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Sample records for cftr frameshift mutation

  1. DNA evolved to minimize frameshift mutations

    OpenAIRE

    Agoni, Valentina

    2013-01-01

    Point mutations can surely be dangerous but what is worst than to lose the reading frame?! Does DNA evolved a strategy to try to limit frameshift mutations?! Here we investigate if DNA sequences effectively evolved a system to minimize frameshift mutations analyzing the transcripts of proteins with high molecular weights.

  2. Evaluation of CFTR gene mutations in Adana

    OpenAIRE

    Ozlem Goruroglu Ozturk; Filiz Kibar; Esin Damla Ziyanoglu Karacor; Salih Cetiner; Gulhan Sahin; Akgun Yaman

    2013-01-01

    ABSTRACT Objective: Cystic fibrosis is the most common autosomal recessive inherited disorder seen in the white populations. It develops in result of mutations of cystic fibrosis transmembrane regulator (CFTR) gene. Rate of these mutations vary in different geographical regions. In this study, we aimed to determine the frequency of CFTR gene mutations in Adana. Methods: DNA samples of 63 subjects (21 women, 42 men) who were diagnosed as cystic fibrosis at Balcali Hospital of Cukurova Universi...

  3. Evaluation of CFTR gene mutations in Adana

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    Ozlem Goruroglu Ozturk

    2013-04-01

    Full Text Available ABSTRACT Objective: Cystic fibrosis is the most common autosomal recessive inherited disorder seen in the white populations. It develops in result of mutations of cystic fibrosis transmembrane regulator (CFTR gene. Rate of these mutations vary in different geographical regions. In this study, we aimed to determine the frequency of CFTR gene mutations in Adana. Methods: DNA samples of 63 subjects (21 women, 42 men who were diagnosed as cystic fibrosis at Balcali Hospital of Cukurova University, were studied for 19 different CFTR mutations by the strip assay method which is based on reverse hybridization. Results: In cystic fibrosis diagnosed patients, 19 mutations were observed of which 9 were homozygous and 10 were heterozygous. ∆F508 frequency was found as 11.9%, and rate of homozygous was found as 66.7%. Mutation frequencies of W1282X and N1303K were found as 2.40% and 4.80% respectively and rate of homozygous mutations were 50% for both. I148T mutation frequency was found as 3.20% and all were heterozygous. For the whole 19 mutations, frequency of mutation in 63 subjects was 22.3%. Conclusion: Detection of CFTR gene mutations by the strip assay method by reverse hybridization is an easy, fast and informative method. However, due to improvability of the common mutations in probable cystic fibrosis patients because of heterogenity in this region, it is still a major problem and does not exclude cystic fibrosis diagnosis. But this problematic issue can be overcome by evaluating the whole exons of CFTR mutations by advanced molecular tecniques. Key words: CFTR, cystic fibrosis, molecular diagnosis, reverse hibridisation [Cukurova Med J 2013; 38(2.000: 202-208

  4. Naturally occurring mutations in the canine CFTR gene

    OpenAIRE

    Spadafora, Domenico; Hawkins, Eleanor C.; Murphy, Keith E.; Clark, Leigh Anne; Ballard, Stephen T.

    2010-01-01

    Naturally occurring cystic fibrosis (CF)-causing mutations in the CFTR gene have not been identified in any nonhuman animal species. Since domestic dogs are known to develop medical conditions associated with atypical CF in humans (e.g., bronchiectasis and pancreatitis), we hypothesized that dogs with these disorders likely have a higher expression rate of CFTR mutations than the at-large population. Temporal temperature-gradient gel electrophoresis (TTGE) was used to screen canine CFTR in 40...

  5. Multi-physiopathological consequences of the c.1392G>T CFTR mutation revealed by clinical and cellular investigations.

    Science.gov (United States)

    Farhat, Raed; El-Seedy, Ayman; El-Moussaoui, Kamal; Pasquet, Marie-Claude; Adolphe, Catherine; Bieth, Eric; Languepin, Jeanne; Sermet-Gaudelus, Isabelle; Kitzis, Alain; Ladevèze, Véronique

    2015-02-01

    This study combines a clinical approach and multiple level cellular analyses to determine the physiopathological consequences of the c.1392G>T (p.Lys464Asn) CFTR exon 10 mutation, detected in a CF patient with a frameshift deletion in trans and a TG(11)T(5) in cis. Minigene experiment, with different TG(m)T(n) alleles, and nasal cell mRNA extracts were used to study the impact of c.1392G>T on splicing in both in cellulo and in vivo studies. The processing and localization of p.Lys464Asn protein were evaluated, in cellulo, by western blotting analyses and confocal microscopy. Clinical and channel exploration tests were performed on the patient to determine the exact CF phenotype profile and the CFTR chloride transport activity. c.1392G>T affects exon 10 splicing by inducing its complete deletion and encoding a frameshift transcript. The polymorphism TG(11)T(5) aggravates the effects of this mutation on aberrant splicing. Analysis of mRNA obtained from parental airway epithelial cells confirmed these in cellulo results. At the protein level the p.Lys464Asn protein showed neither maturated form nor membrane localization. Furthermore, the in vivo channel tests confirmed the absence of CFTR activity. Thus, the c.1392G>T mutation alone or in association with the TG repeats and the poly T tract revealed obvious impacts on splicing and CFTR protein processing and functionality. The c.[T(5); 1392G>T] complex allele contributes to the CF phenotype by affecting splicing and inducing a severe misprocessing defect. These results demonstrate that the classical CFTR mutations classification is not sufficient: in vivo and in cellulo studies of a possible complex allele in a patient are required to provide correct CFTR mutation classification, adequate medical counseling, and adapted therapeutic strategies.

  6. New frameshift CF mutation 3729delAinsTCT in a Tunisian cystic ...

    Indian Academy of Sciences (India)

    More than 1800 CFTR (cystic fibrosis transmembrane con- ductance regulator gene) sequence variations have been identified in the cystic fibrosis (CF) mutation database. (http://www.genet.sickkids.on.ca/cftr/). For North African populations, however, the nature and frequency of the major. CFTR mutations remain unclear, ...

  7. Role of CFTR mutation analysis in the diagnostic algorithm for cystic fibrosis.

    Science.gov (United States)

    Ratkiewicz, Michelle; Pastore, Matthew; McCoy, Karen Sharrock; Thompson, Rohan; Hayes, Don; Sheikh, Shahid Ijaz

    2017-04-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation identification is being used with increased frequency to aid in the diagnosis of cystic fibrosis (CF) in those suspected with CF. Aim of this study was to identify diagnostic outcomes when CFTR mutational analysis was used in CF diagnosis. CFTR mutational analysis results were also compared with sweat chloride results. This study was done on all patients at our institution who had CFTR mutation analysis over a sevenyear period since August 2006. A total of 315 patients underwent CFTR mutational analysis. Fifty-one (16.2%) patients had two mutations identified. Among them 32 had positive sweat chloride levels (≥60 mmol/L), while seven had borderline sweat chloride levels (40-59 mmol/L). An additional 70 patients (22.3%) had only one mutation identified. Among them eight had positive sweat chloride levels, and 17 had borderline sweat chloride levels. Fifty-five patients (17.5%) without CFTR mutations had either borderline (n=45) or positive (n=10) sweat chloride results. Three patients with a CF phenotype had negative CFTR analysis but elevated sweat chloride levels. In eighty-three patients (26.4%) CFTR mutational analysis was done without corresponding sweat chloride testing. Although CFTR mutation analysis has improved the diagnostic capability for CF, its use either as the first step or the only test to diagnose CFTR dysfunction should be discouraged and CF diagnostic guidelines need to be followed.

  8. Mutational patterns in the frameshift-regulating site of HIV-1 selected by protease inhibitors.

    Science.gov (United States)

    Knops, Elena; Brakier-Gingras, Léa; Schülter, Eugen; Pfister, Herbert; Kaiser, Rolf; Verheyen, Jens

    2012-05-01

    Sustained suppression of viral replication in HIV-1 infected patients is especially hampered by the emergence of HIV-1 drug resistance. The mechanisms of drug resistance mainly involve mutations directly altering the interaction of viral enzymes and inhibitors. However, protease inhibitors do not only select for mutations in the protease but also for mutations in the precursor Gag and Pol proteins. In this study, we analysed the frameshift-regulating site of HIV-1 subtype B isolates, which also encodes for Gag and Pol proteins, classified as either treatment-naïve (TN) or protease inhibitor resistant (PI-R). HIV-1 Gag cleavage site mutations (G435E, K436N, I437V, L449F/V) especially correlated with protease inhibitor resistance mutations, but also Pol cleavage site mutations (D05G, D05S) could be assigned to specific protease resistance profiles. Additionally, two Gag non-cleavage site mutations (S440F, H441P) were observed more often in HIV-1 isolates carrying protease resistance mutations. However, in dual luciferase assays, the frameshift efficiencies of specific clones did not reveal any effect from these mutations. Nevertheless, two patterns of mutations modestly increased the frameshift rates in vitro, but were not specifically accumulating in PI-resistant HIV-1 isolates. In summary, HIV-1 Gag cleavage site mutations were dominantly selected in PI-resistant HIV-1 isolates but also Pol cleavage site mutations influenced resistance profiles in the protease. Additionally, Gag non-cleavage site mutations accumulated in PI-resistant HIV-1 isolates, but were not related to an increased frameshift efficiency.

  9. New frameshift CF mutation 3729delAinsTCT in a Tunisian cystic ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 92; Issue 1. New frameshift CF mutation 3729delAinsTCT in a Tunisian cystic fibrosis patient. Sondess Hadj Fredj Monia Boudaya Sabrine Oueslati Safa Sahnoun Chaima Sahli Hajer Siala Khedija Boussetta Amina Bibi Taieb Messaoud. Research Note Volume 92 Issue 1 April ...

  10. New frameshift CF mutation 3729delAinsTCT in a Tunisian cystic

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 92; Issue 1. New frameshift CF mutation 3729delAinsTCT in a Tunisian cystic fibrosis patient. Sondess Hadj Fredj Monia Boudaya Sabrine Oueslati Safa Sahnoun Chaima Sahli Hajer Siala Khedija Boussetta Amina Bibi Taieb Messaoud. Research Note Volume 92 Issue 1 April ...

  11. Three novel mutations in the CFTR gene identified in Galician patients.

    Science.gov (United States)

    Rana-Díez, P; Colón, C; Alonso-Fernández, J R; Solar, A; Barros-Tizón, J C; Barros-Casas, D; Sirvent, J; Carracedo, A; Barros, F

    2008-11-01

    We report three novel CFTR missense mutations detected in Spanish patients from Galicia (North West of Spain). In the first case, a patient homozygous for a novel S1045Y mutation died due to pulmonary problems. In the other two cases, both heterozygous for novel mutations combined with the F508del mutation, clinical symptoms were different depending on the mutation, detected as M595I and A107V.

  12. Heterozygous CAV1 frameshift mutations (MIM 601047 in patients with atypical partial lipodystrophy and hypertriglyceridemia

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    Alston Lindsay

    2008-01-01

    Full Text Available Abstract Background Mice with a deleted Cav1 gene encoding caveolin-1 develop adipocyte abnormalities and insulin resistance. From genomic DNA of patients with atypical lipodystrophy and hypertriglyceridemia who had no mutations in any known lipodystrophy gene, we used DNA sequence analysis to screen the coding regions of human CAV1 (MIM 601047. Results We found a heterozygous frameshift mutation in CAV1, designated I134fsdelA-X137, in a female patient who had atypical partial lipodystrophy, with subcutaneous fat loss affecting the upper part of her body and face, but sparing her legs, gluteal region and visceral fat stores. She had severe type 5 hyperlipoproteinemia, with recurrent pancreatitis. In addition, she had some atypical features, including congenital cataracts and neurological findings. Her father was also heterozygous for this mutation, and had a similar pattern of fat redistribution, hypertriglyceridemia and congenital cataracts, with milder neurological involvement. An unrelated patient had a different heterozygous frameshift mutation in the CAV1 gene, designated -88delC. He also had a partial lipodystrophy phenotype, with subcutaneous fat loss affecting the arms, legs and gluteal region, but sparing his face, neck and visceral fat stores. He also had severe type 5 hyperlipoproteinemia, with recurrent pancreatitis; however he had no clinically apparent neurological manifestations. The mutations were absent from the genomes of 1063 healthy individuals. Conclusion Thus, very rare CAV1 frameshift mutations appear to be associated with atypical lipodystrophy and hypertriglyceridemia.

  13. Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis

    NARCIS (Netherlands)

    Wu, Y; Hofstra, RMW; Scheffer, H; Uitterlinden, AG; Mullaart, E; Buys, CHCM; Vijg, J

    1996-01-01

    The large number of possible disease causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for

  14. Analysis of mutations in the cystic fibrosis transmembrane regulator (CFTR gene in patients with obstructive azoospermia

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    Andrea L.F. Bernardino

    2003-01-01

    Full Text Available Congenital bilateral absence of the vas deferens (CBAVD accounts for 1%-2% of sterility in men. A high incidence of mutations, as well as the involvement of the 5T variant of the T tract length in intron 8 of the cystic fibrosis conductance regulator (CFTR gene, have been previously described in males with CBAVD. Herein we report the screening for mutations and for the 5T variant of the CFTR gene in 17 patients with CBAVD and three others with non-CABVD obstructive azoospermia. In the CBAVD group, three patients (15% were compound heterozygotes for mutations, and five patients (25% had a mutation in one allele and the 5T variant in the other; the 5T variant was also present in two other patients, one of them being homozygous. The most frequent mutation was DF508, present on five chromosomes (12.5%. A novel missense mutation (A399D was detected in a Japanese CBVAD patient. Our results yield further evidence for a strong association between male obstructive azoospermia caused by CBAVD and mutation/5T variant in the CFTR gene. The search for CFTR mutations in such patients is thus recommended for genetic counseling of couples who undergo assisted fertilization due to CBAVD.

  15. Frameshift mutation hotspot identified in Smith-Magenis syndrome: case report and review of literature

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    Dudding Tracy

    2010-10-01

    Full Text Available Abstract Smith-Magenis syndrome (SMS is a complex syndrome involving intellectual disabilities, sleep disturbance, behavioural problems, and a variety of craniofacial, skeletal, and visceral anomalies. While the majority of SMS cases harbor an ~3.5 Mb common deletion on 17p11.2 that encompasses the retinoic acid induced-1 (RAI1 gene, some patients carry small intragenic deletions or point mutations in RAI1. We present data on two cases of Smith-Magenis syndrome with mutation of RAI1. Both cases are phenotypically consistent with SMS and RAI1 mutation but also have other anomalies not previously reported in SMS, including spontaneous pneumothoraces. These cases also illustrate variability in the SMS phenotype not previously shown for RAI1 mutation cases, including hearing loss, absence of self-abusive behaviours, and mild global delays. Sequencing of RAI1 revealed mutation of the same heptameric C-tract (CCCCCCC in exon 3 in both cases (c.3103delC one case and and c.3103insC in the other, resulting in frameshift mutations. Of the seven reported frameshift mutations occurring in poly C-tracts in RAI1, four cases (~57% occur at this heptameric C-tract. Collectively, these results indicate that this heptameric C-tract is a preferential hotspot for single nucleotide insertion/deletions (SNindels and therefore, should be considered a primary target for analysis in patients suspected for mutations in RAI1. We expect that as more patients are sequenced for mutations in RAI1, the incidence of frameshift mutations in this hotspot will become more evident.

  16. Frameshift mutation hotspot identified in Smith-Magenis syndrome: case report and review of literature.

    Science.gov (United States)

    Truong, Hoa T; Dudding, Tracy; Blanchard, Christopher L; Elsea, Sarah H

    2010-10-08

    Smith-Magenis syndrome (SMS) is a complex syndrome involving intellectual disabilities, sleep disturbance, behavioural problems, and a variety of craniofacial, skeletal, and visceral anomalies. While the majority of SMS cases harbor an ~3.5 Mb common deletion on 17p11.2 that encompasses the retinoic acid induced-1 (RAI1) gene, some patients carry small intragenic deletions or point mutations in RAI1. We present data on two cases of Smith-Magenis syndrome with mutation of RAI1. Both cases are phenotypically consistent with SMS and RAI1 mutation but also have other anomalies not previously reported in SMS, including spontaneous pneumothoraces. These cases also illustrate variability in the SMS phenotype not previously shown for RAI1 mutation cases, including hearing loss, absence of self-abusive behaviours, and mild global delays. Sequencing of RAI1 revealed mutation of the same heptameric C-tract (CCCCCCC) in exon 3 in both cases (c.3103delC one case and and c.3103insC in the other), resulting in frameshift mutations. Of the seven reported frameshift mutations occurring in poly C-tracts in RAI1, four cases (~57%) occur at this heptameric C-tract. Collectively, these results indicate that this heptameric C-tract is a preferential hotspot for single nucleotide insertion/deletions (SNindels) and therefore, should be considered a primary target for analysis in patients suspected for mutations in RAI1. We expect that as more patients are sequenced for mutations in RAI1, the incidence of frameshift mutations in this hotspot will become more evident.

  17. A simple, fast and inexpensive method for mutation scanning of CFTR gene.

    Science.gov (United States)

    Figueredo Lago, Juan Emilio; Armas Cayarga, Anny; González González, Yaimé Josefina; Collazo Mesa, Teresa

    2017-05-25

    Mutation scanning methods in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene may not distinguish between a Cystic Fibrosis (CF) causing mutation and a benign variant. We have developed a simple and fast method for scanning 14 selected CF-causing mutations which have high frequency in Latin America. In a group of 35 samples coming from CF patients previously characterized and using two allele-specific real-time multiplex PCRs targeting wild-type and mutant alleles respectively, we detect the presence of mutations by analyzing the Ct variation. Twenty-five samples without mutations considered non-carrier samples, were also included in this study. High Resolution Melting Analysis (HRMA) was performed to confirm the result of the scanning method and in most cases allowed the genotype determination. The results validate this method for CF diagnosis. A least one CFTR gene mutation was detected in the samples of CF patients, as predicted by their ΔCt values. The ΔCt value also indicated the zygosity of the sample according to the distribution of CFTR gene mutations. In most cases, HRMA allowed the identification of the mutation(s), thereby confirming the efficiency of this scanning strategy. This strategy simplifies the detection of CF, reducing the analysis of 14 CF-causing mutations to two parallel reactions and making the procedure compatible with the analysis of a large number of samples. As the method is fast, inexpensive and highly reliable, it is advisable for scanning CFTR gene mutations in newborns, patients with a clinical suspicion of CF as well as in the preconception carrier screening.

  18. A frame-shift mutation of PMS2 is a widespread cause of Lynch syndrome

    DEFF Research Database (Denmark)

    Clendenning, Mark; Senter, Leigha; Hampel, Heather

    2008-01-01

    BACKGROUND: When compared to the other mismatch repair genes involved in Lynch syndrome, the identification of mutations within PMS2 has been limited (syndrome cases...... are caused by PMS2. This disparity is primarily due to complications in the study of this gene caused by interference from pseudogene sequences. METHODS: Using a recently developed method for detecting PMS2 specific mutations, we have screened 99 patients who are likely candidates for PMS2 mutations based...... on immunohistochemical analysis. RESULTS: We have identified a frequently occurring frame-shift mutation (c.736_741del6ins11) in 12 ostensibly unrelated Lynch syndrome patients (20% of patients we have identified with a deleterious mutation in PMS2, n=61). These individuals all display the rare allele (population...

  19. Analysis of Y chromosome microdeletions and CFTR gene mutations as genetic markers of infertility in Serbian men

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    Dinić Jelena

    2007-01-01

    Full Text Available Background/Aim. Impaired fertility of a male partner is the main cause of infertility in up to one half of all infertile couples. At the genetic level, male infertility can be caused by chromosome aberrations or gene mutations. The presence and types of Y chromosome microdeletions and cystic fybrosis transmembrane conductance regulator (CFTR gene mutations as genetic cause of male infertility was tested in Serbian men. The aim of this study was to analyze CFTR gene mutations and Y chromosome microdelations as potential causes of male infertility in Serbian patients, as well as to test the hypothesis that CFTR mutations in infertile men are predominantly located in the several last exons of the gene. Methods. This study has encompassed 33 men with oligo- or azoospermia. The screening for Y chromosome microdeletions in the azoospermia factor (AZF region was performed by multiplex PCR analysis. The screening of the CFTR gene was performed by denaturing gradient gel electrophoresis (DGGE method. Results. Deletions on Y chromosome were detected in four patients, predominantly in AZFc region (four of total six deletions. Mutations in the CFTR gene were detected on eight out of 66 analyzed chromosomes of infertile men. The most common mutation was F508del (six of total eight mutations. Conclusion. This study confirmed that both Y chromosome microdeletions and CFTR gene mutations played important role in etiology of male infertility in Serbian infertile men. Genetic testing for Y chromosome microdeletions and CFTR gene mutations has been introduced in routine diagnostics and offered to couples undergoing assisted reproduction techniques. Considering that both the type of Y chromosome microdeletion and the type of CFTR mutation have a prognostic value, it is recommended that AZF and CFTR genotyping should not only be performed in patients with reduced sperm quality before undergoing assisted reproduction, but also for the purpose of preimplantation and

  20. Frequent alteration of MLL3 frameshift mutations in microsatellite deficient colorectal cancer.

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    Yoshiyuki Watanabe

    Full Text Available MLL3 is a histone 3-lysine 4 methyltransferase with tumor-suppressor properties that belongs to a family of chromatin regulator genes potentially altered in neoplasia. Mutations in MLL3 were found in a whole genome analysis of colorectal cancer but have not been confirmed by a separate study.We analyzed mutations of coding region and promoter methylation in MLL3 using 126 cases of colorectal cancer. We found two isoforms of MLL3 and DNA sequencing revealed frameshift and other mutations affecting both isoforms of MLL3 in colorectal cancer cells and 19 of 134 (14% primary colorectal samples analyzed. Moreover, frameshift mutations were more common in cases with microsatellite instability (31% both in CRC cell lines and primary tumors. The largest isoform of MLL3 is transcribed from a CpG island-associated promoter that has highly homology with a pseudo-gene on chromosome 22 (psiTPTE22. Using an assay which measured both loci simultaneously we found prominent age related methylation in normal colon (from 21% in individuals less than 25 years old to 56% in individuals older than 70, R = 0.88, p<0.001 and frequent hypermethylation (83% in both CRC cell lines and primary tumors. We next studied the two loci separately and found that age and cancer related methylation was solely a property of the pseudogene CpG island and that the MLL3 loci was unmethylated.We found that frameshift mutations of MLL3 in both CRC cells and primary tumor that were more common in cases with microsatellite instability. Moreover, we have shown CpG island-associated promoter of MLL3 gene has no DNA methylation in CRC cells but also primary tumor and normal colon, and this region has a highly homologous of pseudo gene (psiTPTE22 that was age relate DNA methylation.

  1. A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

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    Sergey V. Prykhozhij

    2017-06-01

    Full Text Available Clustered regularly interspaced palindromic repeats (CRISPR/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.

  2. Effects of the umuC36 mutation on ultraviolet-radiation-induced base-change and frameshift mutations in Escherichia coli

    International Nuclear Information System (INIS)

    Kato, T.; Nakano, E.

    1981-01-01

    The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr + or uvr - strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His + , ColE and Spc, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N'-nitro-N-nitrosoguanidine-induced His + mutations. In contrast, only 1 out of 13 trp - frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC + -dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp + mutations in most of the strains regardless of the umu + or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC + -dependent pathway of error-prone repair. (orig.)

  3. Microsatellite instability derived JAK1 frameshift mutations are associated with tumor immune evasion in endometrioid endometrial cancer

    NARCIS (Netherlands)

    Stelloo, Ellen; Versluis, Marco A; Nijman, Hans W; de Bruyn, Marco; Plat, Annechien; Osse, Elisabeth M; van Dijk, Reinhardt H; Nout, Remi A; Creutzberg, Carien L; de Bock, Geertruida H; Smit, Vincent T; Bosse, Tjalling; Hollema, Harry

    2016-01-01

    JAK1 frameshift mutations may promote cancer cell immune evasion by impeding upregulation of the antigen presentation pathway in microsatellite unstable endometrial cancers (ECs). This study investigated the JAK1 mutation frequency, its functional implication in immune evasion and its prognostic

  4. Correlation between CFTR gene mutations in Iranian men with congenital absence of the vas deferens and anatomical genital phenotype.

    Science.gov (United States)

    Radpour, Ramin; Gourabi, Hamid; Gilani, Mohamad Ali Sadighi; Dizaj, Ahmad Vosough

    2008-01-01

    Congenital bilateral absence of the vas deferens (CBAVD) and congenital unilateral absence of the vas deferens (CUAVD) are 2 causes of male sterility; these phenotypes are found in 1%-2% of men investigated for infertility and approximately 10% of men with azoospermia. To study the correlation between genital phenotype and cystic fibrosis genotype in men lacking at least 1 vas deferens, we evaluated the role of different CFTR gene mutations in the morphologic genital phenotype of 119 infertile men with bilateral or unilateral absence of the vas deferens (112 CBAVD and 7 CUAVD patients). Renal, scrotal, and transrectal ultrasonography were systematically performed. CFTR mutations and (TG)m(T)n polymorphism were analyzed, and epididymal and seminal vesicular abnormalities and testicular volume were compared among men with 2, 1, or no CFTR gene mutation, with or without the 5T allele. Our results showed that patients with CBAVD and renal agenesis have the same reproductive tract abnormalities as those with CUAVD, and reproductive tract abnormalities were independent of the subtypes of CFTR genotype in patients with absence of the vas deferens and CFTR gene mutations. Seminal vesicles did not differ between patients with or without CFTR gene mutation, but epididymal abnormalities were more frequent in CBAVD men without the mutation. Low testicular volume was observed in CBAVD men without the CFTR and IVS8-5T mutations, so we can hypothesize that a testicular factor (genetic or environmental) rather than CFTR gene mutations plays a role in determining the phenotype. Further studies using common diagnostic criteria are required to confirm our observations.

  5. Optimizing nasal potential difference analysis for CFTR modulator development: assessment of ivacaftor in CF subjects with the G551D-CFTR mutation.

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    Steven M Rowe

    Full Text Available Nasal potential difference (NPD is used as a biomarker of the cystic fibrosis transmembrane conductance regulator (CFTR and epithelial sodium channel (ENaC activity. We evaluated methods to detect changes in chloride and sodium transport by NPD based on a secondary analysis of a Phase II CFTR-modulator study. Thirty-nine subjects with CF who also had the G551D-CFTR mutation were randomized to receive ivacaftor (Kalydeco™; also known as VX-770 in four doses or placebo twice daily for at least 14 days. All data were analyzed by a single investigator who was blinded to treatment assignment. We compared three analysis methods to determine the best approach to quantify changes in chloride and sodium transport: (1 the average of both nostrils; (2 the most-polarized nostril at each visit; and (3 the most-polarized nostril at screening carried forward. Parameters of ion transport included the PD change with zero chloride plus isoproterenol (CFTR activity, the basal PD, Ringer's PD, and change in PD with amiloride (measurements of ENaC activity, and the delta NPD (measuring CFTR and ENaC activity. The average and most-polarized nostril at each visit were most sensitive to changes in chloride and sodium transport, whereas the most-polarized nostril at screening carried forward was less discriminatory. Based on our findings, NPD studies should assess both nostrils rather than a single nostril. We also found that changes in CFTR activity were more readily detected than changes in ENaC activity, and that rigorous standardization was associated with relatively good within-subject reproducibility in placebo-treated subjects (± 2.8 mV. Therefore, we have confirmed an assay of reasonable reproducibility for detecting chloride-transport improvements in response to CFTR modulation.

  6. Detection of coding microsatellite frameshift mutations in DNA mismatch repair-deficient mouse intestinal tumors.

    Science.gov (United States)

    Woerner, Stefan M; Tosti, Elena; Yuan, Yan P; Kloor, Matthias; Bork, Peer; Edelmann, Winfried; Gebert, Johannes

    2015-11-01

    Different DNA mismatch repair (MMR)-deficient mouse strains have been developed as models for the inherited cancer predisposing Lynch syndrome. It is completely unresolved, whether coding mononucleotide repeat (cMNR) gene mutations in these mice can contribute to intestinal tumorigenesis and whether MMR-deficient mice are a suitable molecular model of human microsatellite instability (MSI)-associated intestinal tumorigenesis. A proof-of-principle study was performed to identify mouse cMNR-harboring genes affected by insertion/deletion mutations in MSI murine intestinal tumors. Bioinformatic algorithms were developed to establish a database of mouse cMNR-harboring genes. A panel of five mouse noncoding mononucleotide markers was used for MSI classification of intestinal matched normal/tumor tissues from MMR-deficient (Mlh1(-/-) , Msh2(-/-) , Msh2(LoxP/LoxP) ) mice. cMNR frameshift mutations of candidate genes were determined by DNA fragment analysis. Murine MSI intestinal tumors but not normal tissues from MMR-deficient mice showed cMNR frameshift mutations in six candidate genes (Elavl3, Tmem107, Glis2, Sdccag1, Senp6, Rfc3). cMNRs of mouse Rfc3 and Elavl3 are conserved in type and length in their human orthologs that are known to be mutated in human MSI colorectal, endometrial and gastric cancer. We provide evidence for the utility of a mononucleotide marker panel for detection of MSI in murine tumors, the existence of cMNR instability in MSI murine tumors, the utility of mouse subspecies DNA for identification of polymorphic repeats, and repeat conservation among some orthologous human/mouse genes, two of them showing instability in human and mouse MSI intestinal tumors. MMR-deficient mice hence are a useful molecular model system for analyzing MSI intestinal carcinogenesis. © 2014 Wiley Periodicals, Inc.

  7. A CNGB1 frameshift mutation in Papillon and Phalene dogs with progressive retinal atrophy.

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    Saija J Ahonen

    Full Text Available Progressive retinal degenerations are the most common causes of complete blindness both in human and in dogs. Canine progressive retinal atrophy (PRA or degeneration resembles human retinitis pigmentosa (RP and is characterized by a progressive loss of rod photoreceptor cells followed by a loss of cone function. The primary clinical signs are detected as vision impairment in a dim light. Although several genes have been associated with PRAs, there are still PRAs of unknown genetic cause in many breeds, including Papillons and Phalènes. We have performed a genome wide association and linkage studies in cohort of 6 affected Papillons and Phalènes and 14 healthy control dogs to map a novel PRA locus on canine chromosome 2, with a 1.9 Mb shared homozygous region in the affected dogs. Parallel exome sequencing of a trio identified an indel mutation, including a 1-bp deletion, followed by a 6-bp insertion in the CNGB1 gene. This mutation causes a frameshift and premature stop codon leading to probable nonsense mediated decay (NMD of the CNGB1 mRNA. The mutation segregated with the disease and was confirmed in a larger cohort of 145 Papillons and Phalènes (PFisher = 1.4×10(-8 with a carrier frequency of 17.2 %. This breed specific mutation was not present in 334 healthy dogs from 10 other breeds or 121 PRA affected dogs from 44 other breeds. CNGB1 is important for the photoreceptor cell function its defects have been previously associated with retinal degeneration in both human and mouse. Our study indicates that a frameshift mutation in CNGB1 is a cause of PRA in Papillons and Phalènes and establishes the breed as a large functional animal model for further characterization of retinal CNGB1 biology and possible retinal gene therapy trials. This study enables also the development of a genetic test for breeding purposes.

  8. Back-translation for discovering distant protein homologies in the presence of frameshift mutations

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    Noé Laurent

    2010-01-01

    Full Text Available Abstract Background Frameshift mutations in protein-coding DNA sequences produce a drastic change in the resulting protein sequence, which prevents classic protein alignment methods from revealing the proteins' common origin. Moreover, when a large number of substitutions are additionally involved in the divergence, the homology detection becomes difficult even at the DNA level. Results We developed a novel method to infer distant homology relations of two proteins, that accounts for frameshift and point mutations that may have affected the coding sequences. We design a dynamic programming alignment algorithm over memory-efficient graph representations of the complete set of putative DNA sequences of each protein, with the goal of determining the two putative DNA sequences which have the best scoring alignment under a powerful scoring system designed to reflect the most probable evolutionary process. Our implementation is freely available at http://bioinfo.lifl.fr/path/. Conclusions Our approach allows to uncover evolutionary information that is not captured by traditional alignment methods, which is confirmed by biologically significant examples.

  9. Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells

    International Nuclear Information System (INIS)

    Grosovsky, A.J.; de Boer, J.G.; de Jong, P.J.; Drobetsky, E.A.; Glickman, B.W.

    1988-01-01

    The relative role of point mutations and large genomic rearrangements in ionizing radiation-induced mutagenesis has been an issue of long-standing interest. Recent studies using Southern blotting analysis permit the partitioning of ionizing radiation-induced mutagenesis in mammalian cells into detectable deletions and major genomic rearrangements and into point mutations. The molecular nature of these point mutations has been left unresolved; they may include base substitutions as well as small deletions, insertions, and frame-shifts below the level of resolution of Southern blotting analysis. In this investigation, we have characterized a collection of ionizing radiation-induced point mutations at the endogenous adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary cells at the DNA sequence level. Base substitutions represented approximately equal to 2/3 of the point mutations analyzed. Although the collection of mutants is relatively small, every possible type of base substitution event has been recovered. These mutations are well distributed throughout the coding sequence with only one multiple occurrence. Small deletions represented the remainder of characterized mutants; no insertions have been observed. Sequence-directed mechanisms mediated by direct repeats could account for some of the observed deletions, while others appear to be directly attributable to radiation-induced strand breakage

  10. A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers

    DEFF Research Database (Denmark)

    Jensen, Line; Hegelund, Josefine Nymark; Olsen, Andreas

    2016-01-01

    BACKGROUND: The phytohormone ethylene plays a central role in development and senescence of climacteric flowers. In ornamental plant production, ethylene sensitive plants are usually protected against negative effects of ethylene by application of chemical inhibitors. In Campanula, flowers...... are sensitive to even minute concentrations of ethylene. RESULTS: Monitoring flower longevity in three Campanula species revealed C. portenschlagiana (Cp) as ethylene sensitive, C. formanekiana (Cf) with intermediate sensitivity and C. medium (Cm) as ethylene insensitive. We identified key elements in ethylene...... and in response to ethylene. In contrast, EIL2 was found only in Cf and Cm. We identified a natural mutation in Cmeil2 causing a frameshift which resulted in difference in expression levels of EIL2, with more than 100-fold change between Cf and Cm in young flowers. CONCLUSIONS: This study shows that the naturally...

  11. Clinical Characteristics of Wolfram Syndrome in Chinese Population and a Novel Frameshift Mutation in WFS1.

    Science.gov (United States)

    Duan, Lian; Li, Qian; Tong, An-Li; Mao, Jiang-Feng; Yu, Miao; Yuan, Tao; Chai, Xiao-Feng; Gu, Feng

    2018-01-01

    Wolfram syndrome (WS) is a rare, degenerative, and hereditary disorder characterized by ear diabetes mellitus (DM) and optic atrophy (OA). We aim to characterize clinical features in Chinese patients who had been poorly studied until now. We performed a retrospective review of patients with WS seen in the Peking Union Medical College Hospital from 2002 to 2017. Data including demographic data, clinical presentations, examination results, family history, and genetic analysis were described. Six patients with WS were identified, meeting the diagnostic criteria of the coincidence of DM and OA before 15 years old or the existence of two WFS1 mutations. All were male, with the median age of 14.5 years (range 10-19 years). Blood glucose impairment, OA, and diabetes insipidus were present in all (100%), hearing impairment in four (66.7%), urological abnormalities in four (66.7%), neurological abnormalities in one (16.7%), and endocrine disorder in one (16.7%). Rare presentation includes cataract, glaucoma, and spina bifida occulta. Diabetes was insulin-dependent and not ketosis onset, with antibody to glutamic acid decarboxylase and islet cell negative. Genetic analysis revealed mutations in WFS1 in three patients. A novel frameshift mutation (p.Asp151Glufs*93) was identified in exon 4 of WFS1 . Our series of WS patients indicated that WS is a degenerative disease with a wide and variable spectrum, characterized by ear non-autoimmune DM and bilateral OA. Genetic analysis is recommended when suspected of WS.

  12. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene mutations in North Egyptian population: implications for the genetic diagnosis in Egypt.

