijnenburg (Joon); A.T. van der Ploeg (Ans); W.W.M.P. Pijnappel (Pim)

    2017-01-01

    textabstractThe most common variant causing Pompe disease is c.-32-13T>G (IVS1) in the acid α-glucosidase (GAA) gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult

  7. Nonfamilial Juvenile Polyposis Syndrome with Exon 5 Novel Mutation in SMAD 4 Gene

    Directory of Open Access Journals (Sweden)

    Amna Ahmed

    2017-01-01

    Full Text Available Juvenile polyposis syndrome (JPS is a rare autosomal dominant hereditary disorder, characterized by multiple juvenile polyps in the gastrointestinal tract and an increased risk of colorectal cancer. JPS is most frequently caused by mutations in the SMAD4 or BMPR1A genes. Herein, we report a child with juvenile polyposis syndrome (JPS with a novel mutation in the SMAD4 gene. An 8-year-old boy presented with recurrent rectal bleeding and was found to have multiple polyps in the entire colon. The histology of the resected polyps was consistent with juvenile polyps. Subsequent genetic screening revealed a novel mutation in SMAD4, exon 5 (p.Ser144Stop. To the best of our knowledge, this mutation has not been reported before. Offering genotypic diagnosis for patients with JPS is an important step for strategic plan of management.

  8. The exon junction complex component Magoh controls brain size by regulating neural stem cell division

    Science.gov (United States)

    Silver, Debra L.; Watkins-Chow, Dawn E.; Schreck, Karisa C.; Pierfelice, Tarran J.; Larson, Denise M.; Burnetti, Anthony J.; Liaw, Hung-Jiun; Myung, Kyungjae; Walsh, Christopher A.; Gaiano, Nicholas; Pavan, William J.

    2010-01-01

    Summary Brain structure and size requires precise division of neural stem cells (NSCs), which self-renew and generate intermediate neural progenitors (INPs) and neurons. The factors that regulate NSCs remain poorly understood, as do mechanistic explanations of how aberrant NSC division causes reduced brain size as seen in microcephaly. Here we demonstrate that Magoh, a component of the exon junction complex (EJC) that binds RNA, controls mouse cerebral cortical size by regulating NSC division. Magoh haploinsufficiency causes microcephaly due to INP depletion and neuronal apoptosis. Defective mitosis underlies these phenotypes as depletion of EJC components disrupts mitotic spindle orientation and integrity, chromosome number, and genomic stability. In utero rescue experiments revealed that a key function of Magoh is to control levels of the microcephaly-associated protein, LIS1, during neurogenesis. This study uncovers new requirements for the EJC in brain development, NSC maintenance, and mitosis, thus implicating this complex in the pathogenesis of microcephaly. PMID:20364144

  9. Not all floating-harbor syndrome cases are due to mutations in exon 34 of SRCAP.

    Science.gov (United States)

    Le Goff, Carine; Mahaut, Clémentine; Bottani, Armand; Doray, Berenice; Goldenberg, Alice; Moncla, Anne; Odent, Sylvie; Nitschke, Patrick; Munnich, Arnold; Faivre, Laurence; Cormier-Daire, Valérie

    2013-01-01

    Floating-Harbor syndrome (FHS) is a rare disorder characterized by short stature, delayed bone age, speech delay, and dysmorphic facial features. We report here the molecular analysis of nine cases, fulfilling the diagnostic criteria for FHS. Using exome sequencing, we identified SRCAP as the disease gene in two cases and subsequently found SRCAP truncating mutations in 6/9 cases. All mutations occurred de novo and were located in exon 34, in accordance with the recent report of Hood et al. However, the absence of SRCAP mutations in 3/9 cases supported genetic heterogeneity of FH syndrome. Importantly, no major clinical differences were observed supporting clinical homogeneity in this series of FHS patients. © 2012 Wiley Periodicals, Inc.

  10. Characterization of six mutations in Exon 37 of neurofibromatosis type 1 gene

    Energy Technology Data Exchange (ETDEWEB)

    Upadhyaya, M.; Osborn, M.; Maynard, J.; Harper, P. [Institute of Medical Genetics, Cardiff, Wales (United Kingdom)

    1996-07-26

    Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders, with an incidence of 1 in 3,000. We screened a total of 320 unrelated NF1 patients for mutations in exon 37 of the NF1 gene. Six independent mutations were identified, of which three are novel, and these include a recurrent nonsense mutation identified in 2 unrelated patients at codon 2281 (G2281X), a 1-bp insertion (6791 ins A) resulting in a change of TAG (tyrosine) to a TAA (stop codon), and a 3-bp deletion (6839 del TAC) which generated a frameshift. Another recurrent nonsense mutation, Y2264X, which was detected in 2 unrelated patients in this study, was also previously reported in 2 NF1 individuals. All the mutations were identified within a contiguous 49-bp sequence. Further studies are warranted to support the notion that this region of the gene contains highly mutable sequences. 17 refs., 2 figs., 1 tab.

  11. SNP identification and polymorphism analysis in exon 2 of the horse myostatin gene.

    Science.gov (United States)

    Baron, E E; Lopes, M S; Mendonça, D; da Câmara Machado, A

    2012-04-01

    The myostatin gene (MSTN) belongs to the TGF-β superfamily of secreted growth and differentiation factors and is responsible for embryonic and adult skeletal muscle development. In this study, exon 2 of the MSTN gene, which encodes part of the TGF-β pro-peptide, was sequenced in 332 horses of 20 different breeds and compared with the horse MSTN gene sequence deposited in GenBank. The sequences obtained revealed the presence of 11 haplotypes represented by 10 variable nucleotide mutations, eight of them corresponding to amino acid sequence changes. This gene shows a high variability when compared with other genes. This might be an indication that some breeds have the same ancestry but different pressures of selection. © 2011 The Authors, Animal Genetics © 2011 Stichting International Foundation for Animal Genetics.

  12. Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation “Memories”

    Directory of Open Access Journals (Sweden)

    Joshua D. Tompkins

    2016-02-01

    Full Text Available The directed differentiation of human cardiomyocytes (CMs from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation “memories” persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors.

  13. Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation “Memories”

    Science.gov (United States)

    Tompkins, Joshua D.; Jung, Marc; Chen, Chang-yi; Lin, Ziguang; Ye, Jingjing; Godatha, Swetha; Lizhar, Elizabeth; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

    2016-01-01

    The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation “memories” persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors. PMID:26981572

  14. Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation "Memories".

    Science.gov (United States)

    Tompkins, Joshua D; Jung, Marc; Chen, Chang-Yi; Lin, Ziguang; Ye, Jingjing; Godatha, Swetha; Lizhar, Elizabeth; Wu, Xiwei; Hsu, David; Couture, Larry A; Riggs, Arthur D

    2016-02-01

    The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation "memories" persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors.

  15. Becker muscular dystrophy with widespread muscle hypertrophy and a non-sense mutation of exon 2.

    Science.gov (United States)

    Witting, N; Duno, M; Vissing, J

    2013-01-01

    Becker muscular dystrophy features progressive proximal weakness, wasting and often focal hypertrophy. We present a patient with pain and cramps from adolescence. Widespread muscle hypertrophy, preserved muscle strength and a 10-20-fold raised CPK were noted. Muscle biopsy was dystrophic, and Western blot showed a 95% reduction of dystrophin levels. Genetic analyses revealed a non-sense mutation in exon 2 of the dystrophin gene. This mutation is predicted to result in a Duchenne phenotype, but resulted in a mild Becker muscular dystrophy with widespread muscle hypertrophy. We suggest that this unusual phenotype is caused by translation re-initiation downstream from the mutation site. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. WNT10A exonic variant increases the risk of keratoconus by decreasing corneal thickness.

    Science.gov (United States)

    Cuellar-Partida, Gabriel; Springelkamp, Henriët; Lucas, Sionne E M; Yazar, Seyhan; Hewitt, Alex W; Iglesias, Adriana I; Montgomery, Grant W; Martin, Nicholas G; Pennell, Craig E; van Leeuwen, Elisabeth M; Verhoeven, Virginie J M; Hofman, Albert; Uitterlinden, André G; Ramdas, Wishal D; Wolfs, Roger C W; Vingerling, Johannes R; Brown, Matthew A; Mills, Richard A; Craig, Jamie E; Klaver, Caroline C W; van Duijn, Cornelia M; Burdon, Kathryn P; MacGregor, Stuart; Mackey, David A

    2015-09-01

    Keratoconus is a degenerative eye condition which results from thinning of the cornea and causes vision distortion. Treatments such as ultraviolet (UV) cross-linking have proved effective for management of keratoconus when performed in early stages of the disease. The central corneal thickness (CCT) is a highly heritable endophenotype of keratoconus, and it is estimated that up to 95% of its phenotypic variance is due to genetics. Genome-wide association efforts of CCT have identified common variants (i.e. minor allele frequency (MAF) >5%). However, these studies typically ignore the large set of exonic variants whose MAF is usually low. In this study, we performed a CCT exome-wide association analysis in a sample of 1029 individuals from a population-based study in Western Australia. We identified a genome-wide significant exonic variant rs121908120 (P = 6.63 × 10(-10)) in WNT10A. This gene is 437 kb from a gene previously associated with CCT (USP37). We showed in a conditional analysis that the WNT10A variant completely accounts for the signal previously seen at USP37. We replicated our finding in independent samples from the Brisbane Adolescent Twin Study, Twin Eye Study in Tasmania and the Rotterdam Study. Further, we genotyped rs121908120 in 621 keratoconus cases and compared the frequency to a sample of 1680 unscreened controls from the Queensland Twin Registry. We found that rs121908120 increases the risk of keratoconus two times (odds ratio 2.03, P = 5.41 × 10(-5)). © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  17. The differential roles of Slit2-exon 15 splicing variants in angiogenesis and HUVEC permeability.

    Science.gov (United States)

    Yang, Yun-Chiu; Chen, Pei-Ni; Wang, Siou-Yu; Liao, Chen-Yi; Lin, Yu-Ying; Sun, Shih-Rhong; Chiu, Chun-Ling; Hsieh, Yih-Shou; Shieh, Jia-Ching; Chang, Jinghua Tsai

    2015-07-01

    Slit2, a secreted glycoprotein, is down-regulated in many cancers. Slit2/Robo signaling pathway plays an important, but controversial, role in angiogenesis. We identified splicing variants of Slit2 at exon 15, Slit2-WT and Slit2-ΔE15, with differential effects on proliferation and invasive capability of lung cancer cells. The aim of this study was to elucidate the differential roles of these exon 15 splicing variants in angiogenesis. Our results revealed that both Slit2-WT and Slit2-ΔE15 inhibit motility of human umbilical vein endothelial cells (HUVECs). The conditioned medium (CM) collected from CL1-5/VC or CL1-5/Slit2-WT lung adenocarcinoma cells blocked HUVEC tube formation and angiogenesis on chorioallantoic membrane (CAM) assay when compared with untreated HUVECs and CAM, respectively. However, CM of CL1-5/Slit2-ΔE15 restored the quality of tubes and the size of vessels. Although both Slit2-WT and Slit2-ΔE15 inhibited permeability induced by CM of cancer cells, Slit2-ΔE15 exhibited stronger effect. These results suggested that Slit2-ΔE15 plays important roles in normalization of blood vessels by enhancing tube quality and tightening endothelial cells, while Slit2-WT only enhances tightening of endothelial cells. It appears that Robo4 is responsible for Slit2 isoform-mediated inhibition of permeability, while neither Robo1 nor Robo4 is required for Slit2-ΔE15-enhanced tube quality. The results of this study suggest that Slit2-ΔE15 splicing form is a promising molecule for normalizing blood vessels around a tumor, which, in turn, may increase efficacy of chemotherapy and radiotherapy.

  18. Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

    Directory of Open Access Journals (Sweden)

    Lacombe Didier

    2011-05-01

    Full Text Available Abstract Background Usher syndrome (USH combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3. Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool. Methods We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3. Results Biallelic mutations were detected in 39 patients (72% and monoallelic mutations in an additional 10 patients (18.5%. In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%, and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48% were novel. Conclusions Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.

  19. An exonic insertion within Tex14 gene causes spermatogenic arrest in pigs

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    Sironen Anu

    2011-12-01

    Full Text Available Abstract Background Male infertility is an increasing problem in all domestic species including man. Localization and identification of genes involved in defects causing male infertility provide valuable information of specific events in sperm development. Sperm development is a complex process, where diploid spermatogonia develop into haploid, highly specialized spermatozoa. Correct expression and function of various genes and their protein products are required for production of fertile sperm. We have identified an infertility defect in Finnish Yorkshire boars caused by spermatogenic arrest. The aim of this study was to locate the disease associated region using genome wide screen with the PorcineSNP60 Beadchip and identify the causal mutation by candidate gene approach. Results In the Finnish Yorkshire pig population the spermatogenic arrest (SA defect appears to be of genetic origin and causes severe degeneration of germ cells and total absence of spermatozoa. Genome wide scan with the PorcineSNP60 Beadchip localized the SA defect to porcine chromosome 12 in a 2 Mbp region. Sequencing of a candidate gene Tex14 revealed a 51 bp insertion within exon 27, which caused differential splicing of the exon and created a premature translation stop codon. The expression of Tex14 was markedly down regulated in the testis of a SA affected boar compared to control boars and no protein product was identified by Western blotting. The SA insertion sequence was also found within intron 27 in all analyzed animals, thus the insertion appears to be a possible duplication event. Conclusion In this study we report the identification of a causal mutation for infertility caused by spermatogenic arrest at an early meiotic phase. Our results highlight the role of TEX14 specifically in spermatogenesis and the importance of specific genomic remodeling events as causes for inherited defects.

  20. Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.

    Science.gov (United States)

    Vulin, Adeline; Barthélémy, Inès; Goyenvalle, Aurélie; Thibaud, Jean-Laurent; Beley, Cyriaque; Griffith, Graziella; Benchaouir, Rachid; le Hir, Maëva; Unterfinger, Yves; Lorain, Stéphanie; Dreyfus, Patrick; Voit, Thomas; Carlier, Pierre; Blot, Stéphane; Garcia, Luis

    2012-11-01

    Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.

  1. Truncating mutations in the last exon of NOTCH3 cause lateral meningocele syndrome.

    Science.gov (United States)

    Gripp, Karen W; Robbins, Katherine M; Sobreira, Nara L; Witmer, P Dane; Bird, Lynne M; Avela, Kristiina; Makitie, Outi; Alves, Daniela; Hogue, Jacob S; Zackai, Elaine H; Doheny, Kimberly F; Stabley, Deborah L; Sol-Church, Katia

    2015-02-01

    Lateral meningocele syndrome (LMS, OMIM%130720), also known as Lehman syndrome, is a very rare skeletal disorder with facial anomalies, hypotonia and meningocele-related neurologic dysfunction. The characteristic lateral meningoceles represent the severe end of the dural ectasia spectrum and are typically most severe in the lower spine. Facial features of LMS include hypertelorism and telecanthus, high arched eyebrows, ptosis, midfacial hypoplasia, micrognathia, high and narrow palate, low-set ears and a hypotonic appearance. Hyperextensibility, hernias and scoliosis reflect a connective tissue abnormality, and aortic dilation, a high-pitched nasal voice, wormian bones and osteolysis may be present. Lateral meningocele syndrome has phenotypic overlap with Hajdu-Cheney syndrome. We performed exome resequencing in five unrelated individuals with LMS and identified heterozygous truncating NOTCH3 mutations. In an additional unrelated individual Sanger sequencing revealed a deleterious variant in the same exon 33. In total, five novel de novo NOTCH3 mutations were identified in six unrelated patients. One had a 26 bp deletion (c.6461_6486del, p.G2154fsTer78), two carried the same single base pair insertion (c.6692_93insC, p.P2231fsTer11), and three individuals had a nonsense point mutation at c.6247A > T (pK2083*), c.6663C > G (p.Y2221*) or c.6732C > A, (p.Y2244*). All mutations cluster into the last coding exon, resulting in premature termination of the protein and truncation of the negative regulatory proline-glutamate-serine-threonine rich PEST domain. Our results suggest that mutant mRNA products escape nonsense mediated decay. The truncated NOTCH3 may cause gain-of-function through decreased clearance of the active intracellular product, resembling NOTCH2 mutations in the clinically related Hajdu-Cheney syndrome and contrasting the NOTCH3 missense mutations causing CADASIL. © 2014 Wiley Periodicals, Inc.

  2. Transcriptome networks in the mouse retina: An exon level BXD RI database.

    Science.gov (United States)

    King, Rebecca; Lu, Lu; Williams, Robert W; Geisert, Eldon E

    2015-01-01

    Differences in gene expression provide diverse retina phenotypes and may also contribute to susceptibility to injury and disease. The present study defines the transcriptome of the retina in the BXD RI strain set, using the Affymetrix Mouse Gene 2.0 ST array to investigate all exons of traditional protein coding genes, non-coding RNAs, and microRNAs. These data are presented in a highly interactive database on the GeneNetwork website. In the Normal Retina Database, the mRNA levels of the transcriptome from retinas was quantified using the Affymetrix Mouse Gene 2.0 ST array. This database consists of data from male and female mice. The data set includes a total of 52 BXD RI strains, the parental strains (C57BL/6J and DBA/2J), and a reciprocal cross. In combination with GeneNetwork, the Department of Defense (DoD) Congressionally Directed Medical Research Programs (CDMRP) Normal Retina Database provides a large resource for mapping, graphing, analyzing, and testing complex genetic networks. Protein-coding and non-coding RNAs can be used to map quantitative trait loci (QTLs) that contribute to expression differences among the BXD strains and to establish links between classical ocular phenotypes associated with differences in the genomic sequence. Using this resource, we extracted transcriptome signatures for retinal cells and defined genetic networks associated with the maintenance of the normal retina. Furthermore, we examined differentially expressed exons within a single gene. The high level of variation in mRNA levels found among the BXD RI strains makes it possible to identify expression networks that underline differences in retina structure and function. Ultimately, we will use this database to define changes that occur following blast injury to the retina.

  3. Evidence for widespread exonic small RNAs in the glaucophyte alga Cyanophora paradoxa.

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    Jeferson Gross

    Full Text Available RNAi (RNA interference relies on the production of small RNAs (sRNAs from double-stranded RNA and comprises a major pathway in eukaryotes to restrict the propagation of selfish genetic elements. Amplification of the initial RNAi signal by generation of multiple secondary sRNAs from a targeted mRNA is catalyzed by RNA-dependent RNA polymerases (RdRPs. This phenomenon is known as transitivity and is particularly important in plants to limit the spread of viruses. Here we describe, using a genome-wide approach, the distribution of sRNAs in the glaucophyte alga Cyanophora paradoxa. C. paradoxa is a member of the supergroup Plantae (also known as Archaeplastida that includes red algae, green algae, and plants. The ancient (>1 billion years ago split of glaucophytes within Plantae suggests that C. paradoxa may be a useful model to learn about the early evolution of RNAi in the supergroup that ultimately gave rise to plants. Using next-generation sequencing and bioinformatic analyses we find that sRNAs in C. paradoxa are preferentially associated with mRNAs, including a large number of transcripts that encode proteins arising from different functional categories. This pattern of exonic sRNAs appears to be a general trend that affects a large fraction of mRNAs in the cell. In several cases we observe that sRNAs have a bias for a specific strand of the mRNA, including many instances of antisense predominance. The genome of C. paradoxa encodes four sequences that are homologous to RdRPs in Arabidopsis thaliana. We discuss the possibility that exonic sRNAs in the glaucophyte may be secondarily derived from mRNAs by the action of RdRPs. If this hypothesis is confirmed, then transitivity may have had an ancient origin in Plantae.

  4. A novel deletion of SNURF/SNRPN exon 1 in a patient with Prader-Willi-like phenotype.

    Science.gov (United States)

    Cao, Yang; AlHumaidi, Susan S; Faqeih, Eissa A; Pitel, Beth A; Lundquist, Patrick; Aypar, Umut

    2017-08-01

    Here we report the smallest deletion involving SNURF/SNRPN that causes major symptoms of Prader-Willi syndrome (PWS), including hypotonia, dysmorphic features, intellectual disability, and obesity. A female patient with the aforementioned and additional features was referred to the Mayo Clinic Cytogenetics laboratory for genetic testing. Chromosomal microarray analysis and subsequent Sanger sequencing identified a de novo 6.4 kb deletion at 15q11.2, containing exon 1 of the SNURF gene and exon 1 of the shortest isoform of the SNRPN gene. SNURF/SNRPN exon 1, which is methylated on the silent maternal allele, is associated with acetylated histones on the expressed paternal allele. This region also overlaps with the PWS-imprinting center (IC). Subsequent molecular methylation analysis was performed using methylation-specific MLPA (MS-MLPA), which characterized that the deletion of SNURF/SNRPN exon 1 was paternal in origin, consistent with the PWS-like phenotype. Since SNURF/SNRPN gene and the PWS-IC are known to regulate snoRNAs, it is likely that the PWS-like phenotype observed in patients with paternal SNURF/SNRPN deletion is due to the disrupted expression of SNORD116 snoRNAs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. CD44 exon 6 expression as a possible early prognostic factor in primary node negative breast carcinoma.

    Science.gov (United States)

    Guriec, N; Marcellin, L; Gairard, B; Caldéroli, H; Wilk, A; Renaud, R; Bergerat, J P; Oberling, F

    1996-10-01

    Breast cancer is the most common female malignancy affecting approximately one woman in eight. Many attempts have been made to define markers which may have potential clinical applications in diagnosis as well as therapy. New isoforms of CD44 with alternative spliced exons have recently been described. We studied the expression of CD44 exon 6 using a semi-quantitative RT-PCR reaction on a panel of 25 normal breast specimens, 10 mammary fibroadenomas, eight cystic samples and 52 primary breast tumors. Significant correlation was found between CD44 exon 6 expression and the overall survival of the N-M-population, P = 0.032, (logrank test by Mantel's method). The same result was also observed for the disease-free survival, P = 0.000002 (logrank test by Mantel's method). CD44 exon 6 expression, as detected by our RT-PCR-based method, might be a useful prognostic indicator of metastasis in breast cancer. However, these preliminary results need to be confirmed by later retrospective and prospective studies on a larger number of patients.

  6. Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.

    Science.gov (United States)

    Wein, Nicolas; Vulin, Adeline; Falzarano, Maria S; Szigyarto, Christina Al-Khalili; Maiti, Baijayanta; Findlay, Andrew; Heller, Kristin N; Uhlén, Mathias; Bakthavachalu, Baskar; Messina, Sonia; Vita, Giuseppe; Passarelli, Chiara; Brioschi, Simona; Bovolenta, Matteo; Neri, Marcella; Gualandi, Francesca; Wilton, Steve D; Rodino-Klapac, Louise R; Yang, Lin; Dunn, Diane M; Schoenberg, Daniel R; Weiss, Robert B; Howard, Michael T; Ferlini, Alessandra; Flanigan, Kevin M

    2014-09-01

    Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.