    Science.gov (United States)

    El-Seedy, A; Pasquet, M C; Shafiek, H; Morsi, T; Kitzis, A; Ladevèze, V

    2016-11-30

    Cystic fibrosis (CF) occurrence in Arab populations is not common and still remains underidentified. Furthermore, the lack of disease awareness and diagnosis facilities have mislead the identification of cystic fibrosis for decades. The knowledge about cystic fibrosis (CF) in Egypt is very limited, and a few reports have drawn attention to the existence of CF or CFTR-related disorders (CFTR-RDs) in the Egyptian population. Therefore a comprehensive genetic analysis of the CFTR gene was realized in patients of North Egypt. DNA samples of 56 Egyptian patients were screened for the CFTR gene mutations. The 27 exons and their flanking regions of the CFTR gene were amplified by PCR, using the published primer pairs, and were studied by automated direct DNA sequencing to detect disease-causing mutations. Moreover, large duplication/deletion was analysed by MLPA technique. CFTR screening revealed the identification of thirteen mutations including four novel ones: c.92G>A (p.Arg31His), c.2782G>C (p.Ala928Pro), c.3718-24G>A, c.4207A>G (p.Arg1403Gly) and nine previously reported mutations: c.454A>T (p.Met152Leu), c.902A>G (p.Tyr301Cys), c.1418delG, c.2620-15C>G, c.2997_3000delAATT, c.3154T>G (p.Phe1052Val), c.3872A>G (p.Gln1291Arg), c.3877G>A (p.Val1293Ile), c.4242+10T>C. Furthermore, eight polymorphisms were found: c.743+40A>G, c.869+11C>T, c.1408A>G, c.1584G>A, c.2562T>G, c.3870A>G, c.4272C>T, c.4389G>A. These mutations and polymorphisms were not previously described in the Egyptian population except for the c.1408A>G polymorphism. Here we demonstrate the importance of the newly discovered mutations in Egyptian patients and the presence of CF, whereas the p.Phe508del mutation is not detected. The identification of CFTR mutations will become increasingly important in undocumented populations. The current findings will help us expand the mutational spectrum of CF and establish the first panel of the CFTR gene mutations in the Egyptian population and design an appropriate

  13. Two new Rett syndrome families and review of the literature: expanding the knowledge of MECP2 frameshift mutations

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    Eiklid Kristin L

    2011-08-01

    Full Text Available Abstract Background Rett syndrome (RTT is an X-linked dominant neurodevelopmental disorder, which is usually caused by de novo mutations in the MECP2 gene. More than 70% of the disease causing MECP2 mutations are eight recurrent C to T transitions, which almost exclusively arise on the paternally derived X chromosome. About 10% of the RTT cases have a C-terminal frameshift deletion in MECP2. Only few RTT families with a segregating MECP2 mutation, which affects female carriers with a phenotype of mental retardation or RTT, have been reported in the literature. In this study we describe two new RTT families with three and four individuals, respectively, and review the literature comparing the type of mutations and phenotypes observed in RTT families with those observed in sporadic cases. Based on these observations we also investigated origin of mutation segregation to further improve genetic counselling. Methods MECP2 mutations were identified by direct sequencing. XCI studies were performed using the X-linked androgen receptor (AR locus. The parental origin of de novo MECP2 frameshift mutations was investigated using intronic SNPs. Results In both families a C-terminal frameshift mutation segregates. Clinical features of the mutation carriers vary from classical RTT to mild mental retardation. XCI profiles of the female carriers correlate to their respective geno-/phenotypes. The majority of the de novo frameshift mutations occur on the paternally derived X chromosome (7/9 cases, without a paternal age effect. Conclusions The present study suggests a correlation between the intrafamilial phenotypic differences observed in RTT families and their respective XCI pattern in blood, in contrast to sporadic RTT cases where a similar correlation has not been demonstrated. Furthermore, we found de novo MECP2 frameshift mutations frequently to be of paternal origin, although not with the same high paternal occurrence as in sporadic cases with C to T

  14. Two new Rett syndrome families and review of the literature: expanding the knowledge of MECP2 frameshift mutations

    Science.gov (United States)

    2011-01-01

    Background Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder, which is usually caused by de novo mutations in the MECP2 gene. More than 70% of the disease causing MECP2 mutations are eight recurrent C to T transitions, which almost exclusively arise on the paternally derived X chromosome. About 10% of the RTT cases have a C-terminal frameshift deletion in MECP2. Only few RTT families with a segregating MECP2 mutation, which affects female carriers with a phenotype of mental retardation or RTT, have been reported in the literature. In this study we describe two new RTT families with three and four individuals, respectively, and review the literature comparing the type of mutations and phenotypes observed in RTT families with those observed in sporadic cases. Based on these observations we also investigated origin of mutation segregation to further improve genetic counselling. Methods MECP2 mutations were identified by direct sequencing. XCI studies were performed using the X-linked androgen receptor (AR) locus. The parental origin of de novo MECP2 frameshift mutations was investigated using intronic SNPs. Results In both families a C-terminal frameshift mutation segregates. Clinical features of the mutation carriers vary from classical RTT to mild mental retardation. XCI profiles of the female carriers correlate to their respective geno-/phenotypes. The majority of the de novo frameshift mutations occur on the paternally derived X chromosome (7/9 cases), without a paternal age effect. Conclusions The present study suggests a correlation between the intrafamilial phenotypic differences observed in RTT families and their respective XCI pattern in blood, in contrast to sporadic RTT cases where a similar correlation has not been demonstrated. Furthermore, we found de novo MECP2 frameshift mutations frequently to be of paternal origin, although not with the same high paternal occurrence as in sporadic cases with C to T transitions. This suggests

  15. A novel frameshift mutation in CX46 associated with hereditary dominant cataracts in a Chinese family

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    Xiu-Kun Cui

    2017-05-01

    Full Text Available AIM: To investigate the genetic mutations that are associated the hereditary autosomal dominant cataract in a Chinese family. METHODS: A Chinese family consisting of 20 cataract patients (including 9 male and 11 female and 2 unaffected individuals from 5 generations were diagnosed to be a typical autosomal dominant cataract pedigree. Genomic DNA samples were extracted from the peripheral blood cells of the participants in this pedigree. Exon sequence was used for genetic mutation screening. In silico analysis was used to study the structure characteristics of connexin 46 (CX46 mutant. Immunoblotting was conduceted for testing the expression of CX46. RESULTS: To determine the involved genetic mutations, 11 well-known cataract-associated genes (cryaa, cryab, crybb1, crybb2, crygc, crygd, Gja3, Gja8, Hsf4, Mip and Pitx3 were chosen for genetic mutation test by using exon sequencing. A novel cytosine insertion at position 1195 of CX46 cDNA (c.1194_1195ins C was found in the samples of 5 tested cataract patients but not in the unaffected 2 individuals nor in normal controls, which resulted in 30 amino acids more extension in CX46C-terminus (cx46fs400 compared with the wild-type CX46. In silico protein structure analysis indicated that the mutant showed distinctive hydrophobicity and protein secondary structure compared with the wild-type CX46. The immunoblot results revealed that CX46 protein, which expressed in the aging cataract lens tissues, was absence in the proband lens. In contrast, CX50, alpha A-crystallin and alphaB-crystallin expressed equally in both proband and aging cataract tissues. Those results revealed that the cx46fs400 mutation could impair CX46 protein expression. CONCLUSION: The insertion of cytosine at position 1195 of CX46 cDNA is a novel mutation site that is associated with the autosomal dominant cataracts in this Chinese family. The C-terminal frameshift mutation is involved in regulating CX46 protein expression.

  16. Distribution of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR Mutations in a Cohort of Patients Residing in Palestine.

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    Issa Siryani

    Full Text Available Cystic fibrosis (CF is an autosomal recessive inherited life-threatening disorder that causes severe damage to the lungs and the digestive system. In Palestine, mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR that contributes to the clinical presentation of CF are ill defined. A cohort of thirty three clinically diagnosed CF patients from twenty one different Palestinian families residing in the central and southern part of Palestine were incorporated in this study. Sweat chloride testing was performed using the Sweat Chek Conductivity Analyzer (ELITECH Group, France to confirm the clinical diagnosis of CF. In addition, nucleic acid from the patients' blood samples was extracted and the CFTR mutation profiles were assessed by direct sequencing of the CFTR 27 exons and the intron-exon boundaries. For patient's DNA samples where no homozygous or two heterozygous CFTR mutations were identified by exon sequencing, DNA samples were tested for deletions or duplications using SALSA MLPA probemix P091-D1 CFTR assay. Sweat chloride testing confirmed the clinical diagnosis of CF in those patients. All patients had NaCl conductivity >60 mmol/l. In addition, nine different CFTR mutations were identified in all 21 different families evaluated. These mutations were c.1393-1G>A, F508del, W1282X, G85E, c.313delA, N1303K, deletion exons 17a-17b-18, deletion exons 17a-17b and Q1100P. c.1393-1G>A was shown to be the most frequent occurring mutation among tested families. We have profiled the underling mutations in the CFTR gene of a cohort of 21 different families affected by CF. Unlike other studies from the Arab countries where F508del was reported to be the most common mutation, in southern/central Palestine, the c.1393-1G>A appeared to be the most common. Further studies are needed per sample size and geographic distribution to account for other possible CFTR genetic alterations and their frequencies. Genotype

  17. Distribution of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutations in a Cohort of Patients Residing in Palestine.

    Science.gov (United States)

    Siryani, Issa; Jama, Mohamed; Rumman, Nisreen; Marzouqa, Hiyam; Kannan, Moein; Lyon, Elaine; Hindiyeh, Musa

    2015-01-01

    Cystic fibrosis (CF) is an autosomal recessive inherited life-threatening disorder that causes severe damage to the lungs and the digestive system. In Palestine, mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that contributes to the clinical presentation of CF are ill defined. A cohort of thirty three clinically diagnosed CF patients from twenty one different Palestinian families residing in the central and southern part of Palestine were incorporated in this study. Sweat chloride testing was performed using the Sweat Chek Conductivity Analyzer (ELITECH Group, France) to confirm the clinical diagnosis of CF. In addition, nucleic acid from the patients' blood samples was extracted and the CFTR mutation profiles were assessed by direct sequencing of the CFTR 27 exons and the intron-exon boundaries. For patient's DNA samples where no homozygous or two heterozygous CFTR mutations were identified by exon sequencing, DNA samples were tested for deletions or duplications using SALSA MLPA probemix P091-D1 CFTR assay. Sweat chloride testing confirmed the clinical diagnosis of CF in those patients. All patients had NaCl conductivity >60 mmol/l. In addition, nine different CFTR mutations were identified in all 21 different families evaluated. These mutations were c.1393-1G>A, F508del, W1282X, G85E, c.313delA, N1303K, deletion exons 17a-17b-18, deletion exons 17a-17b and Q1100P. c.1393-1G>A was shown to be the most frequent occurring mutation among tested families. We have profiled the underling mutations in the CFTR gene of a cohort of 21 different families affected by CF. Unlike other studies from the Arab countries where F508del was reported to be the most common mutation, in southern/central Palestine, the c.1393-1G>A appeared to be the most common. Further studies are needed per sample size and geographic distribution to account for other possible CFTR genetic alterations and their frequencies. Genotype/phenotype assessments are also

  18. Frameshift mutations in dentin phosphoprotein and dependence of dentin disease phenotype on mutation location

    NARCIS (Netherlands)

    Nieminen, P.; Papagiannoulis-Lascarides, L.; Waltimo-Siren, J.; Ollila, P.; Karjalainen, S.; Arte, S.; Veerkamp, J.; Tallon Walton, V.; Chimenos Küstner, E.; Siltanen, T.; Holappa, H.; Lukinmaa, P.L.; Alaluusua, S.

    2011-01-01

    We describe results from a mutational analysis of the region of the dentin sialophosphoprotein (DSPP) gene encoding dentin phosphoprotein (DPP) in 12 families with dominantly inherited dentin diseases. In eight families (five mutations in the N-terminal third of DPP), the clinical and radiologic

  19. Clinical Characteristics of Wolfram Syndrome in Chinese Population and a Novel Frameshift Mutation in WFS1

    Directory of Open Access Journals (Sweden)

    Lian Duan

    2018-02-01

    Full Text Available ObjectiveWolfram syndrome (WS is a rare, degenerative, and hereditary disorder characterized by ear diabetes mellitus (DM and optic atrophy (OA. We aim to characterize clinical features in Chinese patients who had been poorly studied until now.MethodsWe performed a retrospective review of patients with WS seen in the Peking Union Medical College Hospital from 2002 to 2017. Data including demographic data, clinical presentations, examination results, family history, and genetic analysis were described.ResultsSix patients with WS were identified, meeting the diagnostic criteria of the coincidence of DM and OA before 15 years old or the existence of two WFS1 mutations. All were male, with the median age of 14.5 years (range 10–19 years. Blood glucose impairment, OA, and diabetes insipidus were present in all (100%, hearing impairment in four (66.7%, urological abnormalities in four (66.7%, neurological abnormalities in one (16.7%, and endocrine disorder in one (16.7%. Rare presentation includes cataract, glaucoma, and spina bifida occulta. Diabetes was insulin-dependent and not ketosis onset, with antibody to glutamic acid decarboxylase and islet cell negative. Genetic analysis revealed mutations in WFS1 in three patients. A novel frameshift mutation (p.Asp151Glufs*93 was identified in exon 4 of WFS1.ConclusionOur series of WS patients indicated that WS is a degenerative disease with a wide and variable spectrum, characterized by ear non-autoimmune DM and bilateral OA. Genetic analysis is recommended when suspected of WS.

  20. Missense Mutations in SLC26A8, Encoding a Sperm-Specific Activator of CFTR, Are Associated with Human Asthenozoospermia

    Science.gov (United States)

    Dirami, Thassadite; Rode, Baptiste; Jollivet, Mathilde; Da Silva, Nathalie; Escalier, Denise; Gaitch, Natacha; Norez, Caroline; Tuffery, Pierre; Wolf, Jean-Philippe; Becq, Frédéric; Ray, Pierre F.; Dulioust, Emmanuel; Gacon, Gérard; Bienvenu, Thierry; Touré, Aminata

    2013-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is present in mature sperm and is required for sperm motility and capacitation. Both these processes are controlled by ions fluxes and are essential for fertilization. We have shown that SLC26A8, a sperm-specific member of the SLC26 family of anion exchangers, associates with the CFTR channel and strongly stimulates its activity. This suggests that the two proteins cooperate to regulate the anion fluxes required for correct sperm motility and capacitation. Here, we report on three heterozygous SLC26A8 missense mutations identified in a cohort of 146 men presenting with asthenozoospermia: c.260G>A (p.Arg87Gln), c.2434G>A (p.Glu812Lys), and c.2860C>T (p.Arg954Cys). These mutations were not present in 121 controls matched for ethnicity, and statistical analysis on a control population of 8,600 individuals (from dbSNP and 1000 Genomes) showed them to be associated with asthenozoospermia with a power > 95%. By cotransfecting Chinese hamster ovary (CHO)-K1 cells with SLC26A8 variants and CFTR, we showed that the physical interaction between the two proteins was partly conserved but that the capacity to activate CFTR-dependent anion transport was completely abolished for all mutants. Biochemical studies revealed the presence of much smaller amounts of protein for all variants, but these amounts were restored to wild-type levels upon treatment with the proteasome inhibitor MG132. Immunocytochemistry also showed the amounts of SLC26A8 in sperm to be abnormally small in individuals carrying the mutations. These mutations might therefore impair formation of the SLC26A8-CFTR complex, principally by affecting SLC26A8 stability, consistent with an impairment of CFTR-dependent sperm-activation events in affected individuals. PMID:23582645

  1. [Aquagenic palmar keratoderma in a patient heterozygous for the mutation c.3197G>C in the CFTR gene].

    Science.gov (United States)

    Nadal, M; Laudier, B; Malinge, M C; Binois, R; Estève, E

    2015-03-01

    Aquagenic palmar keratoderma is an entity recently described in the literature by English and McCollough in 1996. It is a rare condition affecting young women and is of unknown incidence. It causes a wrinkled and oedematous appearance in the skin of the hands that may be seen a few minutes after immersion in water. This condition may be associated with a heterozygous mutation in CFTR, the gene involved in cystic fibrosis. We report the first case of aquagenic keratoderma associated with a new mutation in the CFTR gene. An 18-year-old patient with no particular history was referred for a painful rash on both palms occurring whenever she showered, and which had been ongoing for several months. The clinical examination was normal except for an appearance of moderate palmar hyperhidrosis. Following a test in which both hands were immersed in cold water for 5minutes, the patient presented itching, burning and pain localized to the hands. The palms were wrinkled and oedematous with white, translucent and confluent papules. A clinical diagnosis of aquagenic palmar keratoderma was made. Since this condition may be associated with mutations in the CFTR gene, a genetic study was performed for this patient and revealed the presence of a new mutation in the CFTR gene for cystic fibrosis in the heterozygous state inherited from her mother: c.3197G>C or p.Arg1066.Pro and a heterozygous polypyrimidic 5T variant inherited from her father. We report a new case of aquagenic palmar keratoderma in a patient heterozygous for a new mutation of the gene involved in cystic fibrosis. Several studies have shown association of aquagenic keratoderma with the CFTR gene for heterozygotes (carriers without cystic fibrosis), for patients with cystic fibrosis and for a patient presenting CFTRopathy with pancreatic insufficiency. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  2. Acute administration of ivacaftor to people with cystic fibrosis and a G551D-CFTR mutation reveals smooth muscle abnormalities

    Science.gov (United States)

    Adam, Ryan J.; Hisert, Katherine B.; Dodd, Jonathan D.; Grogan, Brenda; Launspach, Janice L.; Barnes, Janel K.; Gallagher, Charles G.; Sieren, Jered P.; Gross, Thomas J.; Fischer, Anthony J.; Cavanaugh, Joseph E.; Hoffman, Eric A.; Singh, Pradeep K.; Welsh, Michael J.; McKone, Edward F.; Stoltz, David A.

    2016-01-01

    BACKGROUND. Airflow obstruction is common in cystic fibrosis (CF), yet the underlying pathogenesis remains incompletely understood. People with CF often exhibit airway hyperresponsiveness, CF transmembrane conductance regulator (CFTR) is present in airway smooth muscle (ASM), and ASM from newborn CF pigs has increased contractile tone, suggesting that loss of CFTR causes a primary defect in ASM function. We hypothesized that restoring CFTR activity would decrease smooth muscle tone in people with CF. METHODS. To increase or potentiate CFTR function, we administered ivacaftor to 12 adults with CF with the G551D-CFTR mutation; ivacaftor stimulates G551D-CFTR function. We studied people before and immediately after initiation of ivacaftor (48 hours) to minimize secondary consequences of CFTR restoration. We tested smooth muscle function by investigating spirometry, airway distensibility, and vascular tone. RESULTS. Ivacaftor rapidly restored CFTR function, indicated by reduced sweat chloride concentration. Airflow obstruction and air trapping also improved. Airway distensibility increased in airways less than 4.5 mm but not in larger-sized airways. To assess smooth muscle function in a tissue outside the lung, we measured vascular pulse wave velocity (PWV) and augmentation index, which both decreased following CFTR potentiation. Finally, change in distensibility of <4.5-mm airways correlated with changes in PWV. CONCLUSIONS. Acute CFTR potentiation provided a unique opportunity to investigate CFTR-dependent mechanisms of CF pathogenesis. The rapid effects of ivacaftor on airway distensibility and vascular tone suggest that CFTR dysfunction may directly cause increased smooth muscle tone in people with CF and that ivacaftor may relax smooth muscle. FUNDING. This work was funded in part from an unrestricted grant from the Vertex Investigator-Initiated Studies Program. PMID:27158673

  3. Identification of a novel frameshift mutation in PITX2 gene in a Chinese family with Axenfeld-Rieger syndrome.

    Science.gov (United States)

    Yin, Hou-fa; Fang, Xiao-yun; Jin, Chong-fei; Yin, Jin-fu; Li, Jin-yu; Zhao, Su-juan; Miao, Qi; Song, Feng-wei

    2014-01-01

    Axenfeld-Rieger syndrome (ARS) is phenotypically and genetically heterogeneous. In this study, we identified the underlying genetic defect in a Chinese family with ARS. A detailed family history and clinical data were recorded. The ocular phenotype was documented using slit-lamp photography and systemic anomalies were also documented where available. The genomic DNA was extracted from peripheral blood leukocytes. All coding exons and intron-exon junctions of paired-like homeodomain transcription factor 2 (PITX2) gene and the forkhead box C1 (FOXC1) gene were amplified by polymerase chain reaction (PCR) and screened for mutation by direct DNA sequencing. Variations detected in exon 5 of PITX2 were further evaluated with cloning sequencing. The exon 5 of PITX2 was also sequenced in 100 healthy controls, unrelated to the family, for comparison. Structural models of the wild type and mutant homeodomain of PITX2 were investigated by SWISS-MODEL. Affected individuals exhibited variable ocular phenotypes, whereas the systemic anomalies were similar. After direct sequencing and cloning sequencing, a heterozygous deletion/insertion mutation c.198_201delinsTTTCT (p.M66Ifs*133) was revealed in exon 5 of PITX2. This mutation co-segregated with all affected individuals in the family and was not found either in unaffected family members or in 100 unrelated controls. We detected a novel frameshift mutation p.M66Ifs*133 in PITX2 in a Chinese family with ARS. Although PITX2 mutations and polymorphisms have been reported from various ethnic groups, we report for the first time the identification of a novel deletion/insertion mutation that causes frameshift mutation in the homeodomain of PITX2 protein.

  4. mRNA-based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity.

    Science.gov (United States)

    Felício, V; Ramalho, A S; Igreja, S; Amaral, M D

    2017-03-01

    Even with advent of next generation sequencing complete sequencing of large disease-associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease-causing mutations. Moreover, with the advent of mutation-based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost-effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA-based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

    Science.gov (United States)

    Maletzki, Claudia; Huehns, Maja; Bauer, Ingrid; Ripperger, Tim; Mork, Maureen M; Vilar, Eduardo; Klöcking, Sabine; Zettl, Heike; Prall, Friedrich; Linnebacher, Michael

    2017-07-01

    Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFβR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis. © 2017 Wiley Periodicals, Inc.

  6. SOS1 frameshift mutations cause pure mucosal neuroma syndrome, a clinical phenotype distinct from multiple endocrine neoplasia type 2B.

    Science.gov (United States)

    Owens, Martina; Kivuva, Emma; Quinn, Anthony; Brennan, Paul; Caswell, Richard; Lango Allen, Hana; Vaidya, Bijay; Ellard, Sian

    2016-05-01

    Mucosal neuromas, thickened corneal nerves and marfanoid body habitus are characteristic phenotypic features of multiple endocrine neoplasia type 2B (MEN2B) and often provide an early clue to the diagnosis of the syndrome. Rarely, patients present with typical physical features of MEN2B but without associated endocrinopathies (medullary thyroid carcinoma or pheochromocytoma) or a RET gene mutation; this clinical presentation is thought to represent a distinct condition termed 'pure mucosal neuroma syndrome'. Exome sequencing was performed in two unrelated probands with mucosal neuromas, thickened corneal nerves and marfanoid body habitus, but no MEN2B-associated endocrinopathy or RET gene mutation. Sanger sequencing was performed to confirm mutations detected by exome sequencing and to test in family members and 3 additional unrelated index patients with mucosal neuromas or thickened corneal nerves. A heterozygous SOS1 gene frameshift mutation (c.3266dup or c.3248dup) was identified in each proband. Sanger sequencing showed that proband 1 inherited the c.3266dup mutation from his affected mother, while the c.3248dup mutation had arisen de novo in proband 2. Sanger sequencing also identified one further novel SOS1 mutation (c.3254dup) in one of the 3 additional index patients. Our results demonstrate the existence of pure mucosal neuroma syndrome as a clinical entity distinct from MEN2B that can now be diagnosed by genetic testing. © 2015 John Wiley & Sons Ltd.

  7. Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface.

    Science.gov (United States)

    Chin, Stephanie; Yang, Donghe; Miles, Andrew J; Eckford, Paul D W; Molinski, Steven; Wallace, B A; Bear, Christine E

    2017-02-03

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Spectrum of CFTR gene mutations in Ecuadorian cystic fibrosis patients: the second report of the p.H609R mutation.

    Science.gov (United States)

    Ortiz, Sofía C; Aguirre, Santiago J; Flores, Sofía; Maldonado, Claudio; Mejía, Juan; Salinas, Lilian

    2017-11-01

    High heterogeneity in the CFTR gene mutations disturbs the molecular diagnosis of cystic fibrosis (CF). In order to improve the diagnosis of CF in our country, the present study aims to define a panel of common CFTR gene mutations by sequencing 27 exons of the gene in Ecuadorian Cystic Fibrosis patients. Forty-eight Ecuadorian individuals with suspected/confirmed CF diagnosis were included. Twenty-seven exons of CFTR gene were sequenced to find sequence variations. Prevalence of pathogenic variations were determined and compared with other countries' data. We found 70 sequence variations. Eight of these are CF-causing mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. Also this study is the second report of p.H609R in Ecuadorian population. Mutation prevalence differences between Ecuadorian population and other Latin America countries were found. The panel of mutations suggested as an initial screening for the Ecuadorian population with cystic fibrosis should contain the mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. © 2017 NETLAB Laboratorios Especializados. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

  9. CFTR mutations impart elevated immune reactivity in a murine model of cystic fibrosis related diabetes.

    Science.gov (United States)

    Stalvey, Michael S; Brusko, Todd M; Mueller, Christian; Wasserfall, Clive H; Schatz, Desmond A; Atkinson, Mark A; Flotte, Terence R

    2008-10-01

    Increased life expectancy in cystic fibrosis (CF) is accompanied by an increasing incidence of CF related diabetes (CFRD). Altered immune reactivity occurs in CF, which we hypothesize, is exacerbated by hyperglycemia. Cystic fibrosis transmembrane conductance regulator deficient (CFTR-/-) mice were rendered hyperglycemic by streptozotocin (STZ) to test this hypothesis. CFTR-/-, C57BL/6J, and FVB/NJ mice received either STZ or lactated ringers (LR) (n=5-10). Four weeks later, splenocytes were harvested, mitogen stimulated, and analyzed for cytokine production (IL-2, IL-4, and IL-10) along with stimulation indices (SI). SI of STZ-treated CFTR-/- were elevated compared to LR-treated mice, although both were greater than C57BL/6J and FVB/NJ (p<0.05). Fasting glucose levels of STZ-treated CFTR-/- mice correlated with SI (p<0.003). Stimulated IL-10 concentrations were elevated in STZ-treated CFTR-/- compared to LR-treated animals and controls (p<0.05). IL-2 levels were greater in CFTR-/- mice compared to controls (p<0.05), but unrelated to STZ. Reinforcing generalized cytokine up-regulation in CFTR-/-, IL-4 levels were greater in CFTR-/- mice compared to C57BL/6J, but FVB/NJ mice demonstrated greatest concentrations following STZ. These results suggest that, hyperglycemia may exacerbate the clinical course in CF by impacting immune reactivity. There is clear need to maximize metabolic management in CFRD.

  10. Multiple nevoid basal cell carcinoma syndrome associated with congenital orbital teratoma, caused by a PTCH1 frameshift mutation.

    Science.gov (United States)

    Rodrigues, A L; Carvalho, A; Cabral, R; Carneiro, V; Gilardi, P; Duarte, C P; Puente-Prieto, J; Santos, P; Mota-Vieira, L

    2014-07-25

    Gorlin-Goltz syndrome, or nevoid basal cell carcinoma syndrome (NBCCS), is a rare autosomal dominant disorder caused by mutations in the PTCH1 gene and shows a high level of penetrance and variable expressivity. The syndrome is characterized by developmental abnormalities or neoplasms and is diagnosed with 2 major criteria, or with 1 major and 2 minor criteria. Here, we report a new clinical manifestation associated with this syndrome in a boy affected by NBCCS who had congenital orbital teratoma at birth. Later, at the age of 15 years, he presented with 4 major and 4 minor criteria of NBCCS, including multiple basal cell carcinoma and 2 odontogenic keratocysts of the jaw, both confirmed by histology, more than 5 palmar pits, calcification of the cerebral falx, extensive meningeal calcifications, macrocephaly, hypertelorism, frontal bosses, and kyphoscoliosis. PTCH1 mutation analysis revealed the heterozygous germline mutation c.290dupA. This mutation generated a frameshift within exon 2 and an early premature stop codon (p.Asn97LysfsX43), predicting a truncated protein with complete loss of function. Identification of this mutation is useful for genetic counseling. Although the clinical symptoms are well-known, our case contributes to the understanding of phenotypic variability in NBCCS, highlighting that PTCH1 mutations cannot be used for predicting disease burden and reinforces the need of a multidisciplinary team in the diagnosis, treatment, and follow-up of NBCCS patients.

  11. Sweat chloride and immunoreactive trypsinogen in infants carrying two CFTR mutations and not affected by cystic fibrosis.

    Science.gov (United States)

    Castellani, Carlo; Tridello, Gloria; Tamanini, Anna; Assael, Baroukh M

    2017-07-01

    Newborns with raised immunotrypsinogen levels who have non-pathological sweat chloride values and carry two cystic fibrosis transmembrane regulator ( CFTR ) mutations of which at least one is not acknowledged to be cystic fibrosis (CF)-causing are at risk of developing clinical manifestations consistent with CFTR-related disorders or even CF. It is not known whether newborns with similar genotypes and normal immunoreactive trypsinogen (IRT) may share the same risk. This study found that newborns with these characteristics and normal IRT have lower sweat chloride values than those with raised IRT (p=0.007). Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  12. Frameshift mutations in infectious cDNA clones of Citrus tristeza virus: a strategy to minimize the toxicity of viral sequences to Escherichia coli

    International Nuclear Information System (INIS)

    Satyanarayana, Tatineni; Gowda, Siddarame; Ayllon, Maria A.; Dawson, William O.

    2003-01-01

    The advent of reverse genetics revolutionized the study of positive-stranded RNA viruses that were amenable for cloning as cDNAs into high-copy-number plasmids of Escherichia coli. However, some viruses are inherently refractory to cloning in high-copy-number plasmids due to toxicity of viral sequences to E. coli. We report a strategy that is a compromise between infectivity of the RNA transcripts and toxicity to E. coli effected by introducing frameshift mutations into 'slippery sequences' near the viral 'toxicity sequences' in the viral cDNA. Citrus tristeza virus (CTV) has cDNA sequences that are toxic to E. coli. The original full-length infectious cDNA of CTV and a derivative replicon, CTV-ΔCla, cloned into pUC119, resulted in unusually limited E. coli growth. However, upon sequencing of these cDNAs, an additional uridinylate (U) was found in a stretch of U's between nts 3726 and 3731 that resulted in a change to a reading frame with a stop codon at nt 3734. Yet, in vitro produced RNA transcripts from these clones infected protoplasts, and the resulting progeny virus was repaired. Correction of the frameshift mutation in the CTV cDNA constructs resulted in increased infectivity of in vitro produced RNA transcripts, but also caused a substantial increase of toxicity to E. coli, now requiring 3 days to develop visible colonies. Frameshift mutations created in sequences not suspected to facilitate reading frame shifting and silent mutations introduced into oligo(U) regions resulted in complete loss of infectivity, suggesting that the oligo(U) region facilitated the repair of the frameshift mutation. Additional frameshift mutations introduced into other oligo(U) regions also resulted in transcripts with reduced infectivity similarly to the original clones with the +1 insertion. However, only the frameshift mutations introduced into oligo(U) regions that were near and before the toxicity region improved growth and stability in E. coli. These data demonstrate that

  13. Whole-exome sequencing of a patient with severe and complex hemostatic abnormalities reveals a possible contributing frameshift mutation in C3AR1

    DEFF Research Database (Denmark)

    Leinøe, Eva; Nielsen, Ove Juul; Jønson, Lars

    2016-01-01

    -threatening coagulation disorder causing recurrent venous thromboembolic events, severe thrombocytopenia, and subdural hematomas. Whole-exome sequencing revealed a frameshift mutation (C3AR1 c.355-356dup, p.Asp119Alafs*19) resulting in a premature stop codon in C3AR1 (Complement Component 3a Receptor 1). Based...

  14. Serum zinc concentration in cystic fibrosis patients with CFTR I1234V mutation associated with pancreatic sufficiency.

    Science.gov (United States)

    AbdulWahab, Atqah; Abushahin, Ahmed; Allangawi, Mona; Chandra, Prem; Abdel Rahman, Mohamed Osman; Soliman, Ashraf

    2017-05-01

    To determine serum zinc (Zn) level among cystic fibrosis (CF) patients with homozygous CFTR I1234V mutation associated with pancreatic sufficiency (PS). A cross-sectional study was conducted including both pediatric and adult CF patients. Data on age, weight, height, body mass index (BMI), BMI Z-score, FEV1, and chronic Pseudomonas aeruginosa infection were collected. Serum Zn, albumin, and total proteins were measured and analyzed. Forty-five CF patients with homozygous CFTR I1234V mutation belonging to a large Arab kindred tribe and eight CF patients with other mutations associated with pancreatic insufficiency (PI). Patient's age ranged from 2 to 49 years with a mean age of 15.1 ± 9.1 years and mean plasma Zn of 0.78 ± 0.15 mcg/mL. Seven (13.2%) patients with CFTR I1234V and PS had low Zn levels (<0.6 mcg/mL). Mean age among Zn deficient group was significantly older. The mean FEV1 in the deficient group was found to be insignificant low. Persistent P. aeruginosa colonization was more prevalent in Zn deficient group. BMI Z-scosre of CF patients were positively correlated with Zn levels. Forty-five healthy subjects belonging to the same Arab tribe were selected in order to assess their Zn levels and their mean plasma Zn of 0.84 ± 0.11 mcg/mL (range 0.65-1.1 mcg/mL) with mean age 20.4 ± 10.1 years (range 6-40 years). These findings suggest that Zn deficiency can occur in CF patients with PS. The association of Zn levels and the frequency of P. aeruginosa isolated in CF patients need further investigation. © 2015 The Authors. The Clinical Respiratory Journal Published by John Wiley & Sons Ltd.

  15. De novo MECP2 frameshift mutation in a boy with moderate mental retardation, obesity and gynaecomastia.

    NARCIS (Netherlands)

    Kleefstra, T.; Yntema, H.G.; Oudakker, A.R.; Romein, T.; Sistermans, E.A.; Nillessen, W.; Bokhoven, J.H.L.M. van; Vries, L.B.A. de; Hamel, B.C.J.

    2002-01-01

    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the MECP2 gene, with apparent lethality in male embryos. However, recent studies indicate that mutations in the MECP2 gene can cause congenital encephalopathy, an Angelman-like phenotype and even nonspecific mental

  16. Novel compound heterozygous frameshift mutations of C2orf37 in a ...

    Indian Academy of Sciences (India)

    ganglia, T-wave abnormalities and depressed insulin-like growth factor 1 levels. A mutation in the C2orf37 gene was described as the cause of WSS in 2008 in the Saudi families including the ones originally described by Woodhouse and Sakati (Alazami et al. 2008). Additional mutations in. C2orf37 were also described by ...

  17. A homozygous frameshift mutation in the HOXC13 gene underlies pure hair and nail ectodermal dysplasia in a Syrian family.

    Science.gov (United States)

    Farooq, Muhammad; Kurban, Mazen; Fujimoto, Atsushi; Fujikawa, Hiroki; Abbas, Ossama; Nemer, Georges; Saliba, Jessica; Sleiman, Rima; Tofaili, Mona; Kibbi, Abdul-Ghani; Ito, Masaaki; Shimomura, Yutaka

    2013-04-01

    Pure hair and nail ectodermal dysplasia (PHNED) is a rare genetic disorder characterized by hypotrichosis or complete alopecia, as well as nail dystrophy. Mutations in the type II hair keratin gene KRT85 and the HOXC13 gene on chromosome 12q have recently been identified in families with autosomal-recessive PHNED. In the present study, we have analyzed a consanguineous Syrian family with an affected girl having complete alopecia and nail dystrophy since birth. The family clearly showed linkage to chromosome 12q13.13-12q14.3, which excluded the KRT85 gene. Sequencing of another candidate gene HOXC13 within the linkage interval identified a homozygous frameshift mutation (c.355delC; p.Leu119Trpfs*20). Expression studies in cultured cells revealed that the mutant HOXC13 protein mislocalized within the cytoplasm, and failed to upregulate the promoter activities of its target genes. Our results strongly suggest crucial roles of the HOXC13 gene in the development of hair and nails in humans. © 2013 Wiley Periodicals, Inc.

  18. An exon 53 frameshift mutation in CUBN abrogates cubam function and causes Imerslund-Gräsbeck syndrome in dogs.

    Science.gov (United States)

    Fyfe, John C; Hemker, Shelby L; Venta, Patrick J; Fitzgerald, Caitlin A; Outerbridge, Catherine A; Myers, Sherry L; Giger, Urs

    2013-08-01

    Cobalamin malabsorption accompanied by selective proteinuria is an autosomal recessive disorder known as Imerslund-Gräsbeck syndrome in humans and was previously described in dogs due to amnionless (AMN) mutations. The resultant vitamin B12 deficiency causes dyshematopoiesis, lethargy, failure to thrive, and life-threatening metabolic disruption in the juvenile period. We studied 3 kindreds of border collies with cobalamin malabsorption and mapped the disease locus in affected dogs to a 2.9Mb region of homozygosity on canine chromosome 2. The region included CUBN, the locus encoding cubilin, a peripheral membrane protein that in concert with AMN forms the functional intrinsic factor-cobalamin receptor expressed in ileum and a multi-ligand receptor in renal proximal tubules. Cobalamin malabsorption and proteinuria comprising CUBN ligands were demonstrated by radiolabeled cobalamin uptake studies and SDS-PAGE, respectively. CUBN mRNA and protein expression were reduced ~10 fold and ~20 fold, respectively, in both ileum and kidney of affected dogs. DNA sequencing demonstrated a single base deletion in exon 53 predicting a translational frameshift and early termination codon likely triggering nonsense mediated mRNA decay. The mutant allele segregated with the disease in the border collie kindred. The border collie disorder indicates that a CUBN mutation far C-terminal from the intrinsic factor-cobalamin binding site can abrogate receptor expression and cause Imerslund-Gräsbeck syndrome. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. A Newly Described Bovine Type 2 Scurs Syndrome Segregates with a Frame-Shift Mutation in TWIST1

    Science.gov (United States)

    Capitan, Aurélien; Grohs, Cécile; Weiss, Bernard; Rossignol, Marie-Noëlle; Reversé, Patrick; Eggen, André

    2011-01-01

    The developmental pathways involved in horn development are complex and still poorly understood. Here we report the description of a new dominant inherited syndrome in the bovine Charolais breed that we have named type 2 scurs. Clinical examination revealed that, despite a strong phenotypic variability, all affected individuals show both horn abnormalities similar to classical scurs phenotype and skull interfrontal suture synostosis. Based on a genome-wide linkage analysis using Illumina BovineSNP50 BeadChip genotyping data from 57 half-sib and full-sib progeny, this locus was mapped to a 1.7 Mb interval on bovine chromosome 4. Within this region, the TWIST1 gene encoding a transcription factor was considered as a strong candidate gene since its haploinsufficiency is responsible for the human Saethre-Chotzen syndrome, characterized by skull coronal suture synostosis. Sequencing of the TWIST1 gene identified a c.148_157dup (p.A56RfsX87) frame-shift mutation predicted to completely inactivate this gene. Genotyping 17 scurred and 20 horned founders of our pedigree as well as 48 unrelated horned controls revealed a perfect association between this mutation and the type 2 scurs phenotype. Subsequent genotyping of 32 individuals born from heterozygous parents showed that homozygous mutated progeny are completely absent, which is consistent with the embryonic lethality reported in Drosophila and mouse suffering from TWIST1 complete insufficiency. Finally, data from previous studies on model species and a fine description of type 2 scurs symptoms allowed us to propose different mechanisms to explain the features of this syndrome. In conclusion, this first report on the identification of a potential causal mutation affecting horn development in cattle offers a unique opportunity to better understand horn ontogenesis. PMID:21814570

  20. A frameshift mutation in MC1R and a high frequency of somatic reversions cause black spotting in pigs.

    Science.gov (United States)

    Kijas, J M; Moller, M; Plastow, G; Andersson, L

    2001-06-01

    Black spotting on a red or white background in pigs is determined by the E(P) allele at the MC1R/Extension locus. A previous comparison of partial MC1R sequences revealed that E(P) shares a missense mutation (D121N) with the E(D2) allele for dominant black color. Sequence analysis of the entire coding region now reveals a second mutation in the form of a 2-bp insertion at codon 23 (nt67insCC). This mutation expands a tract of six C nucleotides to eight and introduces a premature stop codon at position 56. This frameshift mutation is expected to cause a recessive red color, which was in fact observed in some breeds with the E(P) allele present (Tamworth and Hereford). RT-PCR analyses were conducted using skin samples taken from both spotted and background areas of spotted pigs. The background red area had transcript only from the mutant nt67insCC MC1R allele, whereas the black spot also contained a transcript without the 2-bp insertion. This indicates that black spots are due to somatic reversion events that restore the frame and MC1R function. The phenotypic expression of the E(P) allele is highly variable and the associated coat color ranges from red, red with black spots, white with black spots, to almost completely solid black. In several breeds of pigs the phenotypic manifestation of this allele has been modified by selection for or against black spots.