  7. Systemic Antisense Therapeutics for Dystrophin and Myostatin Exon Splice Modulation Improve Muscle Pathology of Adult mdx Mice.

    Science.gov (United States)

    Lu-Nguyen, Ngoc; Malerba, Alberto; Popplewell, Linda; Schnell, Fred; Hanson, Gunnar; Dickson, George

    2017-03-17

    Antisense-mediated exon skipping is a promising approach for the treatment of Duchenne muscular dystrophy (DMD), a rare life-threatening genetic disease due to dystrophin deficiency. Such an approach can restore the disrupted reading frame of dystrophin pre-mRNA, generating a truncated form of the protein. Alternatively, antisense therapy can be used to induce destructive exon skipping of myostatin pre-mRNA, knocking down myostatin expression to enhance muscle strength and reduce fibrosis. We have reported previously that intramuscular or intraperitoneal antisense administration inducing dual exon skipping of dystrophin and myostatin pre-mRNAs was beneficial in mdx mice, a mouse model of DMD, although therapeutic effects were muscle type restricted, possibly due to the delivery routes used. Here, following systemic intravascular antisense treatment, muscle strength and body activity of treated adult mdx mice increased to the levels of healthy controls. Importantly, hallmarks of muscular dystrophy were greatly improved in mice receiving the combined exon-skipping therapy, as compared to those receiving dystrophin antisense therapy alone. Our results support the translation of antisense therapy for dystrophin restoration and myostatin inhibition into the clinical setting for DMD. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex.

    Science.gov (United States)

    Saulière, Jérôme; Murigneux, Valentine; Wang, Zhen; Marquenet, Emélie; Barbosa, Isabelle; Le Tonquèze, Olivier; Audic, Yann; Paillard, Luc; Roest Crollius, Hugues; Le Hir, Hervé

    2012-11-01

    The exon junction complex (EJC) is a central effector of the fate of mRNAs, linking nuclear processing to mRNA transport, translation and surveillance. However, little is known about its transcriptome-wide targets. We used cross-linking and immunoprecipitation methods coupled to high-throughput sequencing (CLIP-seq) in human cells to identify the binding sites of the DEAD-box helicase eIF4AIII, an EJC core component. CLIP reads form peaks that are located mainly in spliced mRNAs. Most expressed exons harbor peaks either in the canonical EJC region, located ~24 nucleotides upstream of exonic junctions, or in other noncanonical regions. Notably, both of these types of peaks are preferentially associated with unstructured and purine-rich sequences containing the motif GAAGA, which is a potential binding site for EJC-associated factors. Therefore, EJC positions vary spatially and quantitatively between exons. This transcriptome-wide mapping of human eIF4AIII reveals unanticipated aspects of the EJC and broadens its potential impact on post-transcriptional regulation.

  9. Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA

    DEFF Research Database (Denmark)

    Andersen, Christian Brix Folsted; Ballut, Lionel; Johansen, Jesper Sanderhoff

    2006-01-01

    In higher eukaryotes, a multiprotein exon junction complex is deposited on spliced messenger RNAs. The complex is organized around a stable core, which serves as a binding platform for numerous factors that influence messenger RNA function. Here, we present the crystal structure of a tetrameric e...

  10. Molecular analysis of exons 6 and 7 of phenylalanine hydroxylase gene mutations in Phenylketonuria patients in Western Iran

    Science.gov (United States)

    Moradi, Keyvan; Alibakhshi, Reza; Ghadiri, Keyghobad; Khatami, Saeid Reza; Galehdari, Hamid

    2012-01-01

    BACKGROUND: Phenylketonuria (PKU) is an inborn error of amino acid metabolism that results from a deficiency of phenylalanine hydroxylase (PAH). According to PAH database, exons 6 and 7 and their flanking introns of PAH gene contain the greatest number of mutant alleles. Therefore, as a preliminary study, nucleotide sequence analysis of exons 6 and 7 of the PAH gene has been performed in 25 PKU patients whose ancestors lived in Kermanshah province of Iran. To date, there has been no mutation data describing the genotypes of the PKU disease in this Kurdish ethnic region background. MATERIALS AND METHODS: Twenty-five patients (aged between 2 and 23 years) participated in this study. The DNA fragments containing two exons of the PAH gene [6 and 7] and their exon-flanking intronic sequences were amplified and sequenced. RESULTS: The total of detected mutations were R261X (8%), R176X (4%), R243Q (4%), R243X (2%) and R261Q (2%), as they accounted for 20% of all mutant alleles in this study. The identified polymorphisms are: IVS5 -54 G > A (22%), Q232Q (8%) and V245V (4%). All of the detected mutations in this study are related to CpG dinucleotides in the PAH gene sequence. CONCLUSION: The frequency of R261X, the most common mutation in our study, in Iranian population is Iran. Therefore, it may be necessary to study the PAH gene mutations in other provinces of Iran separately. PMID:23716935

  11. A DNMT3B alternatively spliced exon and encoded peptide are novel biomarkers of human pluripotent stem cells.

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    Sailesh Gopalakrishna-Pillai

    Full Text Available A major obstacle in human stem cell research is the limited number of reagents capable of distinguishing pluripotent stem cells from partially differentiated or incompletely reprogrammed derivatives. Although human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs express numerous alternatively spliced transcripts, little attention has been directed at developing splice variant-encoded protein isoforms as reagents for stem cell research. In this study, several genes encoding proteins involved in important signaling pathways were screened to detect alternatively spliced transcripts that exhibited differential expression in pluripotent stem cells (PSCs relative to spontaneously differentiated cells (SDCs. Transcripts containing the alternatively spliced exon 10 of the de novo DNA methyltransferase gene, DNMT3B, were identified that are expressed in PSCs. To demonstrate the utility and superiority of splice variant specific reagents for stem cell research, a peptide encoded by DNMT3B exon 10 was used to generate an antibody, SG1. The SG1 antibody detects a single DNMT3B protein isoform that is expressed only in PSCs but not in SDCs. The SG1 antibody is also demonstrably superior to other antibodies at distinguishing PSCs from SDCs in mixed cultures containing both pluripotent stem cells and partially differentiated derivatives. The tightly controlled down regulation of DNMT3B exon 10 containing transcripts (and exon 10 encoded peptide upon spontaneous differentiation of PSCs suggests that this DNMT3B splice isoform is characteristic of the pluripotent state. Alternatively spliced exons, and the proteins they encode, represent a vast untapped reservoir of novel biomarkers that can be used to develop superior reagents for stem cell research and to gain further insight into mechanisms controlling stem cell pluripotency.

  12. Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy.

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    Johann Böhm

    2013-06-01

    Full Text Available Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM, a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM. However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD. Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.

  13. Evolution of the histones: free play with exon shuffling Evolucion de las histonas: Juego libre con reordenamiento de exones al azar

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    G. CECILIA TORO

    2001-03-01

    Full Text Available In higher eukaryotes, the nuclear DNA is organized for transcription, replication and mitosis in competent chromatin and chromosomes. The basic unit of chromatin is the nucleosome. This entity is formed by 168 base pairs of DNA wound around an octamer of histones, this octamer of histones consist of two copies of H2A, H2B, H3 and H4. The DNA is sealed in its input and output point by a histone linker: histone H1. Histones were supposed to be very conserved proteins. However, during the past few years it was found that these proteins present a high degree of divergency in several lower eukaryotes. In Trypanosoma, it was found that histones H3 and H4, which are at the center of the nucleosomal organization, showed more than 30 % of divergency, while histone H1 corresponded to only one of the three peptide domains present in higher eukaryotes. These features of Trypanosoma histones may explain, at least in part, the unability of chromatin to condense into chromosomes during the cell division in these parasites. Evolution of histones was usually considered as peculiar, with several proposals which are difficult to reconcile with experimental data. In the present work, it is proposed that histones followed the same evolutionary route as many other proteins. Considering that exons code for structural and functional domains in proteins and that, at the origin of eukaryotes, the histones, as other proteins, could be formed by "units" (mecano theory, it was expected that these units or domains eventually would be found in living organisms exhibiting primitive features. Furthermore, those units could work independently. Our results on the structure of Trypanosoma cruzi histone genes and proteins as well as the analysis of other histones from different species fit with this proposalEn los eucariontes superiores, el DNA nuclear se organiza en cromatina competente y en cromosomas para la transcripción, replicación y mitosis. La unidad básica de la

  14. Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences.

    Science.gov (United States)

    Günther, Katharina; Rust, Mareike; Leers, Joerg; Boettger, Thomas; Scharfe, Maren; Jarek, Michael; Bartkuhn, Marek; Renkawitz, Rainer

    2013-03-01

    The heterogeneous collection of nucleosome remodelling and deacetylation (NuRD) complexes can be grouped into the MBD2- or MBD3-containing complexes MBD2-NuRD and MBD3-NuRD. MBD2 is known to bind to methylated CpG sequences in vitro in contrast to MBD3. Although functional differences have been described, a direct comparison of MBD2 and MBD3 in respect to genome-wide binding and function has been lacking. Here, we show that MBD2-NuRD, in contrast to MBD3-NuRD, converts open chromatin with euchromatic histone modifications into tightly compacted chromatin with repressive histone marks. Genome-wide, a strong enrichment for MBD2 at methylated CpG sequences is found, whereas CpGs bound by MBD3 are devoid of methylation. MBD2-bound genes are generally lower expressed as compared with MBD3-bound genes. When depleting cells for MBD2, the MBD2-bound genes increase their activity, whereas MBD2 plus MBD3-bound genes reduce their activity. Most strikingly, MBD3 is enriched at active promoters, whereas MBD2 is bound at methylated promoters and enriched at exon sequences of active genes.

  15. Characterization of an Equine α-S2-Casein Variant Due to a 1.3 kb Deletion Spanning Two Coding Exons.

    Science.gov (United States)

    Brinkmann, Julia; Koudelka, Tomas; Keppler, Julia K; Tholey, Andreas; Schwarz, Karin; Thaller, Georg; Tetens, Jens

    2015-01-01

    The production and consumption of mare's milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows' milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplication. Findings at the DNA-level have been verified by cDNA sequencing from horse milk of mares with different genotypes. At the protein-level, we were able to show by SDS-page and in-gel digestion with subsequent LC-MS analysis that both proteins are actually expressed. The comparison with published sequences of other equids revealed that the deletion has probably occurred before the ancestor of present-day asses and zebras diverged from the horse lineage.

  16. Characterization of an Equine α-S2-Casein Variant Due to a 1.3 kb Deletion Spanning Two Coding Exons.

    Directory of Open Access Journals (Sweden)

    Julia Brinkmann

    Full Text Available The production and consumption of mare's milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows' milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplication. Findings at the DNA-level have been verified by cDNA sequencing from horse milk of mares with different genotypes. At the protein-level, we were able to show by SDS-page and in-gel digestion with subsequent LC-MS analysis that both proteins are actually expressed. The comparison with published sequences of other equids revealed that the deletion has probably occurred before the ancestor of present-day asses and zebras diverged from the horse lineage.

  17. Expanded spectrum of exon 33 and 34 mutations in SRCAP and follow-up in patients with Floating-Harbor syndrome.

    Science.gov (United States)

    Seifert, Wenke; Meinecke, Peter; Krüger, Gabriele; Rossier, Eva; Heinritz, Wolfram; Wüsthof, Achim; Horn, Denise

    2014-11-30

    Floating-Harbor syndrome is a rare autosomal dominant short stature syndrome with retarded speech development, intellectual disability and dysmorphic facial features. Recently dominant mutations almost exclusively located in exon 34 of the Snf2-related CREBBP activator protein gene were identified to cause FHS. Here we report the genetic analysis of 5 patients fulfilling the diagnostic criteria of FHS obtained by Sanger sequencing. All of them presented with short stature, speech delay as well as psychomotor delay and typical facial dysmorphism. Three patients showed a good response to growth hormone treatment. Two patients demonstrate novel, heterozygous de novo frameshift mutations in exon 34 (c.7396delA and c.7218dupT) leading to premature stop mutations in SRCAP (p.Val2466Tyrfs*9 and p.Gln2407Serfs*36, respectively). In two further patients we found already known SRCAP mutations in exon 34, c.7330C > T and c.7303C > T, respectively, which also lead to premature stop codons: p.Arg2444* and p.Arg2435*. In one patient, we identified a novel de novo stop mutation in exon 33 (c.6985C > T, p.Arg2329*) demonstrating that not all FHS cases are caused by mutations in exon 34 of SRCAP. Our data confirm a mutational hot spot in the final exon of SRCAP in the majority of FHS patients but also show that exon 33 of this gene can be affected.

  18. Germline mutations of BRCA1 gene exon 11 are not associated with platinum response neither with survival advantage in patients with primary ovarian cancer: understanding the clinical importance of one of the biggest human exons. A study of the Tumor Bank Ovarian Cancer (TOC) Consortium.

    Science.gov (United States)

    Dimitrova, Desislava; Ruscito, Ilary; Olek, Sven; Richter, Rolf; Hellwag, Alexander; Türbachova, Ivana; Woopen, Hannah; Baron, Udo; Braicu, Elena Ioana; Sehouli, Jalid

    2016-09-01

    Germline mutations in BRCA1 gene have been reported in up to 20 % of epithelial ovarian cancer (EOC) patients. Distinct clinical characteristics have been attributed to this special EOC population. We hypothesized that mutations in different BRCA1 gene exons may differently affect the clinical course of the disease. The aim of this study was to analyze, in a large cohort of primary EOCs, the clinical impact of mutations in BRCA1 gene exon 11, the largest exon of the gene sequence encoding the 60 % of BRCA1 protein. Two hundred sixty-three primary EOC patients, treated between 2000 and 2008 at Charité University Hospital of Berlin, were included. Patients' blood samples were obtained from the Tumor Ovarian Cancer (TOC) Network ( www.toc-network.de ). Direct sequencing of BRCA1 gene exon 11 was performed for each patient to detect mutations. Based on their BRCA1 exon 11 mutational status, patients were compared regarding clinico-pathological variables and survival. Mutations in BRCA1 exon 11 were found in 18 out of 263 patients (6.8 %). Further 10/263 (3.8 %) cases showed variants of uncertain significance (VUS). All exon 11 BRCA1-positive tumors (100 %) were Type 2 ovarian carcinomas (p = 0.05). Age at diagnosis was significantly younger in Type 2 exon 11 mutated patients (p = 0.01). On multivariate analysis, BRCA1 exon 11 mutational status was not found to be an independent predictive factor for optimal cytoreduction, platinum response, or survival. Mutations in BRCA1 gene exon 11 seem to predispose women to exclusively develop a Type 2 ovarian cancer at younger age. Exon 11 BRCA1-mutated EOC patients showed distinct clinico-pathological features but similar clinical outcome with respect to sporadic EOC patients.

  19. Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

    Science.gov (United States)

    Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

    2015-01-01

    In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene. © 2015 by the Association of Clinical Scientists, Inc.

  20. Somatic mutations of the ret Protooncogene in sporadic medullary thyroid carcinoma are not restricted to exon 16 and are associated with tumor recurrence

    Energy Technology Data Exchange (ETDEWEB)

    Romei, C.; Elisei, R.; Pinchera, A. [Univ. of Pisa (Italy)] [and others

    1996-04-01

    Germline point mutations in exons 10, 11, and 16 of the ret protooncogene have been identified as causative in multiple endocrine neoplasia type 2 and in familial medullary thyroid carcinoma (MTC). Somatic point mutations of the same gene, exclusively associated with codon 918 of exon 16, have also been reported in few cases of sporadic medullary thyroid carcinoma. We analyzed the blood and tumor DNA of 19 patients with sporadic MTC and 6 patients with primary parathyroid adenoma for point mutations at exons 10, 11, and 16 of the ret protooncogene by restriction analysis of the PCR-amplified product and by sequence analysis of exons 10 and 11. A Cys{sup 634}{r_arrow}Tyr mutation was found in both the tumoral and blood DNA of one patient, indicating that he was affected by an hereditary form of MTC, erroneously considered sporadic. In the other 18 patients with MTC, somatic point mutations of ret were found in 8 cases (44.4%). In 5 cases the mutation affected exon 16 (Met{sup 918}{r_arrow}Thr), and in 3 cases it affected exon 11 (Cys{sup 634}{r_arrow}Arg in 1 and Cys{sup 634}{r_arrow}Trp in 2); these 3 mutations were confirmed by sequence analysis. The remaining 10 patients had no mutation in exon 10 by either restriction analysis or sequence analysis. Clinical data showed that 75% of the patients whose tumor carried ret mutation had tumor recurrence and/or increased serum calcitonin concentrations during the postsurgical follow-up period as opposed to 10% of the patients without mutations (P < 0.02, by {chi}{sup 2} analysis). No ret mutation was found in the tumoral DNA from parathyroid adenomas. Our findings indicate that the somatic ret point mutation frequently found in sporadic MTC may affect not only exon 16 but also exon 11 and is associated with less favorable clinical outcome. 14 refs., 2 figs., 3 tabs.

  1. One missense mutation in exon 2 of the PAX5 gene in Iran.

    Science.gov (United States)

    Yazdanparast, S; Khatami, S R; Galehdari, H; Jaseb, K

    2015-12-22

    The PAX5 gene, which encodes the B-cell specific activator protein, is one of the most important factors in determination of B-cell development. This gene is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). For example, point mutations, deletions, as well as other gene rearrangements may lead to several forms of B-cell malignancy. In this study, we obtained 50 blood samples from patients diagnosed with ALL, and screened for PAX5 mutations using sequencing in exons 1, 2 and 3. We found a heterozygous germline variant, c.113G>A (p.Arg38His), which affects the paired domain of PAX5. It seems that this mutation is pathogenic, but is recessive. Our findings suggest that this mutation in a single allele of the PAX5 gene is not sufficient to cause disease, and it is possible that other alleles are also involved in the onset of B-ALL.

  2. The survey of association between Polymorphism of CTLA-4 Exon 1 with Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Mahdieh Shojaa

    2015-01-01

    Full Text Available Background & aim: Cytotoxic lymphocyte antigen-4 (CTLA-4 plays an important role in inhibition of T cell activation and resulting in prevention of autoimmune disorder such as systemic lupus erythematosus (SLE. The purpose of the present study was to investigate the relationship between AG 49's polymorphisms in exon 1with systemic lupus erythematosus. Methods: The present case-control study was conducted on 180 patients and 304 healthy controls who were matched in age and ethnicity to the similar individual patient. After DNA extraction from blood samples, polymerase chain reaction (PCR was used to analyze the genotype and allele frequencies of 49AG polymorphism of CTLA-4 gene. The collected Data was analyzed by SPSS software and Chi-square and Fisher’s exact test. Results: The results indicated that AA genotype was found in 67.2% of patients. A significant difference was seen compared to the control group (p = 0.0001. While the AG genotype with a frequency of 49.7% in healthy subjects compared with patients frequency of 27.8% and G allele with a frequency of 9.2% in healthy subjects and 5% in patients were significantly more common (p = 0.0001. Although the A allele in 81.1 % of patients and in 66% of control group were seen but no significant difference observed. Conclusion: The results showed that the AG 49 polymorphism played an important role in the pathogenesis of systemic lupus erythematosus.

  3. Sequence polymorphism of PrP exon 3 gene in Istrian and crossbred sheep

    Directory of Open Access Journals (Sweden)

    Ino Curik

    2010-01-01

    Full Text Available Polymorphisms in sheep PrP (prion protein gene are known for scrapie susceptibility. We sequenced part of PrP exon 3 gene in 92 autochthonous Istrian (IS and 38 crossbred sheep (CBS. ARQ, ARR and AHQ alleles were predominant with frequency of 0.674 (0.526, 0.228 (0.132 and 0.082 (0.263 in IS (CBS, respectively, while VRQ (0.011 in IS and ARH (0.005 in IS and 0.079 in CBS alleles were rare. We also found non-synonymous mutations at codons 112 (M→T, 127 (G→S and 143 (H→R, and synonymous mutations at codons 231 (R and 237 (L. Additional mutations were associated only with AHQ, ARH and ARQ alleles. The polymorphism of PrP gene in IS was not critical with respect to scrapie susceptibility and with some efforts number of “favourable” genotypes can be increased.

  4. Polymorphism in exon2 of BMP15 gene in Iranian sangsari sheep

    Directory of Open Access Journals (Sweden)

    zana pirkhezranian

    2015-04-01

    Full Text Available Fertility rate is an economically important trait in sheep, which is influenced by genetic and environment. So far, three genes have been identified that affects this trait, one of them would be the BMP family, the most famous one is BMP15. Different mutations in the BMP15 gene, increases reproductive performance and growth rate in sheep. The aim of this study was to investigate the genetic and phylogenetic of BMP15 gene sequence in Iranian Sangsari sheep. For this purpose, the blood samples from 20 animal of Damghan station were collected. After DNA extracting, a segment of 222 bp of exon 2 of BMP15 gene was amplified using polymerase chain reaction. Then, all of the PCR products were sequenced. The results showed existence of four haplotypes and three significant mutations of the gene that which one of them was seen for first. In order to determine the genetic distance of Sansari sheep with other animals especially sheep breeds about 103 sequences were taken from Genebank, Then, phylogenetic trees were drawn. Genetic distances and nucleotide differences were calculated. The results showed that goat, cattle and buffalo have minimum genetic distance and monkey, human and mouse have maximum distance with Sangsari sheep and native Hindi and Kashmiri sheep have not any differences with Iranian Sangsari sheep.

  5. Laing distal myopathy with a novel mutation in exon 34 of the MYH7 gene.

    Science.gov (United States)

    Ferbert, A; Zibat, A; Rautenstrauß, B; Kress, W; Hügens-Penzel, M; Weis, J; Shah, Y; Roth, C

    2016-09-01

    We investigated a four-generation family of German ancestry with distal myopathy. Four individuals in two generations were affected. Foot and toe extensor paresis progressing very slowly over decades was the core neurological sign, reflected by fatty infiltration of the lower leg extensor muscles on muscle MRI. Additionally, finger extensor paresis was present in two patients and quadriceps muscle paresis in one. Distal sensory signs had initially given rise to the diagnosis of axonal Charcot-Marie-Tooth (CMT) disease. Two patients had extended verrucae of their foot sole, which may or may not be part of the disease spectrum. All four patients had a novel c.4645G > C mutation in exon 34 of the MYH7 gene that was not present in three clinically unaffected family members. Muscle biopsy of one patient revealed a myopathic pattern associated with type 1 muscle fibre atrophy and core-like lesions in many muscle fibres consistent with a myosin-related myopathy. We conclude that some of the typical clinical signs such as extensor weakness of the big toe and the little finger may only develop in the further course of the disease. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Exon first nucleotide mutations in splicing: evaluation of in silico prediction tools.

    Science.gov (United States)

    Grodecká, Lucie; Lockerová, Pavla; Ravčuková, Barbora; Buratti, Emanuele; Baralle, Francisco E; Dušek, Ladislav; Freiberger, Tomáš

    2014-01-01

    Mutations in the first nucleotide of exons (E(+1)) mostly affect pre-mRNA splicing when found in AG-dependent 3' splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, in silico prediction tools are commonly used to score 3' splice sites. In this study, we have assessed the ability of sequence features and in silico prediction tools to discriminate between the splicing-affecting and non-affecting E(+1) variants. For this purpose, we newly tested 16 substitutions in vitro and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting.

  7. SEQUENCING AND SEQUENCE ANALYSIS OF MYOSTATIN GENE IN THE EXON 1 OF THE CAMEL (CAMELUS DROMEDARIUS

    Directory of Open Access Journals (Sweden)

    M. G. SHAH, A. S. QURESHI1, M. REISSMANN2 AND H. J. SCHWARTZ3

    2006-10-01

    Full Text Available Myostatin, also called growth differentiation factor-8 (GDF-8, is a member of the mammalian growth transforming family (TGF-beta superfamily, which is expressed specifically in developing an adult skeletal muscle. Muscular hypertrophy allele (mh allele in the double muscle breeds involved mutation within the myostatin gene. Genomic DNA was isolated from the camel hair using NucleoSpin Tissue kit. Two animals of each of the six breeds namely, Marecha, Dhatti, Larri, Kohi, Sakrai and Cambelpuri were used for sequencing. For PCR amplification of the gene, a primer pair was designed from homolog regions of already published sequences of farm animals from GenBank. Results showed that camel myostatin possessed more than 90% homology with that of cattle, sheep and pig. Camel formed separate cluster from the pig in spite of having high homology (98% and showed 94% homology with cattle and sheep as reported in literature. Sequence analysis of the PCR amplified part of exon 1 (256 bp of the camel myostatin was identical among six camel breeds.

  8. A genome-wide analysis of putative functional and exonic variation associated with extremely high intelligence.

    Science.gov (United States)

    Spain, S L; Pedroso, I; Kadeva, N; Miller, M B; Iacono, W G; McGue, M; Stergiakouli, E; Davey Smith, G; Putallaz, M; Lubinski, D; Meaburn, E L; Plomin, R; Simpson, M A

    2016-08-01

    Although individual differences in intelligence (general cognitive ability) are highly heritable, molecular genetic analyses to date have had limited success in identifying specific loci responsible for its heritability. This study is the first to investigate exome variation in individuals of extremely high intelligence. Under the quantitative genetic model, sampling from the high extreme of the distribution should provide increased power to detect associations. We therefore performed a case-control association analysis with 1409 individuals drawn from the top 0.0003 (IQ >170) of the population distribution of intelligence and 3253 unselected population-based controls. Our analysis focused on putative functional exonic variants assayed on the Illumina HumanExome BeadChip. We did not observe any individual protein-altering variants that are reproducibly associated with extremely high intelligence and within the entire distribution of intelligence. Moreover, no significant associations were found for multiple rare alleles within individual genes. However, analyses using genome-wide similarity between unrelated individuals (genome-wide complex trait analysis) indicate that the genotyped functional protein-altering variation yields a heritability estimate of 17.4% (s.e. 1.7%) based on a liability model. In addition, investigation of nominally significant associations revealed fewer rare alleles associated with extremely high intelligence than would be expected under the null hypothesis. This observation is consistent with the hypothesis that rare functional alleles are more frequently detrimental than beneficial to intelligence.

  9. Multiple non-coding exons and alternative splicing in the mouse Mas protooncogene.

    Science.gov (United States)

    Alenina, Natalia; Böhme, Ilka; Bader, Michael; Walther, Thomas

    2015-09-01

    The Mas protooncogene encodes a G protein-coupled receptor with the common seven transmembrane domains, expressed mainly in the testis and brain. We provided evidence that Mas is a functional angiotensin-(1-7) receptor and can interact with the angiotensin II type 1 (AT1) receptor. The gene is transcriptionally regulated during development in the brain and testis, but its structure was unresolved. In this study we used 5'- and 3'-RACE, RT-PCR, and RNase-protection assays to elucidate the complete Mas gene structure and organization. We identified 12 exons in the mouse Mas gene with 11 in the 5' untranslated mRNA, which can be alternatively spliced. We also showed that Mas transcription can start from 4 tissue-specific promoters, whereby testis-specific Mas mRNA is transcribed from two upstream promoters, and the expression of Mas in the brain starts from two downstream promoters. Alternative splicing and multiple promoter usage result in at least 12 Mas transcripts in which different 5' untranslated regions are fused to a common coding sequence. Moreover, termination of Mas mRNA is regulated by two different polyadenylation signals. The gene spans approximately 27 kb, and the longest detected mRNA contains 2,451 bp. Thus, our results characterize the Mas protooncogene as the gene with the most complex gene structure of all described members of the gene family coding for G protein-coupled receptors. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Personality, dopamine receptor D4 exon III polymorphisms, and academic achievement in medical students.

    Science.gov (United States)

    Ham, Byung-Joo; Lee, Young-Mee; Kim, Meyoung-Kon; Lee, Juneyoung; Ahn, Duck-Sun; Choi, Myoung-Jin; Lyoo, In Kyoon; Choi, Ihn-Geun; Lee, Min-Soo

    2006-01-01

    This study investigated the relationships between genetic polymorphisms, personality traits, and academic achievement in medical school students. Study subjects were 220 1st-year medical students at Korea University Medical School during two consecutive academic years (2003-2004). Grade-point averages (GPA) during the second semester of the 1st year of the medical school were obtained as a measure of academic achievement. In addition, all participants completed the Temperament and Character Inventory and questionnaires on depression and anxiety. The polymorphisms in exon III of the dopamine D4 receptor (DRD4) were determined using the polymerase chain reaction. Our results revealed that both male and female subjects with a higher GPA may be characterized as having higher persistence and lower novelty seeking traits. In addition, male subjects with high GPA had higher scores in self directedness and female subjects with a higher GPA may be characterized as having higher scores in harm avoidance. Male students with 4-repeat alleles had a significantly lower GPA than male students without 4-repeat alleles. This relationship also remained after controlling for the personality variables, none of which showed a relationship with the polymorphism after Bonferroni correction. For females, however, no associations could be found between GPA and the polymorphism. Thus, the present study demonstrated for the first time a possible influence of the DRD4 48 bp variable number of tandem repeats polymorphism on academic achievement and proved that this was not mediated by performance-associated personality traits.

  11. Deletion of exon 26 of the dystrophin gene is associated with a mild Becker muscular dystrophy phenotype

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, Morten; Vissing, John

    2011-01-01

    With the possible introduction of exon skipping therapy in Duchenne muscular dystrophy, it has become increasingly important to know the role of each exon of the dystrophin gene to protein expression, and thus the phenotype. In this report, we present two related men with an unusually mild BMD...... skipping therapy for Duchenne muscular dystrophy. This report also shows that BMD may present with a normal CK....... showed dystrophic changes. He had comorbidity with dystonia, slight mental retardation, low stature and neuropathy. The brother of the proband's mother came to medical attention when he was 43 years old. He complained about muscle pain. On examination, a MRC grade 4+ hip extention palsy and a discrete...

  12. Identification of two point mutations and a one base deletion in exon 19 of the dystrophin gene by heteroduplex formation.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartello, C; Mendell, J R

    1993-03-01

    Two thirds of the Duchenne muscular dystrophy population have either gene deletions or duplications. The nondeletion/duplication cases are most likely the result of point mutations or small deletions and duplications that cannot be easily identified by current strategies. The major obstacle in identifying small mutations is due to the large size of the dystrophin gene. We selectively screened 5 DMD exons containing CpG dinucleotides in 110 DMD patients without detectable deletions or duplications. Nonsenses mutations are frequently due to a C- to -T transition within a CG dinucleotide pair. To screen for the nonsense mutations, we used the heteroduplex method. Utilizing this approach, we identified 2 different nonsense mutations and a single base deletion all occurring in exon 19. This is the first report of a clustering of small mutations in the dystrophin gene.

  13. A synonymous polymorphic variation in ACADM exon 11 affects splicing efficiency and may affect fatty acid oxidation

    DEFF Research Database (Denmark)

    Bruun, Gitte Hoffmann; Doktor, Thomas Koed; Andresen, Brage Storstein

    2013-01-01

    beta-oxidation of medium-chain fatty acids. We examined the functional basis for this association and identified linkage between rs211718 and the intragenic synonymous polymorphic variant c.1161A>G in ACADM exon 11 (rs1061337). Employing minigene studies we show that the c.1161A allele is associated...... with exon 11 missplicing, and that the c.1161G allele corrects this missplicing. This may result in production of more full length MCAD protein from the c.1161G allele. Our analysis suggests that the improved splicing of the c.1161G allele is due to changes in the relative binding of splicing regulatory......, perhaps due to improved splicing. This study is a proof of principle that synonymous SNPs are not neutral. By changing the binding sites for splicing regulatory proteins they can have significant effects on pre-mRNA splicing and thus protein function. In addition, this study shows that for a sequence...

  14. Deletion of exon 26 of the dystrophin gene is associated with a mild Becker muscular dystrophy phenotype

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, Morten; Vissing, John

    2011-01-01

    With the possible introduction of exon skipping therapy in Duchenne muscular dystrophy, it has become increasingly important to know the role of each exon of the dystrophin gene to protein expression, and thus the phenotype. In this report, we present two related men with an unusually mild BMD...... skipping therapy for Duchenne muscular dystrophy. This report also shows that BMD may present with a normal CK....... calf hypertrophy was noted. Creatine kinase was normal or raised maximally to 500 U/l. The muscle biopsy was myopathic with increased fiber size variation and many internal nuclei, but no dystrophy. No comorbidity was found. In both cases, western blot showed a reduced dystrophin band. Genetic...

  15. To Screen Inactivation Mutation of Exon 1 of FSHR Gene in Polycystic Ovarian Syndrome: A South Indian Cohort Study

    Science.gov (United States)

    Sekar, Nishu; Yeole, Samiksha; Pradeep, Rashmi; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

    2017-11-01

    Polycystic ovary syndrome is an endocrine disorder. Irregular menstrual cycle, acne, facial hair and elevated androgen levels are the most common signs for PCOS. PCOS has an estimated prevalence of 4-12% among reproductive age women, thus making it a forerunner in female infertility. FSHR plays an important role in FSH signaling pathway making it an important gene for PCOS. In this study, we aim to focus on any association between the FSHR gene and PCOS. Our study was to evaluate any polymorphism of exon 1 of FSHR gene associated with PCOS.PCR-RFLP technique was performed on the PCOS samples. Hormonal changes were found in the patients. Exon 1 inactivation mutation of FSHR gene was not observed in the patient sample. A study of this association needs to be done using large sample size.

  16. Mutations in MYH9 exons 1, 16, 26, and 30 are infrequently found in Japanese patients with nonsyndromic deafness.

    Science.gov (United States)

    Kunishima, Shinji; Matsunaga, Tatsuo; Ito, Yoshimi; Saito, Hidehiko

    2009-10-01

    Mutations in MYH9 result in the autosomal dominant giant platelet disorders with leukocyte inclusion bodies with varying degrees of Alport manifestations, including nephritis, deafness, and cataracts. A specific MYH9 mutation in exon 16, R705H, causes nonsyndromic deafness DFNA17. We searched for mutations in MYH9 exons 1, 16, 26, and 30 in a total of 157 Japanese patients with nonsyndromic deafness without known cause of hearing loss, but no mutations were found. We conclude that mutations in MYH9 are infrequently found in patients with nonsyndromic deafness and suggest that MYH9 mutations infrequently cause isolated sensorineural hearing loss. Thus, MYH9 may not currently be a good candidate gene for efficient screening of genetic causes in nonsyndromic deafness.

  17. Partial correlation analysis indicates causal relationships between GC-content, exon density and recombination rate in the human genome

    OpenAIRE

    Li Wentian; Yang Yaning; Wang Mingyi; Freudenberg Jan

    2009-01-01

    Abstract Background Several features are known to correlate with the GC-content in the human genome, including recombination rate, gene density and distance to telomere. However, by testing for pairwise correlation only, it is impossible to distinguish direct associations from indirect ones and to distinguish between causes and effects. Results We use partial correlations to construct partially directed graphs for the following four variables: GC-content, recombination rate, exon density and ...

  18. Severe congenital lipodystrophy and a progeroid appearance: Mutation in the penultimate exon of FBN1 causing a recognizable phenotype.

    Science.gov (United States)

    Takenouchi, Toshiki; Hida, Mariko; Sakamoto, Yoshiaki; Torii, Chiharu; Kosaki, Rika; Takahashi, Takao; Kosaki, Kenjiro

    2013-12-01

    Recently, three marfanoid patients with congenital lipodystrophy and a neonatal progeroid appearance were reported. Although their phenotype was distinct from that of classic Marfan syndrome, they all had a truncating mutation in the penultimate exon, i.e., exon 64, of FBN1, the causative gene for Marfan syndrome. These patients might represent a new entity, but the exact phenotypic and genotypic spectrum remains unknown. Here, we report on a girl born prematurely who exhibited severe congenital lipodystrophy and a neonatal progeroid appearance. The patient exhibited a characteristic growth pattern consisting of an accelerated growth in height with a discrepant poor weight gain. She had a characteristic facial appearance with craniosynostosis. A mutation analysis identified c.8175_8182del8bp, p.Arg2726Glufs*9 in exon 64 of the FBN1 gene. A review of similar, recently reported patients revealed that the cardinal features of these patients include (1) congenital lipodystrophy, (2) premature birth with an accelerated linear growth disproportionate to the weight gain, and (3) a progeroid appearance with distinct facial features. Lines of molecular evidence suggested that this new progeroid syndrome represents a neomorphic phenotype caused by truncated transcripts with an extremely charged protein motif that escapes from nonsense-mediated mRNA decay, altering FBN1-TGF beta signaling, rather than representing the severe end of the hypomorphic phenotype of the FBN1-TGF beta disorder spectrum. We propose that this marfanoid entity comprised of congenital lipodystrophy, a neonatal progeroid appearance, and a peculiar growth profile and caused by rare mutations in the penultimate exon of FBN1, be newly referred to as marfanoid-progeroid syndrome. © 2013 Wiley Periodicals, Inc.

  19. CD33: increased inclusion of exon 2 implicates the Ig V-set domain in Alzheimer's disease susceptibility

    Science.gov (United States)

    Raj, Towfique; Ryan, Katie J.; Replogle, Joseph M.; Chibnik, Lori B.; Rosenkrantz, Laura; Tang, Anna; Rothamel, Katie; Stranger, Barbara E.; Bennett, David A.; Evans, Denis A.; De Jager, Philip L.; Bradshaw, Elizabeth M.

    2014-01-01

    We previously demonstrated that the Alzheimer's disease (AD) associated risk allele, rs3865444C, results in a higher surface density of CD33 on monocytes. Here, we find alternative splicing of exon 2 to be the primary mechanism of the genetically driven differential expression of CD33 protein. We report that the risk allele, rs3865444C, is associated with greater cell surface expression of CD33 in both subjects of European and African–American ancestry and that there is a single haplotype influencing CD33 surface expression. A meta-analysis of the two populations narrowed the number of significant SNPs in high linkage disequilibrium (LD) (r2 > 0.8) with rs3865444 to just five putative causal variants associated with increased protein expression. Using gene expression data from flow-sorted CD14+CD16− monocytes from 398 healthy subjects of three populations, we show that the rs3865444C risk allele is strongly associated with greater expression of CD33 exon 2 (pMETA = 2.36 × 10−60). Western blotting confirms increased protein expression of the full-length CD33 isoform containing exon 2 relative to the rs3865444C allele (P < 0.0001). Of the variants in strong LD with rs3865444, rs12459419, which is located in a putative SRSF2 splice site of exon 2, is the most likely candidate to mediate the altered alternative splicing of CD33's Immunoglobulin V-set domain 2 and ultimately influence AD susceptibility. PMID:24381305

  20. The Effect of 6-Thioguanine on Alternative Splicing and Antisense-Mediated Exon Skipping Treatment for Duchenne Muscular Dystrophy

    OpenAIRE

    Verhaart, Ingrid E. C.; Aartsma-Rus, Annemieke

    2012-01-01

    The severe muscle wasting disorder Duchenne muscular dystrophy (DMD) is caused by genetic defects in the DMD gene, leading to a complete absence of dystrophin protein. Of the therapeutic approaches addressing the underlying genetic defect, exon skipping through antisense oligonucleotides (AONs) is the closest to clinical application. Several strategies to improve the efficiency of this approach are currently being investigated, such as the use of small chemical compounds that improve AONmedia...

  1. In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

    OpenAIRE

    Wagner, Stefan; Chiosea, Simion; Ivshina, Maria; Nickerson, Jeffrey A.

    2004-01-01

    We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)–labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)–dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splic...

  2. Identification of MSH2 inversion of exons 1-7 in clinical evaluation of families with suspected Lynch syndrome.

    Science.gov (United States)

    Mork, Maureen E; Rodriguez, Andrea; Taggart, Melissa W; Rodriguez-Bigas, Miguel A; Lynch, Patrick M; Bannon, Sarah A; You, Y Nancy; Vilar, Eduardo

    2017-07-01

    Traditional germline sequencing and deletion/duplication analysis does not detect Lynch syndrome-causing mutations in all individuals whose colorectal or endometrial tumors demonstrate mismatch repair (MMR) deficiency. Unique inversions and other rearrangements of the MMR genes have been reported in families with Lynch syndrome. In 2014, a recurrent inversion of MSH2 exons 1-7 was identified in five families suspected to have Lynch syndrome. We aimed to describe our clinical experience in identifying families with this specific inversion. Four probands whose Lynch syndrome-associated tumors demonstrated absence of MSH2/MSH6 staining and who had negative MMR germline testing were evaluated for the MSH2 inversion of exons 1-7, offered during initial genetic workup or upon routine clinical follow-up. All four probands tested positive for the MSH2 inversion. Proband cancer diagnoses included colon and endometrial adenocarcinoma and sebaceous adenoma. A variety of Lynch syndrome-associated cancers were reported in the family histories, although only one family met Amsterdam II criteria. Thirteen at-risk relatives underwent predictive testing. MSH2 inversion of exons 1-7 was found in four probands previously suspected to have Lynch syndrome based on family history and tumor testing. This testing should be offered routinely to patients with tumors demonstrating loss of MSH2/MSH6 staining.

  3. A Novel Nonsense Mutation in Exon 5 of KIND1 Gene in an Iranian Family with Kindler Syndrome.

    Science.gov (United States)

    Heidari, Mohammad Mehdi; Khatami, Mehri; Kargar, Saeed; Azari, Mojdeh; Hoseinzadeh, Hassan; Fallah, Hamedeh

    2016-06-01

    Kindler syndrome (KS) is an autosomal recessive skin disease characterized by actual blistering, photosensitivity and a progressive poikiloderma. The disorder results from rare mutations in the KIND1 gene. This gene contains 15 exons and expresses two kindlin-1 isoforms. The aim of this investigation was to analyze mutations in the exons 1 to 15 of KIND1 gene in an Iranian family clinically affected with Kindler syndrome. The mutations analysis of 15 coding exons of KIND1 gene was performed with PCR-SSCP and direct sequencing in 14 subjects from one Iranian family clinically affected with Kindler syndrome. We identified eight new nucleotide changes in KIND1 in this family. These changes were found in g.3892delA, g.3951T>C, g.3962T>G, g.4190G>T, g.7497G>A, g.11076T>C, g.11102C>T and g.13177C>T positions. Among them, the g.13177C>T mutation resulting in the formation of a premature stop codon (Q226X) was detected only in seven affected family individuals as homozygous but was not present in 100 unrelated healthy controls. This study suggests that nonsense mutation may lead to incomplete and non-functional protein products and is pathogenic and has meaningful implications for the diagnosis of patients with Kindler syndrome.

  4. Mutation analysis in exons 22 and 24 of SCN4A gene in Iranian patients with non-dystrophic myotonia.

    Science.gov (United States)

    Heidari, Mohammad Mehdi; Khatami, Mehri; Nafissi, Shahriar; Hesami-Zokai, Faezeh; Khorrami, Afshin

    2015-10-07

    Non-dystrophic myotonias are a heterogeneous set of skeletal, muscular channelopathies, which have been associated with point mutations within sodium channel α-subunit (SCN4A) gene. Because exons 22 and 24 of SCN4A gene are recognized as hot spots for this disease, the purpose of the study is to identify mutation in exons 22 and 24 of SCN4A gene in Iranian non-dystrophic myotonias patients. In this study, 28 Iranian patients with non-dystrophic myotonia analyzed for the mutation scanning in exons 22 and 24 of SCN4A gene by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and sequencing. We found 29073G>C substitution in SCN4A gene in one case and 31506A>G substitution in seven cases. The 29073G>C substitution causes a missense mutation G1306A, located in the conserved cytoplasmic loop connecting repeat III and IV of the SCN4A channel but, 31506A>G substitution do not alter amino acid in SCN4A protein. G1306A residue is located in functionally important protein region. In "hinged-lid model" for Na(+) channel inactivation in which glycines(1306) act as the hinge of the lid occluding the channel pore. Mutation in this region slowed fast inactivation. Therefore, it might be a pathogenic mutation. The causal relationship of this mutation with the disease is an object for further discussion.

  5. Fibril polymorphism affects immobilized non-amyloid flanking domains of huntingtin exon1 rather than its polyglutamine core

    Science.gov (United States)

    Lin, Hsiang-Kai; Boatz, Jennifer C.; Krabbendam, Inge E.; Kodali, Ravindra; Hou, Zhipeng; Wetzel, Ronald; Dolga, Amalia M.; Poirier, Michelle A.; van der Wel, Patrick C. A.

    2017-05-01

    Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington's disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells.

  6. Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

    Science.gov (United States)

    Horan, Lucas; Yasuhara, Jiro C.; Kohlstaedt, Lori A.; Rio, Donald C.

    2015-01-01

    Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites. PMID:26545814

  7. De novo disruption of promoter and exon 1 of STAR gene reveals essential role for gonadal development

    Directory of Open Access Journals (Sweden)

    Anil Piya

    2017-03-01

    Full Text Available Cholesterol transport into the mitochondria is required for synthesis of the first steroid, pregnenolone. Cholesterol is transported by the steroidogenic acute regulatory protein (STAR, which acts at the outer mitochondrial membrane prior to its import. Mutations in the STAR protein result in lipoid congenital adrenal hyperplasia (CAH. Although the STAR protein consists of seven exons, biochemical analysis in nonsteroidogenic COS-1 cells showed that the first two were not essential for pregnenolone synthesis. Here, we present a patient with ambiguous genitalia, salt-lossing crisis within two weeks after birth and low cortisol levels. Sequence analysis of the STAR, including the exon–intron boundaries, showed the complete deletion of exon 1 as well as more than 50 nucleotides upstream of STAR promoter. Mitochondrial protein import with the translated protein through synthesis cassette of the mutant STAR lacking exon 1 showed protein translation, but it is less likely to have synthesized without a promoter in our patient. Thus, a full-length STAR gene is necessary for physiological mitochondrial cholesterol transport in vivo.