  1. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cystic fibrosis transmembrane conductance... DEVICES Immunological Test Systems § 866.5900 Cystic fibrosis transmembrane conductance regulator (CFTR... intended as an aid in confirmatory diagnostic testing of individuals with suspected cystic fibrosis (CF...

  2. Frequency of CFTR, SPINK1, and Cathepsin B Gene Mutation in North Indian Population: Connections between Genetics and Clinical Data

    Directory of Open Access Journals (Sweden)

    Shweta Singh

    2014-01-01

    Full Text Available Objectives. Genetic mutations and polymorphisms have been correlated with chronic pancreatitis (CP. This study aims to investigate the association of genetic variants of cystic fibrosis transmembrane conductance regulator (CFTR and serine protease inhibitor Kazal type 1 (SPINK-1 genes and Cathepsin B gene polymorphisms with CP and to associate genetic backgrounds with clinical phenotypes. Methods. 150 CP patients and 150 normal controls were enrolled consecutively. We analyzed SPINK-1 N34S and IVS3+2T>C gene mutations by PCR-restriction-fragment length polymorphism (RFLP. The identification of DF508, G551D, G542X, R117H, and W1282X mutations was carried out by ARMS-PCR. S549N mutation, IVS8 polyTn polymorphism, and Cathepsin B Lec26Val were analysed by PCR-RFLP, nested PCR, and PCR-RFLP plus sequencing, respectively. Results. We found a significant association of SPINK1 (N34S gene polymorphism. IVS1−37T>C polymorphism shows linkage with 101A>G. 300 chromosomes belonging to the CFTR subgroup exhibited minor allele frequency of 0.04, 0.03, 0.03, 0.013, 0.006, and 0.02 for DF508, G452X, G551D, S549N, R117H, and IVS8 T5, respectively. Except for R117H and IVS8 T5 polymorphisms, all other mutations showed significant variation. Conclusion. Analysis of potential susceptibility variants is needed to support nature of the genes and environment in pancreatitis. This data may help establish genetic screening and prenatal setup for Indian population.

  3. Frequency of CFTR, SPINK1, and cathepsin B gene mutation in North Indian population: connections between genetics and clinical data.

    Science.gov (United States)

    Singh, Shweta; Choudhuri, Gourdas; Agarwal, Sarita

    2014-01-01

    Genetic mutations and polymorphisms have been correlated with chronic pancreatitis (CP). This study aims to investigate the association of genetic variants of cystic fibrosis transmembrane conductance regulator (CFTR) and serine protease inhibitor Kazal type 1 (SPINK-1) genes and Cathepsin B gene polymorphisms with CP and to associate genetic backgrounds with clinical phenotypes. 150 CP patients and 150 normal controls were enrolled consecutively. We analyzed SPINK-1 N34S and IVS3+2T>C gene mutations by PCR-restriction-fragment length polymorphism (RFLP). The identification of DF508, G551D, G542X, R117H, and W1282X mutations was carried out by ARMS-PCR. S549N mutation, IVS8 polyTn polymorphism, and Cathepsin B Lec26Val were analysed by PCR-RFLP, nested PCR, and PCR-RFLP plus sequencing, respectively. We found a significant association of SPINK1 (N34S) gene polymorphism. IVS1-37T>C polymorphism shows linkage with 101A>G. 300 chromosomes belonging to the CFTR subgroup exhibited minor allele frequency of 0.04, 0.03, 0.03, 0.013, 0.006, and 0.02 for DF508, G452X, G551D, S549N, R117H, and IVS8 T5, respectively. Except for R117H and IVS8 T5 polymorphisms, all other mutations showed significant variation. Analysis of potential susceptibility variants is needed to support nature of the genes and environment in pancreatitis. This data may help establish genetic screening and prenatal setup for Indian population.

  4. Novel frameshift mutation in the KCNQ1 gene responsible for Jervell and Lange-Nielsen syndrome

    Directory of Open Access Journals (Sweden)

    Azam Amirian

    2018-01-01

    Full Text Available Objective(s: Jervell and Lange–Nielsen syndrome is an autosomal recessive disorder caused by mutations in KCNQ1 or KCNE1 genes. The disease is characterized by sensorineural hearing loss and long QT syndrome. Methods: Here we present a 3.5-year-old female patient, an offspring of consanguineous marriage, who had a history of recurrent syncope and congenital sensorineural deafness. The patient and the family members were screened for mutations in KCNQ1 gene by linkage analysis and DNA sequencing. Results: DNA sequencing showed a c.1532_1534delG (p. A512Pfs*81 mutation in the KCNQ1 gene in homozygous form. The results of short tandem repeat (STR markers showed that the disease in the family is linked to the KCNQ1 gene. The mutation was confirmed in the parents in heterozygous form. Conclusion: This is the first report of this variant in KCNQ1 gene in an Iranian family. The data of this study could be used for early diagnosis of the condition in the family and genetic counseling.

  5. Identification of a novel frameshift mutation in the ILDR1 gene in a UAE family, mutations review and phenotype genotype correlation.

    Directory of Open Access Journals (Sweden)

    Abdelaziz Tlili

    Full Text Available Autosomal recessive non-syndromic hearing loss is one of the most common monogenic diseases. It is characterized by high allelic and locus heterogeneities that make a precise diagnosis difficult. In this study, whole-exome sequencing was performed for an affected patient allowing us to identify a new frameshift mutation (c.804delG in the Immunoglobulin-Like Domain containing Receptor-1 (ILDR1 gene. Direct Sanger sequencing and segregation analysis were performed for the family pedigree. The mutation was homozygous in all affected siblings but heterozygous in the normal consanguineous parents. The present study reports a first ILDR1 gene mutation in the UAE population and confirms that the whole-exome sequencing approach is a robust tool for the diagnosis of monogenic diseases with high levels of allelic and locus heterogeneity. In addition, by reviewing all reported ILDR1 mutations, we attempt to establish a genotype phenotype correlation to explain the phenotypic variability observed at low frequencies.

  6. A novel frameshift mutation in the sterol 27-hydroxylase gene in an Egyptian family with cerebrotendinous xanthomatosis without cataract.

    Science.gov (United States)

    Abdel-Hamid, Mohamed S; Issa, Mahmoud Y; Otaify, Ghada A; Zaki, Maha S

    2017-04-01

    Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive lipid storage disorder caused by deficiency of the mitochondrial cytochrome P450 sterol 27-hydroxylase enzyme encoded by CYP27A1 gene. CTX is characterized by tendon xanthomas, juvenile cataracts and multiple progressive neurological symptoms. Here we report on the clinical and molecular findings of a 35-years old Egyptian patient with CTX without cataract. Parents were first cousins with family history of two deceased sibs with mild impaired cognitive functions and epilepsy without appearance of tendon xanthomas. Our proband had learning disabilities and developed seizures at 9 years old. Tendon xanthomata appeared at the age of 16 and his neurological symptoms remained stationary till 28 years followed by progressive cerebello-pyramidal signs, dementia and psychiatric disturbance. Cataract was not evident in our patient. Brain MRI showed the characteristic focal lesions appeared as xanthomas in cerebellum and occipital horns of lateral ventricles. Molecular study identified a novel homozygous frameshift mutation in CYP27A1 gene, c.1169delT (p.K391Rfs*17). Our study emphasizes the important role of early genetic testing in prevention of morbidity and mortality of the disease and proper counseling. Moreover, it shows that the absence of cataract should not rule out the diagnosis of CTX.

  7. Identification of a novel COL1A1 frameshift mutation, c.700delG, in a Chinese osteogenesis imperfecta family

    Science.gov (United States)

    Wang, Xiran; Pei, Yu; Dou, Jingtao; Lu, Juming; Li, Jian; Lv, Zhaohui

    2015-01-01

    Osteogenesis imperfecta (OI) is a family of genetic disorders associated with bone loss and fragility. Mutations associated with OI have been found in genes encoding the type I collagen chains. People with OI type I often produce insufficient α1-chain type I collagen because of frameshift, nonsense, or splice site mutations in COL1A1 or COL1A2. This report is of a Chinese daughter and mother who had both experienced two bone fractures. Because skeletal fragility is predominantly inherited, we focused on identifying mutations in COL1A1 and COL1A2 genes. A novel mutation in COL1A1, c.700delG, was detected by genomic DNA sequencing in the mother and daughter, but not in their relatives. The identification of this mutation led to the conclusion that they were affected by mild OI type I. Open reading frame analysis indicated that this frameshift mutation would truncate α1-chain type I collagen at residue p263 (p.E234KfsX264), while the wild-type protein would contain 1,464 residues. The clinical data were consistent with the patients’ diagnosis of mild OI type I caused by haploinsufficiency of α1-chain type I collagen. Combined with previous reports, identification of the novel mutation COL1A1-c.700delG in these patients suggests that additional genetic and environmental factors may influence the severity of OI. PMID:25983617

  8. A CHRNB1 frameshift mutation is associated with familial arthrogryposis multiplex congenita in Red dairy cattle.

    Science.gov (United States)

    Agerholm, Jørgen S; McEvoy, Fintan J; Menzi, Fiona; Jagannathan, Vidhya; Drögemüller, Cord

    2016-06-30

    Bovine arthrogryposis multiplex congenita (AMC) is a syndromic term for a congenital condition characterized by multiple joint contractures. Rare inherited forms of bovine AMC have been reported in different breeds. For AMC in Angus cattle a causative genomic deletion encompassing the agrin (AGRN) gene, encoding an essential neural regulator that induces the aggregation of acetylcholine receptors (AChRs), is known. In 2015, three genetically related cases of generalized AMC affecting Red dairy calves were diagnosed in Denmark. The family history of three affected calves suggested an autosomal recessive inheritance. Single nucleotide polymorphism (SNP) genotyping showed a single genomic region of extended homozygosity of 21.5 Mb on chromosome 19. Linkage analysis revealed a maximal parametric LOD score of 1.8 at this region. By whole genome re-sequencing of the three cases, two private homozygous non-synonymous variants were detected in the critical interval. Both variants, located in the myosin phosphatase Rho interacting protein (MPRIP) and the cholinergic receptor nicotinic beta 1 subunit gene (CHRNB1), were perfectly associated with the AMC phenotype. Previously described CHRNB1 variants in humans lead to a congenital myasthenic syndrome with impaired neuromuscular transmission. The cattle variant represents a single base deletion in the first exon of CHRNB1 (c.55delG) introducing a premature stop codon (p.Ala19Profs47*) in the second exon, truncating 96 % of the protein. This study provides the first phenotypically and genetically characterized example of a bovine AMC phenotype that represents an inherited neuromuscular disorder corresponding to human congenital myasthenic syndrome. The identified CHRNB1 loss of function variant is predicted to have a deleterious effect on fetal AChR function, which could explain the lethal phenotype reported in this study. The identification of this candidate causative mutation thus widens the known phenotypic spectrum of

  9. Novel CFTR missense mutations in Brazilian patients with congenital absence of vas deferens: counseling issues Mutações novas no gene CFTR de pacientes brasileiros portadores de agenesia dos vasos deferentes: dificuldades no aconselhamento

    Directory of Open Access Journals (Sweden)

    Patricia de Campos Pieri

    2007-01-01

    Full Text Available PURPOSE: Screening for mutations in the entire Cystic Fibrosis gene (CFTR of Brazilian infertile men with congenital absence of vas deferens, in order to prevent transmission of CFTR mutations to offspring with the use of assisted reproductive technologies. METHOD: Specific polymerase chain reaction (PCR primers were designed to each of the 27 exons and splicing sites of interest followed by single strand conformational polymorphism and Heteroduplex Analysis (SSCP-HA in precast 12.5% polyacrylamide gels at 7ºC and 20ºC. Fragments with abnormal SSCP migration pattern were sequenced. RESULTS: Two novel missense mutations (S753R and G149W were found in three patients (two brothers together with the IVS8-5T allele in hetrozygosis. CONCLUSION: The available screenings for CF mutations do not include the atypical mutations associated to absence of vas deferens and thus, when these tests fail to find mutations, there is still a genetic risk of affected children with the help of assisted reproduction. We recommend the screening of the whole CFTR gene for these infertile couples, as part of the work-up before assisted reproduction.OBJETIVO: Pesquisar mutações em toda a extensão do gene que causa a Fibrose Cística (CFTR de homens brasileiros inférteis por agenesia congênita dos vasos deferentes, com a finalidade de prevenir a transmissão de mutações em CFTR à prole com o uso das tecnologias de reprodução assistida. MÉTODOS: Foram desenhados oligonucleotídeos específicos para realização de reação de polimerização em cadeia (PCR para cada um dos 27 exons e sítios de processamento de interesse no gene CFTR. O PCR foi seguido pela técnica de SSCP-HA (polimorfismos de conformação no DNA de fita simples e na formação de heteroduplexes em géis pré-fabricados de poliacrilamida a 12,5% em duas temperaturas, 7ºC e 20ºC. Os fragmentos com padrão alterado na migração do SSCP foram submetidos a seqüenciamento automatizado

  10. Mutation Analysis of Exons 10 and 17a of CFTR Gene in Patients with Cystic Fibrosis in Kermanshah Province, Western Iran.

    Science.gov (United States)

    Sahami, Abbas; Alibakhshi, Reza; Ghadiri, Keyghobad; Sadeghi, Hamid

    2014-01-01

    Cystic fibrosis (CF) is the most common genetic disorder with autosomal recessive inheritance among Caucasian populations. So far, more than 1950 different mutations were identified in cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR gene has 27 exons. The type and distribution of mutations vary widely among different countries and/or ethnic groups. Therefore, a comprehensive analysis was performed on exon10 and exon17a of CFTR gene in CF patients in the Kermanshah province, western Iran. We tested 27 patients admitted to the medical genetics laboratory of Kermanshah University of Medical Sciences. The patients were from different cities of Kermanshah province. All the patients had the clinical signals and two positive sweat tests. After filling agreement forms and questionnaire, the peripheral blood sampling and DNA extraction were done. DNA samples were extracted. PCR and sequencing special PCR were done. Finally analysis of the results with DNA sequencing analysis version 5.2 software was performed. CFTR mutations analysis identified 4 different mutations in our CF patients. The disease-causing mutations were p.F508del (ΔF508) (14.81%), p.S466X (1.85%), and p.T1036I (1.85%). M470V polymorphism with frequency of 74.1% was found in 23 patients (17 homozygous and 6 heterozygous). Three disease-causing mutations in CF patients in the present study account for approximately 18.51% of mutations. The frequency of p.F508del, the most common mutation was 16-18.1% in Iranian population. The results of the present study can be applied for genetic counseling, population screening and prenatal diagnosis.

  11. Impact of the CFTR-potentiator ivacaftor on airway microbiota in cystic fibrosis patients carrying a G551D mutation.

    Directory of Open Access Journals (Sweden)

    Cédric Bernarde

    Full Text Available Airway microbiota composition has been clearly correlated with many pulmonary diseases, and notably with cystic fibrosis (CF, an autosomal genetic disorder caused by mutation in the CF transmembrane conductance regulator (CFTR. Recently, a new molecule, ivacaftor, has been shown to re-establish the functionality of the G551D-mutated CFTR, allowing significant improvement in lung function.The purpose of this study was to follow the evolution of the airway microbiota in CF patients treated with ivacaftor, using quantitative PCR and pyrosequencing of 16S rRNA amplicons, in order to identify quantitative and qualitative changes in bacterial communities. Three G551D children were followed up longitudinally over a mean period of more than one year covering several months before and after initiation of ivacaftor treatment.129 operational taxonomy units (OTUs, representing 64 genera, were identified. There was no significant difference in total bacterial load before and after treatment. Comparison of global community composition found no significant changes in microbiota. Two OTUs, however, showed contrasting dynamics: after initiation of ivacaftor, the relative abundance of the anaerobe Porphyromonas 1 increased (p<0.01 and that of Streptococcus 1 (S. mitis group decreased (p<0.05, possibly in relation to the anti-Gram-positive properties of ivacaftor. The anaerobe Prevotella 2 correlated positively with the pulmonary function test FEV-1 (r=0.73, p<0.05. The study confirmed the presumed positive role of anaerobes in lung function.Several airway microbiota components, notably anaerobes (obligate or facultative anaerobes, could be valuable biomarkers of lung function improvement under ivacaftor, and could shed light on the pathophysiology of lung disease in CF patients.

  12. N-acetoxy-N-2-acetylaminofluorene induced frameshift mutations: a comparison between the DNA modification spectrum and the mutation spectrum

    International Nuclear Information System (INIS)

    Fuchs, R.P.P.; Koffel-Schwartz, N.; Daune, M.

    1983-01-01

    We describe the analysis of forward mutations induced in the tetracycline resistance gene of the plasmid pBR322 by directing the reaction of the carcinogen N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) to a small restriction fragment (BamHI, SalI) that is located in the proximal part of the antibiotic-resistance gene. Mutant plasmids obtained both in wild type and excision repair deficient (uvrA) bacterial cells are compared. Preliminary data showing the distribution of the -AAF adducts along this restriction fragments are discussed in relation to the observed spectrum of mutations. 20 references, 4 figures

  13. Lumacaftor/ivacaftor, a novel agent for the treatment of cystic fibrosis patients who are homozygous for the F580del CFTR mutation.

    Science.gov (United States)

    Bulloch, Marilyn N; Hanna, Cameron; Giovane, Richard

    2017-10-01

    Cystic Fibrosis (CF) is an autosomal recessive disease affecting up to 90,000 people worldwide. Approximately 73% of patients are homozygous for the F508del cystic fibrosis transmembrane conductance regulator [CFTR] mutation. Traditionally treatment has only included supportive care. Therefore, there is a need for safe and effective novel therapies targeting the underlying molecular defects seen with CF. Areas covered: In 2016, the Food and Drug Administration and the European Commission approved LUM/IVA (Orkambi), a CFTR modulator that includes both a CFTR corrector and potentiator, for CF patients homozygous for the F508del CFTR mutation. This article reviews the pharmacologic features, clinical efficacy, and safety of LUM/IVA and summarize the available pre-clinical and clinical data of LUM/IVA use. Expert commentary: LUM/IVA showed modest, but significant improvements from baseline in percent predicted FEV 1 (ppFEV 1 ) as well as a reduction in pulmonary exacerbations by 35% It was shown to be safe for short- and long-term use. Currently, LUM/IVA is the only oral agent in its class available and represents a milestone the development of therapies for the management of CF. Nonetheless, pharmacoeconomic data are necessary to justify its high cost before is use becomes standard of care.

  14. A Frameshift Mutation in the Cubilin Gene (CUBN) in Border Collies with Imerslund-Gräsbeck Syndrome (Selective Cobalamin Malabsorption)

    Science.gov (United States)

    Owczarek-Lipska, Marta; Jagannathan, Vidhya; Drögemüller, Cord; Lutz, Sabina; Glanemann, Barbara

    2013-01-01

    Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene (CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies. PMID:23613799

  15. Phenotypic variability in a seven-generation Swedish family segregating autosomal dominant hearing impairment due to a novel EYA4 frameshift mutation

    DEFF Research Database (Denmark)

    Frykholm, Carina; Klar, Joakim; Arnesson, Hanna

    2015-01-01

    Linkage to an interval overlapping the DFNA10 locus on chromosome 6q22-23 was found through genome wide linkage analysis in a seven-generation Swedish family segregating postlingual, autosomal dominant nonsyndromic sensorineural hearing impairment. A novel heterozygous frame-shift mutation (c.579......-sectional and longitudinal deterioration of pure tone average (PTA) once the process of hearing deterioration had started, and no gender, parent-of-origin or family branch differences on PTA could be found. Age at onset varied between the family branches. In summary, this is the ninth published genetically verified DFNA10...

  16. Rare F311L CFTR gene mutation in a child presented with recurrent electrolyte abnormalities and metabolic alkalosis: case report.

    Science.gov (United States)

    Lumpaopong, Adisorn; Thirakhupt, Prapaipim; Srisuwan, Konggrapun; Chulamokha, Yupapin

    2009-05-01

    Delta F508 mutation is recognized as the most common genotype of cystic fibrosis (CF) however, there are small numbers of CF patients having Delta F508/F311L. In the present study, the authors report a 2-year-old Thai boy, originating from North India, presenting with recurrent episodes of febrile illness, hyponatremia, hypokalemia, and metabolic alkalosis since 4 months of age. He was transferred to our hospital for further investigation. Blood chemistry revealed the following serum electrolytes, sodium 122, potassium 3.69, chloride 79.7, and bicarbonate 33.8 mEq/L, and the following urine electrolytes, sodium metabolic alkalosis improved DNA sequencing analysis of his blood demonstrates compound mutation for Delta F508 and F311L in CFTR gene. In conclusion, the authors report a rare case of CF with Delta F508/F311L genotype presented with recurrent hyponatremia and metabolic alkalosis. Awareness of electrolyte abnormalities during febrile illness, proper genetic counseling, and long-term follow up are necessary in this patient.

  17. A novel frameshift mutation of SMPX causes a rare form of X-linked nonsyndromic hearing loss in a Chinese family.

    Directory of Open Access Journals (Sweden)

    Zhijie Niu

    Full Text Available X-linked hearing impairment is the rarest form of genetic hearing loss (HL and represents only a minor fraction of all cases. The aim of this study was to investigate the cause of X-linked inherited sensorineural HL in a four-generation Chinese family. A novel duplication variant (c.217dupA, p.Ile73Asnfs*5 in SMPX was identified by whole-exome sequencing. The frameshift mutation predicted to result in the premature truncation of the SMPX protein was co-segregated with the HL phenotype and was absent in 295 normal controls. Subpopulation screening of the coding exons and flanking introns of SMPX was further performed for 338 Chinese patients with nonsydromic HL by Sanger sequencing, and another two potential causative substitutions (c.238C>A and c.55A>G in SMPX were identified in additional sporadic cases of congenital deafness. Collectively, this study is the first to report the role of SMPX in Chinese population and identify a novel frameshift mutation in SMPX that causes not only nonsyndromic late-onset progressive HL, but also congenital hearing impairment. Our findings extend the mutation and phenotypic spectrum of the SMPX gene.

  18. A Novel Frameshift Mutation (c.5387_5388insTT) in NIPBL in Cornelia de Lange Syndrome with Severe Phenotype.

    Science.gov (United States)

    Kang, Min Jae; Ahn, Soo Min; Hwang, Il Tae

    2018-01-01

    Cornelia de Lange syndrome (CdLS) is a developmental disorder which is characterized by typical facial features, upper extremity malformations, and growth and cognitive delays. The genes involved in CdLS encode the cohesin complex and its associated proteins; and NIPBL mutations, which account for half of the cases, result in severe CdLS phenotypes. We describe a girl with CdLS, presenting with typical facial dysmorphism, cleft palate, hypertrichosis, upper limb hypertonicity, flexion contracture of elbows, micromelia, bilateral hearing loss, gastroesophageal reflux, and severe pyloric stenosis. We detected a heterozygous frameshift mutation in NIPBL (c.5387_5388ins(TT), p.Leu1796Phefs*8) which is a novel mutation. © 2018 by the Association of Clinical Scientists, Inc.

  19. Postmortem diagnosis of Marfan syndrome in a case of sudden death due to aortic rupture: Detection of a novel FBN1 frameshift mutation.

    Science.gov (United States)

    Wang, Yunyun; Chen, Shu; Wang, Rongshuai; Huang, Sizhe; Yang, Mingzhen; Liu, Liang; Liu, Qian

    2016-04-01

    To investigate the sudden death of a 36-year-old Chinese man, a medicolegal autopsy was performed, combining forensic pathological examinations and genetic sequencing analysis to diagnose the cause of death. Genomic DNA samples were extracted from blood and subjected to high-throughput sequencing. Major findings included a dilated aortic root with a ruptured and dissected aorta and consequent tamponade of the pericardial sac. Moreover, arachnodactyly and other skeletal deformities were noted. By sequencing the fibrillin-1 gene (FBN1), five genetic variations were found, including four previously known single nucleotide polymorphisms (SNPs) and a novel frameshift mutation, leading to the diagnosis of Marfan syndrome. The frameshift mutation (c.4921delG, p.glu1641llysFsX9) detected in exon 40 led to a stop codon after the next 8 amino acids. The four SNPs included a splice site mutation (c.3464-5 G>A, rs11853943), a synonymous mutation (p.Asn625Asn, rs25458), and two missense mutations (p.Pro1148Ala, rs140598; p.Cys472Tyr, rs4775765). Genetic screening was recommended for the relatives as it was reported that the father and brother of the deceased had died at the ages of 40 and 25, respectively, from sudden cardiac failure. The son of the deceased lacked the relevant mutations. This report emphasizes the important contribution of medicolegal postmortem analysis on the molecular pathogenesis study of Marfan syndrome and early diagnosis of at-risk relatives. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Identification of a novel frameshift mutation in the DMD gene as the cause of muscular dystrophy in a Norfolk terrier dog.

    Science.gov (United States)

    Jenkins, Christopher A; Forman, Oliver P

    2015-01-01

    A Norfolk terrier was referred to the Animal Health Trust neurology department with suspected dystrophin-deficient muscular dystrophy (DD-MD), which was confirmed by clinical workup and immunohistochemistry. Exon resequencing of the canine Duchenne Muscular Dystrophy (DMD) gene was undertaken to screen for potential disease causing mutations. The sequence data generated from all coding DMD exons revealed a 1 bp deletion in exon 22, causing a frameshift and premature termination of the coding sequence. Gene expression analysis indicated reduced levels of dystrophin transcript in the DD-MD case and western blot confirmed the absence of full length protein. The finding represents a novel mutation causing DD-MD in the dog.

  1. A disease-associated frameshift mutation in caveolin-1 disrupts caveolae formation and function through introduction of a de novo ER retention signal.

    Science.gov (United States)

    Copeland, Courtney A; Han, Bing; Tiwari, Ajit; Austin, Eric D; Loyd, James E; West, James D; Kenworthy, Anne K

    2017-11-01

    Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the CAV1 gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in CAV1 , P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in Cav1 -/- MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in Cav1 -/- MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly. © 2017 Copeland, Han, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  2. Epistatic participation of REV1 and REV3 in the formation of UV-induced frameshift mutations in cell cycle-arrested yeast cells

    International Nuclear Information System (INIS)

    Heidenreich, Erich; Eisler, Herfried; Steinboeck, Ferdinand

    2006-01-01

    Mutations arising in times of cell cycle arrest may provide a selective advantage for unicellular organisms adapting to environmental changes. For multicellular organisms, however, they may pose a serious threat, in that such mutations in somatic cells contribute to carcinogenesis and ageing. The budding yeast Saccharomyces cerevisiae presents a convenient model system for studying the incidence and the mechanisms of stationary-phase mutation in a eukaryotic organism. Having studied the emergence of frameshift mutants after several days of starvation-induced cell cycle arrest, we previously reported that all (potentially error-prone) translesion synthesis (TLS) enzymes identified in S. cerevisiae did not contribute to the basal level of spontaneous stationary-phase mutations. However, we observed that an increased frequency of stationary-phase frameshift mutations, brought about by a defective nucleotide excision repair (NER) pathway or by UV irradiation, was dependent on Rev3p, the catalytic subunit of the TLS polymerase zeta (Pol ζ). Employing the same two conditions, we now examined the effect of deletions of the genes coding for polymerase eta (Pol η) (RAD30) and Rev1p (REV1). In a NER-deficient strain background, the increased incidence of stationary-phase mutations was only moderately influenced by a lack of Pol η but completely reduced to wild type level by a knockout of the REV1 gene. UV-induced stationary-phase mutations were abundant in wild type and rad30Δ strains, but substantially reduced in a rev1Δ as well as a rev3Δ strain. The similarity of the rev1Δ and the rev3Δ phenotype and an epistatic relationship evident from experiments with a double-deficient strain suggests a participation of Rev1p and Rev3p in the same mutagenic pathway. Based on these results, we propose that the response of cell cycle-arrested cells to an excess of exo- or endogenously induced DNA damage includes a novel replication-independent cooperative function of Rev1p and

  3. Modeling cystic fibrosis disease progression in patients with the rare CFTR mutation P67L.

    Science.gov (United States)

    MacKenzie, Isobel E R; Paquette, Valerie; Gosse, Frances; George, Sheenagh; Chappe, Frederic; Chappe, Valerie

    2017-05-01

    The progression of cystic fibrosis (CF) in patients with the rare mutation P67L was examined to determine if it induced a milder form of CF compared to the common severe ΔF508 mutation. Parameters of lung function, level of bacterial infection, nutritional status and hospitalization were used to represent CF progression. Age at diagnosis and pancreatic status were used to assess CF presentation. Analysis of data from the CF Canada Registry collected over a 15-year period included 266 ΔF508/ΔF508 homozygote patients from CF clinics in Atlantic Canada and 26 compound heterozygote patients with the rare P67L mutation from clinics across Canada. Late age at diagnosis, high incidence of pancreatic sufficiency, maintained Body Mass Index (BMI) with age, delayed life-threatening bacterial infection, and fewer days in hospital were observed for P67L heterozygote patients included in this study. Although the decline of lung function did not differ from ΔF508 homozygotes, the fact that a greater proportion of P67L heterozygotes live to an older age suggests that lung function is not the primary factor determining CF progression for P67L heterozygote patients. The P67L mutation is associated with a mild disease, even when combined with the severe ΔF508 mutation. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  4. Comprehensive CFTR gene analysis of the French cystic fibrosis screened newborn cohort: implications for diagnosis, genetic counseling, and mutation-specific therapy.

    Science.gov (United States)

    Audrézet, Marie Pierre; Munck, Anne; Scotet, Virginie; Claustres, Mireille; Roussey, Michel; Delmas, Dominique; Férec, Claude; Desgeorges, Marie

    2015-02-01

    Newborn screening (NBS) for cystic fibrosis (CF) was implemented throughout France in 2002. It involves a four-tiered procedure: immunoreactive trypsin (IRT)/DNA/IRT/sweat test [corrected] was implemented throughout France in 2002. The aim of this study was to assess the performance of molecular CFTR gene analysis from the French NBS cohort, to evaluate CF incidence, mutation detection rate, and allelic heterogeneity. During the 8-year period, 5,947,148 newborns were screened for cystic fibrosis. The data were collected by the Association Française pour le Dépistage et la Prévention des Handicaps de l'Enfant. The mutations identified were classified into four groups based on their potential for causing disease, and a diagnostic algorithm was proposed. Combining the genetic and sweat test results, 1,160 neonates were diagnosed as having cystic fibrosis. The corresponding incidence, including both the meconium ileus (MI) and false-negative cases, was calculated at 1 in 4,726 live births. The CF30 kit, completed with a comprehensive CFTR gene analysis, provides an excellent detection rate of 99.77% for the mutated alleles, enabling the identification of a complete genotype in 99.55% of affected neonates. With more than 200 different mutations characterized, we confirmed the French allelic heterogeneity. The very good sensitivity, specificity, and positive predictive value obtained suggest that the four-tiered IRT/DNA/IRT/sweat test procedure may provide an effective strategy for newborn screening for cystic fibrosis.

  5. Molecular characterization of WFS1 in an Iranian family with Wolfram syndrome reveals a novel frameshift mutation associated with early symptoms.

    Science.gov (United States)

    Sobhani, Maryam; Tabatabaiefar, Mohammad Amin; Rajab, Asadollah; Kajbafzadeh, Abdol-Mohammad; Noori-Daloii, Mohammad Reza

    2013-10-10

    Wolfram syndrome (WS) is a rare autosomal recessive neurodegenerative disorder that represents a likely source of childhood diabetes especially among countries in the consanguinity belt. The main responsible gene is WFS1 for which over one hundred mutations have been reported from different ethnic groups. The aim of this study was to identify the molecular etiology of WS and to perform a possible genotype-phenotype correlation in Iranian kindred. An Iranian family with two patients was clinically studied and WS was suspected. Genetic linkage analysis via 5 STR markers was carried out. For identification of mutations, DNA sequencing of WFS1 including all the exons, exon-intron boundaries and the promoter was performed. Linkage analysis indicated linkage to the WFS1 region. After DNA sequencing of WFS1, one novel pathogenic mutation, which causes frameshift alteration c.2177_2178insTCTTC (or c.2173_2177dupTCTTC) in exon eight, was found. The genotype-phenotype correlation analysis suggests that the presence of the homozygous mutation may be associated with early onset of disease symptoms. This study stresses the necessity of considering the molecular analysis of WFS1 in childhood diabetes with some symptoms of WS. © 2013 Elsevier B.V. All rights reserved.

  6. A novel COL4A1 frameshift mutation in familial kidney disease: the importance of the C-terminal NC1 domain of type IV collagen.

    Science.gov (United States)

    Gale, Daniel P; Oygar, D Deren; Lin, Fujun; Oygar, P Derin; Khan, Nadia; Connor, Thomas M F; Lapsley, Marta; Maxwell, Patrick H; Neild, Guy H

    2016-11-01

    Hereditary microscopic haematuria often segregates with mutations of COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts. We used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses. We identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested. Missense mutations of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome (hereditary angiopathy, nephropathy, aneurysms and cramps). Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the α1 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans. © The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA.

  7. A frame-shift mutation of PMS2 is a widespread cause of Lynch syndrome

    DEFF Research Database (Denmark)

    Clendenning, Mark; Senter, Leigha; Hampel, Heather

    2008-01-01

    BACKGROUND: When compared to the other mismatch repair genes involved in Lynch syndrome, the identification of mutations within PMS2 has been limited (... are caused by PMS2. This disparity is primarily due to complications in the study of this gene caused by interference from pseudogene sequences. METHODS: Using a recently developed method for detecting PMS2 specific mutations, we have screened 99 patients who are likely candidates for PMS2 mutations based...

  8. Tigecycline resistance in Acinetobacter baumannii mediated by frameshift mutation in plsC, encoding 1-acyl-sn-glycerol-3-phosphate acyltransferase.

    Science.gov (United States)

    Li, X; Liu, L; Ji, J; Chen, Q; Hua, X; Jiang, Y; Feng, Y; Yu, Y

    2015-03-01

    Acinetobacter baumannii is an important pathogen of healthcare-associated infections and shows multidrug resistance. With the increasing application of tigecycline, isolates resistant to this antibiotic are of growing concern clinically. However, the definitive mechanism of tigecycline resistance remains unclear. To explore the mechanism of tigecycline resistance in A. baumannii, a tigecycline-resistant strain was obtained by increasing the concentration of the antimicrobial in liquid culture. Three mutations were identified by the whole genome comparison, including one synonymous substitution in a hypothetical protein and a frameshift mutation in plsC and omp38. The plsC gene was confirmed to cause decreased susceptibility to tigecycline by a complementation experiment and cellular membrane change was detected by flow cytometry. By measuring the relative growth rate, the fitness cost of plsC was estimated to be approximately 8 %. In conclusion, plsC was found to play an important role in tigecycline resistance in A. baumannii. The minor fitness cost of plsC indicates a high risk of the emergence and development of tigecycline resistance in A. baumannii.

  9. Novel frameshift mutation in theKCNQ1gene responsible for Jervell and Lange-Nielsen syndrome.

    Science.gov (United States)

    Amirian, Azam; Dalili, Seyed Mohammad; Zafari, Zahra; Saber, Siamak; Karimipoor, Morteza; Akbari, Vahid; Fazelifar, Amir Farjam; Zeinali, Sirous

    2018-01-01

    Jervell and Lange-Nielsen syndrome is an autosomal recessive disorder caused by mutations in KCNQ1 or KCNE1 genes. The disease is characterized by sensorineural hearing loss and long QT syndrome. Here we present a 3.5-year-old female patient, an offspring of consanguineous marriage, who had a history of recurrent syncope and congenital sensorineural deafness. The patient and the family members were screened for mutations in KCNQ1 gene by linkage analysis and DNA sequencing. DNA sequencing showed a c.1532_1534delG (p. A512Pfs*81) mutation in the KCNQ1 gene in homozygous form. The results of short tandem repeat (STR) markers showed that the disease in the family is linked to the KCNQ1 gene. The mutation was confirmed in the parents in heterozygous form. This is the first report of this variant in KCNQ1 gene in an Iranian family. The data of this study could be used for early diagnosis of the condition in the family and genetic counseling.