  8. Novel P2 promoter-derived HNF4{alpha} isoforms with different N-terminus generated by alternate exon insertion

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jianmin, E-mail: jmhuang@partners.org [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States); Levitsky, Lynne L. [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States); Rhoads, David B., E-mail: rhoads@helix.mgh.harvard.edu [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States)

    2009-04-15

    Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a critical transcription factor for pancreas and liver development and functions in islet {beta} cells to maintain glucose homeostasis. Mutations in the human HNF4A gene lead to maturity onset diabetes of the young (MODY1) and polymorphisms are associated with increased risk for type 2 diabetes mellitus (T2DM). Expression of six HNF4{alpha} variants, three each from two developmentally regulated promoters, has been firmly established. We have now detected a new set of HNF4{alpha} variants designated HNF4{alpha}10-12 expressed from distal promoter P2. These variants, generated by inclusion of previously undetected exon 1E (human = 222 nt, rodent = 136 nt) following exon 1D have an altered N-terminus but identical remaining reading frame. HNF4{alpha}10-{alpha}12 are expressed in pancreatic islets (and liver) and exhibit transactivation potentials similar to the corresponding {alpha}7-{alpha}9 isoforms. DNA-binding analyses implied much higher protein levels of HNF4{alpha}10-{alpha}12 in liver than expected from the RT-PCR data. Our results provide evidence for a more complex expression pattern of HNF4{alpha} than previously appreciated. We recommend inclusion of exon 1E and nearby DNA sequences in screening for HNF4{alpha} mutations and polymorphisms in genetic analyses of MODY1 and T2DM.

  9. Polymorphisms within Exon 9, But Not Intron 8, of the Vitamin D Receptor Gene Are Associated with Asthma

    Directory of Open Access Journals (Sweden)

    Mohammad Kazemi Arababadi

    2011-05-01

    Full Text Available AbstractObjective(sDeregulation of the immune system through allied factors and cytokine responses are thought to be important contributors to the pathogenesis of asthma. Vitamin D3 and its nuclear receptor appear to be factors that maybe involved in regulating immune responses during the progression of asthma. The aim of this study was to investigate the association between polymorphisms in intron 8 and exon 9 of the vitamin D receptor (VDR and this disease.Materials and MethodsThis study was performed on 100 asthmatic patients and 100 healthy controls. PCR-RFLP was performed to examine polymorphisms in intron 8 and exon 9 of VDR gene. ResultsOur results showed a statistically significant difference in the Taq-1 evaluated genotypes of exon 9 of the VDR gene when comparing healthy patients to asthmatic patients. ConclusionBased on our results, it can be concluded that VDR and its functional polymorphisms may play an important role in the pathogenesis of asthma.

  10. CRIPT exonic deletion and a novel missense mutation in a female with short stature, dysmorphic features, microcephaly, and pigmentary abnormalities.

    Science.gov (United States)

    Leduc, Magalie S; Niu, Zhiyv; Bi, Weimin; Zhu, Wenmiao; Miloslavskaya, Irene; Chiang, Theodore; Streff, Haley; Seavitt, John R; Murray, Stephen A; Eng, Christine; Chan, Audrey; Yang, Yaping; Lalani, Seema R

    2016-08-01

    Mutations in CRIPT encoding cysteine-rich PDZ domain-binding protein are rare, and to date have been reported in only two patients with autosomal recessive primordial dwarfism and distinctive facies. Here, we describe a female with biallelic mutations in CRIPT presenting with postnatal growth retardation, global developmental delay, and dysmorphic features including frontal bossing, high forehead, and sparse hair and eyebrows. Additional clinical features included high myopia, admixed hyper- and hypopigmented macules primarily on the face, arms, and legs, and syndactyly of 4-5 toes bilaterally. Using whole exome sequencing (WES) and chromosomal microarray analysis (CMA), we detected a c.8G>A (p.C3Y) missense variant in exon 1 of the CRIPT gene inherited from the mother and a 1,331 bp deletion encompassing exon 1, inherited from the father. The c.8G>A (p.C3Y) missense variant in CRIPT was apparently homozygous in the proband due to the exon 1 deletion. Our findings illustrate the clinical utility of combining WES with copy number variant (CNV) analysis to provide a molecular diagnosis to patients with rare Mendelian disorders. Our findings also illustrate the clinical spectrum of CRIPT related mutations. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. A Novel Homozygous Frameshift Mutation in Exon 2 of LEP Gene Associated with Severe Obesity: A Case Report.

    Science.gov (United States)

    Altawil, Ashwaq Shukri; Mawlawi, Horia Ahmad; Alghamdi, Khalid Ateeq; Almijmaj, Faten Fohaid

    2016-01-01

    Monogenic obesity is a rare type of obesity caused by a mutation in a single gene. Patients with monogenic obesity may develop early onset of obesity and severe metabolic abnormalities. A two-and-half-year-old girl was presented to our clinic because of excessive weight gain and hyperphagia. She was born at full term, by normal vaginal delivery with birth weight of 2.82 kg and no complications during pregnancy. The patient was the second child of two healthy, non-obese Saudis with known consanguinity. She gained weight rapidly leading to obesity at the age of three months. The demographic data and clinical features were recorded. Blood samples were collected and tested for endocrine and metabolic characteristics and genetic studies. Mutations of the LEP gene were screened. The coding exons 2 and 3 and the corresponding exon-intron boundaries were amplified by polymerase chain reaction using specific primers, analyzed by direct sequencing using an ABI sequencer 3500 xL GA (Applied Biosystems), and evaluated using the JSI SeqPilot software. The resulting sequence data were compared with the reference MM_0002302. We report a novel homozygous frameshift mutation c.144delin TAC (G1n49Thrfs*23) in exon 2 of the LEP gene associated with extreme obesity.

  12. Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches.

    Science.gov (United States)

    Lorain, Stéphanie; Peccate, Cécile; Le Hir, Maëva; Griffith, Graziella; Philippi, Susanne; Précigout, Guillaume; Mamchaoui, Kamel; Jollet, Arnaud; Voit, Thomas; Garcia, Luis

    2013-09-01

    RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans-splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre-trans-splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans-splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv, illustrating that a given PTM can be suitable for a variety of mutations.

  13. Long-term Exon Skipping Studies With 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in Dystrophic Mouse Models

    Directory of Open Access Journals (Sweden)

    Christa L Tanganyika-de Winter

    2012-01-01

    Full Text Available Antisense-mediated exon skipping for Duchenne muscular dystrophy (DMD is currently tested in phase 3 clinical trials. The aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts, allowing the synthesis of a partly functional dystrophin protein. Studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides (AONs. Here, we tested the safety and efficacy of subcutaneously administered 2′-O-methyl phosphorothioate AON at 200 mg/kg/week for up to 6 months in mouse models with varying levels of disease severity: mdx mice (mild phenotype and mdx mice with one utrophin allele (mdx/utrn+/−; more severe phenotype. Long-term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals. Notably, in the more severely affected mdx/utrn+/− mice the therapeutic effect was larger: creatine kinase (CK levels were more decreased and rotarod running time was more increased. This suggests that the mdx/utrn+/− model may be a more suitable model to test potential therapies than the regular mdx mouse. Our results also indicate that long-term subcutaneous treatment in dystrophic mouse models with these AONs is safe and beneficial.

  14. A method to identify RNA A-to-I editing targets using I-specific cleavage and exon array analysis.

    Science.gov (United States)

    Tseng, Chao-Neng; Chang, Hsueh-Wei; Stocker, Joel; Wang, Hui-Chun; Lu, Chiu-Chin; Wu, Cheng-Hsuan; Yang, Jyuer-Ger; Cho, Chung-Lung; Huang, Hurng-Wern

    2013-02-01

    RNA A-to-I editing is the most common single-base editing in the animal kingdom. Dysregulations of RNA A-to-I editing are associated with developmental defects in mouse and human diseases. Mouse knockout models deficient in ADAR activities show lethal phenotypes associated with defects in nervous system, failure of hematopoiesis and reduced tolerance to stress. While several methods of identifying RNA A-to-I editing sites are currently available, most of the critical editing targets responsible for the important biological functions of ADARs remain unknown. Here we report a method to systematically analyze RNA A-to-I editing targets by combining I-specific cleavage and exon array analysis. Our results show that I-specific cleavage on editing sites causes more than twofold signal reductions in edited exons of known targets such as Gria2, Htr2c, Gabra3 and Cyfip2 in mice. This method provides an experimental approach for genome-wide analysis of RNA A-to-I editing targets with exon-level resolution. We believe this method will help expedite inquiry into the roles of RNA A-to-I editing in various biological processes and diseases. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Exon 8-17 deletions of SPAST in a Chinese family with hereditary spastic paraplegia: a case report and literature review.

    Science.gov (United States)

    Wang, Kang; Zhao, Guohua

    2015-10-15

    Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders. SPG4 is the most common autosomal dominant form of HSP subtypes and is caused by mutations of the SPAST gene. Here we reported a Chinese family with HSP caused by deletion of exons 8-17 of the SPAST gene and reviewed the clinical phenotypes of patients with exon deletion that were reported in literatures. The patients with deletions of exons in the SPAST gene showed pure HSP, and the age at onset showed interfamily and intrafamily variations. This study suggests that exon deletion should be examined routinely in patients who are clinically diagnosed with HSP. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we...... compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs...... and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little...

  17. Deletion of exons 3-9 encompassing a mutational hot spot in the DMD gene presents an asymptomatic phenotype, indicating a target region for multiexon skipping therapy.

    Science.gov (United States)

    Nakamura, Akinori; Fueki, Noboru; Shiba, Naoko; Motoki, Hirohiko; Miyazaki, Daigo; Nishizawa, Hitomi; Echigoya, Yusuke; Yokota, Toshifumi; Aoki, Yoshitsugu; Takeda, Shin'ichi

    2016-07-01

    Few cases of dystrophinopathy show an asymptomatic phenotype with mutations in the 5' (exons 3-7) hot spot in the Duchenne muscular dystrophy (DMD) gene. Our patient showed increased serum creatine kinase levels at 12 years of age. A muscle biopsy at 15 years of age led to a diagnosis of Becker muscular dystrophy. The patient showed a slight decrease in cardiac function at the age of 21 years and was administered a β-blocker, but there was no muscle involvement even at the age of 27 years. A deletion of exons 3-9 encompassing a mutational hot spot in the DMD gene was detected, and dystrophin protein expression was ∼15% that of control level. We propose that in-frame deletion of exons 3-9 may produce a functional protein, and that multiexon skipping therapy targeting these exons may be feasible for severe dystrophic patients with a mutation in the 5' hot spot of the DMD gene.

  18. Exon 5 Polymorphisms in the O6-Alkylguanine DNA Alkyltransferase Gene and Lung Cancer Risk in Non–Smokers Exposed to Second-Hand Smoke

    National Research Council Canada - National Science Library

    Catherine Cohet; Stephane Borel; Fredrik Nyberg; Anush Mukeria; Irene Brüske-Hohlfeld; Vali Constantinescu; Simone Benhamou; Paul Brennan; Janet Hall; Paolo Boffetta

    2004-01-01

    .... Experimental Design: Exon 5 of the AGT gene was sequenced in genomic DNA from 136 cases and 133 hospital- or population-based controls for whom questionnaire information on second-hand smoke and diet was available...

  19. Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy

    NARCIS (Netherlands)

    Bornert, Olivier; Kuhl, Tobias; Bremer, Jeroen; van den Akker, Peter C.; Pasmooij, Anna M. G.; Nystrom, Alexander

    Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)-a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The

  20. Biochemical characterization of patients with in-frame or out-of-frame DMD deletions pertinent to exon 44 or 45 skipping.

    Science.gov (United States)

    Anthony, Karen; Arechavala-Gomeza, Virginia; Ricotti, Valeria; Torelli, Silvia; Feng, Lucy; Janghra, Narinder; Tasca, Giorgio; Guglieri, Michela; Barresi, Rita; Armaroli, Annarita; Ferlini, Alessandra; Bushby, Katherine; Straub, Volker; Ricci, Enzo; Sewry, Caroline; Morgan, Jennifer; Muntoni, Francesco

    2014-01-01

    In Duchenne muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)-mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than others. Patients with exon 44 or 45 skippable deletions (AOs in clinical development) have a less predictable phenotype than those skippable for exon 51, a group in advanced clinical trials. A way to predict the potential of AOs is the study of patients with BMD who have deletions that naturally mimic those that would be achieved by exon skipping. To quantify dystrophin messenger RNA (mRNA) and protein expression in patients with DMD deletions treatable by, or mimicking, exon 44 or 45 skipping. Retrospective study of nondystrophic controls (n = 2), patients with DMD (n = 5), patients with intermediate muscular dystrophy (n = 3), and patients with BMD (n = 13) at 4 university-based academic centers and pediatric hospitals. Biochemical analysis of existing muscle biopsies was correlated with the severity of the skeletal muscle phenotype. Dystrophin mRNA and protein expression. Patients with DMD who have out-of-frame deletions skippable for exon 44 or 45 had an elevated number of revertant and trace dystrophin expression (approximately 19% of control, using quantitative immunohistochemistry) with 4 of 9 patients presenting with an intermediate muscular dystrophy phenotype (3 patients) or a BMD-like phenotype (1 patient). Corresponding in-frame deletions presented with predominantly mild BMD phenotypes and lower dystrophin levels (approximately 42% of control) than patients with BMD modeling exon 51 skipping (approximately 80% of control). All 12 patients with in-frame deletions had a stable transcript compared with 2 of 9 patients with out-of-frame deletions (who had intermediate muscular dystrophy and BMD phenotypes). Exon

  1. An Exon-Based Comparative Variant Analysis Pipeline to Study the Scale and Role of Frameshift and Nonsense Mutation in the Human-Chimpanzee Divergence

    OpenAIRE

    Yu, GongXin

    2009-01-01

    Chimpanzees and humans are closely related but differ in many deadly human diseases and other characteristics in physiology, anatomy, and pathology. In spite of decades of extensive research, crucial questions about the molecular mechanisms behind the differences are yet to be understood. Here I report ExonVar, a novel computational pipeline for Exon-based human-chimpanzee comparative Variant analysis. The objective is to comparatively analyze mutations specifically those that caused th...

  2. Missense mutation in exon 2 of SLC36A1 responsible for champagne dilution in horses.

    Directory of Open Access Journals (Sweden)

    Deborah Cook

    2008-09-01

    Full Text Available Champagne coat color in horses is controlled by a single, autosomal-dominant gene (CH. The phenotype produced by this gene is valued by many horse breeders, but can be difficult to distinguish from the effect produced by the Cream coat color dilution gene (CR. Three sires and their families segregating for CH were tested by genome scanning with microsatellite markers. The CH gene was mapped within a 6 cM region on horse chromosome 14 (LOD = 11.74 for theta = 0.00. Four candidate genes were identified within the region, namely SPARC [Secreted protein, acidic, cysteine-rich (osteonectin], SLC36A1 (Solute Carrier 36 family A1, SLC36A2 (Solute Carrier 36 family A2, and SLC36A3 (Solute Carrier 36 family A3. SLC36A3 was not expressed in skin tissue and therefore not considered further. The other three genes were sequenced in homozygotes for CH and homozygotes for the absence of the dilution allele (ch. SLC36A1 had a nucleotide substitution in exon 2 for horses with the champagne phenotype, which resulted in a transition from a threonine amino acid to an arginine amino acid (T63R. The association of the single nucleotide polymorphism (SNP with the champagne dilution phenotype was complete, as determined by the presence of the nucleotide variant among all 85 horses with the champagne dilution phenotype and its absence among all 97 horses without the champagne phenotype. This is the first description of a phenotype associated with the SLC36A1 gene.

  3. Differential methylation of the promoter and first exon of the RASSF1A gene in hepatocarcinogenesis

    Science.gov (United States)

    Jain, Surbhi; Xie, Lijia; Boldbaatar, Batbold; Lin, Selena Y.; Hamilton, James P.; Meltzer, Stephen J.; Chen, Shun-Hua; Hu, Chi-Tan; Block, Timothy M.; Song, Wei; Su, Ying-Hsiu

    2015-01-01

    Aim Aberrant methylation of the promoter, P2, and the first exon, E1, regions of the tumor suppressor gene RASSF1A, have been associated with hepatocellular carcinoma (HCC), albeit with poor specificity. This study analyzed the methylation profiles of P1, P2 and E1 regions of the gene to identify the region of which methylation most specifically corresponds to HCC and to evaluate the potential of this methylated region as a biomarker in urine for HCC screening. Methods Bisulfite DNA sequencing and quantitative methylation-specific polymerase chain reaction assays were performed to compare methylation of the 56 CpG sites in regions P1, P2 and E1 in DNA isolated from normal, hepatitic, cirrhotic, adjacent non-HCC, and HCC liver tissue and urine samples for the characterization of hypermethylation of the RASSF1A gene as a biomarker for HCC screening. Results In tissue, comparing HCC (n = 120) with cirrhosis and hepatitis together (n = 70), methylation of P1 had an area under the receiver operating characteristics curve (AUROC) of 0.90, whereas methylation of E1 and P2 had AUROC of 0.84 and 0.72, respectively. At 90% sensitivity, specificity for P1 methylation was 72.9% versus 38.6% for E1 and 27.1% for P2. Methylated P1 DNA was detected in urine in association with cirrhosis and HCC. It had a sensitivity of 81.8% for α-fetoprotein negative HCC. Conclusion Among the three regions analyzed, methylation of P1 is the most specific for HCC and holds great promise as a DNA marker in urine for screening of cirrhosis and HCC. PMID:25382672

  4. Proteins Associated with the Exon Junction Complex Also Control the Alternative Splicing of Apoptotic Regulators

    Science.gov (United States)

    Michelle, Laetitia; Cloutier, Alexandre; Toutant, Johanne; Shkreta, Lulzim; Thibault, Philippe; Durand, Mathieu; Garneau, Daniel; Gendron, Daniel; Lapointe, Elvy; Couture, Sonia; Le Hir, Hervé; Klinck, Roscoe; Elela, Sherif Abou; Prinos, Panagiotis

    2012-01-01

    Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-xS splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level. PMID:22203037

  5. Functional interconnections of Arabidopsis exon junction complex proteins and genes at multiple steps of gene expression.

    Science.gov (United States)

    Mufarrege, Eduardo F; Gonzalez, Daniel H; Curi, Graciela C

    2011-10-01

    The exon junction complex (EJC) is deposited on mRNA after splicing and participates in several aspects of RNA metabolism, from intracellular transport to translation. In this work, the functional and molecular interactions of Arabidopsis homologues of Mago, Y14, and PYM, three EJC components that participate in intron-mediated enhancement of gene expression in animals, have been analysed. AtMago, AtY14, and AtPYM are encoded by single genes that show similar expression patterns and contain common regulatory elements, known as site II, that are required for expression. AtPYM and AtY14 are phosphorylated by plant extracts and this modification regulates complex formation between both proteins. In addition, overexpression of AtMago and AtY14 in plants produces an increase in AtPYM protein levels, while overexpression of AtPYM results in increased formation of a complex that contains the three proteins. The effect of AtMago and AtY14 on AtPYM expression is most likely to be due to intron-mediated enhacement of AtPYM expression, since the AtPYM gene contains a leader intron that is required for expression. Indeed, transient transformation asssays indicated that the three proteins are able to increase expression from reporter constructs that contain leader introns required for the expression of different genes. The results indicate that the plant homologues of Mago, Y14, and PYM are closely interconnected, not only through their function as EJC components but also at different steps of their own gene expression mechanisms, probably reflecting the importance of their interaction for the correct expression of plant genes.

  6. Probable effects of glutatione s-transferase p1 gene exon-6 (ala114val polimorphism on the etiology of lung cancer

    Directory of Open Access Journals (Sweden)

    Etem Akbaş

    2012-09-01

    Full Text Available Objectives: Genetic susceptibility also has significanteffects on the etiology of lung cancer, besides smoking.Previously it has been reported that some genetic polymorphismshave important roles; especially Glutatyon STransferazP1 (GSTP1 gene for the development of lungcancer. GSTP1 gene has a role in phase II of xenobioticmetabolism. GSTP1 Exon-6 polymorphisms have functionaleffects on gene production and causes differencesin enzyme activity.The aim of this study was to investigateprobable effects of GSTP1gene exon-6 (ala114val polimorphismon the etiology of lung cancer.Materials and methods: Our research population consistsedof 160 subjects; 80 as control group and 80 sufferingfrom lung cancer. Genomic DNA was extractedfrom peripheral blood leukocytes and the genotypes havebeen determined by using PCR and RFLP methods.Results: No effect of exon 6 (ala114val polymorphismgenotype of GSTP1 gene were found on etiology of lungcancer in present study. This study showed that smoking,old age and being male are important risk factors forlung cancer. Additionally, our sample’s GSTP1 gene exon6 (ala114val polymorphism genotype frequencies weredetermined.Conclusions: Our data derived from present study didnot suggest an effect of GSTP1gene exon-6 (ala114valpolimorphism on the etiology of lung cancer.Key words: Polimorphism of GSTP1 - exon-6 (ala114val- lung cancer -

  7. A missense mutation in the APC tumor suppressor gene disrupts an ASF/SF2 splicing enhancer motif and causes pathogenic skipping of exon 14.

    Science.gov (United States)

    Gonçalves, Vânia; Theisen, Patrícia; Antunes, Ofélia; Medeira, Ana; Ramos, José Silva; Jordan, Peter; Isidro, Glória

    2009-03-09

    A missense mutation at codon 640 in the APC gene was identified in a familial adenomatous polyposis (FAP) patient, however, its pathological consequence remained unclear. Here we found that this missense mutation interferes at the nucleotide level with an exonic splicing regulatory element and leads to aberrant splicing of the mutant APC transcript rather than exerting its effect through the observed amino acid change. Analysis of the patient RNA revealed complete skipping of exon 14 in transcripts from the mutant APC allele, leading to a frameshift and a premature stop codon. When cloned into a splicing reporter minigene and transfected into colorectal cell lines, the exon 14 point mutation c.1918C>G (pR640G) was found sufficient to cause the observed exon skipping. Bioinformatic analysis predicted the mutation to change SRp55, hnRNP A1 or ASF/SF2 splicing factor binding sites. Using RNA interference methodology these predictions were experimentally validated and revealed that only ASF/SF2 was required for exon 14 inclusion. These research data identify APC mutation c.1918C>G (pR640G) as pathogenic and indicate a mechanism involving disruption of an ASF/SF2 exonic splicing enhancer element. The results allow genetic diagnosis of a hereditary tumour predisposition but also illustrate the need to complement in silico prediction by splicing reporter assays.

  8. Ehlers-Danlos syndrome type VII: a single base change that causes exon skipping in the type I collagen alpha 2(I) chain.