  10. 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

    -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...... in our study suggest that truncation of the C-terminal domain of DVL1, a protein hypothesized to have a downstream role in the Wnt-5a non-canonical pathway, is a common cause of DRS....

  11. Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface*

    OpenAIRE

    Chin, Stephanie; Yang, Donghe; Miles, Andrew J.; Eckford, Paul D. W.; Molinski, Steven; Wallace, B. A.; Bear, Christine E.

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein...

  12. Report on the p.Ser489X (p.Ser489*) CFTR mutation, a variant with severe associated phenotype and high prevalence in a Quebec French-Canadian cystic fibrosis patient population.

    Science.gov (United States)

    De Bie, Isabelle; Agatep, Ron; Scott, Patrick; Ruchon, Andrea

    2012-10-01

    This study reports on the phenotype of cystic fibrosis patients identified to be carriers of the p.Ser489X (p.Ser489*; c.1466C>A) cystic fibrosis transmembrane conductance regulator (CFTR) mutation, a variant rarely described in the cystic fibrosis literature, as well as on its allelic frequency in a French-Canadian cystic fibrosis patient cohort. Reported phenotypes and allelic frequency of this variant were collected based on the data from a large French-Canadian cystic fibrosis patient cohort. Cystic fibrosis patients found to carry the p.Ser489X variant generally presented with classic gastrointestinal manifestations of this condition in infancy. The allelic frequency of this variant was calculated to be 0.7% for this population. The p.Ser489X CFTR variant is a severe disease-causing CFTR allele that is relatively frequent in the French-Canadian cystic fibrosis patient population, warranting its inclusion into CFTR molecular testing panel for this population.

  13. CFTR2: How will it help care?

    Science.gov (United States)

    Castellani, Carlo

    2013-05-01

    The Clinical and Functional Translation of CFTR (CFTR2) project presents a novel approach to clinical and functional annotation of mutations identified in disease-causing genes. Phenotype and genotype information on approximately 40,000 cystic fibrosis (CF) patients were collected from registries and large clinics. The disease-liability of the 160 most frequently reported mutations was evaluated by means of a multistage process which involved clinical (sweat chloride average), functional (expression in cell-based systems) and epidemiological (mutation analysis in obligate heterozygotes) steps. The results of this analysis can be consulted in a dedicated website. Data originated by CFTR2 may be valuable in several facets of CF care, including diagnosis, newborn screening, carrier testing, genotype/phenotype correlation and mutation-specific therapeutics. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. [New therapies for cystic fibrosis targeting the CFTR gene or the CFTR protein].

    Science.gov (United States)

    Hubert, D; Bui, S; Marguet, C; Colomb-Jung, V; Murris-Espin, M; Corvol, H; Munck, A

    2016-10-01

    The treatment of cystic fibrosis has been symptom-based for a number of years. New therapies that aim to improve CFTR protein function are now emerging. The results of gene therapy has been modest but a recent clinical trial shows a positive effect on FEV1. Recent research has focused primarily on CFTR protein function. Significant respiratory improvement (an average 10% FEV1 increase and a decrease in the frequency of exacerbations) has been achieved with ivacaftor, a CFTR potentiator, in patients with gating mutations, resulting in its marketing authorization (in 2012 for the G551D mutation and in 2015 for rarer mutations). In phe508del homozygous patients, the combination of ivacaftor with a CFTR corrector (lumacaftor) has also led to respiratory improvement, albeit less impressive. The effectiveness of ataluren in patients with nonsense mutations is being evaluated. New CFTR correctors and potentiators are being developed. CFTR protein therapy could change the course of the disease but cost/effectiveness issues should not be overlooked. Ivacaftor can be prescribed in CF patients with a class 3 mutation from the age of 6 years. The Orkambi ® will soon be available for homozygous phe508del patients from the age of 12 years. Copyright © 2015 SPLF. Published by Elsevier Masson SAS. All rights reserved.

  15. CFTR activity and mitochondrial function

    Directory of Open Access Journals (Sweden)

    Angel Gabriel Valdivieso

    2013-01-01

    Full Text Available Cystic Fibrosis (CF is a frequent and lethal autosomal recessive disease, caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR. Before the discovery of the CFTR gene, several hypotheses attempted to explain the etiology of this disease, including the possible role of a chloride channel, diverse alterations in mitochondrial functions, the overexpression of the lysosomal enzyme α-glucosidase and a deficiency in the cytosolic enzyme glucose 6-phosphate dehydrogenase. Because of the diverse mitochondrial changes found, some authors proposed that the affected gene should codify for a mitochondrial protein. Later, the CFTR cloning and the demonstration of its chloride channel activity turned the mitochondrial, lysosomal and cytosolic hypotheses obsolete. However, in recent years, using new approaches, several investigators reported similar or new alterations of mitochondrial functions in Cystic Fibrosis, thus rediscovering a possible role of mitochondria in this disease. Here, we review these CFTR-driven mitochondrial defects, including differential gene expression, alterations in oxidative phosphorylation, calcium homeostasis, oxidative stress, apoptosis and innate immune response, which might explain some characteristics of the complex CF phenotype and reveals potential new targets for therapy.

  16. A cis-eQTL of HLA-DRB1 and a frameshift mutation of MICA contribute to the pattern of association of HLA alleles with cervical cancer.

    Science.gov (United States)

    Chen, Dan; Gyllensten, Ulf

    2014-04-01

    The association of classic human leukocyte antigen (HLA) alleles with risk of cervical cancer has been extensively studied, and a protective effect has consistently been found for DRB1*1301, DQA1*0103, and/or DQB1*0603 (these three alleles are in perfect linkage disequilibrium [LD] and often occur on the same haplotype in Europeans), while reports have differed widely with respect to the effect of HLA-B*07, DRB1*1501, and/or DQB1*0602 (the last two alleles are also in perfect LD in Europeans). It is not clear whether the reported HLA alleles are responsible for the differences in cervical cancer susceptibility, or if functional variants at other locations within the major histocompatibility complex (MHC) region may explain the effect. In order to assess the relative contribution of both classic HLA alleles and single-nucleotide polymorphisms (SNPs) within the MHC region to cervical cancer susceptibility, we have imputed classic HLA alleles in 1034 cervical cancer patients and 3948 controls in a Swedish population for an integrated analysis. We found that the protective haplotype DRB1*1301-DQA1*0103-DQB1*0603 has a direct effect on cervical cancer and always occurs together with the C allele of a HLA-DRB1 cis-eQTL (rs9272143), which increases the expression of HLA-DRB1. The haplotype rs9272143C-DRB1*1301-DQA1*0103-DQB1*0603 conferred the strongest protection against cervical cancer (odds ratio [OR] = 0.41, 95% confidence interval [CI] = 0.32-0.52, P = 6.2 × 10(-13)). On the other hand, the associations with HLA-B*0702 and DRB1*1501-DQB1*0602 are attributable to the joint effects of both the HLA-DRB1 cis-eQTL (rs9272143) and a frameshift mutation (G inserion of rs67841474, also known as A5.1) of the MHC class I polypeptide-related sequence A gene (MICA). Variation in LD between the classic HLA loci, rs9272143 and rs67841474 between populations may explain the different associations of HLA-B*07 and DRB1*1501-DQB1*0602 with cervical cancer between studies. The

  17. Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa [version 2; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Shamsudheen Karuthedath Vellarikkal

    2016-07-01

    Full Text Available Dystrophic epidermolysis bullosa simplex (DEB is a phenotypically diverse inherited skin fragility disorder. It is majorly manifested by appearance of epidermal bullae upon friction caused either by physical or environmental trauma. The phenotypic manifestations also include appearance of milia, scarring all over the body and nail dystrophy. DEB can be inherited in a recessive or dominant form and the recessive form of DEB (RDEB is more severe. In the present study, we identify a novel p.G2254fs mutation in COL7A1 gene causing a sporadic case of RDEB by whole exome sequencing (WES. Apart from adding a novel frameshift Collagen VII mutation to the repertoire of known mutations reported in the disease, to the best of our knowledge, this is the first report of a genetically characterized case of DEB from India.

  18. Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa [version 1; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Shamsudheen Karuthedath Vellarikkal

    2016-05-01

    Full Text Available Dystrophic epidermolysis bullosa simplex (DEB is a phenotypically diverse inherited skin fragility disorder. It is majorly manifested by appearance of epidermal bullae upon friction caused either by physical or environmental trauma. The phenotypic manifestations also include appearance of milia, scarring all over the body and nail dystrophy. DEB can be inherited in a recessive or dominant form and the recessive form of DEB (RDEB is more severe. In the present study, we identify a novel p.G2254fs mutation in COL7A1 gene causing a sporadic case of RDEB by whole exome sequencing (WES. Apart from adding a novel frameshift Collagen VII mutation to the repertoire of known mutations reported in the disease, to the best of our knowledge, this is the first report of a genetically characterized case of DEB from India.

  19. Recovery of lung function following a pulmonary exacerbation in patients with cystic fibrosis and the G551D-CFTR mutation treated with ivacaftor.

    Science.gov (United States)

    Flume, Patrick A; Wainwright, Claire E; Elizabeth Tullis, D; Rodriguez, Sally; Niknian, Minoo; Higgins, Mark; Davies, Jane C; Wagener, Jeffrey S

    2018-01-01

    Pulmonary exacerbations (PEx) are associated with acute loss of lung function that is often not recovered after treatment. We investigated lung function recovery following PEx for ivacaftor- and placebo-treated subjects. Short- and long-term pulmonary function recovery data after PEx were summarized from a placebo-controlled trial in 161 cystic fibrosis patients≥12years old with the G551D-CFTR mutation (NCT00909532). Short-term recovery was measured 2 to 8weeks after treatment, and long-term recovery was determined at the end-of-study, both compared with baseline measured just prior to the PEx. Fewer patients receiving ivacaftor experienced a PEx than patients receiving placebo (33.7% vs. 56.4%; P=0.004) and had a lower adjusted incidence rate of PEx (0.589 vs. 1.382; P<0.001). The proportion of PEx followed by full short-term recovery of percent predicted forced expiratory volume in 1s was similar (ivacaftor vs. placebo, 57.1% vs. 53.7), as was the proportion of patients having long-term recovery (46.4% vs. 47.7%). Ivacaftor treatment reduces the frequency of PEx but does not improve on the rate of complete lung function recovery after PEx when compared with placebo. Copyright © 2017 European Cystic Fibrosis Society. All rights reserved.

  20. Functional architecture of the CFTR chloride channel.

    Science.gov (United States)

    Linsdell, Paul

    2014-02-01

    Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) family of membrane transport proteins. CFTR is unique among ABC proteins in that it functions not as an active transporter but as an ATP-gated Cl(-) channel. As an ion channel, the function of the CFTR transmembrane channel pore that mediates Cl(-) movement has been studied in great detail. On the other hand, only low resolution structural data is available on the transmembrane parts of the protein. The structure of the channel pore has, however, been modeled on the known structure of active transporter ABC proteins. Currently, significant barriers exist to building a unified view of CFTR pore structure and function. Reconciling functional data on the channel with indirect structural data based on other proteins with very different transport functions and substrates has proven problematic. This review summarizes current structural and functional models of the CFTR Cl(-) channel pore, including a comprehensive review of previous electrophysiological investigations of channel structure and function. In addition, functional data on the three-dimensional arrangement of pore-lining helices, as well as contemporary hypotheses concerning conformational changes in the pore that occur during channel opening and closing, are discussed. Important similarities and differences between different models of the pore highlight current gaps in our knowledge of CFTR structure and function. In order to fill these gaps, structural and functional models of the membrane-spanning pore need to become better integrated.

  1. Applying Cystic Fibrosis Transmembrane Conductance Regulator Genetics and CFTR2 Data to Facilitate Diagnoses.

    Science.gov (United States)

    Sosnay, Patrick R; Salinas, Danieli B; White, Terry B; Ren, Clement L; Farrell, Philip M; Raraigh, Karen S; Girodon, Emmanuelle; Castellani, Carlo

    2017-02-01

    As a Mendelian disease, genetics plays an integral role in the diagnosis of cystic fibrosis (CF). The identification of 2 disease-causing mutations in the CF transmembrane conductance regulator (CFTR) in an individual with a phenotype provides evidence that the disease is CF. However, not all variations in CFTR always result in CF. Therefore, for CFTR genotype to provide the same level of evidence of CFTR dysfunction as shown by direct tests such as sweat chloride or nasal potential difference, the mutations identified must be known to always result in CF. The use of CFTR genetics in CF diagnosis, therefore, relies heavily on mutation interpretation. Progress that has been made on mutation interpretation and annotation was reviewed at the recent CF Foundation Diagnosis Consensus Conference. A modified Delphi method was used to identify consensus statements on the use of genetic analysis in CF diagnosis. The largest recent advance in CF genetics has come through the Clinical and Functional Translation of CFTR (CFTR2) project. This undertaking seeks to characterize CFTR mutations from patients with CF around the world. The project also established guidelines for the clinical, functional, and population/penetrance criteria that can be used to interpret mutations not yet included in CFTR2's review. The use of CFTR genetics to aid in diagnosis of CF requires that the mutations identified have a known disease liability. The demonstration of 2 in trans mutations known to always result in CF is satisfactory evidence of CFTR dysfunction. However, if the identified mutations are known to be associated with variable outcomes, or have unknown consequence, that genotype may not result in a CF phenotype. In these cases, other tests of CFTR function may help. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Mutations in CFTR gene and clinical correlation in Argentine patients with congenital bilateral absence of the vas deferens Correlación de las características clínicas con mutaciones del gen CFTR en pacientes argentinos con ausencia bilateral congénita de vasos deferentes

    Directory of Open Access Journals (Sweden)

    Estrella M Levy

    2004-06-01

    Full Text Available Congenital bilateral absence of the vas deferens (CBAVD is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR gene have been identified. Here we identify different mutations of CFTR and the poly-T variant of intron 8 (IVS8 in Argentine patients and analyze sweat test values and clinical characteristic related to Cystic Fibrosis (CF. For counseling purposes the two most frequent mutations in Argentine CF population: DF508 and G542X were screened in wives. In all cases, it was possible to reduce the risk of CF/CBAVD descendants in these couples because none of the mutation were found in the 36 samples. Eight patients (23% showed abnormal chloride values (> 60 mmol/l. A second group of 6 patients (18% had borderline values of sweat chloride (40-59 mmol/l. We defined another group with 6 patients (18%, with normal sweat chloride levels (30-39 mmo/l and a fourth group of 14 (41% patients with sweat chloride below 30 mmol/l. DF508, the most frequent CF mutation in the Argentine population, was found on 15 of the 72 chromosomes (21%, R117H mutation was detected on 2 of 62 chromosomes (3%. Only one R347P allele was found on 28 chromosomes analyzed (2%. On a sample of 27 patients, IVS8 analysis showed a frequency of 6/56 chromosomes (11% of 5T allele. Even though these findings present an improvement in the detection of mutations related to clinical correlations in Argentine CBAVD population, the search for other common and uncommon mutations should be continued.La ausencia bilateral congénita de vasos deferentes (CBAVD es una forma de infertilidad masculina en la que se han identificado mutaciones en el gen de la conductancia transmembrana de la fibrosis quística (CFTR. Hemos estudiado en pacientes argentinos diferentes mutaciones en el CFTR y la variante poli T del intron 8 (IVS8 y analizado los valores de test del sudor y las características clínicas relacionadas a la Fibrosis Qu

  3. Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

    OpenAIRE

    LaRusch, Jessica; Jung, Jinsei; General, Ignacio J.; Lewis, Michele D.; Park, Hyun Woo; Brand, Randall E.; Gelrud, Andres; Anderson, Michelle A.; Banks, Peter A.; Conwell, Darwin; Lawrence, Christopher; Romagnuolo, Joseph; Baillie, John; Alkaade, Samer; Cote, Gregory

    2014-01-01

    CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev ) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize tha...

  4. Rattlesnake Phospholipase A2 Increases CFTR-Chloride Channel Current and Corrects ∆F508CFTR Dysfunction: Impact in Cystic Fibrosis.

    Science.gov (United States)

    Faure, Grazyna; Bakouh, Naziha; Lourdel, Stéphane; Odolczyk, Norbert; Premchandar, Aiswarya; Servel, Nathalie; Hatton, Aurélie; Ostrowski, Maciej K; Xu, Haijin; Saul, Frederick A; Moquereau, Christelle; Bitam, Sara; Pranke, Iwona; Planelles, Gabrielle; Teulon, Jacques; Herrmann, Harald; Roldan, Ariel; Zielenkiewicz, Piotr; Dadlez, Michal; Lukacs, Gergely L; Sermet-Gaudelus, Isabelle; Ollero, Mario; Corringer, Pierre-Jean; Edelman, Aleksander

    2016-07-17

    Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmembrane regulator (CFTR; a cyclic AMP-regulated chloride channel) is the most frequent mutation associated with cystic fibrosis. This mutation affects the maturation and gating of CFTR protein. The search for new high-affinity ligands of CFTR acting as dual modulators (correctors/activators) presents a major challenge in the pharmacology of cystic fibrosis. Snake venoms are a rich source of natural multifunctional proteins, potential binders of ion channels. In this study, we identified the CB subunit of crotoxin from Crotalus durissus terrificus as a new ligand and allosteric modulator of CFTR. We showed that CB interacts with NBD1 of both wild type and ∆F508CFTR and increases their chloride channel currents. The potentiating effect of CB on CFTR activity was demonstrated using electrophysiological techniques in Xenopus laevis oocytes, in CFTR-HeLa cells, and ex vivo in mouse colon tissue. The correcting effect of CB was shown by functional rescue of CFTR activity after 24-h ΔF508CFTR treatments with CB. Moreover, the presence of fully glycosylated CFTR was observed. Molecular docking allowed us to propose a model of the complex involving of the ABCβ and F1-like ATP-binding subdomains of ΔF508-NBD1. Hydrogen-deuterium exchange analysis confirmed stabilization in these regions, also showing allosteric stabilization in two other distal regions. Surface plasmon resonance competition studies showed that CB disrupts the ∆F508CFTR-cytokeratin 8 complex, allowing for the escape of ∆F508CFTR from degradation. Therefore CB, as a dual modulator of ΔF508CFTR, constitutes a template for the development of new anti-CF agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. A new compound heterozygous frameshift mutation in the type II 3{beta}-hydroxysteroid dehydrogenase 3{beta}-HSD gene causes salt-wasting 3{beta}-HSD deficiency congenital adrenal hyperplasia

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, L.; Sakkal-Alkaddour, S.; Chang, Ying T.; Yang, Xiaojiang; Songya Pang [Univ. of Illinois, Chicago, IL (United States)

    1996-01-01

    We report a new compound heterozygous frameshift mutation in the type II 3{Beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) gene in a Pakistanian female child with the salt-wasting form of 3{Beta}-HSD deficiency congenital adrenal hyperplasia. The etiology for her congenital adrenal hyperplasia was not defined. Although the family history suggested possible 3{beta}-HSd deficiency disorder, suppressed adrenal function caused by excess glucocorticoid therapy in this child at 7 yr of age did not allow hormonal diagnosis. To confirm 3{beta}-HSD deficiency, we sequenced the type II 3{beta}-HSD gene in the patient, her family, and the parents of her deceased paternal cousins. The type II 3{beta}-HSD gene region of a putative promotor, exons I, II, III, and IV, and exon-intron boundaries were amplified by PCR and sequenced in all subjects. The DNA sequence of the child revealed a single nucleotide deletion at codon 318 [ACA(Thr){r_arrow}AA] in exon IV in one allele, and two nucleotide deletions at codon 273 [AAA(Lys){r_arrow}A] in exon IV in the other allele. The remaining gene sequences were normal. The codon 318 mutation was found in one allele from the father, brother, and parents of the deceased paternal cousins. The codon 273 mutation was found in one allele of the mother and a sister. These findings confirmed inherited 3{beta}-HSD deficiency in the child caused by the compound heterozygous type II 3{beta}-HSD gene mutation. Both codons at codons 279 and 367, respectively, are predicted to result in an altered and truncated type II 3{beta}-HSD protein, thereby causing salt-wasting 3{beta}-HSD deficiency in the patient. 21 refs., 2 figs., 1 tab.

  6. Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

    Directory of Open Access Journals (Sweden)

    Michael S Stalvey

    Full Text Available Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF. CF-related bone disease (CFBD is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR, the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/- mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+ littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl mRNA was detected, significantly less osteoprotegerin (Opg was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt

  7. HRCT in cystic fibrosis in patients with CFTR I1234V mutation: Assessment of scoring systems with low dose technique using multidetector system and correlation with pulmonary function tests

    International Nuclear Information System (INIS)

    Bhat, Venkatraman; Wahab, Atiqa Abdul; Garg, Kailash C; Janahi, Ibrahim; Singh, Rajvir

    2015-01-01

    Pulmonary changes in patients with cystic fibrosis (CF) with CFTR I1234V mutation have not been extensively documented. Impact of geographic influence on phenotypical expression is largely unknown. This descriptive clinical study presents the high-resolution computed tomography (HRCT) pulmonary findings and computed tomography (CT) scoring with respect to pulmonary function tests (PFT) in a small subset of CF group. We examined 29 patients between 2 and 31 years of age with CFTR I1234V mutation. HRCT and PFT were performed within 2 weeks of each other. Imaging abnormalities on HRCT were documented and analyzed by utilizing the scoring system described by Bhalla et al., Brody et al., Helbich et al.,and Santamaria et al. Efficacy of the scoring system with respect to PFT was compared. Inter-observer reliability of the scoring systems was tested using intraclass correlation (ICC) between the two observers. Spearman correlation coefficients were calculated between the scoring systems and between the scoring systems and PFT results. In our study, right upper and middle lobes were the most frequently involved sites of involvement. Bronchiectasis and peribronchial thickening were the most frequent imaging findings. Scores with all four scoring systems were reproducible, with good ICC coefficient of 0.69. There was good agreement between senior radiologists in all scoring systems. We noted pulmonary imaging abnormalities in a large majority (96%) of our CF patients. There was no significant difference in the CT scores observed from various systems. The CT evaluation system by Broody is detailed and time consuming, and is ideal for research and academic setup. On the other hand, the systems by Bhalla and Santamaria are easy to use, quick, and equally informative. We found the scoring system by Santamaria preferable over that of Bhalla by virtue of additional points of evaluation and ease of use, and therefore better suited for busy clinical practice

  8. Applicability and Efficiency of NGS in Routine Diagnosis: In-Depth Performance Analysis of a Complete Workflow for CFTR Mutation Analysis.

    Directory of Open Access Journals (Sweden)

    Adrien Pagin

    Full Text Available Actually, about 2000 sequence variations have been documented in the CFTR gene requiring extensive and multi-step genetic testing in the diagnosis of cystic fibrosis and CFTR-related disorders. We present a two phases study, with validation and performance monitoring, of a single experiment methodology based on multiplex PCR and high throughput sequencing that allows detection of all variants, including large rearrangements, affecting the coding regions plus three deep intronic loci.A total of 340 samples, including 257 patients and 83 previously characterized control samples, were sequenced in 17 MiSeq runs and analyzed with two bioinformatic pipelines in routine diagnostic conditions. We obtained 100% coverage for all the target regions in every tested sample.We correctly identified all the 87 known variants in the control samples and successfully confirmed the 62 variants identified among the patients without observing false positive results. Large rearrangements were identified in 18/18 control samples. Only 17 patient samples showed false positive signals (6.6%, 12 of which showed a borderline result for a single amplicon. We also demonstrated the ability of the assay to detect allele specific dropout of amplicons when a sequence variation occurs at a primer binding site thus limiting the risk for false negative results.We described here the first NGS workflow for CFTR routine analysis that demonstrated equivalent diagnostic performances compared to Sanger sequencing and multiplex ligation-dependent probe amplification. This study illustrates the advantages of NGS in term of scalability, workload reduction and cost-effectiveness in combination with an improvement of the overall data quality due to the simultaneous detection of SNVs and large rearrangements.

  9. 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...

  10. Increased bone turnover, osteoporosis, progressive tibial bowing, fractures, and scoliosis in a patient with a final-exon SATB2 frameshift mutation.

    Science.gov (United States)

    Boone, Philip M; Chan, Yiu Man; Hunter, Jill V; Pottkotter, Louis E; Davino, Nelson A; Yang, Yaping; Beuten, Joke; Bacino, Carlos A

    2016-11-01

    Haploinsufficiency of SATB2 causes cleft palate, intellectual disability with deficient speech, facial and dental abnormalities, and other variable features known collectively as SATB2-associated syndrome. This phenotype was accompanied by osteoporosis, fractures, and tibial bowing in two previously reported adult patients; each possessed SATB2 mutations either predicted or demonstrated to escape nonsense-mediated decay, suggesting that the additional bone defects result from a dominant negative effect and/or age-dependent penetrance. These hypotheses remain to be confirmed, as do the specific downstream defects causing bone abnormalities. We report a SATB2 mutation (c.2018dupA; p.(H673fs)) in a 15-year-old patient whose SATB2-associated syndrome phenotype is accompanied by osteoporosis, fractures, progressive tibial bowing, and scoliosis. As this homeodomain-disrupting and predicted truncating mutation resides within the final exon of SATB2, escape from nonsense-mediated decay is likely. Thus, we provide further evidence of bone phenotypes beyond those typically associated with SATB2-associated syndrome in individuals with potential dominant-negative SATB2 alleles, as well as evidence for age-dependence of bone features. Elevations in alkaline phosphatase, urinary N-telopeptide/creatinine ratio, and osteocalcin in the patient indicate increased bone turnover. We propose surveillance and treatment with osteoclast inhibitors to prevent fractures and to slow progressive bone deformities. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

    International Nuclear Information System (INIS)

    Ebner, Andreas; Hinterdorfer, Peter; Nikova, Dessy; Lange, Tobias; Bruns, Reimer; Oberleithner, Hans; Schillers, Hermann; Haeberle, Johannes; Falk, Sabine; Duebbers, Angelika

    2008-01-01

    CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl - ) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients

  12. An Exon-Based Comparative Variant Analysis Pipeline to Study the Scale and Role of Frameshift and Nonsense Mutation in the Human-Chimpanzee Divergence

    Directory of Open Access Journals (Sweden)

    GongXin Yu

    2009-01-01

    important biological processes such as T cell lineage development, the pathogenesis of inflammatory diseases, and antigen induced cell death. A “less-is-more” model was previously established to illustrate the role of the gene inactivation and disruptions during human evolution. Here this analysis suggested a different model where the chimpanzee-specific exon-disrupting mutations may act as additional evolutionary force that drove the human-chimpanzee divergence. Finally, the analysis revealed a number of sequencing errors in the chimpanzee and human genome sequences and further illustrated that they could be corrected without resequencing.

  13. Adana İlinde CFTR Gen Mutasyonlarının Değerlendirilmesi

    OpenAIRE

    Öztürk, Özlem Görüroğlu; Kibar, Filiz; Karaçor, Esin Damla Ziyanoğlu; Çetiner, Salih; Şahin, Gülhan; Yaman, Akgün

    2014-01-01

    ABSTRACT Objective: Cystic fibrosis is the most common autosomal recessive inherited disorder seen in the white populations. It develops in result of mutations of cystic fibrosis transmembrane regulator (CFTR) gene. Rate of these mutations vary in different geographical regions. In this study, we aimed to determine the frequency of CFTR gene mutations in Adana. Methods: DNA samples of 63 subjects (21 women, 42 men) who were diagnosed as cystic fibrosis at Balcalı Hospital of Çukurova Unive...

  14. Alternative splicing and tissue-specific elastin misassembly act as biological modifiers of human elastin gene frameshift mutations associated with dominant cutis laxa.

    Science.gov (United States)

    Sugitani, Hideki; Hirano, Eiichi; Knutsen, Russell H; Shifren, Adrian; Wagenseil, Jessica E; Ciliberto, Christopher; Kozel, Beth A; Urban, Zsolt; Davis, Elaine C; Broekelmann, Thomas J; Mecham, Robert P

    2012-06-22

    Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway.

  15. Alternative Splicing and Tissue-specific Elastin Misassembly Act as Biological Modifiers of Human Elastin Gene Frameshift Mutations Associated with Dominant Cutis Laxa*

    Science.gov (United States)

    Sugitani, Hideki; Hirano, Eiichi; Knutsen, Russell H.; Shifren, Adrian; Wagenseil, Jessica E.; Ciliberto, Christopher; Kozel, Beth A.; Urban, Zsolt; Davis, Elaine C.; Broekelmann, Thomas J.; Mecham, Robert P.

    2012-01-01

    Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway. PMID:22573328

  16. Expandiendo el espectro mutacional en pacientes chilenos con fibrosis quística Expanding the CFTR mutation spectrum in Chilean patients with cystic fibrosis

    OpenAIRE

    Marcos Vásquez D; Guillermo Lay-Son R; Felipe Vial U; María Lina Boza C; Ilse Contreras E; Jaime Lozano C; Daniel Zenteno A; María Gabriela Repetto L

    2012-01-01

    Introducción: La fibrosis quística (FQ) es una enfermedad con herencia autosómica recesiva, que presenta una incidencia de 1 en 8.000 a 9.000 recién nacidos en Chile. A la fecha se han descrito más de 1.800 mutaciones diferentes en el gen CFTR. El diagnóstico molecular disponible consiste en el análisis de las 36 mutaciones presentes con mayor frecuencia en población caucásica, donde se describe una tasa de detección de un 85%. Sin embargo, en Chile el rendimiento corresponde a un 42%. Por es...

  17. Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR

    DEFF Research Database (Denmark)

    Wainwright, Claire E; Elborn, J Stuart; Ramsey, Bonnie W

    2015-01-01

    BACKGROUND: Cystic fibrosis is a life-limiting disease that is caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) protein activity. Phe508del is the most common CFTR mutation. METHODS: We conducted two phase 3, randomized, double-blind, placebo......-controlled studies that were designed to assess the effects of lumacaftor (VX-809), a CFTR corrector, in combination with ivacaftor (VX-770), a CFTR potentiator, in patients 12 years of age or older who had cystic fibrosis and were homozygous for the Phe508del CFTR mutation. In both studies, patients were randomly......-ivacaftor and placebo groups. The rate of discontinuation due to an adverse event was 4.2% among patients who received lumacaftor-ivacaftor versus 1.6% among those who received placebo. CONCLUSIONS: These data show that lumacaftor in combination with ivacaftor provided a benefit for patients with cystic fibrosis...

  18. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators.

    Directory of Open Access Journals (Sweden)

    Wei Wang

    Full Text Available W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO 0.9 when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5% due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.

  19. Stimulation of Airway and Intestinal Mucosal Secretion by Natural Coumarin CFTR Activators.

    Science.gov (United States)

    Yang, Hong; Xu, Li-Na; Sui, Yu-Jie; Liu, Xin; He, Cheng-Yan; Fang, Rou-Yu; Liu, Jia; Hao, Feng; Ma, Tong-Hui

    2011-01-01

    Mutations of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) cause lethal hereditary disease CF that involves extensive destruction and dysfunction of serous epithelium. Possible pharmacological therapy includes correction of defective intracellular processing and abnormal channel gating. In a previous study, we identified five natural coumarin potentiators of ΔF508-CFTR including osthole, imperatorin, isopsoralen, praeruptorin A, and scoparone. The present study was designed to determine the activity of these coumarine compounds on CFTR activity in animal tissues as a primary evaluation of their therapeutic potential. In the present study, we analyzed the affinity of these coumarin potentiators in activating wild-type CFTR and found that they are all potent activators. Osthole showed the highest affinity with K(d) values coumarin compounds for the treatment of CF and other CFTR-related diseases.

  20. Side chain and backbone contributions of Phe508 to CFTR folding

    Energy Technology Data Exchange (ETDEWEB)

    Thibodeau, Patrick H.; Brautigam, Chad A.; Machius, Mischa; Thomas, Philip J. (U. of Texas-SMED)

    2010-12-07

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

  1. CFTR Genotype and Maximal Exercise Capacity in Cystic Fibrosis: A Cross-sectional Study.

    Science.gov (United States)

    Radtke, Thomas; Hebestreit, Helge; Gallati, Sabina; Schneiderman, Jane E; Braun, Julia; Stevens, Daniel; Hulzebos, Erik Hj; Takken, Tim; Boas, Steven R; Urquhart, Don S; Lands, Larry C; Tejero, Sergio; Sovtic, Aleksandar; Dwyer, Tiffany; Petrovic, Milos; Harris, Ryan A; Karila, Chantal; Savi, Daniela; Usemann, Jakob; Mei-Zahav, Meir; Hatziagorou, Elpis; Ratjen, Felix; Kriemler, Susi

    2018-02-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human skeletal muscle cells. Variations of CFTR dysfunction among patients with cystic fibrosis may be an important determinant of maximal exercise capacity in cystic fibrosis. Previous studies on the relationship between CFTR genotype and maximal exercise capacity are scarce and contradictory. This study was designed to explore factors influencing maximal exercise capacity, expressed as peak oxygen uptake (V.O2peak), with a specific focus on CFTR genotype in children and adults with cystic fibrosis. In an international, multicenter, cross-sectional study, we collected data on CFTR genotype and cardiopulmonary exercise tests in patients with cystic fibrosis who were ages 8 years and older. CFTR mutations were classified into functional classes I–V. The final analysis included 726 patients (45% females; age range, 8–61 yr; forced expiratory volume in 1 s, 16 to 123% predicted) from 17 cystic fibrosis centers in North America, Europe, Australia, and Asia, all of whom had both valid maximal cardiopulmonary exercise tests and complete CFTR genotype data. Overall, patients exhibited exercise intolerance (V.O2peak, 77.3 ± 19.1% predicted), but values were comparable among different CFTR classes. We did not detect an association between CFTR genotype functional classes I–III and either V.O2peak (percent predicted) (adjusted β = −0.95; 95% CI, −4.18 to 2.29; P = 0.57) or maximum work rate (Wattmax) (adjusted β = −1.38; 95% CI, −5.04 to 2.27; P = 0.46) compared with classes IV–V. Those with at least one copy of a F508del-CFTR mutation and one copy of a class V mutation had a significantly lower V.O2peak (β = −8.24%; 95% CI, −14.53 to −2.99; P = 0.003) and lower Wattmax (adjusted β = −7.59%; 95% CI, −14.21 to −0.95; P = 0.025) than those with two copies of a class II mutation. On the basis of linear regression analysis adjusted for

  2. Biophysical characterisation of calumenin as a charged F508del-CFTR folding modulator.

    Directory of Open Access Journals (Sweden)

    Rashmi Tripathi

    Full Text Available The cystic fibrosis transmembrane regulator (CFTR is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs. We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with

  3. CFTR Modulators: Shedding Light on Precision Medicine for Cystic Fibrosis

    Science.gov (United States)

    Lopes-Pacheco, Miquéias

    2016-01-01

    Cystic fibrosis (CF) is the most common life-threatening monogenic disease afflicting Caucasian people. It affects the respiratory, gastrointestinal, glandular and reproductive systems. The major cause of morbidity and mortality in CF is the respiratory disorder caused by a vicious cycle of obstruction of the airways, inflammation and infection that leads to epithelial damage, tissue remodeling and end-stage lung disease. Over the past decades, life expectancy of CF patients has increased due to early diagnosis and improved treatments; however, these patients still present limited quality of life. Many attempts have been made to rescue CF transmembrane conductance regulator (CFTR) expression, function and stability, thereby overcoming the molecular basis of CF. Gene and protein variances caused by CFTR mutants lead to different CF phenotypes, which then require different treatments to quell the patients’ debilitating symptoms. In order to seek better approaches to treat CF patients and maximize therapeutic effects, CFTR mutants have been stratified into six groups (although several of these mutations present pleiotropic defects). The research with CFTR modulators (read-through agents, correctors, potentiators, stabilizers and amplifiers) has achieved remarkable progress, and these drugs are translating into pharmaceuticals and personalized treatments for CF patients. This review summarizes the main molecular and clinical features of CF, emphasizes the latest clinical trials using CFTR modulators, sheds light on the molecular mechanisms underlying these new and emerging treatments, and discusses the major breakthroughs and challenges to treating all CF patients. PMID:27656143

  4. Investigating CFTR and KCa3.1 Protein/Protein Interactions.

    Directory of Open Access Journals (Sweden)

    Hélène Klein

    Full Text Available In epithelia, Cl- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl- ions and electrolytes needs however to be coupled to an increase in K+ conductance in order to recycle K+ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K+ efflux is ensured by K+ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400 interact with the NBD2 segment (G1237-Y1420 and C- region of CFTR (residues T1387-L1480, respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca2+ concentration. Altogether, these results suggest 1 that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2 that KCa3.1 and CFTR form a dynamic complex, the formation of which

  5. Anchored PDE4 regulates chloride conductance in wild-type and ΔF508-CFTR human airway epithelia

    Science.gov (United States)

    Blanchard, Elise; Zlock, Lorna; Lao, Anna; Mika, Delphine; Namkung, Wan; Xie, Moses; Scheitrum, Colleen; Gruenert, Dieter C.; Verkman, Alan S.; Finkbeiner, Walter E.; Conti, Marco; Richter, Wito

    2014-01-01

    Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impair its expression and/or chloride channel function. Here, we provide evidence that type 4 cyclic nucleotide phosphodiesterases (PDE4s) are critical regulators of the cAMP/PKA-dependent activation of CFTR in primary human bronchial epithelial cells. In non-CF cells, PDE4 inhibition increased CFTR activity under basal conditions (ΔISC 7.1 μA/cm2) and after isoproterenol stimulation (increased ΔISC from 13.9 to 21.0 μA/cm2) and slowed the return of stimulated CFTR activity to basal levels by >3-fold. In cells homozygous for ΔF508-CFTR, the most common mutation found in CF, PDE4 inhibition alone produced minimal channel activation. However, PDE4 inhibition strongly amplified the effects of CFTR correctors, drugs that increase expression and membrane localization of CFTR, and/or CFTR potentiators, drugs that increase channel gating, to reach ∼25% of the chloride conductance observed in non-CF cells. Biochemical studies indicate that PDE4s are anchored to CFTR and mediate a local regulation of channel function. Taken together, our results implicate PDE4 as an important determinant of CFTR activity in airway epithelia, and support the use of PDE4 inhibitors to potentiate the therapeutic benefits of CFTR correctors and potentiators.—Blanchard, E., Zlock, L., Lao, A., Mika, D., Namkung, W., Xie, M., Scheitrum, C., Gruenert, D.C., Verkman, A.S., Finkbeiner, W.E., Conti, M., Richter, W. Anchored PDE4 regulates chloride conductance in wild type and ΔF508-CFTR human airway epithelia. PMID:24200884

  6. Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium.

    Science.gov (United States)

    Walker, Nancy M; Liu, Jinghua; Stein, Sydney R; Stefanski, Casey D; Strubberg, Ashlee M; Clarke, Lane L

    2016-01-15

    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl(-) and HCO3 (-) efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3 (-))-loading proteins and upregulation of the basolateral membrane HCO3 (-)-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl(-)/HCO3 (-) exchange with maximized gradients, it also had increased intracellular Cl(-) concentration relative to wild-type. Pharmacological reduction of intracellular Cl(-) concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl(-) and HCO3 (-) efflux, which impairs pHi regulation by Ae2. Retention of Cl(-) and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. Copyright © 2016 the American Physiological Society.

  7. Current insights into the role of PKA phosphorylation in CFTR channel activity and the pharmacological rescue of cystic fibrosis disease-causing mutants.

    Science.gov (United States)

    Chin, Stephanie; Hung, Maurita; Bear, Christine E

    2017-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.

  8. The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients.

    Directory of Open Access Journals (Sweden)

    Eva K Roth

    Full Text Available BACKGROUND: The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF. Previous studies demonstrated that the K⁺ channel opener 1-ethyl-2-benzimidazolone (1-EBIO potentiates CFTR-mediated Cl⁻ secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. METHODS: We studied the effects of 1-EBIO on CFTR-mediated Cl⁻ secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl⁻ secretion. RESULTS: Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl⁻ secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl⁻ secretion by 39.2±6.7% (P<0.001 via activation of basolateral Ca²⁺-activated and clotrimazole-sensitive KCNN4 K⁺ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl⁻ secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001, but had no effect on tissues lacking CFTR-mediated Cl⁻ conductance. CONCLUSIONS: We conclude that 1-EBIO potentiates Cl⁻secretion in native CF tissues expressing CFTR mutants with residual Cl⁻ channel function by activation of basolateral KCNN4 K⁺ channels that increase the driving force for luminal Cl⁻ exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

  9. The HDAC inhibitor SAHA does not rescue CFTR membrane expression in Cystic Fibrosis.

    Science.gov (United States)

    Bergougnoux, Anne; Petit, Aurélie; Knabe, Lucie; Bribes, Estelle; Chiron, Raphaël; De Sario, Albertina; Claustres, Mireille; Molinari, Nicolas; Vachier, Isabelle; Taulan-Cadars, Magali; Bourdin, Arnaud

    2017-07-01

    The development of suitable Cystic Fibrosis (CF) models for preclinical bench tests of therapeutic candidates is challenging. Indeed, the validation of molecules to rescue the p.Phe508del-CFTR channel (encoded by the Cystic Fibrosis Transmembrane conductance Regulator gene carrying the p.Phe508del mutation) requires taking into account their overall effects on the epithelium. Suberoylanilide Hydroxamic Acid (SAHA), a histone deacetylase inhibitor (HDACi), was previously shown to be a CFTR corrector via proteostasis modulation in CFTR-deficient immortalized cells. Here, we tested SAHA effects on goblet cell metaplasia using an ex vivo model based on the air-liquid interface (ALI) culture of differentiated airway epithelial cells obtained by nasal scraping from CF patients and healthy controls. Ex vivo epithelium grew successfully in ALI cultures with significant rise in the expression of CFTR and of markers of airway epithelial differentiation compared to monolayer cell culture. SAHA decreased CFTR transcript and protein levels in CF and non-CF epithelia. Whereas SAHA induced lysine hyperacetylation, it did not change histone modifications at the CFTR promoter. SAHA reduced MUC5AC and MUC5B expression and inhibited goblet epithelial cell differentiation. Similar effects were obtained in CF and non-CF epithelia. All the effects were fully reversible within five days from SAHA withdrawal. We conclude that, ex vivo, SAHA modulate the structure of airway epithelia without specific effect on CFTR gene and protein suggesting that HDACi cannot be useful for CF treatment. Copyright © 2017. Published by Elsevier Ltd.

  10. The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy

    KAUST Repository

    Jourdain, P.

    2013-12-11

    The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

  11. Evidence that CFTR is expressed in rat tracheal smooth muscle cells and contributes to bronchodilation

    Directory of Open Access Journals (Sweden)

    Mettey Yvette

    2006-08-01

    Full Text Available Abstract Background The airway functions are profoundly affected in many diseases including asthma, chronic obstructive pulmonary disease (COPD and cystic fibrosis (CF. CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR gene, which normally encodes a multifunctional and integral membrane protein, the CF transmembrane conductance regulator (CFTR expressed in airway epithelial cells. Methods To demonstrate that CFTR is also expressed in tracheal smooth muscle cells (TSMC, we used iodide efflux assay to analyse the chloride transports in organ culture of rat TSMC, immunofluorescence study to localize CFTR proteins and isometric contraction measurement on isolated tracheal rings to observe the implication of CFTR in the bronchodilation. Results We characterized three different pathways stimulated by the cAMP agonist forskolin and the isoflavone agent genistein, by the calcium ionophore A23187 and by hypo-osmotic challenge. The pharmacology of the cAMP-dependent iodide efflux was investigated in detail. We demonstrated in rat TSMC that it is remarkably similar to that of the epithelial CFTR, both for activation (using three benzo [c]quinolizinium derivatives and for inhibition (glibenclamide, DPC and CFTRinh-172. Using rat tracheal rings, we observed that the activation of CFTR by benzoquinolizinium derivatives in TSMC leads to CFTRinh-172-sensitive bronchodilation after constriction with carbachol. An immunolocalisation study confirmed expression of CFTR in tracheal myocytes. Conclusion Altogether, these observations revealed that CFTR in the airways of rat is expressed not only in the epithelial cells but also in tracheal smooth muscle cells leading to the hypothesis that this ionic channel could contribute to bronchodilation.

  12. Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR).

    Science.gov (United States)

    Rosenberg, Mark F; Kamis, Alhaji Bukar; Aleksandrov, Luba A; Ford, Robert C; Riordan, John R

    2004-09-10

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis. Here we report the purification and first crystallization of wild-type human CFTR. Functional characterization of the material showed it to be highly active. Electron crystallography of negatively stained two-dimensional crystals of CFTR has revealed the overall architecture of this channel for two different conformational states. These show a strong structural homology to two conformational states of another eukaryotic ATP-binding cassette transporter, P-glycoprotein. In contrast to P-glycoprotein, however, both conformational states can be observed in the presence of a nucleotide, which may be related to the role of CFTR as an ion channel rather than a transporter. The hypothesis that the two conformations could represent the "open" and "closed" states of the channel is considered.

  13. Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.

    Science.gov (United States)

    LaRusch, Jessica; Jung, Jinsei; General, Ignacio J; Lewis, Michele D; Park, Hyun Woo; Brand, Randall E; Gelrud, Andres; Anderson, Michelle A; Banks, Peter A; Conwell, Darwin; Lawrence, Christopher; Romagnuolo, Joseph; Baillie, John; Alkaade, Samer; Cote, Gregory; Gardner, Timothy B; Amann, Stephen T; Slivka, Adam; Sandhu, Bimaljit; Aloe, Amy; Kienholz, Michelle L; Yadav, Dhiraj; Barmada, M Michael; Bahar, Ivet; Lee, Min Goo; Whitcomb, David C

    2014-07-01

    CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<0.0001). WNK1-SPAK pathway-activated increases in CFTR

  14. Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.

    Directory of Open Access Journals (Sweden)

    Jessica LaRusch

    2014-07-01

    Full Text Available CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev cause complete loss of CFTR function and result in cystic fibrosis (CF, a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002. Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005 and male infertility (OR 395, p<<0.0001. WNK1-SPAK pathway-activated increases in

  15. Individualized medicine using intestinal responses to CFTR potentiators and correctors

    NARCIS (Netherlands)

    Beekman, Jeffrey M.

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) modulators that target the mutant CFTR protein are being introduced for treatment of cystic fibrosis. Stratification of subjects based on their CFTR genotype has been proven essential to demonstrate clinical efficacy of these novel

  16. Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

    Science.gov (United States)

    Ehrhardt, Annette; Chung, W Joon; Pyle, Louise C; Wang, Wei; Nowotarski, Krzysztof; Mulvihill, Cory M; Ramjeesingh, Mohabir; Hong, Jeong; Velu, Sadanandan E; Lewis, Hal A; Atwell, Shane; Aller, Steve; Bear, Christine E; Lukacs, Gergely L; Kirk, Kevin L; Sorscher, Eric J

    2016-01-22

    In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. CFTR impairment upregulates c-Src activity through IL-1β autocrine signaling.

    Science.gov (United States)

    Massip-Copiz, María Macarena; Clauzure, Mariángeles; Valdivieso, Ángel Gabriel; Santa-Coloma, Tomás Antonio

    2017-02-15

    Cystic Fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Previously, we found several genes showing a differential expression in CFDE cells (epithelial cells derived from a CF patient). One corresponded to c-Src; its expression and activity was found increased in CFDE cells, acting as a signaling molecule between the CFTR activity and MUC1 overexpression. Here we report that bronchial IB3-1 cells (CF cells) also showed increased c-Src activity compared to 'CFTR-corrected' S9 cells. In addition, three different Caco-2 cell lines, each stably transfected with a different CFTR-specific shRNAs, displayed increased c-Src activity. The IL-1β receptor antagonist IL1RN reduced the c-Src activity of Caco-2/pRS26 cells (expressing a CFTR-specific shRNA). In addition, increased mitochondrial and cellular ROS levels were detected in Caco-2/pRS26 cells. ROS levels were partially reduced by incubation with PP2 (c-Src inhibitor) or IL1RN, and further reduced by using the NOX1/4 inhibitor GKT137831. Thus, IL-1β→c-Src and IL-1β→NOX signaling pathways appear to be responsible for the production of cellular and mitochondrial ROS in CFTR-KD cells. In conclusion, IL-1β constitutes a new step in the CFTR signaling pathway, located upstream of c-Src, which is stimulated in cells with impaired CFTR activity. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Potentiators of Defective ΔF508-CFTR Gating that Do Not Interfere with Corrector Action.

    Science.gov (United States)

    Phuan, Puay-Wah; Veit, Guido; Tan, Joseph A; Finkbeiner, Walter E; Lukacs, Gergely L; Verkman, A S

    2015-10-01

    Combination drug therapies under development for cystic fibrosis caused by the ∆F508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) include a "corrector" to improve its cellular processing and a "potentiator" to improve its chloride channel function. Recently, it was reported that the approved potentiator N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (Ivacaftor) reduces ∆F508-CFTR cellular stability and the efficacy of investigational correctors, including 3-(6-[([1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl) amino]-3-methyl-2-pyridinyl)-benzoic acid and 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-(1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl), which might contribute to the modest reported efficacy of combination therapy in clinical trials. Here, we report the identification and characterization of potentiators that do not interfere with ∆F508-CFTR stability or corrector action. High-throughput screening and structure-activity analysis identified several classes of potentiators that do not impair corrector action, including tetrahydrobenzothiophenes, thiooxoaminothiazoles, and pyrazole-pyrrole-isoxazoles. The most potent compounds have an EC(50) for ∆F508-CFTR potentiation down to 18 nM and do not reduce corrector efficacy in heterologous ∆F508-CFTR-expressing cells or primary cultures of ∆F508/∆F508 human bronchial epithelia. The ΔF508-CFTR potentiators also activated wild-type and G551D CFTR, albeit weakly. The efficacy of combination therapy for cystic fibrosis caused by the ∆F508 mutation may be improved by replacement of Ivacaftor with a potentiator that does not interfere with corrector action. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  19. Les vésicules extracellulaires comme vecteurs de macromolécules bioactives : modèle du transporteur ABCC7 (CFTR) et application à la biothérapie de la mucoviscidose

    OpenAIRE

    Vituret, Cyrielle

    2015-01-01

    Cystic fibrosis is a genetic disease in which its prognosis depends on the lung damage. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), resulting in a dysfunctional CFTR protein normally located at the plasma membrane of epithelial cells. This thesis is a study of a novel therapeutic approach to use extracellular vesicles (EVs), microvesicles and exosomes, as transfer vectors for CFTR mRNA and protein to target cells. The proof of concept for ...

  20. CFTR (ABCC7) is a hydrolyzable-ligand-gated channel.

    Science.gov (United States)

    Aleksandrov, Andrei A; Aleksandrov, Luba A; Riordan, John R

    2007-02-01

    As the product of the gene mutated in cystic fibrosis, the most common genetic disease of Caucasians, CFTR is an atypical ABC protein. From an evolutionary perspective, it is apparently a relatively young member of the ABC family, present only in metazoans where it plays a critical role in epithelial salt and fluid homeostasis. Functionally, the membrane translocation process it mediates, the passive bidirectional diffusion of small inorganic anions, is simpler than the vectorial transport of larger more complex substrates ("allocrites") by most ABC transporters. However, the control of the permeation pathway which cannot go unchecked is necessarily more stringent than in the case of the transporters. There is tight regulation by the phosphorylation/dephosphorylation of the unique CFTR R domain superimposed on the basic ABC regulation mode of ATP binding and hydrolysis at the dual nucleotide binding sites. As with other ABCC subfamily members, only the second of these sites is hydrolytic in CFTR. The phosphorylation and ATP binding/hydrolysis events do not strongly influence each other; rather, R domain phosphorylation appears to enable transduction of the nucleotide binding allosteric signal to the responding channel gate. ATP hydrolysis is not required for either the opening or closing gating transitions but efficiently clears the ligand-binding site enabling a new gating cycle to be initiated.

  1. Interaction between Hb E and Hb Yala (HBB:c.129delT); a novel frameshift beta globin gene mutation, resulting in Hemoglobin E/β0 thalassemia.

    Science.gov (United States)

    Ekwattanakit, Supachai; Riolueang, Suchada; Viprakasit, Vip

    2018-03-01

    There are more than 200 known mutations found in patients with β-thalassemia, a possibility to identify an unknown or novel mutation becomes less possible. Here, we report a novel mutation in a patient from Thailand who presented with chronic hemolytic anemia. A comprehensive hematology and DNA analysis was applied in the index patient and her mother. Hematological and hemoglobin analyses were consistent with the clinical diagnosis of Hb E/β 0 -thalassemia. However, we could find only Hb E heterozygous mutation using our common polymerase chain reaction-based mutation detection of the β-globin genes. Furthermore, the molecular analysis demonstrated a novel T-deletion at codon 42 of the second exon of the β-globin gene which we named 'Hb Yala' according to the origin of this index family. This mutation was assumed to generate a truncated β-globin chain terminating at codon 60 with possible unstable variant leading to a 'null' or β 0 -thalassemia. However, the clinical phenotype was surprisingly mild and no other ameliorating genetic factors, including co-inheritance of α-thalassemia and high propensity of Hb F by Xmn I polymorphism, were found. This report has provided evidence that genotype-phenotype correlation in thalassemia syndromes is highly complex and a correct clinical severity classification of thalassemia should be mainly based on clinical evaluation.

  2. In vivo and in vitro ivacaftor response in cystic fibrosis patients with residual CFTR function: N-of-1 studies.

    Science.gov (United States)

    McGarry, Meghan E; Illek, Beate; Ly, Ngoc P; Zlock, Lorna; Olshansky, Sabrina; Moreno, Courtney; Finkbeiner, Walter E; Nielson, Dennis W

    2017-04-01

    Ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, decreases sweat chloride concentration, and improves pulmonary function in 6% of cystic fibrosis (CF) patients with specific CFTR mutations. Ivacaftor increases chloride transport in many other CFTR mutations in non-human cells, if CFTR is in the epithelium. Some CF patients have CFTR in the epithelium with residual CFTR function. The effect of ivacaftor in these patients is unknown. This was a series of randomized, crossover N-of-1 trials of ivacaftor and placebo in CF patients ≥8 years old with potential residual CFTR function (intermediate sweat chloride concentration, pancreatic sufficient, or mild bronchiectasis on chest CT). Human nasal epithelium (HNE) was obtained via nasal brushing and cultured. Sweat chloride concentration change was the in vivo outcome. Chloride current change in HNE cultures with ivacaftor was the in vitro outcome. Three subjects had decreased sweat chloride concentration (-14.8 to -40.8 mmol/L, P < 0.01). Two subjects had unchanged sweat chloride concentration. Two subjects had increased sweat chloride concentration (+23.8 and +27.3 mmol/L, P < 0.001); both were heterozygous for A455E and pancreatic sufficient. Only subjects with decreased sweat chloride concentration had increased chloride current in HNE cultures. Some CF patients with residual CFTR function have decreased sweat chloride concentration with ivacaftor. Increased chloride current in HNE cultures among subjects with decreased sweat chloride concentrations may predict clinical response to ivacaftor. Ivacaftor can increase sweat chloride concentration in certain mutations with unclear clinical effect. Pediatr Pulmonol. 2017;52:472-479. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  3. Functional interaction between CFTR and the sodium-phosphate co-transport type 2a in Xenopus laevis oocytes.

    Directory of Open Access Journals (Sweden)

    Naziha Bakouh

    Full Text Available A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR. CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis.

  4. Increased susceptibility of Cftr_/_ mice to LPS-induced lung remodeling

    NARCIS (Netherlands)

    Bruscia, E.M. (Emanuela M.); Zhang, P.-X. (Ping-Xia); Barone, C. (Christina); B.J. Scholte (Bob); Homer, R. (Robert); Krause, D.S. (Diane S.); Egan, M.E. (Marie E.)

    2016-01-01

    textabstractCystic fibrosis (CF) is caused by homozygous mutations of the CF transmembrane conductance regulator (CFTR) Cl_ channel, which result in chronic pulmonary infection and inflammation, the major cause of morbidity and mortality. Although these processes are clearly related to each other,

  5. Dynamic pathways of -1 translational frameshifting.

    Science.gov (United States)

    Chen, Jin; Petrov, Alexey; Johansson, Magnus; Tsai, Albert; O'Leary, Seán E; Puglisi, Joseph D

    2014-08-21

    Spontaneous changes in the reading frame of translation are rare (frequency of 10(-3) to 10(-4) per codon), but can be induced by specific features in the messenger RNA (mRNA). In the presence of mRNA secondary structures, a heptanucleotide 'slippery sequence' usually defined by the motif X XXY YYZ, and (in some prokaryotic cases) mRNA sequences that base pair with the 3' end of the 16S ribosomal rRNA (internal Shine-Dalgarno sequences), there is an increased probability that a specific programmed change of frame occurs, wherein the ribosome shifts one nucleotide backwards into an overlapping reading frame (-1 frame) and continues by translating a new sequence of amino acids. Despite extensive biochemical and genetic studies, there is no clear mechanistic description for frameshifting. Here we apply single-molecule fluorescence to track the compositional and conformational dynamics of individual ribosomes at each codon during translation of a frameshift-inducing mRNA from the dnaX gene in Escherichia coli. Ribosomes that frameshift into the -1 frame are characterized by a tenfold longer pause in elongation compared to non-frameshifted ribosomes, which translate through unperturbed. During the pause, interactions of the ribosome with the mRNA stimulatory elements uncouple EF-G catalysed translocation from normal ribosomal subunit reverse-rotation, leaving the ribosome in a non-canonical intersubunit rotated state with an exposed codon in the aminoacyl-tRNA site (A site). tRNA(Lys) sampling and accommodation to the empty A site and EF-G action either leads to the slippage of the tRNAs into the -1 frame or maintains the ribosome into the 0 frame. Our results provide a general mechanistic and conformational framework for -1 frameshifting, highlighting multiple kinetic branchpoints during elongation.

  6. Important role of platelets in modulating endotoxin-induced lung inflammation in CFTR-deficient mice.

    Directory of Open Access Journals (Sweden)

    Caiqi Zhao

    Full Text Available Mutation of CFTR (cystic fibrosis transmembrane conductance regulator leads to cystic fibrosis (CF. Patients with CF develop abnormalities of blood platelets and recurrent lung inflammation. However, whether CFTR-mutated platelets play a role in the development of lung inflammation is elusive. Therefore, we intratracheally challenged wildtype and F508del (a common type of CFTR mutation mice with LPS to observe changes of F508del platelets in the peripheral blood and indexes of lung inflammation (BAL neutrophils and protein levels. Furthermore, we investigated whether or not and how F508del platelets modulate the LPS-induced acute lung inflammation by targeting anti-platelet aggregation, depletion of neutrophils, reconstitution of bone marrow or neutrophils, blockade of P-selectin glycoprotein ligand-1 (PSGL-1, platelet activating factor (PAF, and correction of mutated CFTR trafficking. We found that LPS-challenged F508del mice developed severe thrombocytopenia and had higher levels of plasma TXB2 coincided with neutrophilic lung inflammation relative to wildtype control. Inhibition of F508del platelet aggregation or depletion of F508del neutrophils diminished the LPS-induced lung inflammation in the F508del mice. Moreover, wildtype mice reconstituted with either F508del bone marrow or neutrophils developed worse thrombocytopenia. Blocking PSGL-1, platelet activating factor (PAF, or rectifying trafficking of mutated CFTR in F508del mice diminished and alveolar neutrophil transmigration in the LPS-challenged F508del mice. These findings suggest that F508del platelets and their interaction with neutrophils are requisite for the development of LPS-induced lung inflammation and injury. As such, targeting platelets might be an emerging strategy for dampening recurrent lung inflammation in cystic fibrosis patients.

  7. Whole-gene CFTR sequencing combined with digital RT-PCR improves genetic diagnosis of cystic fibrosis.

    Science.gov (United States)

    Straniero, Letizia; Soldà, Giulia; Costantino, Lucy; Seia, Manuela; Melotti, Paola; Colombo, Carla; Asselta, Rosanna; Duga, Stefano

    2016-12-01

    Despite extensive screening, 1-5% of cystic fibrosis (CF) patients lack a definite molecular diagnosis. Next-generation sequencing (NGS) is making affordable genetic testing based on the identification of variants in extended genomic regions. In this frame, we analyzed 23 CF patients and one carrier by whole-gene CFTR resequencing: 4 were previously characterized and served as controls; 17 were cases lacking a complete diagnosis after a full conventional CFTR screening; 3 were consecutive subjects referring to our centers, not previously submitted to any screening. We also included in the custom NGS design the coding portions of the SCNN1A, SCNN1B and SCNN1G genes, encoding the subunits of the sodium channel ENaC, which were found to be mutated in CF-like patients. Besides 2 novel SCNN1B missense mutations, we identified 22 previously-known CFTR mutations, including 2 large deletions (whose breakpoints were precisely mapped), and novel deep-intronic variants, whose role on splicing was excluded by ex-vivo analyses. Finally, for 2 patients, compound heterozygotes for a CFTR mutation and the intron-9c.1210-34TG [11-12] T 5 allele-known to be associated with decreased CFTR mRNA levels-the molecular diagnosis was implemented by measuring the residual level of wild-type transcript by digital reverse transcription polymerase chain reaction performed on RNA extracted from nasal brushing.

  8. Hypertonic saline increases lung epithelial lining fluid glutathione and thiocyanate: two protective CFTR-dependent thiols against oxidative injury

    Directory of Open Access Journals (Sweden)

    Gould Neal S

    2010-08-01

    Full Text Available Abstract Background Cystic fibrosis is a debilitating lung disease due to mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR and is associated with chronic infections resulting in elevated myeloperoxidase activity and generation of hypochlorous acid (HOCl. CFTR mutations lead to decreased levels of glutathione (GSH and thiocyanate (SCN in the epithelial lining fluid (ELF. Hypertonic saline is used to improve lung function however the mechanism is uncertain. Methods In the present study, the effect of GSH and SCN on HOCl-mediated cell injury and their changes in the ELF after hypertonic saline nebulization in wild type (WT and CFTR KO mice was examined. CFTR sufficient and deficient lung cells were assessed for GSH, SCN and corresponding sensitivity towards HOCl-mediated injury, in vitro. Results CFTR (- cells had lower extracellular levels of both GSH and SCN and were more sensitive to HOCl-mediated injury. In vivo, hypertonic saline increased ELF GSH in the WT and to a lesser extent in the CFTR KO mice but only SCN in the WT ELF. Finally, potential protective effects of GSH and SCN at concentrations found in the ELF against HOCl toxicity were examined in vitro. Conclusions While the concentrations of GSH and SCN associated with the WT ELF protect against HOCl toxicity, those found in the CFTR KO mice were less sufficient to inhibit cell injury. These data suggests that CFTR has important roles in exporting GSH and SCN which are protective against oxidants and that hypertonic saline treatment may have beneficial effects by increasing their levels in the lung.

  9. Characterization of nasal potential difference in cftr knockout and F508del-CFTR mice.

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    Emilie Lyne Saussereau

    Full Text Available BACKGROUND: Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF. Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V(TE to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V(TE in CF mice must be well characterized for correct interpretation. METHODS: We performed V(TE measurements in large-scale studies of two mouse models of CF--B6;129 cftr knockout and FVB F508del-CFTR--and their respective wild-type (WT littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice. RESULTS: We determined the typical V(TE values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl(- solution was considered to indicate a normal response. CONCLUSIONS: These data will make it possible to interpret changes in nasal V(TE in mouse models of CF, in future preclinical studies.

  10. Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR.

    Science.gov (United States)

    Kim, Jiyoon; Noh, Shin Hye; Piao, He; Kim, Dong Hee; Kim, Kuglae; Cha, Jeong Seok; Chung, Woo Young; Cho, Hyun-Soo; Kim, Joo Young; Lee, Min Goo

    2016-07-01

    Induction of endoplasmic reticulum (ER)-to-Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)-mediated, Golgi-independent unconventional cell-surface trafficking of the folding-deficient ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation-dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508-CFTR, such as induction of ER-to-Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C-terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation-mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation-inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER-retained ΔF508-CFTR and mediates the ER stress-induced unconventional secretion pathway. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

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    Guido Veit

    2016-05-01

    Full Text Available The most common cystic fibrosis (CF causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del, results in functional expression defect of the CF transmembrane conductance regulator (CFTR at the apical plasma membrane (PM of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER. Deletion of phenylalanine 670 (ΔF670 in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

  12. The silent codon change I507-ATC->ATT contributes to the severity of the ΔF508 CFTR channel dysfunction.

    Science.gov (United States)

    Lazrak, Ahmed; Fu, Lianwu; Bali, Vedrana; Bartoszewski, Rafal; Rab, Andras; Havasi, Viktoria; Keiles, Steve; Kappes, John; Kumar, Ranjit; Lefkowitz, Elliot; Sorscher, Eric J; Matalon, Sadis; Collawn, James F; Bebok, Zsuzsanna

    2013-11-01

    The most common disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the out-of-frame deletion of 3 nucleotides (CTT). This mutation leads to the loss of phenylalanine-508 (ΔF508) and a silent codon change (SCC) for isoleucine-507 (I507-ATC→ATT). ΔF508 CFTR is misfolded and degraded by endoplasmic reticulum-associated degradation (ERAD). We have demonstrated that the I507-ATC→ATT SCC alters ΔF508 CFTR mRNA structure and translation dynamics. By comparing the biochemical and functional properties of the I507-ATT and I507-ATC ΔF508 CFTR, we establish that the I507-ATC→ATT SCC contributes to the cotranslational misfolding, ERAD, and to the functional defects associated with ΔF508 CFTR. We demonstrate that the I507-ATC ΔF508 CFTR is less susceptible to the ER quality-control machinery during translation than the I507-ATT, although 27°C correction is necessary for sufficient cell-surface expression. Whole-cell patch-clamp recordings indicate sustained, thermally stable cAMP-activated Cl(-) transport through I507-ATC and unstable function of the I507-ATT ΔF508 CFTR. Single-channel recordings reveal improved gating properties of the I507-ATC compared to I507-ATT ΔF508 CFTR (NPo=0.45±0.037 vs. NPo=0.09±0.002; P<0.001). Our results signify the role of the I507-ATC→ATT SCC in the ΔF508 CFTR defects and support the importance of synonymous codon choices in determining the function of gene products.

  13. A case of cerebrotendinous xanthomatosis mimicking the clinical phenotype of mitochondrial disease with a novel frame-shift mutation (c. 43_44 delGG) in CYP27A1 gene exon 1.

    Science.gov (United States)

    Koge, Junpei; Hayashi, Shintaro; Yamaguchi, Hiroo; Tateishi, Takahisa; Murai, Hiroyuki; Kira, Jun-Ichi

    2016-10-28

    A 37-old-male with a history of early childhood mental retardation was admitted to our hospital. He experienced recurrent syncopes at 23 years old, and at age 35 gait disturbance and hearing impairment developed gradually and worsened over time. His grandparents were in a consanguineous marriage. He was of short stature and absent of tendon xanthomas. Neurological examinations revealed scanning speech, dysphagia, right sensorineural hearing loss, spasticity in both upper and lower extremities, and spastic gait. Tendon reflexes were brisk throughout, and Babinski and Chaddock reflexes were both positive bilaterally. Laboratory tests revealed elevated lactate and pyruvate concentrations in both serum and cerebrospinal fluid. Fluid attenuated inversion recovery magnetic resonance imaging showed high intensity lesions in the bilateral cerebellar hemispheres, pyramidal tracts in the brainstem, and internal capsules symmetrically. Brain magnetic resonance spectroscopy measurements revealed an elevated lactate/creatine plus phosphocreatine ratio and a decreased N-acetyl-aspartate/creatine plus phosphocreatine ratio in the cerebellum. At this point, mitochondrial diseases, particularly myoclonic epilepsy with ragged-red fibers (MERRF), to be the most likely cause. We performed a biopsy of his left biceps brachii muscle, showing variations in fiber size with occasional central nuclei and very few ragged-red fibers. Blood mitochondrial respiratory enzyme assays showed normal values with elevated citrate synthase activity, and mitochondrial DNA analyses for MERRF revealed no pathogenic mutations. We then explored other possibilities and detected an elevated serum cholestanol concentration of 20.4 μg/ml (reference value CYP27A1 gene exon1, leading to a diagnosis of cerebrotendinous xanthomatosis (CTX). This case emphasizes importance of awareness of CTX as a possibility when patients present with clinical phenotypes mimicking mitochondrial diseases, but with negative

  14. All azoospermic males should be screened for cystic fibrosis mutations before intracytoplasmic sperm injection.

    LENUS (Irish Health Repository)

    Mocanu, Edgar

    2010-11-01

    We assessed the frequency of CFTR mutations in groups with varying degrees of sub-fertility and compared these groups to a fertile male group with proven paternity. Screening for CFTR mutations should be routine for all azoospermic males, irrespective of obstructive or non-obstructive etiology, prior to proposing ICSI treatment. CFTR testing has no value in the investigation of non-azoospermic infertile males.

  15. All azoospermic males should be screened for cystic fibrosis mutations before intracytoplasmic sperm injection.

    LENUS (Irish Health Repository)

    Mocanu, Edgar

    2012-02-01

    We assessed the frequency of CFTR mutations in groups with varying degrees of sub-fertility and compared these groups to a fertile male group with proven paternity. Screening for CFTR mutations should be routine for all azoospermic males, irrespective of obstructive or non-obstructive etiology, prior to proposing ICSI treatment. CFTR testing has no value in the investigation of non-azoospermic infertile males.

  16. A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor.

    Directory of Open Access Journals (Sweden)

    Jessica E Char

    Full Text Available To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (- ivacaftor, 3 only (+ ivacaftor and 3 (+/- ivacaftor (1-5 tests per condition. The total number of gland measurements was 852 (- ivacaftor and 906 (+ ivacaftor. A healthy control was tested 4 times (51 glands. For each gland we measured both CFTR-independent (M-sweat and CFTR-dependent (C-sweat; C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects. By contrast, 6/6 subjects (113/342 glands produced C-sweat in the (+ ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+ ivacaftor  = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

  17. Haplotype identity between individuals who share a CFTR mutation allele ''identical by descent'' : Demonstration of the usefulness of the haplotype-sharing concept for gene mapping in real populations

    NARCIS (Netherlands)

    deVries, HG; vanderMeulen, MA; Rozen, R; Halley, DJJ; Scheffer, H; tenKate, LP; Buys, CHCM; teMeerman, GJ

    Cystic fibrosis (CF) patients with the A455E mutation, in both the French Canadian and the Dutch population, share a common haplotype over distances of up to 25 cM. French Canadian patients with the 621+1G-->T mutation share a common haplotype of more than 14 cM. In contrast, haplotypes containing

  18. Haplotype identity between individuals who share a CFTR mutation allele 'identical by descent': Demonstration of the usefulness of the haplotype-sharing concept for gene mapping in real populations

    NARCIS (Netherlands)

    H.G. de Vries (Hendrik); M.A. van der Meulen (Martin); R. Rozen (Rima); D.J.J. Halley (Dicky); H. Scheffer (Hans); L.P. ten Kate; C.H.C.M. Buys; G.J. Te Meerman (Gerard)

    1996-01-01

    markdownabstractAbstract Cystic fibrosis (CF) patients with the A455E mutation, in both the French Canadian and the Dutch population, share a common haplotype over distances of up to 25 cM. French Canadian patients with the 621+1G→T mutation share a common haplotype of more than 14 cM. In

  19. Haplotype identity between individuals who share a CFTR mutation allele "identical by descent" : demonstration of the usefulness of the haplotype-sharing concept for gene mapping in real populations

    NARCIS (Netherlands)

    de Vries, H G; van der Meulen, M A; Rozen, R; Halley, D J; Scheffer, H; ten Kate, L P; Buys, C H; te Meerman, G J

    Cystic fibrosis (CF) patients with the A455E mutation, in both the French Canadian and the Dutch population, share a common haplotype over distances of up to 25 cM. French Canadian patients with the 621+1G-->T mutation share a common haplotype of more than 14 cM. In contrast, haplotypes containing

  20. Thymosin α-1 does not correct F508del-CFTR in cystic fibrosis airway epithelia

    Science.gov (United States)

    Tomati, Valeria; Caci, Emanuela; Ferrera, Loretta; Pesce, Emanuela; Sondo, Elvira; Cholon, Deborah M.; Quinney, Nancy L.; Boyles, Susan E.; Armirotti, Andrea; Ravazzolo, Roberto; Galietta, Luis J.V.; Gentzsch, Martina

    2018-01-01

    In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. Considering the numerous effects of the F508del mutation on the assembly and processing of CFTR protein, combination therapy with several pharmacological correctors is likely to be required to treat CF patients. Recently, it has been reported that thymosin α-1 (Tα-1) has multiple beneficial effects that could lead to a single-molecule-based therapy for CF patients with F508del. Such effects include suppression of inflammation, improvement in F508del-CFTR maturation and gating, and stimulation of chloride secretion through the calcium-activated chloride channel (CaCC). Given the importance of such a drug, we aimed to characterize the underlying molecular mechanisms of action of Tα-1. In-depth analysis of Tα-1 effects was performed using well-established microfluorimetric, biochemical, and electrophysiological techniques on epithelial cell lines and primary bronchial epithelial cells from CF patients. The studies, which were conducted in 2 independent laboratories with identical outcome, demonstrated that Tα-1 is devoid of activity on mutant CFTR as well as on CaCC. Although Tα-1 may still be useful as an antiinflammatory agent, its ability to target defective anion transport in CF remains to be further investigated. PMID:29415893

  1. Increased susceptibility of Cftr?/? mice to LPS-induced lung remodeling

    OpenAIRE

    Bruscia, Emanuela M.; Zhang, Ping-Xia; Barone, Christina; Scholte, Bob J.; Homer, Robert; Krause, Diane S.; Egan, Marie E.

    2016-01-01

    Cystic fibrosis (CF) is caused by homozygous mutations of the CF transmembrane conductance regulator (CFTR) Cl? channel, which result in chronic pulmonary infection and inflammation, the major cause of morbidity and mortality. Although these processes are clearly related to each other, each is likely to contribute to the pathology differently. Understanding the contribution of each of these processes to the overall pathology has been difficult, because they are usually so intimately connected...