    Science.gov (United States)

    Nicholls, A C; Oliver, J; Renouf, D V; McPheat, J; Palan, A; Pope, F M

    1991-06-01

    We have examined the procollagens and collagens produced by skin fibroblasts from a patient with Ehlers-Danlos syndrome type VII. The patient was heterozygous for an abnormal alpha 2(I) chain migrating with the approximate size of pN alpha 2(I) chains after pepsin digestion. Peptide mapping suggested that the abnormality was located at the amino-terminus of the alpha 2(I) chain. Quantitative analysis of the alpha 2(I) mRNA indicated loss of the exon 6 sequences, and subsequent polymerase chain reaction amplification of cDNA demonstrated a deletion of the 54 bp of exon 6 from some of the alpha 2(I) mRNA. Analysis of genomic DNA from the patient revealed a single base change in one COL1A2 allele, substituting an A for a G as the first base of intron 6. This change mutates the obligate GT-dinulceotide splicing signal to AT and leads to exon skipping with splicing from exon 5 to exon 7. Loss of exon 6 sequences results in the loss of the procollagen-N-propeptidase cleavage site and a lysine residue that normally participates in covalent intermolecular crosslinking within collagen fibres.

  9. CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Hidehito Kuroyanagi

    Full Text Available An enormous number of alternative pre-mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a-4c and 7a-7b, of the Caenorhabditis elegans uncoordinated (unc-32 gene, encoding the a subunit of V0 complex of vacuolar-type H(+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA-binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA-binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive

  10. CELF Family RNA–Binding Protein UNC-75 Regulates Two Sets of Mutually Exclusive Exons of the unc-32 Gene in Neuron-Specific Manners in Caenorhabditis elegans

    Science.gov (United States)

    Kuroyanagi, Hidehito; Watanabe, Yohei; Hagiwara, Masatoshi

    2013-01-01

    An enormous number of alternative pre–mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a–4c and 7a–7b, of the Caenorhabditis elegans uncoordinated (unc)-32 gene, encoding the a subunit of V0 complex of vacuolar-type H+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA–binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA–binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive exons of

  11. CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans.

    Science.gov (United States)

    Kuroyanagi, Hidehito; Watanabe, Yohei; Hagiwara, Masatoshi

    2013-01-01

    An enormous number of alternative pre-mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a-4c and 7a-7b, of the Caenorhabditis elegans uncoordinated (unc)-32 gene, encoding the a subunit of V0 complex of vacuolar-type H(+)-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA-binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA-binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive exons of the unc-32

  12. Single base mutation in the pro. alpha. 2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame pro. alpha. 2(I) chain

    Energy Technology Data Exchange (ETDEWEB)

    Tromp, G.; Prockop, D.J. (Thomas Jefferson Univ., Philadelphia, PA (USA))

    1988-07-01

    Previous observations demonstrated that a lethal variant of osteogenesis imperfecta had two altered alleles for pro{alpha}2(I) chains of type I procollagen. One mutation produced a nonfunctioning allele in that there was synthesis of mRNA but no detectable synthesis of pro{alpha}2(I) chains from the allele. The mutation in the other allele caused synthesis of shortened pro{alpha}2(I) chains that lacked most or all of the 18 amino acids encoded by exon 28. Subclones of the pro{alpha}2(I) gene were prepared from the proband's DNA and the DNA sequence was determined for a 582-base-pair (bp) region that extended from the last 30 bp of intervening sequence 26 to the first 26 bp of intervening sequence 29. Data from six independent subclones demonstrated that all had the same sequence as a previously isolated normal clone for the pro{alpha}2(I) gene except that four subclones had a single base mutation at the 3{prime} end of intervening sequence 27. The mutation was a substitution of guanine for adenine that changed the universal consensus sequence for the 3{prime} splicing site of RNA from -AG- to -GG-. S1 nuclease experiments demonstrated that about half the pro{alpha}2(I) mRNA in the proband's fibroblasts was abnormally spliced and that the major species of abnormal pro{alpha}2(I) mRNA was completely spliced from the last codon of exon 27 to the first codon of exon 29. The mutation is apparently unique among RNA splicing mutations of mammalian systems in producing a shortened polypeptide chain that is in-frame in terms of coding sequences, that is used in the subunit assembly of a protein, and that contributes to a lethal phenotype.

  13. TAT gene mutation analysis in three Palestinian kindreds with oculocutaneous tyrosinaemia type II; characterization of a silent exonic transversion that causes complete missplicing by exon 11 skipping

    DEFF Research Database (Denmark)

    Maydan, G; Andresen, Brage Storstein; Madsen, Pia Pinholt

    2006-01-01

    therefore seek prenatal diagnosis, but this is not possible by biochemical assays as tyrosine does not accumulate in amniotic fluid and TAT is not expressed in chorionic villi or amniocytes. Molecular analysis is therefore the only possible approach for prenatal diagnosis and carrier detection. To this end....... Homozygosity for a c.1249C > T (R417X) exon 12 nonsense mutation (previously reported in a French patient) was identified in both patients from the third kindred, enabling successful prenatal diagnosis of an unaffected fetus using chorionic villous tissue....

  14. Increased Levels of β-catenin, LEF-1, and HPA-1 Correlate with Poor Prognosis for Acral Melanoma with Negative BRAF and NRAS Mutation in BRAF Exons 11 and 15 and NRAS Exons 1 and 2

    Science.gov (United States)

    Xu, Sanxiong; Zhang, Jinyu; Jiang, Yongxin; Chen, Yongbin; Li, Hongjun; Liu, Xuefeng; Xu, Da; Chen, Yanjin; Yang, Yihao; Zhang, Ya; Li, Dongxu; Xia, Junfeng

    2015-01-01

    To determine the expression of β-catenin, lymphoid enhancer-binding protein-1 (LEF-1), and heparanase-1 (HPA-1) and to evaluate these proteins' potential prognostic values in malignant acral melanoma without mutations in BRAF exons 11 and 15 and NRAS exons 1 and 2, specimens from 90 patients with wild-type BRAF and NRAS were assessed and analyzed by immunohistochemistry and western blotting. The positive expression of β-catenin, lymphoid enhancer-binding protein-1, and heparanase-1 was observed in 36 (72%), 31 (62%), and 32 (64%) of the detected acral melanomas, respectively. The expression of β-catenin, lymphoid enhancer-binding protein-1, and heparanase-1 was not correlated with gender, age, or diseased body parts (p>0.05), but was significantly positively correlated with the tumor node metastasis (TNM) stage and metastasis (correlation=0.406 and 0.716, 0.397 and 0.582, 0.353 and 0.579; p=0.040 and 0.0001, 0.0040 and 0.0001, 0.0120 and 0.0001, respectively). We also observed that the increased expression of β-catenin, lymphoid enhancer-binding protein-1, and heparanase-1 was significantly correlated with decreased survival and poor prognosis (p=0.001, 0.010, and 0.023, respectively). A multifactorial analysis using Cox's regression model revealed that β-catenin, lymphoid enhancer-binding protein-1, heparanase-1, and the TNM stage were all independent factors in malignant melanoma (risk ratios were 7.294, 5.550, 5.622, and 4.794; p-values were 0.007, 0.018, 0.018, and 0.029, respectively). This study may provide the basis for the use of β-catenin, lymphoid enhancer-binding protein-1, and heparanase-1 as novel targets in the treatment of malignant invasion and metastasis in acral melanoma cancer. The expression of β-catenin, LEF-1, and HPA-1 was assessed and compared in malignant melanoma with that of peritumoral tissue and benign nevus in the patients with negative mutations in BRAF exons 11 and 15 and NRAS exons 1 and 2. The study may provide the basis for

  15. Comparison of Mutation Profiles in the Duchenne Muscular Dystrophy Gene among Populations: Implications for Potential Molecular Therapies

    Directory of Open Access Journals (Sweden)

    Luz Berenice López-Hernández

    2015-03-01

    Full Text Available Novel therapeutic approaches are emerging to restore dystrophin function in Duchenne Muscular Dystrophy (DMD, a severe neuromuscular disease characterized by progressive muscle wasting and weakness. Some of the molecular therapies, such as exon skipping, stop codon read-through and internal ribosome entry site-mediated translation rely on the type and location of mutations. Hence, their potential applicability worldwide depends on mutation frequencies within populations. In view of this, we compared the mutation profiles of the populations represented in the DMD Leiden Open-source Variation Database with original data from Mexican patients (n = 162 with clinical diagnosis of the disease. Our data confirm that applicability of exon 51 is high in most populations, but also show that differences in theoretical applicability of exon skipping may exist among populations; Mexico has the highest frequency of potential candidates for the skipping of exons 44 and 46, which is different from other populations (p < 0.001. To our knowledge, this is the first comprehensive comparison of theoretical applicability of exon skipping targets among specific populations.

  16. A novel exon 15-deleted, splicing variant of Slit2 shows potential for growth inhibition in addition to invasion inhibition in lung cancer.

    Science.gov (United States)

    Lin, Yu-Ying; Yang, Chun-Hung; Sheu, Gwo-Tarng; Huang, Chi-Ying F; Wu, Yu-Chung; Chuang, Shu-Ming; Fann, Ming-Ji; Chang, Han; Lee, Huei; Chang, Jinghua Tsai

    2011-08-01

    The axon guidance cue molecule Slit2 has been shown to suppress cancer cell invasion. However, the role of Slit2 in growth inhibition is still controversial. The authors identified a novel exon 15 (AKEQYFIP)-deleted slit2, located at the end of the second leucine-rich repeat (LRR2). Because LRR2 interacts with Robo1 receptor to inhibit invasion, they hypothesized that exon 15 plays an important role in modulating Slit2 function. Slit2 expression was assessed via microarray analysis in 27 lung adenocarcinomas. Exon 15-deleted slit2 (slit2-ΔE15) and exon 15-containing slit2 (slit2-WT) were cloned and expressed in the CL1-5 lung cancer cell line. The effect of exon 15 on Slit2-mediated cell growth was evaluated by a xenografted model and in vitro cell growth assays. The effect of exon 15 on Slit2-mediated invasion was analyzed with a modified Boyden chamber in vitro. Tumor growth from CL1-5/Slit2-WT cells was comparable to that from CL1-5 cells bearing empty vector. However, tumor size from CL1-5/Slit2-ΔE15 cells was much smaller than that from Slit2-WT cells or vector control cells in the xenografted model. In vitro analyses demonstrated that Slit2-WT inhibits invasion of CL1-5 cells. In addition to inhibiting invasion, Slit2-ΔE15 greatly suppresses cell growth. The data demonstrated that exon 15 modulates Slit2 function in growth inhibition of lung cancer cells. Because slit2-ΔE15 splice variant is present in low invasive cancer cells and nontumor lung tissues, loss of this splice variant is an important event in tumor progression and invasion. Copyright © 2011 American Cancer Society.

  17. CR1 exon variants are associated with lowered CR1 expression and increased susceptibility to SLE in a Plasmodium falciparum endemic population.

    Science.gov (United States)

    Panda, Aditya K; Ravindran, Balachandran; Das, Bidyut K

    2016-01-01

    Complement receptor 1 (CR1) plays an important role in immune complex clearance by opsonisation and possibly protects subjects from development of autoantibodies. Lower CR1 expression has been associated with susceptibility to systemic lupus erythematosus (SLE). In contrast, subjects displaying lower CR1 expression are protected against severe manifestations of falciparum malaria. This study is the first of its kind to investigate the association of CR1 variants with development of SLE in a P. falciparum endemic population from Odisha, India. CR1 polymorphisms (intron 27 (A>T), exon 22 (A>G) and exon 33 (G>C)) were typed by PCR and restriction length polymorphism in 297 cases of female patients with SLE and 300 age-matched and sex-matched healthy controls from malaria endemic areas in Odisha, India. CR1 expression on monocytes was quantified by flow cytometry. The homozygous mutants of CR1 exon 22 (GG) and exon 33 (GG) and their minor alleles were associated with susceptibility to SLE. Furthermore, patients with SLE who harboured the GG genotype of the exon 33 polymorphism had a 3.12-fold higher chance of developing lupus nephritis. CR1 exon (22 and 33) variants were associated with lowered CR1 expression on monocytes in patients with SLE and in healthy controls. Patients with lupus nephritis showed significantly diminished CR1 expression than those without renal involvement (p=0.01). The results of the present study demonstrate that common CR1 exon variants are associated with diminished CR1 expression on monocytes and increased susceptibility to development of SLE and lupus nephritis in a malaria endemic area.

  18. Structural organization of the human type VII collagen gene (COL7A1), composed of more exons than any previously characterized gene

    Energy Technology Data Exchange (ETDEWEB)

    Christiano, A.M.; Chung-Honet, L.C.; Greenspan, D.S.; Hoffman, G.G.; Lee, S.; Cheng, W. (Univ. of Wisconsin, Madison, WI (United States)); Uitto, J. (Thomas Jefferson Univ., Philadelphia, PA (United States))

    1994-05-01

    The human type VII collagen (COL7A1) gene is the locus for mutations in at least some cases of dystrophic epidermolysis bullosa. Here the authors describe the entire intron/exon organization of COL7A1, which is shown to have 118 exons, more than any previously described gene. Despite this complexity, COL7A1 is compact. Consisting of 31,132 bp from transcription start site to polyadenylation site, it is only about three times the size of type VII collagen mRNA. Thus, COL7A1 introns are small. A 71-nucleotide COL7A1 intron is the smallest intron yet reported in a collagen gene, and only one COL7A1 intron is greater than 1 kb in length. All exons in the COL7A1 triple helix coding region that do not begin with sequences corresponding to imperfections of the triple helix begin with intact codons for Gly residues of Gly-X-Y repeats. This is reminiscent of the structure of fibrillar rather than other nonfibrillar collagen genes. In addition, the COL7A1 triple helix coding region contains many exons of recurring sizes (e.g., 25 exons are 36 bp, 12 exons are 45 bp, 8 exons are 63 bp), suggesting an evolutionary origin distinct from those of other nonfibrillar collagen genes. Sequences from the 5[prime] portion of COL7A1 are presented along with the 3766-bp intergenic sequence, which separated COL7A1 from the upstream gene encoding the core I protein of the cytochrome bc[sub 1] complex. The COL7A1 promoter region is found to lack extensive homologies with promoter regions of other genes expressed primarily in skin. 60 refs., 5 figs., 1 tab.

  19. Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter

    Energy Technology Data Exchange (ETDEWEB)

    Yanagawa, H.; Nishio, H.; Takeshima, Y. [Kobe Univ. School of Medicine (Japan)] [and others

    1994-09-01

    The dystrophin gene, which is muted in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously-unidentified alternative promoter. The case study presented is that of patient with Duchenne muscular dystrophy who had a deletion extending from 5{prime} end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5{prime} part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5{prime} end of the new transcript indicated that the 5{prime} end of exon 3 was extended by 9 codons, only the last (most 3{prime}) of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5{prime} of the previously-identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

  20. [Relationship between R236C site in exon 7 of SP-B gene and respiratory distress syndrome in Han newborns in western Inner Mongolia].

    Science.gov (United States)

    Wang, Jing; Mei, Hua; Liu, Chun-Zhi; Zhang, Ya-Yu; Liu, Chun-Li; Song, Dan; Zhang, Yu-Heng

    2016-09-01

    To detect and analyze the genetic variation in exon 7 of lung surfactant protein B (SP-B), and to investigate the relationship between the genetic variation and the incidence of neonatal respiratory distress syndrome (NRDS) in Han populations in western Inner Mongolia. In the case-control study, 47 Han infants with NRDS were assigned to case group. All the 47 patients had the last three generations of their ancestors reside in western Inner Mongolia. Forty-seven Han newborns without NRDS were assigned to control group. PCR-based gene analysis was used to determine the mutation in exon 7 of SP-B gene and genotype and allele frequencies of the R236C site in exon 7 of SP-B gene. In Han newborns in western Inner Mongolia, there was no mutation in exon 7 of SP-B gene; two genotypes, CC and CT, were identified in the R236C site in exon 7 of SP-B gene. No TT genotype was found in the two groups. There were no significant differences in the genotype frequency of CC or CT as well as the allele frequency of C or T between the case and control groups (CC: 72% vs 85%, P>0.05; CT: 28% vs 15%, P>0.05; C: 85% vs 93%, P>0.05; T: 15% vs 7%, P>0.05). There is no mutation in exon 7 of SP-B gene in Han infants with NRDS in western Inner Mongolia. There is no significant association between the gene polymorphism of the R236C site in exon 7 of SP-B gene and the incidence of NRDS in Han populations in that region.

  1. Allelic Frequency of a 24-bp Duplication in Exon 10 of the CHIT1 Gene in the General Iranian Population.

    Science.gov (United States)

    Motlagh, Behrooz; Taghikhani, Mohammad; Khatami, Shohreh; Zamanfar, Daniel

    2016-01-01

    The human chitinase chitotriosidase enzyme, which is encoded by the CHIT1 gene, is produced by macrophages, and may be important in immune responses to chitin-containing organisms, such as fungi. Plasma chitotriosidase activity is used to diagnose and monitor some forms of lysosomal storage disorders, such as Gaucher's and Niemann-Pick disease. However, homozygous duplication of a 24-bp region in exon 10 of the CHIT1 gene eliminates enzyme activity and may complicate disease monitoring. The high prevalence of this mutation highlights the need to determine its frequency in different populations and screen patients for this mutation to verify whether chitotriosidase activity is a reliable marker of lysosomal storage disease. This study investigated the allele frequency of the 24-bp duplication in the general Iranian population. To identify the 24-bp duplication in exon 10 of the CHIT1 gene (H allele), genotyping of DNA extracted from peripheral blood leukocytes of 577 healthy Iranians was performed using polymerase chain reaction (PCR) amplification and high resolution melting (HRM) PCR techniques. In this study, heterozygous and homozygous duplications were detected in 183 (31.7%) and 35 (6.1%) subjects, respectively. In addition, the allelic frequency was 21.9% (95% confidence interval). Our study indicates that genotype analysis by HRM-PCR is a fast, reliable, and highly accurate screening approach for identifying the 24-bp duplication in CHIT1 exon 10. Due to the wide range of duplication frequencies among different ethnic groups, new biomarkers are necessary for assessing genetic characteristics of lysosomal storage disorders in different populations.

  2. Full-exon pyrosequencing screening of BRCA germline mutations in Mexican women with inherited breast and ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Felipe Vaca-Paniagua

    Full Text Available Hereditary breast cancer comprises 10% of all breast cancers. The most prevalent genes causing this pathology are BRCA1 and BRCA2 (breast cancer early onset 1 and 2, which also predispose to other cancers. Despite the outstanding relevance of genetic screening of BRCA deleterious variants in patients with a history of familial cancer, this practice is not common in Latin American public institutions. In this work we assessed mutations in the entire exonic and splice-site regions of BRCA in 39 patients with breast and ovarian cancer and with familial history of breast cancer or with clinical features suggestive for BRCA mutations by massive parallel pyrosequencing. First we evaluated the method with controls and found 41-485 reads per sequence in BRCA pathogenic mutations. Negative controls did not show deleterious variants, confirming the suitability of the approach. In patients diagnosed with cancer we found 4 novel deleterious mutations (c.2805_2808delAGAT and c.3124_3133delAGCAATATTA in BRCA1; c.2639_2640delTG and c.5114_5117delTAAA in BRCA2. The prevalence of BRCA mutations in these patients was 10.2%. Moreover, we discovered 16 variants with unknown clinical significance (11 in exons and 5 in introns; 4 were predicted as possibly pathogenic by in silico analyses, and 3 have not been described previously. This study illustrates how massive pyrosequencing technology can be applied to screen for BRCA mutations in the whole exonic and splice regions in patients with suspected BRCA-related cancers. This is the first effort to analyse the mutational status of BRCA genes on a Mexican-mestizo population by means of pyrosequencing.

  3. Single nucleotide polymorphism-specific regulation of matrix metalloproteinase-9 by multiple miRNAs targeting the coding exon.

    Science.gov (United States)

    Duellman, Tyler; Warren, Christopher; Yang, Jay

    2014-05-01

    Microribonucleic acids (miRNAs) work with exquisite specificity and are able to distinguish a target from a non-target based on a single nucleotide mismatch in the core nucleotide domain. We questioned whether miRNA regulation of gene expression could occur in a single nucleotide polymorphism (SNP)-specific manner, manifesting as a post-transcriptional control of expression of genetic polymorphisms. In our recent study of the functional consequences of matrix metalloproteinase (MMP)-9 SNPs, we discovered that expression of a coding exon SNP in the pro-domain of the protein resulted in a profound decrease in the secreted protein. This missense SNP results in the N38S amino acid change and a loss of an N-glycosylation site. A systematic study demonstrated that the loss of secreted protein was due not to the loss of an N-glycosylation site, but rather an SNP-specific targeting by miR-671-3p and miR-657. Bioinformatics analysis identified 41 SNP-specific miRNA targeting MMP-9 SNPs, mostly in the coding exon and an extension of the analysis to chromosome 20, where the MMP-9 gene is located, suggesting that SNP-specific miRNAs targeting the coding exon are prevalent. This selective post-transcriptional regulation of a target messenger RNA harboring genetic polymorphisms by miRNAs offers an SNP-dependent post-transcriptional regulatory mechanism, allowing for polymorphic-specific differential gene regulation. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Genetic variation in exon 10 of the BPI gene is associated with Escherichia coli F18 susceptibility in Sutai piglets.

    Science.gov (United States)

    Liu, Lu; Wang, Jing; Zhao, Qiaohui; Zi, Chen; Wu, Zhengchang; Su, Xianmin; Huo, Yongjiu; Zhu, Guoqiang; Wu, Shenglong; Bao, Wenbin

    2013-07-01

    Our aim was to investigate the effect of the porcine bactericidal/permeability-increasing protein (BPI) on the susceptibility to enterotoxigenic Escherichia coli F18 (ETEC F18). Specifically, we wanted to determine whether the HpaII restriction polymorphism in exon 10 of BPI mediates susceptibility to ETEC F18. Thirty verified ETEC F18-resistant and thirty susceptible Sutai (Duroc×Taihu) piglets were identified using the receptor binding assay. Exon 10 of the BPI gene produced the AA, BB, and AB genotypes after HpaII digestion. The genotype distribution among ETEC F18-resistant piglets was significantly different from that among susceptible piglets. Among piglets with the AA genotype, 90% were ETEC F18-resistant; this percentage of resistant piglets was significantly higher than the percentage of resistant piglets with the AB (57.1%) and BB genotypes (17.4%). There was high expression only in the tissues of the duodenum and jejunum, wherein the expression levels in the ETEC F18-resistant group were significantly higher than those in the susceptible group (P<0.05). The average expression levels in individuals with the AA genotype were significantly higher than those in individuals with the AB or BB genotype (P<0.05), while the results of Western blot show the same evidences as real time PCR. These results indicate that the upregulation of porcine BPI gene expression in the small intestines plays a direct role in resistance to ETEC F18 infection. The AA genotype for the HpaII site in exon 10 of the porcine BPI gene was demonstrated to be an anti-ETEC F18 marker and could be used for selective breeding to enhance ETEC F18 resistance. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Dynamics of co-transcriptional pre-mRNA folding influences the induction of dystrophin exon skipping by antisense oligonucleotides.

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    Keng Boon Wee

    Full Text Available Antisense oligonucleotides (AONs mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a "window of analysis" that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered "engaged" if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency of 94% of 176 previously reported AONs. Four novel insights are inferred: (1 the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2 engaged nucleotides at 3' or 5' ends of the target site attenuate AON performance more than at other sites; (3 the performance of longer AONs is less attenuated by engaged nucleotides at 3' or 5' ends of the target site compared to shorter AONs; (4 engaged nucleotides at 3' end of a short target site attenuates AON efficiency more than at 5' end.

  6. Technology to accelerate pangenomic scanning for unknown point mutations in exonic sequences: cycling temperature capillary electrophoresis (CTCE

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    Bjørheim Jens

    2007-08-01

    Full Text Available Abstract Background Rapid means to discover and enumerate unknown mutations in the exons of human genes on a pangenomic scale are needed to discover the genes carrying inherited risk for common diseases or the genes in which somatic mutations are required for clonal diseases such as atherosclerosis and cancers. The method of constant denaturing capillary electrophoresis (CDCE permitted sensitive detection and enumeration of unknown point mutations but labor-intensive optimization procedures for each exonic sequence made it impractical for application at a pangenomic scale. Results A variant denaturing capillary electrophoresis protocol, cycling temperature capillary electrophoresis (CTCE, has eliminated the need for the laboratory optimization of separation conditions for each target sequence. Here are reported the separation of wild type mutant homoduplexes from wild type/mutant heteroduplexes for 27 randomly chosen target sequences without any laboratory optimization steps. Calculation of the equilibrium melting map of each target sequence attached to a high melting domain (clamp was sufficient to design the analyte sequence and predict the expected degree of resolution. Conclusion CTCE provides practical means for economical pangenomic detection and enumeration of point mutations in large-scale human case/control cohort studies. We estimate that the combined reagent, instrumentation and labor costs for scanning the ~250,000 exons and splice sites of the ~25,000 human protein-coding genes using automated CTCE instruments in 100 case cohorts of 10,000 individuals each are now less than U.S. $500 million, less than U.S. $500 per person.

  7. A consensus CaMK IV-responsive RNA sequence mediates regulation of alternative exons in neurons

    OpenAIRE

    Xie, J. Y.; Jan, C.; Stoilov, P; Park, J.; Black, D L

    2005-01-01

    Neurons make extensive use of alternative pre-mRNA splicing to regulate gene expression and diversify physiological responses. We showed previously in a pituitary cell line that the Ca++/calmodulin-dependent protein kinase CaMK IV specifically repressed splicing of the BK channel STREX exon. This repression is dependent on a CaMK IV-responsive RNA element (CaRRE) within the STREX 3′ splice site. Here, we report that similar Ca++ regulation of splicing, mediated by L-type calcium channels and ...

  8. Uncoupling of Expression of an Intronic MicroRNA and Its Myosin Host Gene by Exon Skipping▿

    OpenAIRE

    Bell, Matthew L; Buvoli, Massimo; Leinwand, Leslie A.

    2010-01-01

    The ancient MYH7b gene, expressed in striated muscle and brain, encodes a sarcomeric myosin and the intronic microRNA miR-499. We find that skipping of an exon introduces a premature termination codon in the transcript that downregulates MYH7b protein production without affecting microRNA expression. Among other genes, endogenous miR-499 targets the 3′ untranslated region of the transcription factor Sox6, which in turn acts as a repressor of MYH7b transcriptional activity. Thus, concerted tra...

  9. Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon.

    Science.gov (United States)

    Spena, Silvia; Tenchini, Maria Luisa; Buratti, Emanuele

    2006-06-01

    In this work we report the identification of a strong SF2/ASF binding site within exon 7 of the human fibrinogen Bbeta-chain gene (FGB). Its disruption in the wild-type context has no effect on exon recognition. However, when the mutation IVS7 + 1G>T--initially described in a patient suffering from congenital afibrinogenemia--is present, this SF2/ASF binding site is critical for cryptic 5'ss (splice site) definition. These findings, besides confirming and extending previous results regarding the effect of SF2/ASF on cryptic splice site activation, identify for the first time an enhancer sequence in the FGB gene specific for cryptic splice site usage. Taken together, they suggest the existence of a splicing-regulatory network that is normally silent in the FGB natural splicing environment but which can nonetheless influence splicing decisions when local contexts allow. On a more general note, our conclusions have implications for the evolution of alternative splicing processes and for the development of methods to control aberrant splicing in the context of disease-causing mutations.

  10. A novel BAT3 sequence generated by alternative RNA splicing of exon 11B displays cell type-specific expression and impacts on subcellular localization.

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    Nadine Kämper

    Full Text Available The human lymphocyte antigen (HLA encoded BAT3/BAG6 recently attracted interest as a regulator of protein targeting and degradation, a function that could be exerted in the cytosol and in the nucleus. The BAT3 gene was described to consist of 25 exons. Diversity of transcripts can be generated by alternative RNA splicing, which may control subcellular distribution of BAT3.By cDNA sequencing we identified a novel alternatively spliced sequence of the BAT3 gene located between exons 11 and 12, which was designated as exon 11B. Using PCR and colony hybridization we identified six cDNA variants, which were produced by RNA splicing of BAT3 exons 5, 11B and 24. In four examined cell types the content of BAT3 splice variants was examined. Most of the cDNA clones from monocyte-derived dendritic cells contain exon 11B, whereas this sequence was almost absent in the B lymphoma Raji. Exon 5 was detected in most and exon 24 in approximately half of the cDNA clones. The subcellular distribution of endogenous BAT3 largely correlates with a cell type specific splicing pattern. In cells transfected with BAT3 variants, full-length and Δ24 BAT3 displayed nearly exclusive nuclear staining, whereas variants deleted of exon 11B showed substantial cytosolic expression. We show here that BAT3 is mainly expressed in the cytosol of Raji cells, while other cell types displayed both cytosolic and nuclear staining. Export of BAT3 from the nucleus to the cytosol is inhibited by treatment with leptomycin B, indicating that the Crm1 pathway is involved. Nuclear expression of BAT3 containing exon 11B suggests that this sequence plays a role for nuclear retention of the protein.Cell type-specific subcellular expression of BAT3 suggests distinct functions in the cytosol and in the nucleus. Differential expression of BAT3 variants may reconcile the multiple roles described for BAT3.

  11. Association of Exon 10A and 10B inactivating mutation of follicle stimulating hormone receptor gene (FSHR) and Polycystic Ovarian Syndrome in Vellore cohort

    Science.gov (United States)

    Sekar, Nishu; Kulkarni, Rucha; Ozalkar, Sharvari; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

    2017-11-01

    Polycystic ovarian syndrome is the most common heterogenous endocrine disorder in women. Follicle stimulating hormone receptor is associated with normal development as well as maturation of follicles and triggers estrogen production in granulosa cells of the ovary. Inactivating mutation in FSHR gene correlated with reduction of ovarian function in women is due to damage to receptor function. This study aims to investigate whether inactivating mutations, in follicle stimulating hormone receptor gene is related to polycystic ovarian morphology in women with PCOS. Genomic DNA isolated from 15 subjects from Sandhya Hospital, Vellore (10 patients with PCOS and 5 healthy controls) was taken for this study. Patient data included a clinical report, hormonal levels, and ovarian morphological details. DNA isolation was followed by DNA amplification by polymerase chain reaction using Exon 10 A and Exon 10 B primers. The PCR-RFLP analysis was performed using Dde1 restriction enzyme. Here we discuss inactivating mutation found in Exon 10 of FSHR gene in patients with PCOS.The absence of inactivating mutation was observed through PCR-RFLP study on Exon 10A and Exon 10B.

  12. hnRNP H enhances skipping of a nonfunctional exon P3A in CHRNA1 and a mutation disrupting its binding causes congenital myasthenic syndrome.

    Science.gov (United States)

    Masuda, Akio; Shen, Xin-Ming; Ito, Mikako; Matsuura, Tohru; Engel, Andrew G; Ohno, Kinji

    2008-12-15

    In humans and great apes, CHRNA1 encoding the muscle nicotinic acetylcholine receptor alpha subunit carries an inframe exon P3A, the inclusion of which yields a nonfunctional alpha subunit. In muscle, the P3A(-) and P3A(+) transcripts are generated in a 1:1 ratio but the functional significance and regulation of the alternative splicing remain elusive. An intronic mutation (IVS3-8G>A), identified in a patient with congenital myasthenic syndrome, disrupts an intronic splicing silencer (ISS) and results in exclusive inclusion of the downstream P3A exon. We found that the ISS-binding splicing trans-factor was heterogeneous nuclear ribonucleoprotein (hnRNP) H and the mutation attenuated the affinity of hnRNP for the ISS approximately 100-fold. We next showed that direct placement of hnRNP H to the 3' end of intron 3 silences, and siRNA-mediated downregulation of hnRNP H enhances recognition of exon P3A. Analysis of the human genome suggested that the hnRNPH-binding UGGG motif is overrepresented close to the 3' ends of introns. Pursuing this clue, we showed that alternative exons of GRIP1, FAS, VPS13C and NRCAM are downregulated by hnRNP H. Our findings imply that the presence of the hnRNP H-binding motif close to the 3' end of an intron is an essential but underestimated splicing regulator of the downstream exon.

  13. A novel point mutation within the EDA gene causes an exon dropping in mature RNA in Holstein Friesian cattle breed affected by X-linked anhidrotic ectodermal dysplasia

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    Pariset Lorraine

    2011-07-01

    Full Text Available Abstract Background X-linked anhidrotic ectodermal dysplasia is a disorder characterized by abnormal development of tissues and organs of ectodermal origin caused by mutations in the EDA gene. The bovine EDA gene encodes the ectodysplasin A, a membrane protein expressed in keratinocytes, hair follicles and sweat glands, which is involved in the interactions between cell and cell and/or cell and matrix. Four mutations causing ectodermal dysplasia in cattle have been described so far. Results We identified a new single nucleotide polymorphism (SNP at the 9th base of exon 8 in the EDA gene in two calves of Holstein Friesian cattle breed affected by ectodermal dysplasia. This SNP is located in the exonic splicing enhancer (ESEs recognized by SRp40 protein. As a consequence, the spliceosome machinery is no longer able to recognize the sequence as exonic and causes exon skipping. The mutation determines the deletion of the entire exon (131 bp in the RNA processing, causing a severe alteration of the protein structure and thus the disease. Conclusion We identified a mutation, never described before, that changes the regulation of alternative splicing in the EDA gene and causes ectodermal dysplasia in cattle. The analysis of the SNP allows the identification of carriers that can transmit the disease to the offspring. This mutation can thus be exploited for a rational and efficient selection of unequivocally healthy cows for breeding.

  14. Identification of polymorphism in exons 7 and 12 of lactoferrin gene and its association with incidence of clinical mastitis in Murrah buffalo.

    Science.gov (United States)

    Dinesh, Krishanender; Verma, Archana; Das Gupta, Ishwar; Thakur, Yash Pal; Verma, Nishant; Arya, Ashwani

    2015-04-01

    Lactoferrin gene is one of the important candidate genes for mastitis resistance. The gene is located on chromosome BTA 22 and consists of 17 exons spanning over 34.5 kb of genomic DNA. The present study was undertaken with the objectives to identify allelic variants in exons 7 and 12 of lactoferrin gene and to analyze association between its genetic variants and incidence of clinical mastitis in Murrah buffalo. The amplification of exons 7 and 12 of lactoferrin gene yielded amplicons of 232- and 461-bp sizes. PCR-restriction fragment length polymorphism (RFLP) analysis of 232-bp amplicon using BccI restriction enzyme revealed three genotypes (AA, AB, and BB) with frequencies of 0.62, 0.22, and 0.16, respectively. The frequencies of two alleles, A and B, were estimated as 0.73 and 0.27. Hpy188I-RFLP for 461-bp amplicon revealed polymorphism with three genotypes, CC, CD, and DD, with respective frequencies of 0.06, 0.39, and 0.56, whereas frequencies for C and D alleles were 0.25 and 0.75. The chi-square (χ(2)) analysis revealed a significant association between incidence of clinical mastitis and genetic variants of exon 7, and animals of AA genotype of exon 7 were found to be least susceptible to mastitis. The findings indicate potential scope for incorporation of lactoferrin gene in selection and breeding of Murrah buffaloes for improved genetic resistance to mastitis.

  15. [Whole exon 5 and intron 5 replaced by RHD/CE in partial D phenotype DVa (Hus)].

    Science.gov (United States)

    Zhou, Yi-Yan; Xiong, Wen; Shao, Chao-Peng

    2005-02-01

    The study was purposed to analyze DNA and allele structure of the partial D phenotypes D(Va) and D(VI) of the Rhesus blood group in Chinese. Through polymerase chain reaction (PCR) and direct genomic DNA sequencing, the RHD gene was detected in three weak D individuals identified serologically. The results showed that among the three weak D individuals, one was identified as partial D phenotype D(Va) (Hus) type and genotyped DccEe; another two were testified as D(VI) III type and genotyped DCcee. Moreover, the breakpoints of the replaced region by RHCE in D(Va) (Hus) were 5' end of the exon 5 and 3' end of the intron 5, and there were 7 novel polymorphisms in intron 5: 23-25(GCA)2, 98G>A, 168-169insG, 205-206insT, 494-495insA, 1256-1257insC, 1347G>T. In conclusion the whole exon 5 and intron 5 are replaced by RHCE in D(Va) (Hus) detected in Chinese.

  16. Restoring dystrophin expression in duchenne muscular dystrophy muscle progress in exon skipping and stop codon read through.

    Science.gov (United States)

    Hoffman, Eric P; Bronson, Abby; Levin, Arthur A; Takeda, Shin'ichi; Yokota, Toshifumi; Baudy, Andreas R; Connor, Edward M

    2011-07-01

    The identification of the Duchenne muscular dystrophy gene and protein in the late 1980s led to high hopes of rapid translation to molecular therapeutics. These hopes were fueled by early reports of delivering new functional genes to dystrophic muscle in mouse models using gene therapy and stem cell transplantation. However, significant barriers have thwarted translation of these approaches to true therapies, including insufficient therapeutic material (eg, cells and viral vectors), challenges in systemic delivery, and immunological hurdles. An alternative approach is to repair the patient's own gene. Two innovative small-molecule approaches have emerged as front-line molecular therapeutics: exon skipping and stop codon read through. Both approaches are in human clinical trials and aim to coax dystrophin protein production from otherwise inactive mutant genes. In the clinically severe dog model of Duchenne muscular dystrophy, the exon-skipping approach recently improved multiple functional outcomes. We discuss the status of these two methods aimed at inducing de novo dystrophin production from mutant genes and review implications for other disorders. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  17. Transforming pluripotency: an exon-level study of malignancy-specific transcripts in human embryonal carcinoma and embryonic stem cells.

    Science.gov (United States)

    Alagaratnam, Sharmini; Harrison, Neil; Bakken, Anne Cathrine; Hoff, Andreas M; Jones, Mark; Sveen, Anita; Moore, Harry D; Andrews, Peter W; Lothe, Ragnhild A; Skotheim, Rolf I

    2013-04-01

    To circumvent difficulties of isolating pure populations of cancer stem cells (CSCs) for the purpose of identifying malignancy-specific gene expression, we have compared exon-resolution transcriptomic profiles of 5 embryonal carcinoma (EC) cell lines, a histological subtype of germ cell tumor (GCT), to their nonmalignant caricature, specifically 6 human embryonic stem (ES) cell lines. Both cell types are readily accessible, and were purified for undifferentiated cells only. We identified a set of 28 differentially expressed genes, many of which had cancer and stemness roles. Overexpression of the recently discovered pluripotency gene NR5A2 in malignant EC cells revealed an intriguing indication of how WNT-mediated dysregulation of pluripotency is involved with malignancy. Expression of these 28 genes was further explored within 2 publically available data sets of primary EC tumors and normal testis. At the exon-level, alternative splicing events were detected in ZNF195, DNMT3B, and PMF1, and alternative promoters were detected for ASH2L and ETV5. These events were validated by reverse transcriptase-polymerase chain reaction-based methods in EC and ES lines, where the alternative splicing event in the de novo DNA methyltransferase DNMT3B may have functional consequences. In conclusion, we have identified malignancy-specific gene expression differences within a rigorous pluripotent stem cell context. These findings are of particular interest for both GCT and ES cell biology, and, in general, to the concept of CSCs.

  18. Chromosomal Aberrations and Exon 1 Mutation in the AKR1B1 Gene in Patients with Diabetic Neuropathy.

    Science.gov (United States)

    Saraswathy, Radha; Anand, Sudhaa; Kunnumpurath, Sree Kumar; Kurian, R Jones; Kaye, Alan David; Vadivelu, Nalini

    2014-01-01

    Recent decades have seen an increase in our understanding of a number of pathophysiological processes associated with type 2 diabetes mellitus (DM). Despite increases in understanding and treatment options, diabetic neuropathy remains a significant problem and is associated with tremendous morbidity and mortality. In this regard, oxidative DNA damage is postulated to play a role in diabetes-mediated neuropathic pathogenesis. In this pilot investigation, we studied the extent of chromosomal damage utilizing chromosomal aberration (CA) assay in cultured lymphocytes of patients in 3 subgroups: patients with diabetic neuropathy, patients with type 2 DM and no neuropathy, and a control group. The patients with diabetic neuropathy showed a statistically significantly higher rate of CA (Pneuropathy (0.03 ± 0.02). Samples from subjects with diabetic neuropathy were evaluated to check for mutations in the AKR1B1 gene (exon 1). A significant number of mutations appeared after DNA sequencing within the AKR1B1 gene. Of 50 diabetic neuropathy patient samples analyzed, 10 revealed mutations. Our results suggest that painful diabetic neuropathy is a condition with enhanced genomic instability characterized by increased CA and possible mutations. Exon 1 of the gene AKR1B1 showed significant mutations in patients with painful diabetic neuropathy.

  19. A Novel Homozygous Frameshift Mutation in Exon 2 of Gene Associated with Severe Obesity: A Case Report

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    Ashwaq Shukri Altawil

    2016-01-01

    Full Text Available Background Monogenic obesity is a rare type of obesity caused by a mutation in a single gene. Patients with monogenic obesity may develop early onset of obesity and severe metabolic abnormalities. Case Presentation A two-and-half-year-old girl was presented to our clinic because of excessive weight gain and hyperphagia. She was born at full term, by normal vaginal delivery with birth weight of 2.82 kg and no complications during pregnancy. The patient was the second child of two healthy, non-obese Saudis with known consanguinity. She gained weight rapidly leading to obesity at the age of three months. Methods The demographic data and clinical features were recorded. Blood samples were collected and tested for endocrine and metabolic characteristics and genetic studies. Mutations of the LEP gene were screened. The coding exons 2 and 3 and the corresponding exon–intron boundaries were amplified by polymerase chain reaction using specific primers, analyzed by direct sequencing using an ABI sequencer 3500 xL GA (Applied Biosystems, and evaluated using the JSI SeqPilot software. The resulting sequence data were compared with the reference MM_0002302. Conclusion We report a novel homozygous frameshift mutation c.144delin TAC (G1n49Thrfs * 23 in exon 2 of the LEP gene associated with extreme obesity.

  20. Methyl-Cytosine-Driven Structural Changes Enhance Adduction Kinetics of an Exon 7 fragment of the p53 Gene

    Science.gov (United States)

    Malla, Spundana; Kadimisetty, Karteek; Fu, You-Jun; Choudhary, Dharamainder; Schenkman, John B.; Rusling, James F.

    2017-01-01

    Methylation of cytosine (C) at C-phosphate-guanine (CpG) sites enhances reactivity of DNA towards electrophiles. Mutations at CpG sites on the p53 tumor suppressor gene that can result from these adductions are in turn correlated with specific cancers. Here we describe the first restriction-enzyme-assisted LC-MS/MS sequencing study of the influence of methyl cytosines (MeC) on kinetics of p53 gene adduction by model metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using methodology applicable to correlate gene damage sites for drug and pollutant metabolites with mutation sites. This method allows direct kinetic measurements by LC-MS/MS sequencing for oligonucleotides longer than 20 base pairs (bp). We used MeC and non-MeC (C) versions of a 32 bp exon 7 fragment of the p53 gene. Methylation of 19 cytosines increased the rate constant 3-fold for adduction on G at the major reactive CpG in codon 248 vs. the non-MeC fragment. Rate constants for non-CpG codons 244 and 243 were not influenced significantly by MeC. Conformational and hydrophobicity changes in the MeC-p53 exon 7 fragment revealed by CD spectra and molecular modeling increase the BPDE binding constant to G in codon 248 consistent with a pathway in which preceding reactant binding greatly facilitates the rate of covalent SN2 coupling.

  1. An Enhancer Near ISL1 and an Ultraconserved Exon of PCBP2 areDerived from a Retroposon

    Energy Technology Data Exchange (ETDEWEB)

    Bejerano, Gill; Lowe, Craig; Ahituv, Nadav; King, Bryan; Siepel,Adam; Salama, Sofie; Rubin, Edward M.; Kent, W. James; Haussler, David

    2005-11-27

    Hundreds of highly conserved distal cis-regulatory elementshave been characterized to date in vertebrate genomes1. Many thousandsmore are predicted based on comparative genomics2,3. Yet, in starkcontrast to the genes they regulate, virtually none of these regions canbe traced using sequence similarity in invertebrates, leaving theirevolutionary origin obscure. Here we show that a class of conserved,primarily non-coding regions in tetrapods originated from a novel shortinterspersed repetitive element (SINE) retroposon family that was activein Sarcopterygii (lobe-finned fishes and terrestrial vertebrates) in theSilurian at least 410 Mya4, and, remarkably, appears to be recentlyactive in the "living fossil" Indonesian coelacanth, Latimeriamenadoensis. We show that one copy is a distal enhancer, located 500kbfrom the neuro-developmental gene ISL1. Several others represent new,possibly regulatory, alternatively spliced exons in the middle ofpre-existing Sarcopterygian genes. One of these is the>200bpultraconserved region5, 100 percent identical in mammals, and 80 percentidentical to the coelacanth SINE, that contains a 31aa alternativelyspliced exon of the mRNA processing gene PCBP26. These add to a growinglist of examples7 in which relics of transposable elements have acquireda function that serves their host, a process termed "exaptation"8, andprovide an origin for at least some of the highly-conservedvertebrate-specific genomic sequences recently discovered usingcomparative genomics.

  2. SH3BP2-encoding exons involved in cherubism are not associated with central giant cell granuloma.

    Science.gov (United States)

    Teixeira, R C; Horz, H P; Damante, J H; Garlet, G P; Santos, C F; Nogueira, R L M; Cavalcante, R B; Conrads, G

    2011-08-01

    Central giant cell granuloma (CGCG) is a benign lesion with unpredictable biological behaviour ranging from a slow-growing asymptomatic swelling to an aggressive lesion associated with pain, bone and root resorption and also tooth displacement. The aetiology of the disease is unclear with controversies in the literature on whether it is mainly of reactional, inflammatory, infectious, neoplasic or genetic origin. To test the hypothesis that mutations in the SH3BP2 gene, as the principal cause of cherubism, are also responsible for, or at least associated with, giant cell lesions, 30 patients with CGCG were recruited for this study and subjected to analysis of germ line and/or somatic alterations. In the blood samples of nine patients, one codon alteration in exon 4 was found, but this alteration did not lead to changes at the amino acid level. In conclusion, if a primary genetic defect is the cause for CGCG it is either located in SH3BP2 gene exons not yet related to cherubism or in a different gene. Copyright © 2011 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  3. Mechanism of Deletion Removing All Dystrophin Exons in a Canine Model for DMD Implicates Concerted Evolution of X Chromosome Pseudogenes.