  2. CFTR depletion results in changes in fatty acid composition and promotes lipogenesis in intestinal Caco 2/15 cells.

    Directory of Open Access Journals (Sweden)

    Geneviève Mailhot

    2010-05-01

    Full Text Available Abnormal fatty acid composition (FA in plasma and tissue lipids frequently occurs in homozygous and even in heterozygous carriers of cystic fibrosis transmembrane conductance regulator (CFTR mutations. The mechanism(s underlying these abnormalities remained, however, poorly understood despite the potentially CFTR contributing role.The aim of the present study was to investigate the impact of CFTR depletion on FA uptake, composition and metabolism using the intestinal Caco-2/15 cell line. shRNA-mediated cftr gene silencing induced qualitative and quantitative modifications in FA composition in differentiated enterocytes as determined by gas-liquid chromatography. With the cftr gene disruption, there was a 1,5 fold increase in the total FA amount, largely attributable to monounsaturated and saturated FA compared to controls. The activity of delta-7 desaturase, estimated by the 16:1(n-7/16:0, was significantly higher in knockdown cells and consistent with the striking elevation of the n-7 FA family. When incubated with [14C]-oleic acid, CFTR-depleted cells were capable of quick incorporation and export to the medium concomitantly with the high protein expression of L-FABP known to promote intracellular FA trafficking. Accordingly, lipoprotein vehicles (CM, VLDL, LDL and HDL, isolated from CFTR knockdown cells, exhibited higher levels of radiolabeled FA. Moreover, in the presence of [14C]-acetate, knockdown cells exhibited enhanced secretion of newly synthesized phospholipids, triglycerides, cholesteryl esters and free FA, thereby suggesting a stimulation of the lipogenic pathway. Conformably, gene expression of SREBP-1c, a key lipogenic transcription factor, was increased while protein expression of the phosphorylated and inactive form of acetylCoA carboxylase was reduced, confirming lipogenesis induction. Finally, CFTR-depleted cells exhibited lower gene expression of transcription factors (PPARalpha, LXRalpha, LXRbeta and RXRalpha

  3. Measurements of CFTR-mediated Cl- secretion in human rectal biopsies constitute a robust biomarker for Cystic Fibrosis diagnosis and prognosis.

    Directory of Open Access Journals (Sweden)

    Marisa Sousa

    Full Text Available Cystic Fibrosis (CF is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR gene encoding for a cAMP-regulated chloride (Cl(- channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases.To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl(- secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n=51, individuals with clinical CF suspicion (n=49 and age-matched non-CF controls (n=18. Conclusive measurements were obtained for 96% of cases. Patients with "Classic CF", presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl(- secretion (<5%. Individuals with milder CF disease presented residual CFTR-mediated Cl(- secretion (10-57% and non-CF controls show CFTR-mediated Cl(- secretion ≥ 30-35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in "CF suspicion" individuals allowed to confirm CF in 16/49 individuals (33% and exclude it in 28/49 (57%. Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl(- secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups.Determination of CFTR-mediated Cl(- secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-clinical trials of CFTR-modulator therapies.

  4. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

    2014-07-18

    ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Lipoxin A4 and platelet activating factor are involved in E. coli or LPS-induced lung inflammation in CFTR-deficient mice.

    Directory of Open Access Journals (Sweden)

    Haiya Wu

    Full Text Available CFTR (cystic fibrosis transmembrane conductance regulator is expressed by both neutrophils and platelets. Lack of functional CFTR could lead to severe lung infection and inflammation. Here, we found that mutation of CFTR (F508del or inhibition of CFTR in mice led to more severe thrombocytopenia, alveolar neutrocytosis and bacteriosis, and lower lipoxin A4/MIP-2 (macrophage inhibitory protein-2 or lipoxin A4/neutrophil ratios in the BAL (bronchoalveolar lavage during acute E. coli pneumonia. In vitro, inhibition of CFTR promotes MIP-2 production in LPS-stimulated neutrophils; however, lipoxin A4 could dose-dependently suppress this effect. In LPS-induced acute lung inflammation, blockade of PSGL-1 (P-selectin glycoprotein ligand-1 or P-selectin, antagonism of PAF by WEB2086, or correction of mutated CFTR trafficking by KM11060 could significantly increase plasma lipoxin A4 levels in F508del relevant to wildtype mice. Concurrently, F508del mice had higher plasma platelet activating factor (PAF levels and PAF-AH activity compared to wildtype under LPS challenge. Inhibiting hydrolysis of PAF by a specific PAF-AH (PAF-acetylhydrolase inhibitor, MAFP, could worsen LPS-induced lung inflammation in F508del mice compared to vehicle treated F508del group. Particularly, depletion of platelets in F508del mice could significantly decrease plasma lipoxin A4 and PAF-AH activity and deteriorate LPS-induced lung inflammation compared to control F508del mice. Taken together, lipoxin A4 and PAF are involved in E. coli or LPS-induced lung inflammation in CFTR-deficient mice, suggesting that lipoxin A4 and PAF might be therapeutic targets for ameliorating CFTR-deficiency deteriorated lung inflammation.

  6. An unexpected effect of TNF-α on F508del-CFTR maturation and function [v2; ref status: indexed, http://f1000r.es/5tv

    Directory of Open Access Journals (Sweden)

    Sara Bitam

    2015-09-01

    Full Text Available Cystic fibrosis (CF is a multifactorial disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR, which encodes a cAMP-dependent Cl- channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, with major consequences in the airways of patients with CF, characterized by both fluid transport abnormalities and persistent inflammatory responses. The relationship between the acute phase of inflammation and the expression of wild type (WT CFTR or F508del-CFTR is poorly understood. The aim of the present study was to investigate this effect. The results show that 10 min exposure to TNF-alpha (0.5-50ng/ml of F508del-CFTR-transfected HeLa cells and human bronchial cells expressing F508del-CFTR in primary culture (HBE leads to the maturation of F508del-CFTR and induces CFTR chloride currents. The enhanced CFTR expression and function upon TNFα is sustained, in HBE cells, for at least 24 h. The underlying mechanism of action involves a protein kinase C (PKC signaling pathway, and occurs through insertion of vesicles containing F508del-CFTR to the plasma membrane, with TNFα behaving as a corrector molecule. In conclusion, a novel and unexpected action of TNFα has been discovered and points to the importance of systematic studies on the roles of inflammatory mediators in the maturation of abnormally folded proteins in general and in the context of CF in particular.

  7. An unexpected effect of TNF-α on F508del-CFTR maturation and function [v1; ref status: indexed, http://f1000r.es/5jf

    Directory of Open Access Journals (Sweden)

    Sara Bitam

    2015-07-01

    Full Text Available Cystic fibrosis (CF is a multifactorial disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR, which encodes a cAMP-dependent Cl- channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, with major consequences in the airways of patients with CF, characterized by both fluid transport abnormalities and persistent inflammatory responses. The relationship between the acute phase of inflammation and the expression of wild type (WT CFTR or F508del-CFTR is poorly understood. The aim of the present study was to investigate this effect. The results show that 10 min exposure to TNF-alpha (0.5-50ng/ml of F508del-CFTR-transfected HeLa cells and human bronchial cells expressing F508del-CFTR in primary culture (HBE leads to the maturation of F508del-CFTR and induces CFTR chloride currents. The enhanced CFTR expression and function upon TNFα is sustained, in HBE cells, for at least 24 h. The underlying mechanism of action involves a protein kinase C (PKC signaling pathway, and occurs through insertion of vesicles containing F508del-CFTR to the plasma membrane, with TNFα behaving as a corrector molecule. In conclusion, a novel and unexpected action of TNFα has been discovered and points to the importance of systematic studies on the roles of inflammatory mediators in the maturation of abnormally folded proteins in general and in the context of CF in particular.

  8. Stimulation of airway and intestinal mucosal secretion by natural coumarin CFTR activators

    Directory of Open Access Journals (Sweden)

    Hong eYang

    2011-09-01

    Full Text Available Mutations of cystic fibrosis transmembrane conductance regulator (CFTR cause lethal hereditary disease cystic fibrosis (CF that involves extensive destruction and dysfunction of serous epithelium. Possible pharmacological therapy includes correction of defective intracellular processing and abnormal channel gating. In a previous study, we identified five natural coumarin potentiators of Δ508-CFTR including osthole, imperatorin, isopsoralen, praeruptorin A and scoparone. The present study was designed to determine the activity of these coumarine compounds on CFTR activity in animal tissues as a primary evaluation of their therapeutic potential. In the present study, we analyzed the affinity of these coumarin potentiators in activating wild-type CFTR and found that they are all potent activators. Osthole showed the highest affinity with Kd values <50 nmol/L as determined by Ussing chamber short-circuit current assay. Stimulation of rat colonic mucosal secretion by osthole was tested by the Ussing chamber short-circuit current assay. Osthole reached maximal activation of colonic Cl- secretion at 5 mol/L. Stimulation of mouse tracheal mucosal secretion was analyzed by optical measurement of single gland secretion. Fluid secretion rate of tracheal single submucosal gland stimulated by osthole at 10mol/L was 3-fold more rapid than that in negative control. In both cases the stimulated secretions were fully abolished by CFTRinh-172. In conclusion, the effective stimulation of Cl– and fluid secretion in colonic and tracheal mucosa by osthole suggested the therapeutic potential of natural coumarine compounds for the treatment of cystic fibrosis and other CFTR-related diseases.

  9. Potentiators of Defective ΔF508–CFTR Gating that Do Not Interfere with Corrector Action

    Science.gov (United States)

    Phuan, Puay-Wah; Veit, Guido; Tan, Joseph A.; Finkbeiner, Walter E.; Lukacs, Gergely L.

    2015-01-01

    Combination drug therapies under development for cystic fibrosis caused by the ∆F508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) include a “corrector” to improve its cellular processing and a “potentiator” to improve its chloride channel function. Recently, it was reported that the approved potentiator N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (Ivacaftor) reduces ∆F508-CFTR cellular stability and the efficacy of investigational correctors, including 3-(6-[([1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl) amino]-3-methyl-2-pyridinyl)-benzoic acid and 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-(1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl), which might contribute to the modest reported efficacy of combination therapy in clinical trials. Here, we report the identification and characterization of potentiators that do not interfere with ∆F508-CFTR stability or corrector action. High-throughput screening and structure-activity analysis identified several classes of potentiators that do not impair corrector action, including tetrahydrobenzothiophenes, thiooxoaminothiazoles, and pyrazole-pyrrole-isoxazoles. The most potent compounds have an EC50 for ∆F508-CFTR potentiation down to 18 nM and do not reduce corrector efficacy in heterologous ∆F508-CFTR–expressing cells or primary cultures of ∆F508/∆F508 human bronchial epithelia. The ΔF508-CFTR potentiators also activated wild-type and G551D CFTR, albeit weakly. The efficacy of combination therapy for cystic fibrosis caused by the ∆F508 mutation may be improved by replacement of Ivacaftor with a potentiator that does not interfere with corrector action. PMID:26245207

  10. Evidence for a Rad18-independent frameshift mutagenesis pathway in human cell-free extracts.

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    Régine Janel-Bintz

    Full Text Available Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G and 5'-GGCGCC-3' (NarI site, induces -1 and -2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion.

  11. CFTR-dependent chloride efflux in cystic fibrosis mononuclear cells is increased by ivacaftor therapy.

    Science.gov (United States)

    Guerra, Lorenzo; D'Oria, Susanna; Favia, Maria; Castellani, Stefano; Santostasi, Teresa; Polizzi, Angela M; Mariggiò, Maria A; Gallo, Crescenzio; Casavola, Valeria; Montemurro, Pasqualina; Leonetti, Giuseppina; Manca, Antonio; Conese, Massimo

    2017-07-01

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) potentiator ivacaftor (Kalydeco®) improves clinical outcome in G551D cystic fibrosis (CF) patients. Here, we have investigated whether ivacaftor has a clinical impact on non-G551D gating mutations and function of circulating leukocytes as well. Seven patients were treated with ivacaftor and evaluated at baseline, and at 1-3 and 6 months. Besides clinical and systemic inflammatory parameters, circulating mononuclear cells (MNC) were evaluated for CFTR-dependent chloride efflux by spectrofluorimetry, neutrophils for oxidative burst by cytofluorimetry and HVCN1 mRNA expression by real time PCR. Ivacaftor determined a significant decrease in sweat chloride concentrations at all time points during treatment. Body mass index (BMI), FEV 1 , and FVC showed an increasing trend. While C-reactive protein decreased significantly at 2 months, the opposite behavior was noticed for circulating monocytes. CFTR activity in MNC was found to increase significantly at 3 and 6 months. Neutrophil oxidative burst peaked at 2 months and then decreased to baseline. HVCN1 mRNA expression was significantly higher than baseline at 1-3 months and decreased after 6 months of treatment. The chloride efflux in MNC correlated positively with both FEV 1 and FVC. On the other hand, sweat chloride correlated positively with CRP and WBC, and negatively with both respiratory function tests. A cluster analysis confirmed that sweat chloride, FEV 1 , FVC, BMI, and MNC chloride efflux behaved as a single entity over time. In patients with non-G551D mutations, ivacaftor improved both chloride transport in sweat ducts and chloride efflux in MNC, that is, functions directly imputed to CFTR. © 2017 Wiley Periodicals, Inc.

  12. A novel FLNC frameshift and an OBSCN variant in a family with distal muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Rossi, Daniela; Palmio, Johanna; EvilaÈ, Anni; Galli, Lucia; Barone, Virginia; Caldwell, Tracy A.; Policke, Rachel A.; Aldkheil, Esraa; Berndsen, Christopher E.; Wright, Nathan T.; Malfatti, Edoardo; Brochier, Guy; Pierantozzi, Enrico; Jordanova, Albena; Guergueltcheva, Velina; Romero, Norma Beatriz; Hackman, Peter; Eymard, Bruno; Udd, Bjarne; Sorrentino, Vincenzo (Antwerp); (U. Sofia); (Siena); (Tampere); (J Madison); (Helsinki)

    2017-10-26

    A novel FLNC c.5161delG (p.Gly1722ValfsTer61) mutation was identified in two members of a French family affected by distal myopathy and in one healthy relative. This FLNC c.5161delG mutation is one nucleotide away from a previously reported FLNC mutation (c.5160delC) that was identified in patients and in asymptomatic carriers of three Bulgarian families with distal muscular dystrophy, indicating a low penetrance of the FLNC frameshift mutations. Given these similarities, we believe that the two FLNC mutations alone can be causative of distal myopathy without full penetrance. Moreover, comparative analysis of the clinical manifestations indicates that patients of the French family show an earlier onset and a complete segregation of the disease. As a possible explanation of this, the two French patients also carry a OBSCN c.13330C>T (p.Arg4444Trp) mutation. The p.Arg4444Trp variant is localized within the OBSCN Ig59 domain that, together with Ig58, binds to the ZIg9/ZIg10 domains of titin at Z-disks. Structural and functional studies indicate that this OBSCN p.Arg4444Trp mutation decreases titin binding by ~15-fold. On this line, we suggest that the combination of the OBSCN p.Arg4444Trp variant and of the FLNC c.5161delG mutation, can cooperatively affect myofibril stability and increase the penetrance of muscular dystrophy in the French family.

  13. Deciphering the role of the Gag-Pol ribosomal frameshift signal in HIV-1 RNA genome packaging.

    Science.gov (United States)

    Nikolaitchik, Olga A; Hu, Wei-Shau

    2014-04-01

    A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5' untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5' end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1

  14. Model of the pathway of −1 frameshifting: Long pausing

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    Ping Xie

    2016-03-01

    Full Text Available It has been characterized that the programmed ribosomal −1 frameshifting often occurs at the slippery sequence on the presence of a downstream mRNA pseudoknot. In some prokaryotic cases such as the dnaX gene of Escherichia coli, an additional stimulatory signal—an upstream, internal Shine–Dalgarno (SD sequence—is also necessary to stimulate the efficient −1 frameshifting. However, the molecular and physical mechanism of the −1 frameshifting is poorly understood. Here, we propose a model of the pathway of the −1 translational frameshifting during ribosome translation of the dnaX −1 frameshift mRNA. With the model, the single-molecule fluorescence data (Chen et al. (2014 [29] on the dynamics of the shunt either to long pausing or to normal translation, the tRNA transit and sampling dynamics in the long-paused rotated state, the EF-G sampling dynamics, the mean rotated-state lifetimes, etc., are explained quantitatively. Moreover, the model is also consistent with the experimental data (Yan et al. (2015 [30] on translocation excursions and broad branching of frameshifting pathways. In addition, we present some predicted results, which can be easily tested by future optical trapping experiments.

  15. The silent codon change I507-ATC→ATT contributes to the severity of the ΔF508 CFTR channel dysfunction

    Science.gov (United States)

    Lazrak, Ahmed; Fu, Lianwu; Bali, Vedrana; Bartoszewski, Rafal; Rab, Andras; Havasi, Viktoria; Keiles, Steve; Kappes, John; Kumar, Ranjit; Lefkowitz, Elliot; Sorscher, Eric J.; Matalon, Sadis; Collawn, James F.; Bebok, Zsuzsanna

    2013-01-01

    The most common disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the out-of-frame deletion of 3 nucleotides (CTT). This mutation leads to the loss of phenylalanine-508 (ΔF508) and a silent codon change (SCC) for isoleucine-507 (I507-ATC→ATT). ΔF508 CFTR is misfolded and degraded by endoplasmic reticulum-associated degradation (ERAD). We have demonstrated that the I507-ATC→ATT SCC alters ΔF508 CFTR mRNA structure and translation dynamics. By comparing the biochemical and functional properties of the I507-ATT and I507-ATC ΔF508 CFTR, we establish that the I507-ATC→ATT SCC contributes to the cotranslational misfolding, ERAD, and to the functional defects associated with ΔF508 CFTR. We demonstrate that the I507-ATC ΔF508 CFTR is less susceptible to the ER quality-control machinery during translation than the I507-ATT, although 27°C correction is necessary for sufficient cell-surface expression. Whole-cell patch-clamp recordings indicate sustained, thermally stable cAMP-activated Cl− transport through I507-ATC and unstable function of the I507-ATT ΔF508 CFTR. Single-channel recordings reveal improved gating properties of the I507-ATC compared to I507-ATT ΔF508 CFTR (NPo=0.45±0.037 vs. NPo=0.09±0.002; P<0.001). Our results signify the role of the I507-ATC→ATT SCC in the ΔF508 CFTR defects and support the importance of synonymous codon choices in determining the function of gene products.—Lazrak, A., Fu, L., Bali, V., Bartoszewski, R., Rab, A., Havasi, V., Keiles, S., Kappes, J., Kumar, R., Lefkowitz, E., Sorscher, E. J., Matalon, S., Collawn, J. F., Bebok, Z. The silent codon change I507-ATC→ATT contributes to the severity of the ΔF508 CFTR. PMID:23907436

  16. Cystic fibrosis transmembrane conductance regulator intracellular processing, trafficking, and opportunities for mutation-specific treatment.

    LENUS (Irish Health Repository)

    Rogan, Mark P

    2012-02-01

    Recent advances in basic science have greatly expanded our understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the chloride and bicarbonate channel that is encoded by the gene, which is mutated in patients with CF. We review the structure, function, biosynthetic processing, and intracellular trafficking of CFTR and discuss the five classes of mutations and their impact on the CF phenotype. The therapeutic discussion is focused on the significant progress toward CFTR mutation-specific therapies. We review the results of encouraging clinical trials examining orally administered therapeutics, including agents that promote read-through of class I mutations (premature termination codons); correctors, which overcome the CFTR misfolding that characterizes the common class II mutation F508del; and potentiators, which enhance the function of class III or IV mutated CFTR at the plasma membrane. Long-term outcomes from successful mutation-specific treatments could finally answer the question that has been lingering since and even before the CFTR gene discovery: Will therapies that specifically restore CFTR-mediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease?

  17. Cystic fibrosis transmembrane conductance regulator (CFTR allelic variants relate to shifts in faecal microbiota of cystic fibrosis patients.

    Directory of Open Access Journals (Sweden)

    Serena Schippa

    Full Text Available INTRODUCTION: In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF. CFTR mutations (F508del is the most common lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. METHODS: Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. RESULTS: Patients were classified by two different criteria: 1 presence/absence of F508del mutation; 2 disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum were reduced. CONCLUSIONS: This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a 'systemic disease', linking the lung and the gut in a joined axis.

  18. ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter.

    Science.gov (United States)

    Phuan, Puay-Wah; Veit, Guido; Tan, Joseph-Anthony; Roldan, Ariel; Finkbeiner, Walter E; Haggie, Peter M; Lukacs, Gergely L; Verkman, Alan S

    2018-03-01

    The most common cystic fibrosis-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (∆F508). The ∆F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ∆F508-CFTR modulators that act on NBD1. A biochemical screen for ΔF508-NBD1 cell surface expression was done in Madin-Darby canine kidney cells expressing a chimeric reporter consisting of ΔF508-NBD1, the CD4 transmembrane domain, and an extracellular horseradish peroxidase (HRP) reporter. Using a luminescence readout of HRP activity, the screen was robust with a Z' factor of 0.7. The screening of ~20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased ∆F508-NBD1 cell surface expression by up to 4-fold; for comparison, a 12-fold increased cell surface expression was found for a wild-type NBD1 chimera. While the compounds were inactive as correctors of full-length ΔF508-CFTR, several carboxamide-benzothiophenes had potentiator activity with low micromolar EC 50 . Interestingly, the potentiators did not activate G551D or wild-type CFTR. Our results provide a proof of concept for a cell-based NBD1 domain screen to identify ∆F508-CFTR modulators that target the NBD1 domain.

  19. Interaction between a novel TGFB1 haplotype and CFTR genotype is associated with improved lung function in cystic fibrosis.

    Science.gov (United States)

    Bremer, Lindsay A; Blackman, Scott M; Vanscoy, Lori L; McDougal, Kathryn E; Bowers, Amanda; Naughton, Kathleen M; Cutler, David J; Cutting, Garry R

    2008-07-15

    Cystic fibrosis (CF), the most common lethal single gene disorder in Caucasians, is due to mutations in the CFTR gene. Twin and sibling analysis indicates that modifier genes, rather than allelic variation in CFTR, are responsible for most of the variability in severity of lung disease, the major cause of mortality in CF patients. We used a family-based approach to test for association between lung function and two functional SNPs (rs1800469, '-509' and rs1982073, 'codon 10') in the 5' region of transforming growth factor-beta1 (TGFB1), a putative CF modifier gene. Quantitative transmission disequilibrium testing of 472 CF patient-parent-parent trios revealed that both TGFB1 SNPs showed significant transmission distortion when patients were stratified by CFTR genotype. Although lung function and nutritional status are correlated in CF patients, there was no evidence of association between the TGFB1 SNPs and variation in nutritional status. Additional tagging SNPs (rs8179181, rs2278422, rs8110090, rs4803455 and rs1982072) that capture most of the diversity in TGFB1 were also typed but none showed association with variation in lung function. However, a haplotype composed of the -509 C and codon 10 T alleles along with the C allele of the 3' SNP rs8179181 was highly associated with increased lung function in patients grouped by CFTR genotype. These results demonstrate that TGFB1 is a modifier of CF lung disease and reveal a previously unrecognized beneficial effect of TGFB1 variants upon the pulmonary phenotype.

  20. New insights into interactions between the nucleotide‐binding domain of CFTR and keratin 8

    Science.gov (United States)

    Premchandar, Aiswarya; Kupniewska, Anna; Bonna, Arkadiusz; Faure, Grazyna; Fraczyk, Tomasz; Roldan, Ariel; Hoffmann, Brice; Faria da Cunha, Mélanie; Herrmann, Harald; Lukacs, Gergely L.

    2017-01-01

    Abstract The intermediate filament protein keratin 8 (K8) interacts with the nucleotide‐binding domain 1 (NBD1) of the cystic fibrosis (CF) transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508‐CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for CF patients bearing the ΔF508 mutation. Here, we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen–deuterium exchange coupled with mass spectrometry (HDX‐MS) on recombinant wild‐type (wt) NBD1 and ΔF508‐NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt‐NBD1 and ΔF508‐NBD1. Finally, we performed HDX‐MS analysis of the NBD1 molecules and full‐length K8, revealing hydrogen‐bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508‐NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1. PMID:27870250

  1. Phenylquinoxalinone CFTR activator as potential prosecretory therapy for constipation.

    Science.gov (United States)

    Cil, Onur; Phuan, Puay-Wah; Son, Jung-Ho; Zhu, Jie S; Ku, Colton K; Tabib, Niloufar Akhavan; Teuthorn, Andrew P; Ferrera, Loretta; Zachos, Nicholas C; Lin, Ruxian; Galietta, Luis J V; Donowitz, Mark; Kurth, Mark J; Verkman, Alan S

    2017-04-01

    Constipation is a common condition for which current treatments can have limited efficacy. By high-throughput screening, we recently identified a phenylquinoxalinone activator of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that stimulated intestinal fluid secretion and normalized stool output in a mouse model of opioid-induced constipation. Here, we report phenylquinoxalinone structure-activity analysis, mechanism of action, animal efficacy data in acute and chronic models of constipation, and functional data in ex vivo primary cultured human enterocytes. Structure-activity analysis was done on 175 phenylquinoxalinone analogs, including 15 synthesized compounds. The most potent compound, CFTR act -J027, activated CFTR with EC 50 ∼ 200 nM, with patch-clamp analysis showing a linear CFTR current-voltage relationship with direct CFTR activation. CFTR act -J027 corrected reduced stool output and hydration in a mouse model of acute constipation produced by scopolamine and in a chronically constipated mouse strain (C3H/HeJ). Direct comparison with the approved prosecretory drugs lubiprostone and linaclotide showed substantially greater intestinal fluid secretion with CFTR act -J027, as well as greater efficacy in a constipation model. As evidence to support efficacy in human constipation, CFTR act -J027 increased transepithelial fluid transport in enteroids generated from normal human small intestine. Also, CFTR act -J027 was rapidly metabolized in vitro in human hepatic microsomes, suggesting minimal systemic exposure upon oral administration. These data establish structure-activity and mechanistic data for phenylquinoxalinone CFTR activators, and support their potential efficacy in human constipation. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

    Directory of Open Access Journals (Sweden)

    Suneeth F Mathew

    Full Text Available HIV-1 utilises -1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating -1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the 'intercodon' contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules-eRF1 protein or a cognate suppressor tRNA-were able to access and decode the intercodon prior to -1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.

  3. Predominant constitutive CFTR conductance in small airways

    Directory of Open Access Journals (Sweden)

    Lytle Christian

    2005-01-01

    Full Text Available Abstract Background The pathological hallmarks of chronic obstructive pulmonary disease (COPD are inflammation of the small airways (bronchiolitis and destruction of lung parenchyma (emphysema. These forms of disease arise from chronic prolonged infections, which are usually never present in the normal lung. Despite the fact that primary hygiene and defense of the airways presumably requires a well controlled fluid environment on the surface of the bronchiolar airway, very little is known of the fluid and electrolyte transport properties of airways of less than a few mm diameter. Methods We introduce a novel approach to examine some of these properties in a preparation of minimally traumatized porcine bronchioles of about 1 mm diameter by microperfusing the intact bronchiole. Results In bilateral isotonic NaCl Ringer solutions, the spontaneous transepithelial potential (TEP; lumen to bath of the bronchiole was small (mean ± sem: -3 ± 1 mV; n = 25, but when gluconate replaced luminal Cl-, the bionic Cl- diffusion potentials (-58 ± 3 mV; n = 25 were as large as -90 mV. TEP diffusion potentials from 2:1 NaCl dilution showed that epithelial Cl- permeability was at least 5 times greater than Na+ permeability. The anion selectivity sequence was similar to that of CFTR. The bionic TEP became more electronegative with stimulation by luminal forskolin (5 μM+IBMX (100 μM, ATP (100 μM, or adenosine (100 μM, but not by ionomycin. The TEP was partially inhibited by NPPB (100 μM, GlyH-101* (5–50 μM, and CFTRInh-172* (5 μM. RT-PCR gave identifying products for CFTR, α-, β-, and γ-ENaC and NKCC1. Antibodies to CFTR localized specifically to the epithelial cells lining the lumen of the small airways. Conclusion These results indicate that the small airway of the pig is characterized by a constitutively active Cl- conductance that is most likely due to CFTR.

  4. Mutasi titik hingga mutasi frameshift gen INSR exon 22 pada pasien penderita diabetes mellitus

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    Fatchiyah Fatchiyah

    2012-02-01

    Full Text Available Mutations of human insulin and insulin receptor family can lead autosomal dominant syndrome on diabetes, fasting hyperinsulinemia, and insulin receptor family can lead autosomal dominant syndrome on diabetes, fasting hyperinsulinemia, and insulin resistant. The aim of this research was to identify mutation types of hINSR gene exon 22 which mutation hot spot region. To analyze hINSR gene exon 22 of DM patient and control, we isolated DNA from their blood. DNA was then amplified by PCR using a set of primer for exon 22. PCR product was sequenced by Sequencer and nucleotide sequence analyzed by BLAST analysis. According to Gene Bank database, hINSR gene has two variant with Gene ID 3643, at chromosome 19p13.3-p13.2, and has 22 exons with mRNA 4200bp. The result of research showed that the mutation types of hINS gene exon 22 of DM patients are point mutation, single base deletion and substitution. We found mutation of single deletion at Met eletion at Met1295 Cys1295 and Glut1300 Gly1300, also point mutation are at Met1296 Ser1296 and Trp1299 Ala1299 and Met1389 Iso1389. Because these two deletion are so close, the polypeptids sequence of these changed as frameshift mutation, normal IR has six amino acids -Met Arg Met Cys rp lut- and DM patient has differed the five amino acids - Cys Ala Ser Ala Gly. According to the mutation of DM patient, the IR protein function against tyrosine kinase become abnormal, perhaps its were correlated with genetic syndrom genetic syndrome of insulin resistance. e of insulin resistance.

  5. Amniotic Mesenchymal Stem Cells: A New Source for Hepatocyte-Like Cells and Induction of CFTR Expression by Coculture with Cystic Fibrosis Airway Epithelial Cells

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    Valentina Paracchini

    2012-01-01

    Full Text Available Cystic fibrosis (CF is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic lineages. Moreover, hAMSCs were induced to differentiate into hepatocyte-like cells, as demonstrated by mixed function oxidase activity and expression of albumin, alpha1-antitrypsin, and CK19. We also investigated the CFTR expression in hAMSCs upon isolation and in coculture with CF airway epithelial cells. Freshly isolated hAMSCs displayed low levels of CFTR mRNA, which even decreased with culture passages. Following staining with the vital dye CM-DiI, hAMSCs were mixed with CFBE41o- respiratory epithelial cells and seeded onto permeable filters. Flow cytometry demonstrated that 33–50% of hAMSCs acquired a detectable CFTR expression on the apical membrane, a result confirmed by confocal microscopy. Our data show that amniotic MSCs have the potential to differentiate into epithelial cells of organs relevant in CF pathogenesis and may contribute to partial correction of the CF phenotype.

  6. Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis.

    Directory of Open Access Journals (Sweden)

    Scott Mackenzie Brockman

    Full Text Available Cystic Fibrosis (CF is a genetic disorder caused by mutation(s in the CF-transmembrane conductance regulator (Cftr gene. The most common mutation, ΔF508, leads to accumulation of defective-CFTR protein in aggresome-bodies. Additionally, Pseudomonas aeruginosa (Pa, a common CF pathogen, exacerbates obstructive CF lung pathology. In the present study, we aimed to develop and test a novel strategy to improve the bioavailability and potentially achieve targeted drug delivery of cysteamine, a potent autophagy-inducing drug with anti-bacterial properties, by developing a dendrimer (PAMAM-DEN-based cysteamine analogue.We first evaluated the effect of dendrimer-based cysteamine analogue (PAMAM-DENCYS on the intrinsic autophagy response in IB3-1 cells and observed a significant reduction in Ub-RFP and LC3-GFP co-localization (aggresome-bodies by PAMAM-DENCYS treatment as compared to plain dendrimer (PAMAM-DEN control. Next, we observed that PAMAM-DENCYS treatment shows a modest rescue of ΔF508-CFTR as the C-form. Moreover, immunofluorescence microscopy of HEK-293 cells transfected with ΔF508-CFTR-GFP showed that PAMAM-DENCYS is able to rescue the misfolded-ΔF508-CFTR from aggresome-bodies by inducing its trafficking to the plasma membrane. We further verified these results by flow cytometry and observed significant (p<0.05; PAMAM-DEN vs. PAMAM-DENCYS rescue of membrane-ΔF508-CFTR with PAMAM-DENCYS treatment using non-permeabilized IB3-1 cells immunostained for CFTR. Finally, we assessed the autophagy-mediated bacterial clearance potential of PAMAM-DENCYS by treating IB3-1 cells infected with PA01-GFP, and observed a significant (p<0.01; PAMAM-DEN vs. PAMAM-DENCYS decrease in intracellular bacterial counts by immunofluorescence microscopy and flow cytometry. Also, PAMAM-DENCYS treatment significantly inhibits the growth of PA01-GFP bacteria and demonstrates potent mucolytic properties.We demonstrate here the efficacy of dendrimer-based autophagy

  7. Nutritional Status Improved in Cystic Fibrosis Patients with the G551D Mutation After Treatment with Ivacaftor

    NARCIS (Netherlands)

    Borowitz, Drucy; Lubarsky, Barry; Wilschanski, Michael; Munck, Anne; Gelfond, Daniel; Bodewes, Frank; Schwarzenberg, Sarah Jane

    The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gating mutation G551D prevents sufficient ion transport due to reduced channel-open probability. Ivacaftor, an oral CFTR potentiator, increases the channel-open probability. To further analyze improvements in weight and body mass

  8. Structural diversity of frameshifting signals : reprogramming the programmed

    NARCIS (Netherlands)

    Yu, Chien-Hung

    2011-01-01

    Programmed ribosomal frameshifting (PRF) is one kind of recoding events that is mostly utilized by RNA viruses to synthesize more proteins with defined ratio from their compact genome and it is known that the stoichiometric is critical to virus infection and propagation. Two cis-acting RNA elements

  9. Chemical rescue of ΔF508-CFTR in C127 epithelial cells reverses aberrant extracellular pH acidification to wild-type alkalization as monitored by microphysiometry.

    Science.gov (United States)

    Luckie, Douglas B; Van Alst, Andrew J; Massey, Marija K; Flood, Robert D; Shah, Aashish A; Malhotra, Vishal; Kozel, Bradley J

    2014-09-05

    Cystic fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel expressed in apical membranes of wet epithelia. Since CFTR is permeable to HCO3(-), and may regulate bicarbonate exchangers, it is not surprising evidence of changes in extracellular pH (pHo) have been found in CF. Previously we have shown that tracking pHo can be used to differentiate cells expressing wild-type CFTR from controls in mouse mammary epithelial (C127) and fibroblast (NIH/3T3) cell lines. In this study we characterized forskolin-stimulated extracellular acidification rates in epithelia where chemical correction of mutant ΔF508-CFTR converted an aberrant response in acidification (10%+ increase) to wild-type (25%+ decrease). Thus treatment with corrector (10% glycerol) and the resulting increased expression of ΔF508-CFTR at the surface was detected by microphysiometry as a significant reversal from acidification to alkalization of pHo. These results suggest that CFTR activation as well as correction can be detected by carefully monitoring pHo and support findings in the field that extracellular pH acidification may impact the function of airway surface liquid in CF. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. The CFTR-Associated Ligand Arrests the Trafficking of the Mutant ΔF508 CFTR Channel in the ER Contributing to Cystic Fibrosis

    Directory of Open Access Journals (Sweden)

    Emily Bergbower

    2018-01-01

    Full Text Available Background/Aims: The CFTR-Associated Ligand (CAL, a PDZ domain containing protein with two coiled-coil domains, reduces cell surface WT CFTR through degradation in the lysosome by a well-characterized mechanism. However, CAL’s regulatory effect on ΔF508 CFTR has remained almost entirely uninvestigated. Methods: In this study, we describe a previously unknown pathway for CAL by which it regulates the membrane expression of ΔF508 CFTR through arrest of ΔF508 CFTR trafficking in the endoplasmic reticulum (ER using a combination of cell biology, biochemistry and electrophysiology. Results: We demonstrate that CAL is an ER localized protein that binds to ΔF508 CFTR and is degraded in the 26S proteasome. When CAL is inhibited, ΔF508 CFTR retention in the ER decreases and cell surface expression of mature functional ΔF508 CFTR is observed alongside of enhanced expression of plasma membrane scaffolding protein NHERF1. Chaperone proteins regulate this novel process, and ΔF508 CFTR binding to HSP40, HSP90, HSP70, VCP, and Aha1 changes to improve ΔF508 CFTR cell surface trafficking. Conclusion: Our results reveal a pathway in which CAL regulates the cell surface availability and intracellular retention of ΔF508 CFTR.

  11. High frequency of +1 programmed ribosomal frameshifting in Euplotes octocarinatus

    OpenAIRE

    Wang, Ruanlin; Xiong, Jie; Wang, Wei; Miao, Wei; Liang, Aihua

    2016-01-01

    Programmed ?1 ribosomal frameshifting (?1 PRF) has been identified as a mechanism to regulate the expression of many viral genes and some cellular genes. The slippery site of ?1 PRF has been well characterized, whereas the +1 PRF signal and the mechanism involved in +1 PRF remain poorly understood. Previous study confirmed that +1 PRF is required for the synthesis of protein products in several genes of ciliates from the genus Euplotes. To accurately assess the frequency of genes requiring fr...

  12. An Expanded CAG Repeat in Huntingtin Causes +1 Frameshifting*

    OpenAIRE

    Saffert, Paul; Adamla, Frauke; Schieweck, Rico; Atkins, John F.; Ignatova, Zoya

    2016-01-01

    Maintenance of triplet decoding is crucial for the expression of functional protein because deviations either into the −1 or +1 reading frames are often non-functional. We report here that expression of huntingtin (Htt) exon 1 with expanded CAG repeats, implicated in Huntington pathology, undergoes a sporadic +1 frameshift to generate from the CAG repeat a trans-frame AGC repeat-encoded product. This +1 recoding is exclusively detected in pathological Htt variants, i.e. those with expanded re...

  13. Physiological Adaptation of the Bacterium Lactococcus lactis in Response to the Production of Human CFTR

    NARCIS (Netherlands)

    Steen, Anton; Wiederhold, Elena; Gandhi, Tejas; Breitling, Rainer; Slotboom, Dirk Jan

    Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus

  14. Screening for calreticulin mutations in a cohort of patients suspected ...

    African Journals Online (AJOL)

    Of the 36 types of insertions and deletions identified, type 1 (a. 52-base pair deletion) and type 2 (a 5-base pair insertion) mutations account for >80% of CALR mutations.[7] Phenotypic differences between type 1 and type 2 carriers have been implicated. [3] All recurrent mutations cause a frameshift in the region encoding.