    Science.gov (United States)

    VanBelzen, D Jake; Malik, Alock S; Henthorn, Paula S; Kornegay, Joe N; Stedman, Hansell H

    2017-03-17

    Duchenne muscular dystrophy (DMD) is a lethal, X-linked, muscle-wasting disorder caused by mutations in the large, 2.4-Mb dystrophin gene. The majority of DMD-causing mutations are sporadic, multi-exon, frameshifting deletions, with the potential for variable immunological tolerance to the dystrophin protein from patient to patient. While systemic gene therapy holds promise in the treatment of DMD, immune responses to vectors and transgenes must first be rigorously evaluated in informative preclinical models to ensure patient safety. A widely used canine model for DMD, golden retriever muscular dystrophy, expresses detectable amounts of near full-length dystrophin due to alternative splicing around an intronic point mutation, thereby confounding the interpretation of immune responses to dystrophin-derived gene therapies. Here we characterize a naturally occurring deletion in a dystrophin-null canine, the German shorthaired pointer. The deletion spans 5.6 Mb of the X chromosome and encompasses all coding exons of the DMD and TMEM47 genes. The sequences surrounding the deletion breakpoints are virtually identical, suggesting that the deletion occurred through a homologous recombination event. Interestingly, the deletion breakpoints are within loci that are syntenically conserved among mammals, yet the high homology among this subset of ferritin-like loci is unique to the canine genome, suggesting lineage-specific concerted evolution of these atypical sequence elements.

  4. A missense mutation in a highly conserved alternate exon of dynamin-1 causes epilepsy in fitful mice.

    Directory of Open Access Journals (Sweden)

    Rebecca M Boumil

    2010-08-01

    Full Text Available Dynamin-1 (Dnm1 encodes a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Mice heterozygous for a novel spontaneous Dnm1 mutation--fitful--experience recurrent seizures, and homozygotes have more debilitating, often lethal seizures in addition to severe ataxia and neurosensory deficits. Fitful is a missense mutation in an exon that defines the DNM1a isoform, leaving intact the alternatively spliced exon that encodes DNM1b. The expression of the corresponding alternate transcripts is developmentally regulated, with DNM1b expression highest during early neuronal development and DNM1a expression increasing postnatally with synaptic maturation. Mutant DNM1a does not efficiently self-assemble into higher order complexes known to be necessary for proper dynamin function, and it also interferes with endocytic recycling in cell culture. In mice, the mutation results in defective synaptic transmission characterized by a slower recovery from depression after trains of stimulation. The DNM1a and DNM1b isoform pair is highly conserved in vertebrate evolution, whereas invertebrates have only one isoform. We speculate that the emergence of more specialized forms of DNM1 may be important in organisms with complex neuronal function.

  5. A splice site mutation in the PAX6 gene which induces exon skipping causes autosomal dominant inherited aniridia

    Science.gov (United States)

    Wissinger, Bernd; Gramer, Eugen

    2012-01-01

    Purpose To identify the underlying genetic cause in a two generation German family diagnosed with isolated aniridia. Methods All patients underwent full ophthalmic examination. Mutation screening of the paired box gene 6 (PAX6) was performed by bidirectional Sanger sequencing. A minigene assay was applied to analyze transcript processing of mutant and wildtype PAX6 variants in HEK293 cells. Results We identified a PAX6 sequence variant at the splice donor site (+5) of intron 12. This variant has been described before in another family with aniridia but has not been characterized at the transcript level. We could demonstrate that the mutant allele causes the skipping of exon 12 during transcript processing. The mutation is predicted to result in a ‘run on’ translation past the normal translational stop codon. Conclusions A splice site mutation resulting in exon skipping was found in a family with autosomal dominant aniridia. The mutation is predicted to result in an enlarged protein with an extra COOH-terminal domain. This very likely affects the transactivation properties of the PAX6 protein. PMID:22509105

  6. Analysis of ELA-DQB exon 2 polymorphism in Argentine Creole horses by PCR-RFLP and PCR-SSCP.

    Science.gov (United States)

    Villegas-Castagnasso, E E; Díaz, S; Giovambattista, G; Dulout, F N; Peral-García, P

    2003-08-01

    The second exon of equine leucocyte antigen (ELA)-DQB genes was amplified from genomic DNA of 32 Argentine Creole horses by PCR. Amplified DNA was analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-single-strand conformation polymorphism (SSCP). The PCR-RFLP analysis revealed two HaeIII patterns, four RsaI patterns, five MspI patterns and two HinfI patterns. EcoRI showed no variation in the analysed sample. Additional patterns that did not account for known exon 2 DNA sequences were observed, suggesting the existence of novel ELA-DQB alleles. PCR-SSCP analysis exhibited seven different band patterns, and the number of bands per animal ranged from four to nine. Both methods indicated that at least two DQB genes are present. The presence of more than two alleles in each animal showed that the primers employed in this work are not specific for a unique DQB locus. The improvement of this PCR-RFLP method should provide a simple and rapid technique for an accurate definition of ELA-DQB typing in horses.

  7. Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene

    Directory of Open Access Journals (Sweden)

    Ana Gonçalves

    2017-10-01

    Full Text Available A broad mutational spectrum in the dystrophin (DMD gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD. Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR, and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1, disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD, adding to the diversity of mutational events that give rise to D/BMD.

  8. Distinct intraspecific variations of garlic (Allium sativum L.) revealed by the exon-intron sequences of the alliinase gene.

    Science.gov (United States)

    Endo, Aki; Imai, Yukiko; Nakamura, Mizuho; Yanagisawa, Eri; Taguchi, Takaaki; Torii, Kosuke; Okumura, Hidenobu; Ichinose, Koji

    2014-04-01

    Garlic (Allium sativum L.) has been used worldwide as a food and for medicinal purposes since early times. Garlic cultivars exhibit considerable morphological diversity despite the fact that they are mostly sterile and are grown only by vegetative propagation of cloves. Considerable recombination occurs in garlic genomes, including the genes involved in secondary metabolites. We examined the genomic DNAs (gDNAs) from garlic, encoding alliinase, a key enzyme involved in organosulfur metabolism in Allium plants. The 1.7-kb gDNA fragments, covering three exons (2, 3, and 4) and all four introns, were amplified from total DNAs prepared from garlic samples produced in Asia and Europe, leading to 73 sequences in total: Japan (JPN), China (CHN), India (IND), Spain (ESP), and France (FRA). The exon sequences were highly conserved among all the sequences, probably reflecting the fully functional alliinase associated with the flavor quality. Distinct intraspecific variations were detected for all four intron sequences, leading to the haplotype classifications. A close relationship between JPN and CHN was observed for all four introns, whereas IND showed a more divergent distribution. ESP and FRA afforded clearly different variants compared with those from Asian sequences. The present study provides information that could be useful in the development of an additional molecular marker for garlic authentication and quality control.

  9. Identification of Exonic Nucleotide Variants of the Gene Associated with Carcass Traits and Fatty Acid Composition in Korean Cattle

    Directory of Open Access Journals (Sweden)

    Dong-yep Oh

    2014-10-01

    Full Text Available The thyroid hormone responsive protein (THRSP gene is a functional gene that can be used to indicate the fatty acid compositions. This study investigates the relationships of exonic single nucleotide polymorphisms (SNPs in the THRSP gene and fatty acid composition of muscle fat and marbling score in the 612 Korean cattle. The relationships between fatty acid composition and eight SNPs in the THRSP gene (g.78 G>A, g.173 C>T, g.184 C>T, g.190 C>A, g.194 C>T, g.277 C>G, g.283 T>G and g.290 T>G were investigated, and according to the results, two SNPs (g.78 G>A and g.184 C>T in exon 1 were associated with fatty acid composition. The GG and CC genotypes of g.78 G>A and g.184 C>T had higher unsaturated fatty acid (UFA and monounsaturated fatty acid (MUFA content (pA and g.184 C>T had significantly relationships with UFAs and MUFAs. Two SNPs in the THRSP gene affected fatty acid composition, suggesting that GG and CC genotypes and the ht1*ht1 group (Val/Ala haplotype can be markers to genetically improve the quality and flavor of beef.

  10. The Analysis of the MspI Polymorphism within Exon 3 of the Calpastatin Gene in Slovak Simmental Cattle

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    Michal Gábor

    2012-05-01

    Full Text Available Calpastatin has important function in the tenderization process. The SNP polymorphisms in the calpastatin gene suchas UoG-CAST (intron 5 and CAST-T1 (3´UTR region are used as markers in commercial test for prediction ofanimals with tenderness meat. The others research of the calpastatine gene confirmed the impact of a SNPpolymorphism in exon 3 on fertility and longevity in dairy cattle, too. The aim of this study was to analyse thepopulation of 113 animals of Slovak Simmental (42 bulls and 71 cows for the missense SNP polymorphism in exon3. Bovine genomic DNA was isolated from sperm and blood by commercial kit. The SNP CAST c.283 C>T wasdetected by PCR-RFLP method with restriction endonuclease MspI. The favourable allele C was detected by tworestriction fragments 135 bp and 173 bp and the allele T with the one 308 bp fragment. In the analyzed population ofSlovak Simmental cattle were detected the following frequency of alleles and genotypes for the SNP c.283 C>T ofthe CAST gene. Frequencies of allele C and allele T were 0.6460 and 0.3540 and frequencies of genotypes were0.4336 (genotype CC, 0.4248 (genotype CT and 0.1416 (genotype TT.

  11. Nanoparticle delivery of antisense oligonucleotides and their application in the exon skipping strategy for Duchenne muscular dystrophy.

    Science.gov (United States)

    Falzarano, Maria Sofia; Passarelli, Chiara; Ferlini, Alessandra

    2014-02-01

    Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy.

  12. Use of a Fluorescent Aptamer RNA as an Exonic Sequence to Analyze Self-Splicing Ability of a Group I Intron from Structured RNAs

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    Airi Furukawa

    2016-11-01

    Full Text Available Group I self-splicing intron constitutes an important class of functional RNA molecules that can promote chemical transformation. Although the fundamental mechanism of the auto-excision from its precursor RNA has been established, convenient assay systems for its splicing activity are still useful for a further understanding of its detailed mechanism and of its application. Because some host RNA sequences, to which group I introns inserted form stable three-dimensional (3D structures, the effects of the 3D structures of exonic elements on the splicing efficiency of group I introns are important but not a fully investigated issue. We developed an assay system for group I intron self-splicing by employing a fluorescent aptamer RNA (spinach RNA as a model exonic sequence inserted by the Tetrahymena group I intron. We investigated self-splicing of the intron from spinach RNA, serving as a model exonic sequence with a 3D structure.

  13. IUGR increases chromatin-remodeling factor Brg1 expression and binding to GR exon 1.7 promoter in newborn male rat hippocampus.

    Science.gov (United States)

    Ke, Xingrao; McKnight, Robert A; Gracey Maniar, Lia E; Sun, Ying; Callaway, Christopher W; Majnik, Amber; Lane, Robert H; Cohen, Susan S

    2015-07-15

    Intrauterine growth restriction (IUGR) increases the risk for neurodevelopment delay and neuroendocrine reprogramming in both humans and rats. Neuroendocrine reprogramming involves the glucocorticoid receptor (GR) gene that is epigenetically regulated in the hippocampus. Using a well-characterized rodent model, we have previously shown that IUGR increases GR exon 1.7 mRNA variant and total GR expressions in male rat pup hippocampus. Epigenetic regulation of GR transcription may involve chromatin remodeling of the GR gene. A key chromatin remodeler is Brahma-related gene-1(Brg1), a member of the ATP-dependent SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. Brg1 regulates gene expression by affecting nucleosome repositioning and recruiting transcriptional components to target promoters. We hypothesized that IUGR would increase hippocampal Brg1 expression and binding to GR exon 1.7 promoter, as well as alter nucleosome positioning over GR promoters in newborn male pups. Further, we hypothesized that IUGR would lead to accumulation of specificity protein 1 (Sp1) and RNA pol II at GR exon 1.7 promoter. Indeed, we found that IUGR increased Brg1 expression and binding to GR exon 1.7 promoter. We also found that increased Brg1 binding to GR exon 1.7 promoter was associated with accumulation of Sp1 and RNA pol II carboxy terminal domain pSer-5 (a marker of active transcription). Furthermore, the transcription start site of GR exon 1.7 was located within a nucleosome-depleted region. We speculate that changes in hippocampal Brg1 expression mediate GR expression and subsequently trigger neuroendocrine reprogramming in male IUGR rats. Copyright © 2015 the American Physiological Society.

  14. A statistical framework for genome-wide discovery of biomarker splice variations with GeneChip Human Exon 1.0 ST Arrays.

    Science.gov (United States)

    Yoshida, Ryo; Numata, Kazuyuki; Imoto, Seiya; Nagasaki, Masao; Doi, Atsushi; Ueno, Kazuko; Miyano, Satoru

    2006-01-01

    Alternative splicing is an important regulatory mechanism that generates multiple mRNA transcripts which are transcribed into functionally diverse proteins. According to the current studies, aberrant transcripts due to splicing mutations are known to cause for 15% of genetic diseases. Therefore understanding regulatory mechanism of alternative splicing is essential for identifying potential biomarkers for several types of human diseases. Most recently, advent of GeneChip Human Exon 1.0 ST Array enables us to measure genome-wide expression profiles of over one million exons. With this new microarray platform, analysis of functional gene expressions could be extended to detect not only differentially expressed genes, but also a set of specific-splicing events that are differentially observed between one or more experimental conditions, e.g. tumor or normal control cells. In this study, we address the statistical problems to identify differentially observed splicing variations from exon expression profiles. The proposed method is organized according to the following process: (1) Data preprocessing for removing systematic biases from the probe intensities. (2) Whole transcript analysis with the analysis of variance (ANOVA) to identify a set of loci that cause the alternative splicing-related to a certain disease. We test the proposed statistical approach on exon expression profiles of colorectal carcinoma. The applicability is verified and discussed in relation to the existing biological knowledge. This paper intends to highlight the potential role of statistical analysis of all exon microarray data. Our work is an important first step toward development of more advanced statistical technology. Supplementary information and materials are available from http://bonsai.ims.u-tokyo.ac.jp/~yoshidar/IBSB2006_ExonArray.htm.

  15. High expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice.

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    Yu-Chia Chen

    Full Text Available Spinal muscular atrophy (SMA is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn-/- SMN2 expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-β1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease and a potential target for SMA treatment.

  16. Analysis of 30 putative BRCA1 splicing mutations in hereditary breast and ovarian cancer families identifies exonic splice site mutations that escape in silico prediction.

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    Barbara Wappenschmidt

    Full Text Available Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis.

  17. Splice donor site mutation in the lysosomal neuraminidase gene causing exon skipping and complete loss of enzyme activity in a sialidosis patient.

    Science.gov (United States)

    Penzel, R; Uhl, J; Kopitz, J; Beck, M; Otto, H F; Cantz, M

    2001-07-20

    Sialidosis is a lysosomal storage disease caused by the deficiency of alpha-N-acetylneuraminidase (NEU1; sialidase), the key enzyme for the intralysosomal catabolism of sialylated glycoconjugates. We have identified a homozygous transversion in the last intron (IVSE +1 G>C) in neu1 of a sialidosis patient. Sequencing of the truncated cDNA revealed an alternatively spliced neu1 transcript which lacks the complete sequence of exon 5. Skipping of exon 5 leads to a frameshift and results in a premature termination codon. This is the first description of an intronic point mutation causing a complete deficiency of the lysosomal neuraminidase activity.

  18. Mutations in Exons 9 and 13 of KIT Gene Are Rare Events in Gastrointestinal Stromal Tumors : A Study of 200 Cases

    OpenAIRE

    Lasota, Jerzy; Wozniak, Agnieszka; Sarlomo-Rikala, Maarit; Rys, Janusz; Kordek, Radzislaw; Nassar, Aziza; Sobin, Leslie H.; Miettinen, Markku

    2000-01-01

    Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the gastrointestinal tract, typically express the KIT protein. Activating mutations in the juxtamembrane domain (exon 11) of the c-kit gene have been shown in a subset of GISTs. These mutations lead into ligand-independent activation of the tyrosine kinase of c-kit, and have a transforming effect in vitro. Several groups have studied the clinical implication of the c-kit mutation status of exon 11 in GISTs and a po...

  19. Complexity of a small non-protein coding sequence in chromosomal region 22q11.2: presence of specialized DNA secondary structures and RNA exon/intron motifs.

    Science.gov (United States)

    Delihas, Nicholas

    2015-10-14

    DiGeorge Syndrome is a genetic abnormality involving ~3 Mb deletion in human chromosome 22, termed 22q.11.2. To better understand the non-coding regions of 22q.11.2, a small 10,000 bp non-protein-coding sequence close to the DiGeorge Critical Region 6 gene (DGCR6) was chosen for analysis and functional entities as the homologous sequence in the chimpanzee genome could be aligned and used for comparisons. The GenBank database provided genomic sequences. In silico computer programs were used to find homologous DNA sequences in human and chimpanzee genomes, generate random sequences, determine DNA sequence alignments, sequence comparisons and nucleotide repeat copies, and to predicted DNA secondary structures. At its 5' half, the 10,000 bp sequence has three distinct sections that represent phylogenetically variable sequences. These Variable Regions contain biased mutations with a very high A + T content, multiple copies of the motif TATAATATA and sequences that fold into long A:T-base-paired stem loops. The 3' half of the 10,000 bp unit, highly conserved between human and chimpanzee, has sequences representing exons of lncRNA genes and segments of introns of protein genes. Central to the 10,000 bp unit are the multiple copies of a sequence that originates from the flanking 5' end of the translocation breakpoint Type A sequence. This breakpoint flanking sequence carries the exon and intron motifs. The breakpoint Type A sequence seems to be a major player in the proliferation of these RNA motifs, as well as the proliferation of Variable Regions in the 10,000 bp segment and other regions within 22q.11.2. The data indicate that a non-coding region of the chromosome may be reserved for highly biased mutations that lead to formation of specialized sequences and DNA secondary structures. On the other hand, the highly conserved nucleotide sequence of the non-coding region may form storage sites for RNA motifs.

  20. Molecular genetic analysis of cereal β-amylase genes using exon-primed intron-crossing (EPIC PCR

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    Stratula Olga

    2014-01-01

    Full Text Available The proteins encoded by cereal β-amylase genes Bamy1 and Bamy2 genes play an important role in seedling germination and in the brewing process. Here, we use exon-primed intron-crossing (EPIC to analyse Bamy1 and Bamy2 genetic diversity among 38 accessions belonging to six Poaceae tribes. DNA sequence alignment of multiple Poaceae species β-amylase sequences allowed design of EPIC primers that simultaneously amplify Bamy1 and Bamy2 in all the cereal species investigated. The genetic variation observed in the samples investigated is analysed and discussed, and illustrates the effectiveness of this approach for intra- and interspecific analysis in plant species.

  1. Heterogeneous resistance mechanisms in an EGFR exon 19-mutated non-small cell lung cancer patient treated with erlotinib

    DEFF Research Database (Denmark)

    Santoni-Rugiu, Eric; Grauslund, Morten; Melchior, Linea C

    2017-01-01

    an EGFR exon 19-mutation (p.E746_A750delELREA) and a previously unreported 2bp frame-shift microdeletion in the fibroblast growth factor receptor 3 (FGFR3; p.D785fs*31) gene. Interestingly, FGFR3-mutations have previously been described in other cancer types of Caucasian patients and may represent...... an alternative pathway to EGFR-signaling. The patient received first-line erlotinib but after only 7 weeks showed metastatic pleural effusion, in which transformation to small cell lung cancer (SCLC) that retained the EGFR- and FGFR3-mutations was identified. Consequently, standard carboplatin-etoposide regimen...... for SCLC combined with erlotinib continuation was implemented obtaining significant objective response. However, after completing 6 cycles of this combination, new pulmonary and hepatic metastases appeared and showed persistence of the original EGFR- and FGFR3-mutated ADC phenotype together...

  2. Application of target capture sequencing of exons and conserved non-coding sequences to 20 inbred rat strains

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    Minako Yoshihara

    2016-12-01

    Full Text Available We report sequence data obtained by our recently devised target capture method TargetEC applied to 20 inbred rat strains. This method encompasses not only all annotated exons but also highly conserved non-coding sequences shared among vertebrates. The total length of the target regions covers 146.8 Mb. On an average, we obtained 31.7× depth of target coverage and identified 154,330 SNVs and 24,368 INDELs for each strain. This corresponds to 470,037 unique SNVs and 68,652 unique INDELs among the 20 strains. The sequence data can be accessed at DDBJ/EMBL/GenBank under accession number PRJDB4648, and the identified variants have been deposited at http://bioinfo.sls.kyushu-u.ac.jp/rat_target_capture/20_strains.vcf.gz.

  3. Disease-causing mutations in exon 11 of the medium-chain acyl-CoA dehydrogenase gene

    DEFF Research Database (Denmark)

    Andresen, B S; Jensen, T G; Bross, P

    1994-01-01

    . Our results show that exon 11 is not especially mutation prone. We demonstrate that two of the identified disease-causing mutations can be detected by restriction enzyme digestion of the PCR product from the assay for the G985 mutation, a discovery that makes this assay even more useful than before......Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of the mitochondrial beta-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985....... On the basis of expression of wild-type and mutant MCAD protein in COS-7 cells, we show that the identified mutations abolish MCAD enzyme activity and that they therefore must be disease causing. The M301T, S311R, and K304E mutations are located in helix H, which makes up part of the dimer-dimer interface...

  4. Adeno-Associated Virus (AAV) Mediated Dystrophin Gene Transfer Studies and Exon Skipping Strategies for Duchenne Muscular Dystrophy (DMD).

    Science.gov (United States)

    Kawecka, Klaudia; Theodoulides, Michael; Hasoglu, Yalin; Jarmin, Susan; Kymalainen, Hanna; Le-Heron, Anita; Popplewell, Linda; Malerba, Alberto; Dickson, George; Athanasopoulos, Takis

    2015-01-01

    Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies.

  5. A novel de novo exon 21 DNMT1 mutation causes cerebellar ataxia, deafness, and narcolepsy in a Brazilian patient.

    Science.gov (United States)

    Pedroso, José Luiz; Povoas Barsottini, Orlando Graziani; Lin, Ling; Melberg, Atle; Oliveira, Acary S B; Mignot, Emmanuel

    2013-08-01

    Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN) is caused by DNMT1 mutations. Diagnosing the syndrome can be difficult, as all clinical features may not be present at onset, HLA-DQB1*06:02 is often negative, and sporadic cases occur. We report on clinical and genetic findings in a 31-year-old woman with cerebellar ataxia, deafness, and narcolepsy, and discuss diagnostic challenges. Clinical and genetic investigation in a patient and family members. Ataxia clinic, São Paulo, Brazil. One patient and her family members. N/A. Narcolepsy was supported by polysomnographic and multiple sleep latency testing. HLA-DQB1*06:02 was positive. CSF hypocretin-1 was 191 pg/mL (normal values > 200 pg/mL). Mild brain atrophy was observed on MRI, with cerebellar involvement. The patient, her asymptomatic mother, and 3 siblings gave blood samples for genetic analysis. DNMT1 exons 20 and 21 were sequenced. Haplotyping of polymorphic markers surrounding the mutation was performed. The proband had a novel DNMT1 mutation in exon 21, p.Cys596Arg, c.1786T > C. All 4 parental haplotypes could be characterized in asymptomatic siblings without the mutation, indicating that the mutation is de novo in the patient. The Brazilian patient reported here further adds to the worldwide distribution of ADCA-DN. The mutation is novel, and illustrates a sporadic case with de novo mutation. We believe that many more cases with this syndrome are likely to be diagnosed in the near future, mandating knowledge of this condition and consideration of the diagnosis.

  6. Altered Striatal Synaptic Function and Abnormal Behaviour in Shank3 Exon4-9 Deletion Mouse Model of Autism.

    Science.gov (United States)

    Jaramillo, Thomas C; Speed, Haley E; Xuan, Zhong; Reimers, Jeremy M; Liu, Shunan; Powell, Craig M

    2016-03-01

    Shank3 is a multi-domain, synaptic scaffolding protein that organizes proteins in the postsynaptic density of excitatory synapses. Clinical studies suggest that ∼ 0.5% of autism spectrum disorder (ASD) cases may involve SHANK3 mutation/deletion. Patients with SHANK3 mutations exhibit deficits in cognition along with delayed/impaired speech/language and repetitive and obsessive/compulsive-like (OCD-like) behaviors. To examine how mutation/deletion of SHANK3 might alter brain function leading to ASD, we have independently created mice with deletion of Shank3 exons 4-9, a region implicated in ASD patients. We find that homozygous deletion of exons 4-9 (Shank3(e4-9) KO) results in loss of the two highest molecular weight isoforms of Shank3 and a significant reduction in other isoforms. Behaviorally, both Shank3(e4-9) heterozygous (HET) and Shank3(e4-9) KO mice display increased repetitive grooming, deficits in novel and spatial object recognition learning and memory, and abnormal ultrasonic vocalizations. Shank3(e4-9) KO mice also display abnormal social interaction when paired with one another. Analysis of synaptosome fractions from striata of Shank3(e4-9) KO mice reveals decreased Homer1b/c, GluA2, and GluA3 expression. Both Shank3(e4-9) HET and KO demonstrated a significant reduction in NMDA/AMPA ratio at excitatory synapses onto striatal medium spiny neurons. Furthermore, Shank3(e4-9) KO mice displayed reduced hippocampal LTP despite normal baseline synaptic transmission. Collectively these behavioral, biochemical and physiological changes suggest Shank3 isoforms have region-specific roles in regulation of AMPAR subunit localization and NMDAR function in the Shank3(e4-9) mutant mouse model of autism. © 2015 International Society for Autism Research, Wiley Periodicals, Inc.

  7. Distance-dependent processing of adeno-associated virus type 5 RNA is controlled by 5' exon definition.

    Science.gov (United States)

    Qiu, Jianming; Cheng, Fang; Pintel, David

    2007-08-01

    Adeno-associated virus type 5 (AAV5) is unique among human AAV serotypes in that it uses a polyadenylation site [(pA)p] within the single small intron in the center of the genome. We previously reported that inhibition of polyadenylation at (pA)p, necessary for read-through of P41-generated capsid gene pre-mRNAs which are subsequently spliced, requires binding of U1 snRNP to the upstream donor. Inhibition was reduced as the distance between the cap site and the donor was increased (increasing the size of the 5' exon). Here, we have demonstrated that U1-70K is a key component of U1 snRNP that mediates inhibition of polyadenylation at (pA)p. Furthermore, introduction of a U-rich stretch, predicted to target TIA-1 and thus increase the affinity of U1 snRNP binding to the intervening donor site, significantly augmented inhibition of (pA)p, while depletion of TIA-1 by siRNA increased (pA)p read-through. Finally, artificially tethering the cap binding complex (CBC) components CBP80 and CBP20 upstream of the intron donor increased inhibition of polyadenylation at (pA)p. Our results suggest that interaction with the CBC strengthens U1 snRNP binding to the downstream intron donor in a manner inversely proportional to the size of the 5' exon, thus governing the competition between intron splicing and polyadenylation at (pA)p. This competition must be optimized to program both the levels of polyadenylation of P7- and P19-generated RNA at (pA)p required to produce proper levels of the essential Rep proteins and the splicing of P41-generated RNAs to produce the proper ratio of capsid proteins during AAV5 infection.

  8. Androgen response element of the glycine N-methyltransferase gene is located in the coding region of its first exon.

    Science.gov (United States)

    Lee, Cheng-Ming; Yen, Chia-Hung; Tzeng, Tsai-Yu; Huang, Yu-Zen; Chou, Kuan-Hsien; Chang, Tai-Jay; Arthur Chen, Yi-Ming

    2013-09-17

    Androgen plays an important role in the pathogenesis of PCa (prostate cancer). Previously, we identified GNMT (glycine N-methyltransferase) as a tumour susceptibility gene and characterized its promoter region. Besides, its enzymatic product-sarcosine has been recognized as a marker for prognosis of PCa. The goals of this study were to determine whether GNMT is regulated by androgen and to map its AREs (androgen response elements). Real-time PCR analyses showed that R1881, a synthetic AR (androgen receptor) agonist induced GNMT expression in AR-positive LNCaP cells, but not in AR-negative DU145 cells. In silico prediction showed that there are four putative AREs in GNMT-ARE1, ARE2 and ARE3 are located in the intron 1 and ARE4 is in the intron 2. Consensus ARE motif deduced from published AREs was used to identify the fifth ARE-ARE5 in the coding region of exon 1. Luciferase reporter assay found that only ARE5 mediated the transcriptional activation of R1881. ARE3 overlaps with a YY1 [Yin and Yang 1 (motif (CaCCATGTT, +1118/+1126)] that was further confirmed by antibody supershift and ChIP (chromatin immunoprecipitation) assays. EMSA (electrophoretic mobility shift assay) and ChIP assay confirmed that AR interacts with ARE5 in vitro and in vivo. In summary, GNMT is an AR-targeted gene with its functional ARE located at +19/+33 of the first exon. These results are valuable for the study of the influence of androgen on the gene expression of GNMT especially in the pathogenesis of cancer.

  9. Genetic Variations of TAP1 Gene Exon 3 Affects Gene Expression and Escherichia coli F18 Resistance in Piglets

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    Qiaohui Zhao

    2014-06-01

    Full Text Available Firstly, our research group identified Sutai pigs’ phenotypes that exhibited extreme resistance and susceptibility to the Escherichia coli F18 respectively, and then eight ETEC (Enterotoxigenic Escherichia coli F18-resistant piglets and eight ETEC F18-sensitive piglets were selected. Then, the TAP1 (Transporter associated with antigen processing mRNA relative expression levels were analyzed in 11 tissues of the resistant and susceptible phenotypes. Simultaneously, we detected the genetic variations in exon 3 of the TAP1 gene and evaluated the TAP1 mRNA expression levels among the different genotype pigs to study the effects of the genetic variation on gene expression, and the E. coli F18 resistance. The results revealed higher expression levels in the resistant genotypes than that in the susceptible genotypes in 11 tissues, with significant differences in the spleen, lymph node, lung, thymus, duodenum and jejunum. Furthermore, a G729A mutation was identified in the TAP1 gene exon 3, and this mutation deviates from Hardy-Weinberg equilibrium (p < 0.01. The TAP1 mRNA levels in GG genotype were significantly higher than that in the other two genotypes, with significant differences in the liver, lung, kidney, thymus, lymph node, duodenum and jejunum tissues. We speculated that high expression of the TAP1 gene might confer resistance against the E. coli F18, the G729A mutation had a significant effect on the mRNA expression, and individuals with the GG genotype possessed a stronger ability to resist the E. coli F18 infection.

  10. Imprinting mutations in Angelman syndrome detected by Southern blotting using a probe containing exon {alpha} of SNRPN

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    Beuten, J.; Sutcliffe, J.S.; Nakao, M. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy (UPD), or other mutations. The SNRPN gene maps in this region, is paternally expressed, and is a candidate gene for PWS. Southern blotting using methylation-sensitive enzymes and a genomic DNA probe from the CpG island containing exon {alpha} of the SNRPN gene reveals methylation specific for the maternal allele. In cases of the usual deletions or UPD, the probe detects absence of an unmethylated allele in PWS and absence of a methylated allele in AS. We have analyzed 21 nondeletion/nonUPD AS patients with this probe and found evidence for an imprinting mutation (absence of a methylated allele) in 3 patients. Southern blotting with methylation-sensitive enzymes using the exon {alpha} probe, like use of the PW71 probe, should detect abnormalities in all known PWS cases and in 3 of the 4 forms of AS: deletion, UPD and imprinting mutations. This analysis provides a valuable diagnostic approach for PWS and AS. In efforts to localize the imprinting mutations in AS, one patient was found with failure to inherit a dinucleotide repeat polymorphism near probe 189-1 (D15S13). Analysis of this locus in AS families and CEPH families demonstrates a polymorphism that impairs amplification and a different polymorphism involving absence of hybridization to the 189-1 probe. The functional significance, if any, of deletion of the 189-1 region is unclear.

  11. Exon-level transcriptome profiling in murine breast cancer reveals splicing changes specific to tumors with different metastatic abilities.

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    Amandine Bemmo

    2010-08-01

    Full Text Available Breast cancer is the second most frequent type of cancer affecting women. We are increasingly aware that changes in mRNA splicing are associated with various characteristics of cancer. The most deadly aspect of cancer is metastasis, the process by which cancer spreads from the primary tumor to distant organs. However, little is known specifically about the involvement of alternative splicing in the formation of macroscopic metastases. Our study investigates transcript isoform changes that characterize tumors of different abilities to form growing metastases.To identify alternative splicing events (ASEs that are associated with the fully metastatic phenotype in breast cancer, we used Affymetrix Exon Microarrays to profile mRNA isoform variations genome-wide in weakly metastatic (168FARN and 4T07 and highly metastatic (4T1 mammary carcinomas. Statistical analysis identified significant expression changes in 7606 out of 155,994 (4% exons and in 1725 out of 189,460 (1% intronic regions, which affect 2623 out of 16,654 (16% genes. These changes correspond to putative alternative isoforms-several of which are novel-that are differentially expressed between tumors of varying metastatic phenotypes. Gene pathway analysis showed that 1224 of genes expressing alternative isoforms were involved in cell growth, cell interactions, cell proliferation, cell migration and cell death and have been previously linked to cancers and genetic disorders. We chose ten predicted splice variants for RT-PCR validation, eight of which were successfully confirmed (MED24, MFI2, SRRT, CD44, CLK1 and HNRNPH1. These include three novel intron retentions in CD44, a gene in which isoform variations have been previously associated with the metastasis of several cancers.Our findings reveal that various genes are differently spliced and/or expressed in association with the metastatic phenotype of tumor cells. Identification of metastasis-specific isoforms may contribute to the

  12. The analysis of mutations and exon deletions at TSC2 gene in angiomyolipomas associated with tuberous sclerosis complex.

    Science.gov (United States)

    Yang, Heung-Mo; Choi, Hye-Jung; Hong, Doo-Pyo; Joo, Sung-Yeon; Lee, Na-Eun; Song, Ji-Young; Choi, Yoon-La; Lee, Jeeyun; Choi, Dongil; Kim, BoKyung; Park, Hyo-Jun; Park, Jae-Berm; Kim, Sung Joo

    2014-12-01

    Angiomyolipomas (AMLs) are relatively rare hamartomatous or benign tumors that occasionally occur as part of tuberous sclerosis complex (TSC). Mutations in either of the two genes, TSC1 and TSC2, have been attributed to the development of TSC. Between 1994 and January 2009, 83 patients were diagnosed with AML at the Samsung Medical Center. In that group of patients, 5 (6%) had AML with TSC (AML-TSC). Mutational analysis of the TSC2 gene was performed using 7 samples from the 5 AML-TSC patients and 14 samples from 14 patients with sporadic AML without TSC (AML-non-TSC). From this analysis, mutations in TSC genes were identified in 5 samples from the AML-TSC patients (mutation detection rate=71%) and 3 samples from AML-non-TSC patients (mutation detection rate=21%). In the case of AML-TSC, 6 mutations were found including 3 recurrent mutations and 3 novel mutations, while in the case of AML-non-TSC, 4 mutations were identified once, including 1 novel mutation. Also MLPA analysis of the TSC2 gene showed that TSC2 exon deletion is more frequently observed in AML-TSC patients than in AML-non-TSC patients. This is the first mutation and multiplex ligation-dependent probe amplification (MLPA) analyses of TSC2 in Korean AMLs that focus on TSC. This study provides data that are representative of the distribution of mutations and exon deletions at TSC genes in clinically diagnosed AML-TSC cases of the Korean population. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Lack of mutations of exon 2 of the MEN1 gene in endocrine and nonendocrine sporadic tumors

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    Costa S.C.

    2001-01-01

    Full Text Available In addition to the mutations that underlie most cases of the multiple endocrine neoplasia type 1 (MEN1 syndrome, somatic mutations of the MEN1 gene have also been described in sporadic tumors like gastrinomas, insulinomas and bronchial carcinoid neoplasm. We examined exon 2 of this gene, where most of the mutations have been described, in 148 endocrine and nonendocrine sporadic tumors. DNA was obtained by phenol/chloroform extraction and ethanol precipitation from 92 formalin-fixed, paraffin-embedded samples, and from 40 fresh tumor tissue samples. We used 5 pairs of primers to encompass the complete coding sequence of exon 2 of the MEN1 gene that was screened by the polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP technique in 78 sporadic thyroid cancers: 28 follicular adenomas, 35 papillary carcinomas, 14 follicular carcinomas, and 1 anaplastic thyroid carcinoma. We also examined 46 adrenal lesions (3 hyperplasias, 3 adenomas and 35 adrenocortical carcinomas, 2 pheochromocytomas, 2 ganglioneuroblastomas, and 1 lymphoma and 24 breast cancers (6 noninvasive, 16 infiltrating ductal, and 2 invasive lobular tumors. The PCR product of 5 tumors suspected to present band shifts by SSCP was cloned. Direct sense and antisense sequencing did not identify mutations. These results suggest that the MEN1 gene is not important in breast, thyroid or adrenal sporadic tumorigenesis. Because the frequency of mutations varies significantly among tumor subgroups and allelic deletions are frequently observed at 11q13 in thyroid and adrenal cancers, another tumor suppressor gene residing in this region is likely to be involved in the tumorigenesis of these neoplasms.

  14. Transcription and epigenetic profile of the promoter, first exon and first intron of the human tyrosine hydroxylase gene.

    Science.gov (United States)

    Romano, Gaetano; Macaluso, Marcella; Lucchetti, Chiara; Iacovitti, Lorraine

    2007-05-01

    The transcriptional and chromatin profile of the promoter, first exon and first intron of the human TH gene were analyzed in human neuroblastoma BE(2)-C-16 and human renal carcinoma 293FT cell lines. The latter is a cell culture system that is not permissive for TH gene expression, whereas the former has a 50% cell fraction that tests positive for TH. The engineering of a 6.3 kb recombinant human TH promoter revealed the presence of repressors of transcription between positions (-6,244/-194). The addition of a 1.2 kb fragment of the first intron of the human TH gene (+730/+1,653) enhanced transcriptional activity of the recombinant promoter. However, both constructs were not specific for TH-positive BE(2)-C-16 cells. Chromatin immunoprecipitation (Chip) analysis was carried out on BE(2)-C-16 and 293FT cells to probe sequences of promoter, first exon and first intron of the human TH gene from position (-448/+1,204). The presence of nucleosomes was observed approximately from position (-20/+473) in both cell lines. Chip analysis was then conducted to determine the acetylation of various lysine residues of H3 and H4 in both cell lines. All analyzed lysine residues of H3 and H4 were acetylated in BE(2)-C-16 cells, whereas 293FT cells tested positive for acetylation only in the external lysine residues of the histone tail. Our data are compatible with an active TH gene expression in a 50% cell fraction of BE(2)-C-16 cells. Further analysis of epigenetic programming might lead to the identification of the factors that determine TH gene expression specifically in dopaminergic neurons. (c) 2007 Wiley-Liss, Inc.

  15. Cockayne Syndrome due to a maternally-inherited whole gene deletion of ERCC8 and a paternally-inherited ERCC8 exon 4 deletion.

    Science.gov (United States)

    Ting, T W; Brett, M S; Tan, E S; Shen, Y; Lee, S P; Lim, E C; Vasanwala, R F; Lek, N; Thomas, T; Lim, K W; Tan, E C

    2015-11-10

    Cockayne Syndrome (CS) is an autosomal recessive disorder that causes neurological regression, growth failure and dysmorphic features. We describe a Chinese female child with CS caused by deletions of exon 4 of ERCC8 on one chromosome and exons 1-12 on the other chromosome. By using chromosomal microarray, multiplex ligation-dependant probe analysis and long range PCR, we showed that she inherited a 277 kb deletion affecting the whole ERCC8 gene from the mother and a complex rearrangement resulting in deletion of exon 4 together with a 1,656 bp inversion of intron 4 from the father. A similar complex rearrangement has been reported in four unrelated Japanese CS patients. Analysis of the deletion involving exon 4 identified LINE and other repeat elements that may predispose the region to deletions, insertions and inversions. The patient also had insulin-dependent diabetes mellitus, a rare co-existing feature in patients with CS. More research will be needed to further understand the endocrine manifestations in CS patients. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Microdeletion/duplication at the Xq28 IP locus causes a de novo IKBKG/NEMO/IKKgamma exon4_10 deletion in families with Incontinentia Pigmenti.

    Science.gov (United States)

    Fusco, Francesca; Paciolla, Mariateresa; Pescatore, Alessandra; Lioi, Maria Brigida; Ayuso, Carmen; Faravelli, Francesca; Gentile, Mattia; Zollino, Marcella; D'Urso, Michele; Miano, Maria Giuseppina; Ursini, Matilde Valeria

    2009-09-01

    The Incontinentia Pigmenti (IP) locus contains the IKBKG/NEMO/IKKgamma gene and its truncated pseudogene copy, IKBKGP/deltaNEMO. The major genetic defect in IP is a heterozygous exon4_10 IKBKG deletion (IKBKGdel) caused by a recombination between two consecutive MER67B repeats. We analyzed 91 IP females carrying the IKBKGdel, 59 of whom carrying de novo mutations (65%). In eight parents, we found two recurrent nonpathological variants of IP locus, which were also present as rare polymorphism in control population: the IKBKGPdel, corresponding to the exon4_10 deletion in the pseudogene, and the MER67Bdup, that replicates the exon4_10 region downstream of the normal IKBKG gene. Using quantitative DNA analysis and microsatellite mapping, we established that both variants might promote the generation of the pathological IKBKGdel. Indeed, in family IP-516, the exon4_10 deletion was repositioned in the same allele from the pseudogene to the gene, whereas in family IP-688, the MER67Bdup generated the pathological IKBKGdel by recombination between two direct nonadjacent MER67Bs. Moreover, we found an instance of somatic recombination in a MER67Bdup variant, creating the IKBKGdel in an IP male. Our data suggest that the IP locus undergoes recombination producing recurrent variants that might be "at risk" of generating de novo IKBKGdel by NAHR during either meiotic or mitotic division.

  17. hnRNP A1 and hnRNP F modulate the alternative splicing of exon 11 of the insulin receptor gene.

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    Indrani Talukdar

    Full Text Available Exon 11 of the insulin receptor gene (INSR is alternatively spliced in a developmentally and tissue-specific manner. Linker scanning mutations in a 5' GA-rich enhancer in intron 10 identified AGGGA sequences that are important for enhancer function. Using RNA-affinity purification and mass spectrometry, we identified hnRNP F and hnRNP A1 binding to these AGGGA sites and also to similar motifs at the 3' end of the intron. The hnRNPs have opposite functional effects with hnRNP F promoting and hnRNP A1 inhibiting exon 11 inclusion, and deletion of the GA-rich elements eliminates both effects. We also observed specific binding of hnRNP A1 to the 5' splice site of intron 11. The SR protein SRSF1 (SF2/ASF co-purified on the GA-rich enhancer and, interestingly, also competes with hnRNP A1 for binding to the splice site. A point mutation -3U→C decreases hnRNP A1 binding, increases SRSF1 binding and renders the exon constitutive. Lastly, our data point to a functional interaction between hnRNP F and SRSF1 as a mutant that eliminates SRSF1 binding to exon 11, or a SRSF1 knockdown, which prevents the stimulatory effect of hnRNP F over expression.

  18. Screening for single nucleotide variants, small indels and exon deletions with a next-generation sequencing based gene panel approach for Usher syndrome.

    Science.gov (United States)

    Krawitz, Peter M; Schiska, Daniela; Krüger, Ulrike; Appelt, Sandra; Heinrich, Verena; Parkhomchuk, Dmitri; Timmermann, Bernd; Millan, Jose M; Robinson, Peter N; Mundlos, Stefan; Hecht, Jochen; Gross, Manfred

    2014-09-01

    Usher syndrome is an autosomal recessive disorder characterized both by deafness and blindness. For the three clinical subtypes of Usher syndrome causal mutations in altogether 12 genes and a modifier gene have been identified. Due to the genetic heterogeneity of Usher syndrome, the molecular analysis is predestined for a comprehensive and parallelized analysis of all known genes by next-generation sequencing (NGS) approaches. We describe here the targeted enrichment and deep sequencing for exons of Usher genes and compare the costs and workload of this approach compared to Sanger sequencing. We also present a bioinformatics analysis pipeline that allows us to detect single-nucleotide variants, short insertions and deletions, as well as copy number variations of one or more exons on the same sequence data. Additionally, we present a flexible in silico gene panel for the analysis of sequence variants, in which newly identified genes can easily be included. We applied this approach to a cohort of 44 Usher patients and detected biallelic pathogenic mutations in 35 individuals and monoallelic mutations in eight individuals of our cohort. Thirty-nine of the sequence variants, including two heterozygous deletions comprising several exons of USH2A, have not been reported so far. Our NGS-based approach allowed us to assess single-nucleotide variants, small indels, and whole exon deletions in a single test. The described diagnostic approach is fast and cost-effective with a high molecular diagnostic yield.

  19. Cloning and characterization of promoter and 5 '-UTR of the NMDA receptor subunit epsilon(2) : evidence for alternative splicing of 5 '-non-coding exon

    NARCI