  15. Gene expression patterns of chicken neuregulin 3 in association with copy number variation and frameshift deletion.

    Science.gov (United States)

    Abe, Hideaki; Aoya, Daiki; Takeuchi, Hiro-Aki; Inoue-Murayama, Miho

    2017-07-21

    NRG3 are structural mutations that occurred before the establishment of commercial chicken lines. Our results further suggest that the putative frameshift deletion in exon 2 may potentially affect the expression level of particular isoforms of chicken NRG3.

  16. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    Energy Technology Data Exchange (ETDEWEB)

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun (Cystic); (UAB); (JHU); (Columbia); (Lilly)

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  17. A Frameshift in CSF2RB Predominant Among Ashkenazi Jews Increases Risk for Crohn's Disease and Reduces Monocyte Signaling via GM-CSF.

    Science.gov (United States)

    Chuang, Ling-Shiang; Villaverde, Nicole; Hui, Ken Y; Mortha, Arthur; Rahman, Adeeb; Levine, Adam P; Haritunians, Talin; Evelyn Ng, Sok Meng; Zhang, Wei; Hsu, Nai-Yun; Facey, Jody-Ann; Luong, Tramy; Fernandez-Hernandez, Heriberto; Li, Dalin; Rivas, Manuel; Schiff, Elena R; Gusev, Alexander; Schumm, L Phillip; Bowen, Beatrice M; Sharma, Yashoda; Ning, Kaida; Remark, Romain; Gnjatic, Sacha; Legnani, Peter; George, James; Sands, Bruce E; Stempak, Joanne M; Datta, Lisa W; Lipka, Seth; Katz, Seymour; Cheifetz, Adam S; Barzilai, Nir; Pontikos, Nikolas; Abraham, Clara; Dubinsky, Marla J; Targan, Stephan; Taylor, Kent; Rotter, Jerome I; Scherl, Ellen J; Desnick, Robert J; Abreu, Maria T; Zhao, Hongyu; Atzmon, Gil; Pe'er, Itsik; Kugathasan, Subra; Hakonarson, Hakon; McCauley, Jacob L; Lencz, Todd; Darvasi, Ariel; Plagnol, Vincent; Silverberg, Mark S; Muise, Aleixo M; Brant, Steven R; Daly, Mark J; Segal, Anthony W; Duerr, Richard H; Merad, Miriam; McGovern, Dermot P B; Peter, Inga; Cho, Judy H

    2016-10-01

    Crohn's disease (CD) has the highest prevalence in Ashkenazi Jewish populations. We sought to identify rare, CD-associated frameshift variants of high functional and statistical effects. We performed exome sequencing and array-based genotype analyses of 1477 Ashkenazi Jewish individuals with CD and 2614 Ashkenazi Jewish individuals without CD (controls). To validate our findings, we performed genotype analyses of an additional 1515 CD cases and 7052 controls for frameshift mutations in the colony-stimulating factor 2-receptor β common subunit gene (CSF2RB). Intestinal tissues and blood samples were collected from patients with CD; lamina propria leukocytes were isolated and expression of CSF2RB and granulocyte-macrophage colony-stimulating factor-responsive cells were defined by adenomatous polyposis coli (APC) time-of-flight mass cytometry (CyTOF analysis). Variants of CSF2RB were transfected into HEK293 cells and the expression and functions of gene products were compared. In the discovery cohort, we associated CD with a frameshift mutation in CSF2RB (P = 8.52 × 10(-4)); the finding was validated in the replication cohort (combined P = 3.42 × 10(-6)). Incubation of intestinal lamina propria leukocytes with granulocyte-macrophage colony-stimulating factor resulted in high levels of phosphorylation of signal transducer and activator of transcription (STAT5) and lesser increases in phosphorylation of extracellular signal-regulated kinase and AK straining transforming (AKT). Cells co-transfected with full-length and mutant forms of CSF2RB had reduced pSTAT5 after stimulation with granulocyte-macrophage colony-stimulating factor, compared with cells transfected with control CSF2RB, indicating a dominant-negative effect of the mutant gene. Monocytes from patients with CD who were heterozygous for the frameshift mutation (6% of CD cases analyzed) had reduced responses to granulocyte-macrophage colony-stimulating factor and markedly decreased activity of

  18. CFTR, Mucins, and Mucus Obstruction in Cystic Fibrosis

    Science.gov (United States)

    Kreda, Silvia M.; Davis, C. William; Rose, Mary Callaghan

    2012-01-01

    Mucus pathology in cystic fibrosis (CF) has been known for as long as the disease has been recognized and is sometimes called mucoviscidosis. The disease is marked by mucus hyperproduction and plugging in many organs, which are usually most fatal in the airways of CF patients, once the problem of meconium ileus at birth is resolved. After the CF gene, CFTR, was cloned and its protein product identified as a cAMP-regulated Cl− channel, causal mechanisms underlying the strong mucus phenotype of the disease became obscure. Here we focus on mucin genes and polymeric mucin glycoproteins, examining their regulation and potential relationships to a dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR). Detailed examination of CFTR expression in organs and different cell types indicates that changes in CFTR expression do not always correlate with the severity of CF disease or mucus accumulation. Thus, the mucus hyperproduction that typifies CF does not appear to be a direct cause of a defective CFTR but, rather, to be a downstream consequence. In organs like the lung, up-regulation of mucin gene expression by inflammation results from chronic infection; however, in other instances and organs, the inflammation may have a non-infectious origin. The mucus plugging phenotype of the β-subunit of the epithelial Na+ channel (βENaC)-overexpressing mouse is proving to be an archetypal example of this kind of inflammation, with a dehydrated airway surface/concentrated mucus gel apparently providing the inflammatory stimulus. Data indicate that the luminal HCO3 − deficiency recently described for CF epithelia may also provide such a stimulus, perhaps by causing a mal-maturation of mucins as they are released onto luminal surfaces. In any event, the path between CFTR dysfunction and mucus hyperproduction has proven tortuous, and its unraveling continues to offer its own twists and turns, along with fascinating glimpses into biology. PMID:22951447

  19. Reduced PDZ Interactions of Rescued ΔF508CFTR Increases Its Cell Surface Mobility*

    Science.gov (United States)

    Valentine, Cathleen D.; Lukacs, Gergely L.; Verkman, Alan S.; Haggie, Peter M.

    2012-01-01

    Deletion of phenylalanine 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). Though several maneuvers can rescue endoplasmic reticulum-retained ΔF508CFTR and promote its trafficking to the plasma membrane, rescued ΔF508CFTR remains susceptible to quality control mechanisms that lead to accelerated endocytosis, ubiquitination, and lysosomal degradation. To investigate the role of scaffold protein interactions in rescued ΔF508CFTR surface instability, the plasma membrane mobility of ΔF508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, ΔF508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Knock-down of the plasma membrane quality control proteins CHIP and Hsc70 partially restored ΔF508CFTR-scaffold association. Quantitative comparisons of CFTR cell surface diffusion and endocytosis kinetics suggested an association between reduced scaffold binding and CFTR internalization. Our surface diffusion measurements in live cells indicate defective scaffold interactions of rescued ΔF508CFTR at the cell surface, which may contribute to its defective peripheral processing. PMID:23115232

  20. Mutation induction in Haemophilus influenzae by ICR-191. Pt. 1

    International Nuclear Information System (INIS)

    Perdue, S.W.; Kimball, R.F.; McGray, P.C.; Tennessee Univ., Oak Ridge

    1981-01-01

    The investigation of mutagenic mechanisms in Haemophilus influenzae has been confined until now to mutagens that normally produce mainly base pair substitutions. This paper describes the development of a system suitable for detecting frameshift mutations induced by ICR-191. The system involves reversions from thymidine dependence to thymidine independence. Evidence is presented from a comparison of the responses to ICR-191 and to N-methyl-N'-nitro-N-nitrosoguanidine that the system is specific for frameshift mutations. The genetic recombination involved in transformation leads to a marked increase in spontaneous reversion of the frameshift mutations but not of the base substitution mutations. Presumably, this is a consequence of mispairing, with consequent change in the number of bases, during the recombination. (orig.)

  1. Partial correction of the CFTR-dependent ABPA mouse model with recombinant adeno-associated virus gene transfer of truncated CFTR gene.

    Science.gov (United States)

    Mueller, Christian; Torrez, Daniel; Braag, Sofia; Martino, Ashley; Clarke, Tracy; Campbell-Thompson, Martha; Flotte, Terence R

    2008-01-01

    Recently, we have developed a model of airway inflammation in a CFTR knockout mouse utilizing Aspergillus fumigatus crude protein extract (Af-cpe) to mimic allergic bronchopulmonary aspergillosis (ABPA) 1, an unusual IgE-mediated hypersensitivity syndrome seen in up to 15% of cystic fibrosis (CF) patients and rarely elsewhere. We hypothesized that replacement of CFTR via targeted gene delivery to airway epithelium would correct aberrant epithelial cytokine signaling and ameliorate the ABPA phenotype in CFTR-deficient (CFTR 489X - /-, FABP-hCFTR + / +) mice. CFTR knockout mice underwent intra-tracheal (IT) delivery of recombinant adeno-associated virus serotype 5 (rAAV5Delta-264CFTR) or rAAV5-GFP at 2.58 x 10(12) viral genomes/mouse. All mice were then sensitized with two serial injections (200 microg) of crude Af antigen via the intra-peritoneal (IP) route. Untreated mice were sensitized without virus exposure. Challenges were performed 2 weeks after final sensitization, using a 0.25% solution containing Aspergillus fumigatus crude protein extract delivered by inhalation on three consecutive days. The rAAV5Delta-264CFTR-treated mice had lower total serum IgE levels (172513 ng/ml +/- 1312) than rAAV5-GFP controls (26 892 ng/ml +/- 3715) (p = 0.037) and non-treated, sensitized controls (24 816 +/- 4219 ng/ml). Serum IgG1 levels also were lower in mice receiving the CFTR vector. Interestingly, splenocytes from rAAV5Delta-264CFTR-treated mice secreted less IL-13, INFg, TNFa, RANTES and GM-CSF after ConA stimulation. Gene therapy with rAAV5Delta-264CFTR attenuated the hyper-IgE response in this reproducible CF mouse model of ABPA, with systemic effects also evident in the cytokine response of stimulated splenocytes. (c) 2007 John Wiley & Sons, Ltd.

  2. CFTR expression and organ damage in cystic fibrosis

    Energy Technology Data Exchange (ETDEWEB)

    Tizzano, E. [Hospital de la Santa Creu I Sant Pau, Barcelona (Spain); Chitayat, D.; Buchwald, M. [Hospital for Sick Children, Ontario (Canada)

    1994-09-01

    To assist our understanding of the origin of organ damage caused by cystic fibrosis (CF) disease, we have analyzed the pattern of expression of the CF gene (CFTR). mRNA in situ hybridization analysis was carried out in human fetal, newborn, infant and adult tissues and the abundance of the mRNA was correlated with the known pathology at the various stages of human development. Analysis of the pattern of expression indicates a constitutive level of mRNA in gastrointestinal tissues starting during early development and maintained throughout life. Prenatal respiratory tissues show qualitative and quantitative major differences in comparison to postnatal lung samples. Male reproductive tissues show high levels of expression in the head of the epididymis compared with the rest of the male ducts. Female reproductive tissues show a variable pattern of expression at different stages during fetal development and during puberty probably due to changes in hormonal levels. Gastrointestinal and male reproductive tissues have a consistent pathology at birth, whereas no lung abnormalities have been described in newborns affected by CF. Our results show that there is no exact correlations between organ damage present at birth and the degree of CFTR expression. To explain these observations, we hypothesize that the pathogenesis of organ damage in CF depend on at least three factors: the rate of CFTR-mediated fluid secretion, differences in genotype and environmental factors, such as the amount of macromolecules in the lumen of the ducts. This last element predicts that damage will occur in tissues with high protein loads and low flow rates (e.g. pancreas, epididymis), where the absence of CFTR function leads to obstruction and pathology. Organs that express CFTR but with no significant damage (e.g. prenatal lung, female reproductive tissues), will have a low protein load and a high flow rates.

  3. Efficacy and safety of lumacaftor/ivacaftor combination therapy in patients with cystic fibrosis homozygous for Phe508del CFTR by pulmonary function subgroup: a pooled analysis.

    Science.gov (United States)

    Elborn, J Stuart; Ramsey, Bonnie W; Boyle, Michael P; Konstan, Michael W; Huang, Xiaohong; Marigowda, Gautham; Waltz, David; Wainwright, Claire E

    2016-08-01

    Lumacaftor/ivacaftor combination therapy has shown clinical benefits in patients with cystic fibrosis homozygous for the Phe508del CFTR mutation; however, pretreatment lung function is a confounding factor that potentially affects the efficacy and safety of this therapy. We aimed to assess the efficacy and safety of lumacaftor/ivacaftor therapy in these patients, defined by specific categories of lung function. Both trials (TRAFFIC and TRANSPORT) included in this pooled analysis were multinational, randomised, double-blind, placebo-controlled, parallel-group, phase 3 studies. Eligible patients from 187 participating centres in North America, Australia, and the European Union (both trials) were aged 12 years or older with a confirmed diagnosis of cystic fibrosis, homozygous for the Phe508del CFTR mutation, and with a percent predicted FEV1 (ppFEV1) of 40-90 at the time of screening. Patients were randomly assigned with an interactive web response system (1:1:1) to receive placebo, lumacaftor (600 mg once daily) plus ivacaftor (250 mg every 12 h), or lumacaftor (400 mg every 12 h) plus ivacaftor (250 mg every 12 h) for 24 weeks. Prespecified subgroup analyses of pooled efficacy and safety data by lung function, as measured by ppFEV1, were done for patients with baseline ppFEV1 (cystic fibrosis homozygous for Phe508del CFTR who have varying degrees of lung function impairment. Vertex Pharmaceuticals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Programmed Ribosomal Frameshifting Mediates Expression of the α-Carboxysome.

    Science.gov (United States)

    Chaijarasphong, Thawatchai; Nichols, Robert J; Kortright, Kaitlyn E; Nixon, Charlotte F; Teng, Poh K; Oltrogge, Luke M; Savage, David F

    2016-01-16

    Many bacteria employ a protein organelle, the carboxysome, to catalyze carbon dioxide fixation in the Calvin Cycle. Only 10 genes from Halothiobacillus neapolitanus are sufficient for heterologous expression of carboxysomes in Escherichia coli, opening the door to detailed mechanistic analysis of the assembly process of this complex (more than 200MDa). One of these genes, csoS2, has been implicated in assembly but ascribing a molecular function is confounded by the observation that the single csoS2 gene yields expression of two gene products and both display an apparent molecular weight incongruent with the predicted amino acid sequence. Here, we elucidate the co-translational mechanism responsible for the expression of the two protein isoforms. Specifically, csoS2 was found to possess -1 frameshifting elements that lead to the production of the full-length protein, CsoS2B, and a truncated protein, CsoS2A, which possesses a C-terminus translated from the alternate frame. The frameshifting elements comprise both a ribosomal slippery sequence and a 3' secondary structure, and ablation of either sequence is sufficient to eliminate the slip. Using these mutants, we investigated the individual roles of CsoS2B and CsoS2A on carboxysome formation. In this in vivo formation assay, cells expressing only the CsoS2B isoform were capable of producing intact carboxysomes, while those with only CsoS2A were not. Thus, we have answered a long-standing question about the nature of CsoS2 in this model microcompartment and demonstrate that CsoS2B is functionally distinct from CsoS2A in the assembly of α-carboxysomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Evaluation of point mutations in dystrophin gene in Iranian ...

    Indian Academy of Sciences (India)

    Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants. [Haghshenas M., Akbari M. T., Zare Karizi S., Khordadpoor Deilamani F., Nafissi S. and Salehi Z. 2016 Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular ...

  6. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    International Nuclear Information System (INIS)

    Marasini, Carlotta; Galeno, Lauretta; Moran, Oscar

    2012-01-01

    Highlights: ► CFTR mutations produce cystic fibrosis. ► Chloride transport depends on the regulatory domain phosphorylation. ► Regulatory domain is intrinsically disordered. ► Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and β-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of α-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two conditions, monitoring the changes of the mean residue ellipticity measured at 222 nm as a function of temperature

  7. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    Energy Technology Data Exchange (ETDEWEB)

    Marasini, Carlotta, E-mail: marasini@ge.ibf.cnr.it [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy); Galeno, Lauretta; Moran, Oscar [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer CFTR mutations produce cystic fibrosis. Black-Right-Pointing-Pointer Chloride transport depends on the regulatory domain phosphorylation. Black-Right-Pointing-Pointer Regulatory domain is intrinsically disordered. Black-Right-Pointing-Pointer Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and {beta}-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of {alpha}-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two

  8. Overlapping genetic codes for overlapping frameshifted genes in Testudines, and Lepidochelys olivacea as special case.

    Science.gov (United States)

    Seligmann, Hervé

    2012-12-01

    Mitochondrial genes code for additional proteins after +2 frameshifts by reassigning stops to code for amino acids, which defines overlapping genetic codes for overlapping genes. Turtles recode stops UAR → Trp and AGR → Lys (AGR → Gly in the marine Olive Ridley turtle, Lepidochelys olivacea). In Lepidochelys the +2 frameshifted mitochondrial Cytb gene lacks stops, open reading frames from other genes code for unknown proteins, and for regular mitochondrial proteins after frameshifts according to the overlapping genetic code. Lepidochelys' inversion between proteins coded by regular and overlapping genetic codes substantiates the existence of overlap coding. ND4 differs among Lepidochelys mitochondrial genomes: it is regular in DQ486893; in NC_011516, the open reading frame codes for another protein, the regular ND4 protein is coded by the frameshifted sequence reassigning stops as in other turtles. These systematic patterns are incompatible with Genbank/sequencing errors and DNA decay. Random mixing of synonymous codons, conserving main frame coding properties, shows optimization of natural sequences for overlap coding; Ka/Ks analyses show high positive (directional) selection on overlapping genes. Tests based on circular genetic codes confirm programmed frameshifts in ND3 and ND4l genes, and predicted frameshift sites for overlap coding in Lepidochelys. Chelonian mitochondria adapt for overlapping gene expression: cloverleaf formation by antisense tRNAs with predicted anticodons matching stops coevolves with overlap coding; antisense tRNAs with predicted expanded anticodons (frameshift suppressor tRNAs) associate with frameshift-coding in ND3 and ND4l, a potential regulation of frameshifted overlap coding. Anaeroby perhaps switched between regular and overlap coding genes in Lepidochelys. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Screening for BRCA2 mutations in 81 Dutch breast-ovarian cancer families

    NARCIS (Netherlands)

    Peelen, T.; van Vliet, M.; Bosch, A.; Bignell, G.; Vasen, H. F.; Klijn, J. G.; Meijers-Heijboer, H.; Stratton, M.; van Ommen, G. J.; Cornelisse, C. J.; Devilee, P.

    2000-01-01

    We have analysed 81 families with a history of breast and/or ovarian cancer for the presence of germline mutations in BRCA2 with a number of different mutation screening techniques. The protein truncation test (PTT) for exons 10 and 11 detected four different frame-shifting mutations in six of these

  10. Efficacy and safety of lumacaftor and ivacaftor in patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR: a randomised, placebo-controlled phase 3 trial.

    Science.gov (United States)

    Ratjen, Felix; Hug, Christopher; Marigowda, Gautham; Tian, Simon; Huang, Xiaohong; Stanojevic, Sanja; Milla, Carlos E; Robinson, Paul D; Waltz, David; Davies, Jane C

    2017-07-01

    Lumacaftor and ivacaftor combination treatment showed efficacy in patients aged 12 years or older with cystic fibrosis homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in placebo-controlled studies and patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR in an open-label study. We report efficacy and safety of lumacaftor and ivacaftor in patients with cystic fibrosis aged 6-11 years homozygous for F508del-CFTR. In this phase 3, randomised, double-blind, placebo-controlled, multicentre study, patients were enrolled at 54 hospitals and medical centres in nine countries (the USA, Australia, Belgium, Canada, Denmark, France, Germany, Sweden, and the UK). Eligible patients weighed at least 15 kg, with a confirmed diagnosis of cystic fibrosis, percent predicted forced expiratory volume in 1 s (FEV 1 ) of 70 or more, and lung clearance index 2·5 (LCI 2·5 ) of 7·5 or more at screening (values less than these thresholds were permitted at day 1). All patients were tested for CFTR genotype at screening; eligible patients had to have the F508del-CFTR mutation on both alleles. Exclusion criteria included any comorbidity or laboratory abnormality that might confound the study results or pose additional risk to the patient. Patients were stratified by weight (cystic fibrosis homozygous for F508del-CFTR. The overall safety profile was consistent with previous phase 3 studies of lumacaftor and ivacaftor. Vertex Pharmaceuticals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. A mild form of SLC29A3 disorder: a frameshift deletion leads to the paradoxical translation of an otherwise noncoding mRNA splice variant.

    Directory of Open Access Journals (Sweden)

    Alexandre Bolze

    Full Text Available We investigated two siblings with granulomatous histiocytosis prominent in the nasal area, mimicking rhinoscleroma and Rosai-Dorfman syndrome. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous frameshift deletion in SLC29A3, which encodes human equilibrative nucleoside transporter-3 (hENT3. Germline mutations in SLC29A3 have been reported in rare patients with a wide range of overlapping clinical features and inherited disorders including H syndrome, pigmented hypertrichosis with insulin-dependent diabetes, and Faisalabad histiocytosis. With the exception of insulin-dependent diabetes and mild finger and toe contractures in one sibling, the two patients with nasal granulomatous histiocytosis studied here displayed none of the many SLC29A3-associated phenotypes. This mild clinical phenotype probably results from a remarkable genetic mechanism. The SLC29A3 frameshift deletion prevents the expression of the normally coding transcripts. It instead leads to the translation, expression, and function of an otherwise noncoding, out-of-frame mRNA splice variant lacking exon 3 that is eliminated by nonsense-mediated mRNA decay (NMD in healthy individuals. The mutated isoform differs from the wild-type hENT3 by the modification of 20 residues in exon 2 and the removal of another 28 amino acids in exon 3, which include the second transmembrane domain. As a result, this new isoform displays some functional activity. This mechanism probably accounts for the narrow and mild clinical phenotype of the patients. This study highlights the 'rescue' role played by a normally noncoding mRNA splice variant of SLC29A3, uncovering a new mechanism by which frameshift mutations can be hypomorphic.

  12. Expanding the Mutation Spectrum of Ichthyosis with Confetti.

    Science.gov (United States)

    Lim, Young H; Choate, Keith A

    2016-10-01

    Ichthyosis with confetti is a rare, autosomal dominant disorder caused by frameshift mutations in KRT10 or KRT1 and characterized by the development of white, genetically revertant macules in red, diseased skin. All cases result from mutations affecting the tail domains of keratin-10 or keratin-1, and Suzuki et al. expand the mutation spectrum for ichthyosis with confetti caused by mutations in KRT1, showing that a polyarginine frameshift in the keratin-1 tail can also cause this disorder. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, H.A.; Wang, C.; Zhao, X.; Hamuro, Y.; Conners, K.; Kearins, M.C.; Lu, F.; Sauder, J.M.; Molnar, K.S.; Coales, S.J.; Maloney, P.C.; Guggino, W.B.; Wetmore, D.R.; Weber, P.C.; Hunt, J.F. (SGX); (ExSAR); (Cystic); (JHU-MED); (Columbia)

    2012-04-30

    The {Delta}F508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and {Delta}F508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because {Delta}F508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and {Delta}F508 constructs, and the {Delta}F508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide {sup 1}H/{sup 2}H exchange rates in matched F508 and {Delta}F508 constructs reveal that {Delta}F508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the {Delta}F508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-{Delta}F508 structures but completely solvent exposed in all {Delta}F508 structures. These results reinforce the importance of the perturbation {Delta}F508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased

  14. HIV-1 and Human PEG10 Frameshift Elements Are Functionally Distinct and Distinguished by Novel Small Molecule Modulators.

    Directory of Open Access Journals (Sweden)

    Tony S Cardno

    Full Text Available Frameshifting during translation of viral or in rare cases cellular mRNA results in the synthesis of proteins from two overlapping reading frames within the same mRNA. In HIV-1 the protease, reverse transcriptase, and integrase enzymes are in a second reading frame relative to the structural group-specific antigen (gag, and their synthesis is dependent upon frameshifting. This ensures that a strictly regulated ratio of structural proteins and enzymes, which is critical for HIV-1 replication and viral infectivity, is maintained during protein synthesis. The frameshift element in HIV-1 RNA is an attractive target for the development of a new class of anti HIV-1 drugs. However, a number of examples are now emerging of human genes using -1 frameshifting, such as PEG10 and CCR5. In this study we have compared the HIV-1 and PEG10 frameshift elements and shown they have distinct functional characteristics. Frameshifting occurs at several points within each element. Moreover, frameshift modulators that were isolated by high-throughput screening of a library of 114,000 lead-like compounds behaved differently with the PEG10 frameshift element. The most effective compounds affecting the HIV-1 element enhanced frameshifting by 2.5-fold at 10 μM in two different frameshift reporter assay systems. HIV-1 protease:gag protein ratio was affected by a similar amount in a specific assay of virally-infected cultured cell, but the modulation of frameshifting of the first-iteration compounds was not sufficient to show significant effects on viral infectivity. Importantly, two compounds did not affect frameshifting with the human PEG10 element, while one modestly inhibited rather than enhanced frameshifting at the human element. These studies indicate that frameshift elements have unique characteristics that may allow targeting of HIV-1 and of other viruses specifically for development of antiviral therapeutic molecules without effect on human genes like PEG10 that

  15. Cystic fibrosis in Jews: frequency and mutation distribution.

    Science.gov (United States)

    Kerem, B; Chiba-Falek, O; Kerem, E

    1997-01-01

    The incidence of cystic fibrosis and the frequency of disease causing mutations varies among different ethnic groups and geographical regions around the world. The Jewish population is comprised of two major ethnic groups. Ashkenazi and Non-Ashkenazi. The latter is further classified according to country of origin. An extreme variability in the disease frequency (from 1:2400-1:39,000) was found among the different Jewish ethnic groups. In the entire Jewish CF population, only 12 mutations were identified that altogether enable the identification of 91% of the CF chromosomes. However, in each Jewish ethnic group, the disease is caused by a different repertoire of a small number of mutations. In several ethnic groups, there is a major CFTR mutation that accounts for at least 48% of the CF chromosomes. High proportion of the CF chromosomes can be identified in Ashkenazi Jews (95%), Jews originating from Tunisia (100%), Libya (91%), Turkey (90%), and Georgia (88%). High frequencies of CFTR mutations were found among infertile males with CBAVD who might not have additional CF clinical characteristics. Of the Jewish males with CBAVD, 77% carried at least one CFTR mutation. The 5T mutation is the major mutation in Jewish CBAVD affecteds accounting for 32% of the chromosomes among Ashkenazi Jews and 36% among the non-Ashkenazi Jews. Five additional CFTR mutations, W1282X (12%), delta F508 (9%), N1303K (3%), D1152H, (5%)), and R117H (1%) were identified among Ashkenazi Jews with CBAVD. Only two mutations, delta F508 and R117H, were found among non-Ashkenazi males with CBAVD. An increased frequency of the 5T allele was also found among Jewish patients with atypical CF presentation, 18% in Ashkenazi, and 10% in non-Ashkenazi Jews. In summary, we present the required information for genetic counseling of Jewish families with typical and atypical CF and for carrier screening of healthy Jewish individuals.

  16. Recombinant tagging system using ribosomal frameshifting to monitor protein expression.

    Science.gov (United States)

    Han, Se Jong; Cho, Sayeon; Lowehhaupt, Ky; Park, So-Young; Sim, Sang Jun; Kim, Yang-Gyun

    2013-03-01

    For rapid and accurate quantitation of recombinant proteins during expression and after purification, we introduce a new tagging strategy that expresses both target proteins and limitedly tagged target proteins together in a single cell at a constant ratio by utilizing cis-elements of programmed -1 ribosomal frameshifting (-1RFS) as an embedded device. -1RFS is an alternative reading mechanism that effectively controls protein expression by many viruses. When a target gene is fused to the enhanced green fluorescent protein (EGFP) gene with a -1RFS element implanted between them, the unfused target and the target-GFP fusion proteins are expressed at a fixed ratio. The expression ratio between these two protein products is adjustable simply by changing -1RFS signals. This limited-tagging system would be valuable for the real-time monitoring of protein expression when optimizing expression condition for a new protein, and in monitoring large-scale bioprocesses without a large metabolic burden on host cells. Furthermore, this strategy allows for the direct measurement of the quantity of a protein on a chip surface and easy application to proteomewide study of gene products. Copyright © 2012 Wiley Periodicals, Inc.

  17. Correlation between mechanical strength of messenger RNA pseudoknots and ribosomal frameshifting

    DEFF Research Database (Denmark)

    Hansen, Thomas Møller; Reihani, S Nader S; Oddershede, Lene B

    2007-01-01

    downstream from the slippery sequence. Although the mechanism is not well understood, frameshifting is known to be stimulated by an mRNA structure such as a pseudoknot. Here, we show that the efficiency of frameshifting relates to the mechanical strength of the pseudoknot. Two pseudoknots derived from...... of frameshifting required a nearly 2-fold larger unfolding force than the other. The observed energy difference cannot be accounted for by any existing model. We propose that the degree of ribosomal frameshifting is related to the mechanical strength of RNA pseudoknots. Our observations support the "9 A model......" that predicts some physical barrier is needed to force the ribosome into the -1 frame. Also, our findings support the recent observation made by cryoelectron microscopy that mechanical interaction between a ribosome and a pseudoknot causes a deformation of the A-site tRNA. The result has implications...

  18. FSscan: a mechanism-based program to identify +1 ribosomal frameshift hotspots

    OpenAIRE

    Liao, Pei-Yu; Choi, Yong Seok; Lee, Kelvin H.

    2009-01-01

    In +1 programmed ribosomal frameshifting (PRF), ribosomes skip one nucleotide toward the 3?-end during translation. Most of the genes known to demonstrate +1 PRF have been discovered by chance or by searching homologous genes. Here, a bioinformatic framework called FSscan is developed to perform a systematic search for potential +1 frameshift sites in the Escherichia coli genome. Based on a current state of the art understanding of the mechanism of +1 PRF, FSscan calculates scores for a 16-nt...

  19. SHIFT: server for hidden stops analysis in frame-shifted translation.

    Science.gov (United States)

    Gupta, Arun; Singh, Tiratha Raj

    2013-02-23

    Frameshift is one of the three classes of recoding. Frame-shifts lead to waste of energy, resources and activity of the biosynthetic machinery. In addition, some peptides synthesized after frame-shifts are probably cytotoxic which serve as plausible cause for innumerable number of diseases and disorders such as muscular dystrophies, lysosomal storage disorders, and cancer. Hidden stop codons occur naturally in coding sequences among all organisms. These codons are associated with the early termination of translation for incorrect reading frame selection and help to reduce the metabolic cost related to the frameshift events. Researchers have identified several consequences of hidden stop codons and their association with myriad disorders. However the wealth of information available is speckled and not effortlessly acquiescent to data-mining. To reduce this gap, this work describes an algorithmic web based tool to study hidden stops in frameshifted translation for all the lineages through respective genetic code systems. This paper describes SHIFT, an algorithmic web application tool that provides a user-friendly interface for identifying and analyzing hidden stops in frameshifted translation of genomic sequences for all available genetic code systems. We have calculated the correlation between codon usage frequencies and the plausible contribution of codons towards hidden stops in an off-frame context. Markovian chains of various order have been used to model hidden stops in frameshifted peptides and their evolutionary association with naturally occurring hidden stops. In order to obtain reliable and persuasive estimates for the naturally occurring and predicted hidden stops statistical measures have been implemented. This paper presented SHIFT, an algorithmic tool that allows user-friendly exploration, analysis, and visualization of hidden stop codons in frameshifted translations. It is expected that this web based tool would serve as a useful complement for

  20. Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use.

    Science.gov (United States)

    Atkins, John F; Loughran, Gary; Bhatt, Pramod R; Firth, Andrew E; Baranov, Pavel V

    2016-09-06

    Genetic decoding is not 'frozen' as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Common Β- Thalassaemia Mutations in

    Directory of Open Access Journals (Sweden)

    P Azarfam

    2005-01-01

    Full Text Available Introduction: β –Thalassaemia was first explained by Thomas Cooly as Cooly’s anaemia in 1925. The β- thalassaemias are hereditary autosomal disorders with decreased or absent β-globin chain synthesis. The most common genetic defects in β-thalassaemias are caused by point mutations, micro deletions or insertions within the β-globin gene. Material and Methods: In this research , 142 blood samples (64 from childrens hospital of Tabriz , 15 samples from Shahid Gazi hospital of Tabriz , 18 from Urumia and 45 samples from Aliasghar hospital of Ardebil were taken from thalassaemic patients (who were previously diagnosed .Then 117 non-familial samples were selected . The DNA of the lymphocytes of blood samples was extracted by boiling and Proteinase K- SDS procedure, and mutations were detected by ARMS-PCR methods. Results: From the results obtained, eleven most common mutations,most of which were Mediterranean mutations were detected as follows; IVS-I-110(G-A, IVS-I-1(G-A ،IVS-I-5(G-C ,Frameshift Codon 44 (-C,( codon5(-CT,IVS-1-6(T-C, IVS-I-25(-25bp del ,Frameshift 8.9 (+G ,IVS-II-1(G-A ,Codon 39(C-T, Codon 30(G-C the mutations of the samples were defined. The results showed that Frameshift 8.9 (+G, IVS-I-110 (G-A ,IVS-II-I(G-A, IVS-I-5(G-C, IVS-I-1(G-A , Frameshift Codon 44(-C , codon5(-CT , IVS-1-6(T-C , IVS-I-25(-25bp del with a frequency of 29.9%, 25.47%,17.83%, 7.00%, 6.36% , 6.63% , 3.8% , 2.5% , 0.63% represented the most common mutations in North - west Iran. No mutations in Codon 39(C-T and Codon 30(G-C were detected. Cunclusion: The frequency of the same mutations in patients from North - West of Iran seems to be different as compared to other regions like Turkey, Pakistan, Lebanon and Fars province of Iran. The pattern of mutations in this region is more or less the same as in the Mediterranean region, but different from South west Asia and East Asia.

  2. Cftr Modulates Wnt/β-Catenin Signaling and Stem Cell Proliferation in Murine Intestine

    Directory of Open Access Journals (Sweden)

    Ashlee M. Strubberg

    2018-01-01

    Conclusions: CF intestine shows increased ISC proliferation and Wnt/β-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/β-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors.

  3. TNF Lectin-Like Domain Restores Epithelial Sodium Channel Function in Frameshift Mutants Associated with Pseudohypoaldosteronism Type 1B

    Directory of Open Access Journals (Sweden)

    Anita Willam

    2017-05-01

    Full Text Available Previous in vitro studies have indicated that tumor necrosis factor (TNF activates amiloride-sensitive epithelial sodium channel (ENaC current through its lectin-like (TIP domain, since cyclic peptides mimicking the TIP domain (e.g., solnatide, showed ENaC-activating properties. In the current study, the effects of TNF and solnatide on individual ENaC subunits or ENaC carrying mutated glycosylation sites in the α-ENaC subunit were compared, revealing a similar mode of action for TNF and solnatide and corroborating the previous assumption that the lectin-like domain of TNF is the relevant molecular structure for ENaC activation. Accordingly, TNF enhanced ENaC current by increasing open probability of the glycosylated channel, position N511 in the α-ENaC subunit being identified as the most important glycosylation site. TNF significantly increased Na+ current through ENaC comprising only the pore forming subunits α or δ, was less active in ENaC comprising only β-subunits, and showed no effect on ENaC comprising γ-subunits. TNF did not increase the membrane abundance of ENaC subunits to the extent observed with solnatide. Since the α-subunit is believed to play a prominent role in the ENaC current activating effect of TNF and TIP, we investigated whether TNF and solnatide can enhance αβγ-ENaC current in α-ENaC loss-of-function frameshift mutants. The efficacy of solnatide has been already proven in pathological conditions involving ENaC in phase II clinical trials. The frameshift mutations αI68fs, αT169fs, αP197fs, αE272fs, αF435fs, αR438fs, αY447fs, αR448fs, αS452fs, and αT482fs have been reported to cause pseudohypoaldosteronism type 1B (PHA1B, a rare, life-threatening, salt-wasting disease, which hitherto has been treated only symptomatically. In a heterologous expression system, all frameshift mutants showed significantly reduced amiloride-sensitive whole-cell current compared to wild type αβγ-ENaC, whereas membrane

  4. Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients

    DEFF Research Database (Denmark)

    Petersen, Sanne M; Dandanell, Mette; Rasmussen, Lene J

    2013-01-01

    Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as large genomic...

  5. Enhancement of +1 frameshift by polyamines during translation of polypeptide release factor 2 in Escherichia coli.

    Science.gov (United States)

    Higashi, Kyohei; Kashiwagi, Keiko; Taniguchi, Shiho; Terui, Yusuke; Yamamoto, Kaneyoshi; Ishihama, Akira; Igarashi, Kazuei

    2006-04-07

    Polypeptide release factor 2 (RF2) in Escherichia coli is known to be synthesized by a +1 frameshift at the 26th UGA codon of RF2 mRNA. Polyamines were found to stimulate the +1 frameshift of RF2 synthesis, an effect that was reduced by excess RF2. Polyamine stimulation of +1 frameshift of RF2 synthesis was observed at the early logarithmic phase, which is the important phase in determination of the overall rate of cell growth. A Shine-Dalgarno-like sequence was necessary for an efficient +1 frameshift of RF2 synthesis, but not for polyamine stimulation. Spectinomycin, tetracycline, streptomycin, and neomycin reduced polyamine stimulation of the +1 frameshift of RF2 synthesis. The results suggest that a structural change of the A site on 30 S ribosomal subunits is important for polyamine stimulation of the +1 frameshift. The level of mRNAs of ribosomal proteins and elongation factors having UAA as termination codon was enhanced by polyamines, and OppA synthesis from OppA mRNA having UAA as termination codon was more enhanced by polyamines than that from OppA mRNA having a UGA termination codon. Furthermore, synthesis of ribosomal protein L20 and elongation factor G from the mRNAs having a UAA termination codon was enhanced by polyamines at the level of translation and transcription. The results suggest that some protein synthesis from mRNAs having a UAA termination codon is enhanced at the level of translation through polyamine stimulation of +1 frameshift of RF2 synthesis. It is concluded that prfB encoding RF2 is a new member of the polyamine modulon.

  6. A novel founder MYO15A frameshift duplication is the major cause of genetic hearing loss in Oman.

    Science.gov (United States)

    Palombo, Flavia; Al-Wardy, Nadia; Ruscone, Guido Alberto Gnecchi; Oppo, Manuela; Kindi, Mohammed Nasser Al; Angius, Andrea; Al Lamki, Khalsa; Girotto, Giorgia; Giangregorio, Tania; Benelli, Matteo; Magi, Alberto; Seri, Marco; Gasparini, Paolo; Cucca, Francesco; Sazzini, Marco; Al Khabori, Mazin; Pippucci, Tommaso; Romeo, Giovanni

    2017-02-01

    The increased risk for autosomal recessive disorders is one of the most well-known medical implications of consanguinity. In the Sultanate of Oman, a country characterized by one of the highest rates of consanguineous marriages worldwide, prevalence of genetic hearing loss (GHL) is estimated to be 6/10 000. Families of GHL patients have higher consanguinity rates than the general Omani population, indicating a major role for recessive forms. Mutations in GJB2, the most commonly mutated GHL gene, have been sporadically described. We collected 97 DNA samples of GHL probands, affected/unaffected siblings and parents from 26 Omani consanguineous families. Analyzing a first family by whole-exome sequencing, we identified a novel homozygous frameshift duplication (c.1171_1177dupGCCATCT) in MYO15A, the gene linked to the deafness locus DFNB3. This duplication was then found in a total of 8/26 (28%) families, within a 849 kb founder haplotype. Reconstruction of haplotype structure at MYO15A surrounding genomic regions indicated that the founder haplotype branched out in the past two to three centuries from a haplotype present worldwide. The MYO15A duplication emerges as the major cause of GHL in Oman. These findings have major implications for the design of GHL diagnosis and prevention policies in Oman.

  7. Novel compound heterozygous frameshift mutations of C2orf37 in a ...

    Indian Academy of Sciences (India)

    Woodhouse–Sakati syndrome (WSS, MIM: 241080), a rare autosomal recessive disorder characterized by hypogo- nadism, alopecia, diabetes mellitus, mental retardation and extrapyramidal manifestations, was first described in a few consanguineous Saudi families (Woodhouse and Sakati. 1983) and is now recognized in ...

  8. Transcriptional profile analysis of RPGRORF15 frameshift mutation identifies novel genes associated with retinal degeneration.

    Science.gov (United States)

    Genini, Sem; Zangerl, Barbara; Slavik, Julianna; Acland, Gregory M; Beltran, William A; Aguirre, Gustavo D

    2010-11-01

    To identify genes and molecular mechanisms associated with photoreceptor degeneration in a canine model of XLRP caused by an RPGR exon ORF15 microdeletion. Methods. Expression profiles of mutant and normal retinas were compared by using canine retinal custom cDNA microarrays. qRT-PCR, Western blot analysis, and immunohistochemistry (IHC) were applied to selected genes, to confirm and expand the microarray results. At 7 and 16 weeks, respectively, 56 and 18 transcripts were downregulated in the mutant retinas, but none were differentially expressed (DE) at both ages, suggesting the involvement of temporally distinct pathways. Downregulated genes included the known retina-relevant genes PAX6, CHML, and RDH11 at 7 weeks and CRX and SAG at 16 weeks. Genes directly or indirectly active in apoptotic processes were altered at 7 weeks (CAMK2G, NTRK2, PRKCB, RALA, RBBP6, RNF41, SMYD3, SPP1, and TUBB2C) and 16 weeks (SLC25A5 and NKAP). Furthermore, the DE genes at 7 weeks (ELOVL6, GLOD4, NDUFS4, and REEP1) and 16 weeks (SLC25A5 and TARS2) are related to mitochondrial functions. qRT-PCR of 18 genes confirmed the microarray results and showed DE of additional genes not on the array. Only GFAP was DE at 3 weeks of age. Western blot and IHC analyses also confirmed the high reliability of the transcriptomic data. Several DE genes were identified in mutant retinas. At 7 weeks, a combination of nonclassic anti- and proapoptosis genes appear to be involved in photoreceptor degeneration, whereas at both 7 and 16 weeks, the expression of mitochondria-related genes indicates that they may play a relevant role in the disease process.

  9. A novel frameshift mutation of Chediak-Higashi syndrome and treatment in the accelerated phase

    Directory of Open Access Journals (Sweden)

    X.L. Wu

    Full Text Available Chediak-Higashi syndrome (CHS is a rare autosomal recessive immunodeficiency disease characterized by frequent infections, hypopigmentation, progressive neurologic deterioration and hemophagocytic lymphohistiocytosis (HLH, known as the accelerated phase. There is little experience in the accelerated phase of CHS treatment worldwide. Here, we present a case of a 9-month-old boy with continuous high fever, hypopigmentation of the skin, enlarged lymph nodes, hepatosplenomegaly and lung infection. He was diagnosed with CHS by gene sequencing, and had entered the accelerated phase. After 8 weeks of therapy, the boy had remission and was prepared for allogenic stem cell transplantation.

  10. A novel frameshift mutation of Chediak-Higashi syndrome and treatment in the accelerated phase.

    Science.gov (United States)

    Wu, X L; Zhao, X Q; Zhang, B X; Xuan, F; Guo, H M; Ma, F T

    2017-03-23

    Chediak-Higashi syndrome (CHS) is a rare autosomal recessive immunodeficiency disease characterized by frequent infections, hypopigmentation, progressive neurologic deterioration and hemophagocytic lymphohistiocytosis (HLH), known as the accelerated phase. There is little experience in the accelerated phase of CHS treatment worldwide. Here, we present a case of a 9-month-old boy with continuous high fever, hypopigmentation of the skin, enlarged lymph nodes, hepatosplenomegaly and lung infection. He was diagnosed with CHS by gene sequencing, and had entered the accelerated phase. After 8 weeks of therapy, the boy had remission and was prepared for allogenic stem cell transplantation.

  11. New frameshift CF mutation 3729delAinsTCT in a Tunisian cystic ...

    Indian Academy of Sciences (India)

    fibrosis patient. SONDESS HADJ FREDJ1, MONIA BOUDAYA1, SABRINE OUESLATI1, SAFA SAHNOUN1, CHAIMA SAHLI1,. HAJER SIALA1, KHEDIJA BOUSSETTA2, AMINA BIBI1 and TAIEB MESSAOUD1∗. 1Biochemistry Laboratory and 2Department of Pediatrics, Children's Hospital, Bab Saadoun Square 1007 Tunis, ...

  12. Molecular evaluation of CFTR sequence variants in male infertility of testicular origin.

    Science.gov (United States)

    Larriba, S; Bonache, S; Sarquella, J; Ramos, M D; Giménez, J; Bassas, L; Casals, T

    2005-10-01

    Although the involvement of the CFTR gene has been well established in congenital agenesia of vas deferens, its role in non-obstructive (NOb) infertility is still a matter of debate. In order to definitively define the involvement of the CFTR gene in spermatogenic impairment and a potential synergistic contribution to known genetic and clinical factors, genetic variants in the entire coding sequence and the immediately flanking regions of the CFTR gene, along with a thorough clinical evaluation, were analysed in 83 NOb infertile patients and 87 clinically well-defined fertile individuals as controls. The results of our study showed no statistical difference between CFTR carrier frequency in the infertile and fertile population. Specifically, the IVS8-6(5T) allele carrier frequency was similar in NOb infertile patients when compared with fertile men, but it is noteworthy that, when fertile men were classified into having optimal and suboptimal fertility, no 5T allele was found among the 35 men with optimal fertility parameters. In conclusion, extensive CFTR analysis in infertile individuals and fertile population as adequate control definitively excludes the involvement of the CFTR gene variants in sperm production and stresses the importance of carefully identifying those individuals with obstructive defects, in whom CFTR screening will be beneficial.

  13. Advancing clinical development pathways for new CFTR modulators in cystic fibrosis.

    Science.gov (United States)

    Mayer-Hamblett, Nicole; Boyle, Michael; VanDevanter, Donald

    2016-05-01

    Cystic fibrosis (CF) is a life-shortening genetic disease affecting approximately 70,000 individuals worldwide. Until recently, drug development efforts have emphasised therapies treating downstream signs and symptoms resulting from the underlying CF biological defect: reduced function of the CF transmembrane conductance regulator (CFTR) protein. The current CF drug development landscape has expanded to include therapies that enhance CFTR function by either restoring wild-type CFTR protein expression or increasing (modulating) the function of mutant CFTR proteins in cells. To date, two systemic small-molecule CFTR modulators have been evaluated in pivotal clinical trials in individuals with CF and specific mutant CFTR genotypes that have led to regulatory review and/or approval. Advances in the discovery of CFTR modulators as a promising new class of therapies have been impressive, yet work remains to develop highly effective, disease-modifying modulators for individuals of all CF genotypes. The objectives of this review are to outline the challenges and opportunities in drug development created by systemic genotype-specific CFTR modulators, highlight the advantages of sweat chloride as an established biomarker of CFTR activity to streamline early-phase development and summarise options for later phase clinical trial designs that respond to the adoption of approved genotype-specific modulators into standard of care. An optimal development framework will be needed to move the most promising therapies efficiently through the drug development pipeline and ultimately deliver efficacious and safe therapies to all individuals with CF. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  14. Synergy-based small-molecule screen using a human lung epithelial cell line yields ΔF508-CFTR correctors that augment VX-809 maximal efficacy.

    Science.gov (United States)

    Phuan, Puay-Wah; Veit, Guido; Tan, Joseph; Roldan, Ariel; Finkbeiner, Walter E; Lukacs, Gergely L; Verkman, A S

    2014-07-01

    The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for ΔF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ∆F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 µM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ∆F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors. Copyright © 2014 by The

  15. Unique presentations and chronic complications in adult cystic fibrosis: do they teach us anything about CFTR?

    Directory of Open Access Journals (Sweden)

    Boyle Michael P

    2000-11-01

    Full Text Available Abstract The increase in numbers of adults with cystic fibrosis (CF has allowed us to identify previously unrecognized chronic complications of CF, as well as appreciate unique presentations of cystic fibrosis-related diseases. Do these chronic complications and unique presentations provide us with new insight into cystic fibrosis transmembrane conductance regulator (CFTR function? Current data suggest that the 'chronic complications' reveal mainly the effect of a long-term absence of previously recognized CFTR functions. In contrast, the 'unique presentations' provide new insight into the role of CFTR in different tissues.

  16. Unique presentations and chronic complications in adult cystic fibrosis: do they teach us anything about CFTR?

    Science.gov (United States)

    Boyle, Michael P

    2000-01-01

    The increase in numbers of adults with cystic fibrosis (CF) has allowed us to identify previously unrecognized chronic complications of CF, as well as appreciate unique presentations of cystic fibrosis-related diseases. Do these chronic complications and unique presentations provide us with new insight into cystic fibrosis transmembrane conductance regulator (CFTR) function? Current data suggest that the 'chronic complications' reveal mainly the effect of a long-term absence of previously recognized CFTR functions. In contrast, the 'unique presentations' provide new insight into the role of CFTR in different tissues. PMID:11667976

  17. Molecular analysis using DHPLC of cystic fibrosis: increase of the mutation detection rate among the affected population in Central Italy

    Directory of Open Access Journals (Sweden)

    Nardone Anna

    2004-04-01

    Full Text Available Abstract Background Cystic fibrosis (CF is a multisystem disorder characterised by mutations of the CFTR gene, which encodes for an important component in the coordination of electrolyte movement across of epithelial cell membranes. Symptoms are pulmonary disease, pancreatic exocrine insufficiency, male infertility and elevated sweat concentrations. The CFTR gene has numerous mutations (>1000 and functionally important polymorphisms (>200. Early identification is important to provide appropriate therapeutic interventions, prognostic and genetic counselling and to ensure access to specialised medical services. However, molecular diagnosis by direct mutation screening has proved difficult in certain ethnic groups due to allelic heterogeneity and variable frequency of causative mutations. Methods We applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons and characterise sequence variations in a subgroup of CF Italian patients from the Lazio region (Central Italy characterised by an extensive allelic heterogeneity. Results We have identified a total of 36 different mutations representing 88% of the CF chromosomes. Among these are two novel CFTR mutations, including one missense (H199R and one microdeletion (4167delCTAAGCC. Conclusion Using this approach, we were able to increase our standard power rate of mutation detection of about 11% (77% vs. 88%.

  18. Molecular mechanisms of induced-mutations

    International Nuclear Information System (INIS)

    Kato, Takeshi

    1985-01-01

    The outcome of recent studies on mechanisms of induced-mutations is outlined with particular emphasis on the dependence of recA gene function in Escherichia coli. Genes involved in spontaneous mutation and x-ray- and chemical-induced mutation and genes involved in adaptive response are presented. As for SOS mutagenesis, SOS-induced regulation mechanisms and mutagenic routes are described. Furthermore, specificity of mutagens themselves are discussed in relation to mechanisms of base substitution, frameshift, and deletion mutagenesis. (Namekawa, K.)

  19. Pseudomonas aeruginosa in cystic fibrosis patients with G551D-CFTR treated with ivacaftor.

    Science.gov (United States)

    Heltshe, Sonya L; Mayer-Hamblett, Nicole; Burns, Jane L; Khan, Umer; Baines, Arthur; Ramsey, Bonnie W; Rowe, Steven M

    2015-03-01

    Ivacaftor improves outcomes in cystic fibrosis (CF) patients with the G551D mutation; however, effects on respiratory microbiology are largely unknown. This study examines changes in CF respiratory pathogens with ivacaftor and correlates them with baseline characteristics and clinical response. The G551D Observational Study enrolled a longitudinal observational cohort of US patients with CF aged 6 years and older with at least 1 copy of the G551D mutation. Results were linked with retrospective and prospective culture data in the US Cystic Fibrosis Foundation's National Patient Registry. Pseudomonas aeruginosa infection category in the year before and year after ivacaftor was compared and correlated with clinical findings. Among 151 participants prescribed ivacaftor, 29% (26/89) who were culture positive for P. aeruginosa the year prior to ivacaftor use were culture negative the year following treatment; 88% (52/59) of those P. aeruginosa free remained uninfected. The odds of P. aeruginosa positivity in the year after ivacaftor compared with the year prior were reduced by 35% (odds ratio [OR], 0.65; P < .001). Ivacaftor was also associated with reduced odds of mucoid P. aeruginosa (OR, 0.77; P = .013) and Aspergillus (OR, 0.47; P = .039), but not Staphylococcus aureus or other common CF pathogens. Patients with intermittent culture positivity and higher forced expiratory volume in 1 second (FEV1) were most likely to turn culture negative. Reduction in P. aeruginosa was not associated with change in FEV1, body mass index, or hospitalizations. Pseudomonas aeruginosa culture positivity was significantly reduced following ivacaftor treatment. Efficacious CFTR modulation may contribute to lower frequency of culture positivity for P. aeruginosa and other respiratory pathogens, particularly in patients with less established disease. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For

  20. Novel mutation in CNTNAP1 results in congenital hypomyelinating neuropathy.

    Science.gov (United States)

    Mehta, Paulomi; Küspert, Melanie; Bale, Tejus; Brownstein, Catherine A; Towne, Meghan C; De Girolami, Umberto; Shi, Jiahai; Beggs, Alan H; Darras, Basil T; Wegner, Michael; Piao, Xianhua; Agrawal, Pankaj B

    2017-05-01

    Congenital hypomyelinating neuropathy (CHN) is a rare congenital neuropathy that presents in the neonatal period and has been linked previously to mutations in several genes associated with myelination. A recent study has linked 4 homozygous frameshift mutations in the contactin-associated protein 1 (CNTNAP1) gene with this condition. We report a neonate with CHN who was found to have absent sensory nerve and compound muscle action potentials and hypomyelination on nerve biopsy. On whole exome sequencing, we identified a novel CNTNAP1 homozygous missense mutation (p.Arg388Pro) in the proband, and both parents were carriers. Molecular modeling suggests that this variant disrupts a β-strand to cause an unstable structure and likely significant changes in protein function. This report links a missense CNTNAP1 variant to the disease phenotype previously associated only with frameshift mutations. Muscle Nerve 55: 761-765, 2017. © 2016 Wiley Periodicals, Inc.

  1. CFTR chloride channel as a molecular target of anthraquinone compounds in herbal laxatives

    Science.gov (United States)

    Yang, Hong; Xu, Li-na; He, Cheng-yan; Liu, Xin; Fang, Rou-yu; Ma, Tong-hui

    2011-01-01

    Aim: To clarify whether CFTR is a molecular target of intestinal fluid secretion caused by the anthraquinone compounds from laxative herbal plants. Methods: A cell-based fluorescent assay to measure I− influx through CFTR chloride channel. A short-circuit current assay to measure transcellular Cl− current across single layer FRT cells and freshly isolated colon mucosa. A closed loop experiment to measure colon fluid secretion in vivo. Results: Anthraquinone compounds rhein, aloe-emodin and 1,8-dihydroxyanthraquinone (DHAN) stimulated I− influx through CFTR chloride channel in a dose-dependent manner in the presence of physiological concentration of cAMP. In the short-circuit current assay, the three compound enhanced Cl− currents in epithelia formed by CFTR-expressing FRT cells with EC50 values of 73±1.4, 56±1.7, and 50±0.5μmol/L, respectively, and Rhein also enhanced Cl− current in freshly isolated rat colonic mucosa with a similar potency. These effects were completely reversed by the CFTR selective blocker CFTRinh-172. In in vivo closed loop experiments, rhein 2 mmol/L stimulated colonic fluid accumulation that was largely blocked by CFTRinh-172. The anthraquinone compounds did not elevate cAMP level in cultured FRT cells and rat colonic mucosa, suggesting a direct effect on CFTR activity. Conclusion: Natural anthraquinone compounds in vegetable laxative drugs are CFTR potentiators that stimulated colonic chloride and fluid secretion. Thus CFTR chloride channel is a molecular target of vegetable laxative drugs. PMID:21602836

  2. Frameshifting in the P6 cDNA Phage Display System

    Directory of Open Access Journals (Sweden)

    Veerle Somers

    2010-12-01

    Full Text Available Phage display is a powerful technique that enables easy identification of targets for any type of ligand. Targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins. By means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur. In our studies using C-terminal display of cDNA fragments to phage coat protein p6, we noticed the occasional enrichment of targets that do not contain an open reading frame. This event has previously been described in other phage display studies using N-terminal display of targets to phage coat proteins and was due to uncommon translational events like frameshifting. The aim of this study was to examine if C-terminal display of targets to p6 is also subjected to frameshifting. To this end, an enriched target not containing an open reading frame was selected and an E-tag was coupled at the C-terminus in order to measure target display at the surface of the phage. The tagged construct was subsequently expressed in 3 different reading frames and display of both target and E-tag measured to detect the occurrence of frameshifting. As a result, we were able to demonstrate display of the target both in the 0 and in the +1 reading frame indicating that frameshifting can also take place when C-terminal fusion to minor coat protein p6 is applied.

  3. Ribosome excursions during mRNA translocation mediate broad branching of frameshift pathways.

    Science.gov (United States)

    Yan, Shannon; Wen, Jin-Der; Bustamante, Carlos; Tinoco, Ignacio

    2015-02-26

    Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1 frameshifting occurs on Escherichia coli's dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the -1 frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. A host defense mechanism involving CFTR-mediated bicarbonate secretion in bacterial prostatitis.

    Directory of Open Access Journals (Sweden)

    Chen Xie

    Full Text Available BACKGROUND: Prostatitis is associated with a characteristic increase in prostatic fluid pH; however, the underlying mechanism and its physiological significance have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study a primary culture of rat prostatic epithelial cells and a rat prostatitis model were used. Here we reported the involvement of CFTR, a cAMP-activated anion channel conducting both Cl(- and HCO(3(-, in mediating prostate HCO(3(- secretion and its possible role in bacterial killing. Upon Escherichia coli (E. coli-LPS challenge, the expression of CFTR and carbonic anhydrase II (CA II, along with several pro-inflammatory cytokines was up-regulated in the primary culture of rat prostate epithelial cells. Inhibiting CFTR function in vitro or in vivo resulted in reduced bacterial killing by prostate epithelial cells or the prostate. High HCO(3(- content (>50 mM, rather than alkaline pH, was found to be responsible for bacterial killing. The direct action of HCO(3(- on bacterial killing was confirmed by its ability to increase cAMP production and suppress bacterial initiation factors in E. coli. The relevance of the CFTR-mediated HCO(3(- secretion in humans was demonstrated by the upregulated expression of CFTR and CAII in human prostatitis tissues. CONCLUSIONS/SIGNIFICANCE: The CFTR and its mediated HCO(3(- secretion may be up-regulated in prostatitis as a host defense mechanism.

  5. Mutation rates of TGFBR2 and ACVR2 coding microsatellites in human cells with defective DNA mismatch repair.

    Directory of Open Access Journals (Sweden)

    Heekyung Chung

    Full Text Available Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFbeta family receptors is abrogated in DNA Mismatch repair (MMR-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1-/-, hMSH6-/-, hMSH3-/-, and MMR-proficient to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP gene, allowing a -1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7-35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a -1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2 in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes and M2 (bright, representing full mutants were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91 x 10(-4 and 15 (2.18 x 10(-4 times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was approximately 3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The -1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background.

  6. Birt-Hogg-Dubé syndrome: novel FLCN frameshift deletion in daughter and father with renal cell carcinomas.

    Science.gov (United States)

    Näf, Ernst; Laubscher, Dominik; Hopfer, Helmut; Streit, Markus; Matyas, Gabor

    2016-01-01

    Germline mutation of the FLCN gene causes Birt-Hogg-Dubé syndrome (BHD), a rare autosomal dominant condition characterized by skin fibrofolliculomas, lung cysts, spontaneous pneumothorax and renal tumours. We identified a hitherto unreported pathogenic FLCN frameshift deletion c.563delT (p.Phe188Serfs*35) in a family of a 46-year-old woman presented with macrohematuria due to bilateral chromophobe renal carcinomas. A heritable renal cancer was suspected due to the bilaterality of the tumour and as the father of this woman had suffered from renal cancer. Initially, however, BHD was overlooked by the medical team despite the highly suggestive clinical presentation. We assume that BHD is underdiagnosed, at least partially, due to low awareness of this variable condition and to insufficient use of appropriate genetic testing. Our study indicates that BHD and FLCN testing should be routinely considered in patients with positive family or personal history of renal tumours. In addition, we demonstrate how patients and their families can play a driving role in initiating genetic diagnosis, presymptomatic testing of at-risk relatives, targeted disease management, and genetic counselling of rare diseases such as BHD.

  7. ER-associated complexes (ERACs) containing aggregated cystic fibrosis transmembrane conductance regulator (CFTR) are degraded by autophagy.

    Science.gov (United States)

    Fu, Lianwu; Sztul, Elizabeth

    2009-04-01

    The ubiquitin-proteasome pathway and autophagy are the two major mechanisms responsible for the clearance of cellular proteins. We have used the yeast Saccharomyces cerevisiae as a model system and the cystic fibrosis transmembrane conductance regulator (CFTR) as a model substrate to study the interactive function of these two pathways in the degradation of misfolded proteins. EGFP-tagged human CFTR was introduced into yeast and expressed under a copper-inducible promoter. The localization and degradation of EGFP-CFTR in live cells were monitored by time-lapse imaging following its de novo synthesis. EGFP-CFTR first appears within the perinuclear and sub-cortical ER and is mobile within the plane of the membrane as assessed by fluorescence recovery after photobleaching (FRAP). This pool of EGFP-CFTR is subsequently degraded through a proteasome-dependent pathway that is inhibited in the pre1-1 yeast strain defective in proteasomal degradation. Prolonged expression of EGFP-CFTR leads to the sequestration of EGFP-CFTR molecules into ER structures called ER-associated complexes (ERACs). The sequestration of EGFP-CFTR into ERACs appears to be driven by aggregation since EGFP-CFTR molecules present within ERACs are immobile as measured by FRAP. Individual ERACs are cleared from cells through the autophagic pathway that is blocked in the atg6Delta and atg1Delta yeast strains defective in autophagy. Our results suggest that the proteasomal and the autophagic pathways function together to clear misfolded proteins from the ER.

  8. Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Romain Ferru-Clément

    Full Text Available Cystic fibrosis transmembrane conductance regulator (CFTR is a chloride channel that is expressed on the apical plasma membrane (PM of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o- expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

  9. Laser desorption mass spectrometry for point mutation detection

    Energy Technology Data Exchange (ETDEWEB)

    Taranenko, N.I.; Chung, C.N.; Zhu, Y.F. [Oak Ridge National Lab., TN (United States)] [and others

    1996-10-01

    A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments generated by digestion of a PCR generated target fragment. 21 refs., 10 figs., 2 tabs.

  10. Laser desorption mass spectrometry for point mutation detection

    Energy Technology Data Exchange (ETDEWEB)

    Taranenko, N.I.; Chung, C.N.; Zhu, Y.F. [Oak Ridge National Lab., TN (United States)] [and others

    1996-12-31

    A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments by digestion of a PCR generated target fragment. 21 refs., 10 figs., 2 tabs.

  11. Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot

    Science.gov (United States)

    Su, L.; Chen, L.; Egli, M.; Berger, J. M.; Rich, A.

    1999-01-01

    Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 A crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson-Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

  12. Cigarette smoke activates CFTR through ROS-stimulated cAMP signaling in human bronchial epithelial cells.

    Science.gov (United States)

    Wong, Francis H; AbuArish, Asmahan; Matthes, Elizabeth; Turner, Mark J; Greene, Lana E; Cloutier, Alexandre; Robert, Renaud; Thomas, David Y; Cosa, Gonzalo; Cantin, André M; Hanrahan, John W

    2018-01-01

    Air pollution stimulates airway epithelial secretion through a cholinergic reflex that is unaffected in cystic fibrosis (CF), yet a strong correlation is observed between passive smoke exposure in the home and impaired lung function in CF children. Our aim was to study the effects of low smoke concentrations on cystic fibrosis transmembrane conductance regulator (CFTR) function in vitro. Cigarette smoke extract stimulated robust anion secretion that was transient, mediated by CFTR, and dependent on cAMP-dependent protein kinase activation. Secretion was initiated by reactive oxygen species (ROS) and mediated by at least two distinct pathways: autocrine activation of EP4 prostanoid receptors and stimulation of Ca 2+ store-operated cAMP signaling. The response was absent in cells expressing the most common disease-causing mutant F508del-CFTR. In addition to the initial secretion, prolonged exposure of non-CF bronchial epithelial cells to low levels of smoke also caused a gradual decline in CFTR functional expression. F508del-CFTR channels that had been rescued by the CF drug combination VX-809 (lumacaftor) + VX-770 (ivacaftor) were more sensitive to this downregulation than wild-type CFTR. The results suggest that CFTR-mediated secretion during acute cigarette smoke exposure initially protects the airway epithelium while prolonged exposure reduces CFTR functional expression and reduces the efficacy of CF drugs.

  13. Resveratrol increases F508del-CFTR dependent salivary secretion in cystic fibrosis mice

    Directory of Open Access Journals (Sweden)

    Barbara Dhooghe

    2015-07-01

    Full Text Available Cystic fibrosis (CF is a fatal genetic disease associated with widespread exocrine gland dysfunction. Studies have suggested activating effects of resveratrol, a naturally-occurring polyphenol compound with antioxidant and anti-inflammatory properties, on CF transmembrane conductance regulator (CFTR protein function. We assayed, in F508del-CFTR homozygous (CF and in wild-type mice, the effect of resveratrol on salivary secretion in basal conditions, in response to inhibition by atropine (basal β-adrenergic-dependent component and to stimulation by isoprenaline (CFTR-dependent component. Both components of the salivary secretion were smaller in CF mice than in controls. Two hours after intraperitoneal administration of resveratrol (50 mg/kg dissolved in DMSO, the compound was detected in salivary glands. As in both CF and in wild-type mice, DMSO alone increased the response to isoprenaline in males but not in females, the effect of resveratrol was only measured in females. In wild-type mice, isoprenaline increased secretion by more than half. In CF mice, resveratrol rescued the response to isoprenaline, eliciting a 2.5-fold increase of β-adrenergic-stimulated secretion. We conclude that the salivary secretion assay is suitable to test DMSO-soluble CFTR modulators in female mice. We show that resveratrol applied in vivo to mice reaches salivary glands and increases β-adrenergic secretion. Immunolabelling of CFTR in human bronchial epithelial cells suggests that the effect is associated with increased CFTR protein expression. Our data support the view that resveratrol is beneficial for treating CF. The salivary secretion assay has a potential application to test efficacy of novel CF therapies.

  14. Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

    Directory of Open Access Journals (Sweden)

    Becq Frédéric

    2008-10-01

    Full Text Available Abstract Background In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC stimulation, which generates inositol 1,4,5-trisphosphate (IP3 and 1,2-diacylglycerol (DAG and induces Ca2+ release from endoplasmic reticulum (ER stores. Methods In the present study, we monitored the cytosolic Ca2+ transients using the UV light photolysis technique to uncage caged Ca2+ or caged IP3 into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF and non-CF origin. We compared in these cells the types of Ca2+ receptors present in the ER, and measured their Ca2+ dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin. Results We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP3R dependent-Ca2+ response following both correcting treatments compared to uncorrected cells in such a way that Ca2+ responses (CF+treatment vs wild-type cells were normalized. This normalization of the Ca2+ rate does not affect the activity of Ca2+-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP3R1, we observed a decrease of the implication of IP3R1 in the Ca2+ response in CF corrected cells. We observed a similar Ca2+ mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted. When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP3R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell. Conclusion These results suggest reversal of the IP3R dysfunction in F508del-CFTR epithelial

  15. A very mild form of non-Herlitz junctional epidermolysis bullosa : BP180 rescue by outsplicing of mutated exon 30 coding for the COL15 domain

    NARCIS (Netherlands)

    Pasmooij, AMG; van Zalen, S; Nijenhuis, AM; Kloosterhuis, AJ; Zuiderveen, J; Jonkman, MF; Pas, HH

    Mutations in the gene COL17A1 cause non-Herlitz junctional epidermolysis bullosa. Here, we describe a patient who, despite two heterozygous mutations in COL17A1, has an extremely mild form of the disease missing most of the characteristic clinical features. DNA analysis revealed a frame-shift

  16. A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition

    DEFF Research Database (Denmark)

    Ropero, S; Fraga, MF; Ballestar, E

    2006-01-01

    arising in individuals with hereditary nonpolyposis colorectal cancer syndrome. The presence of the HDAC2 frameshift mutation causes a loss of HDAC2 protein expression and enzymatic activity and renders these cells more resistant to the usual antiproliferative and proapoptotic effects of histone...

  17. 40 CFR 798.5265 - The salmonella typhimurium reverse mutation assay.

    Science.gov (United States)

    2010-07-01

    ... mean number of revertant colonies per plate and standard deviation shall be presented for test chemical... revertant colonies per plate, standard deviation. (vi) Dose-response relationship, if applicable. (g... chemicals which cause base changes or frameshift mutations in the genome of this organism. (b) Definitions...

  18. Novel ELN mutation in a family with supravalvular aortic stenosis and intracranial aneurysm

    DEFF Research Database (Denmark)

    Jelsig, Anne Marie; Urban, Zsolt; Hucthagowder, Vishwanathan

    2017-01-01

    stenosis, various other arterial stenoses, sudden death, and intracranial aneurysms. A frameshift mutation in exon 12, not described before, was detected in the affected family members. This report emphasises the importance of family history, genetic counselling, and demonstrates the great variability...

  19. Senior-Loken syndrome: A novel NPHP5 gene mutation in a family ...

    African Journals Online (AJOL)

    Makia J Marafie

    2014-01-08

    Jan 8, 2014 ... is Senior-Loken syndrome, a hereditary heterogeneous multiorgan disorder, which combines neph- ronophthisis with ... Case report: Here, we are reporting two children from an Arab family with a novel frameshift mutation found in ..... diseases, which are scattered in the Arab world and Middle. East to help ...

  20. Screening of 1331 Danish breast and/or ovarian cancer families identified 40 novel BRCA1 and BRCA2 mutations

    DEFF Research Database (Denmark)

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

    2011-01-01

    and BRCA2 in high risk breast and/or ovarian cancer families. The mutations were detected via pre-screening using dHPLC or high-resolution melting and direct sequencing. We identified 16 variants in BRCA1, including 9 deleterious frame-shift mutations, 2 intronic variants, 4 missense mutations, and 1......Germ-line mutations in the tumour suppressor genes BRCA1 and BRCA2 predispose to breast and ovarian cancer. Since 1999 we have performed mutational screening of breast and/or ovarian cancer patients in East Denmark. During this period we have identified 40 novel sequence variations in BRCA1...... synonymous variant. The remaining 24 variants were identified in BRCA2, including 10 deleterious mutants (6 frame-shift and 4 nonsense), 2 intronic variants, 10 missense mutations and 2 synonymous variants. The frequency of the variants of unknown significance was examined in control individuals. Moreover...

  1. Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S expressing two complementary protein fragments

    Directory of Open Access Journals (Sweden)

    Mortenson Lindsay

    2001-12-01

    Full Text Available Abstract Background The L-type Ca2+ channel formed by the dihydropyridine receptor (DHPR of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1. This channel-to-channel coupling is essential for Ca2+ signaling but poorly understood. We characterized a single-base frame-shift mutant of α1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC coupling by virtue of expressing two complementary hemi-Ca2+ channel fragments. Results Functional analysis of cDNA transfected dysgenic myotubes lacking α1S were carried out using voltage-clamp, confocal Ca2+ indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-α1S expressed the N-terminal half of α1S (M1 to L670 and the C-terminal half starting at M701 separately. The C-terminal fragment was generated by an unexpected restart of translation of the fs-α1S message at M701 and was eliminated by a M701I mutation. Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current. Discussion A premature stop codon in the II-III loop may not necessarily cause a loss of DHPR function due to a restart of translation within the II-III loop, presumably by a mechanism involving leaky ribosomal scanning. In these cases, function is recovered by expression of complementary protein fragments from the same cDNA. DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel.

  2. Analysis of polymorphic variants of CFTR (rs 113993960, IL-4 (rs 2243250, PRSS1 (rs 111033565, SPINK1 (rs ID 6690 and TNF-α (rs 1800629 Genes in Patients with Edematous Pancreatitis Living in Northern Bukovyna region

    Directory of Open Access Journals (Sweden)

    Sergei Ivashchuk

    2016-12-01

    Full Text Available The occurrence of gene mutations affecting the formation of acute pancreatitis or exacerbation of chronic pancreatitis differs in different populations and ethnic groups. The objective of the research was to study the incidence of CFTR (rs 113 993 960, IL-4 (rs 2243250, PRSS1 (rs 111 033 565, SPINK1 (rs ID 6690 and TNF-α (rs 1800629 gene mutations in Northern Bukovyna region and their dependence on etiological factor, sex and type of pancreatitis. Material and methods. Determination of IL-4 (C-590T, TNF-α (G-308A, PRSS1 (R122H, SPINK1 (N34S and CFTR (delF508 genes polymorphisms was performed in 123 patients with acute pancreatitis and the exacerbation of chronic pancreatitis and in 40 healthy individuals. Results. The relative incidence of PRSS1, CFTR, SPINK1 and TNF-α genes polymorphisms in patients with acute pancreatitis and the exacerbation of chronic pancreatitis did not significantly differ. Carriers of CC genotype of IL- 4 gene were present among the patients with acute pancreatitis and in the control group by 22.39% and 21.76% more often than among the patients with the exacerbation of chronic pancreatitis. Acute alcohol-related pancreatitis was observed in men significantly more often than gallstone pancreatitis, namely by 53.58% in carriers of “wild” GG-genotype of PRSS1 gene, by 29.64% in carriers of CC genotype of IL-4 gene, by 42.40% in carriers of NN-genotype of CFTR gene, and by 38.74% in carriers of GG-genotype of SPINK1 gene, respectively. Conclusions. The mutation of CFTR (rs 113 993 960, PRSS1 (rs 111 033 565, SPINK1 (rs ID6690 and TNF-α (rs1800629 gene in the homozygous state among the population of Northern Bukovyna was not detected. Acute alcohol-related pancreatitis was more often diagnosed in men in case of “wild” genotypes of PRSS1, CFTR and SPINK1 genes, whereas gallstone pancreatitis was more often diagnosed in women.

  3. Profile of TP53 gene mutations in sinonasal cancer

    DEFF Research Database (Denmark)

    Holmila, Reetta; Bornholdt, Jette; Suitiala, Tuula

    2010-01-01

    Genetic alterations underlying the development of the cancer of the nose and paranasal sinuses (sinonasal cancer, SNC), a rare cancer that can be included in the group of head and neck cancers, are still largely unknown. We recently reported that TP53 mutations are a common feature of SNC......, with an overall frequency of 77%, and they show association to adenocarcinoma and wood-dust exposure [15]. In this study, we report in detail the sequence change for 159 TP53 mutations identified by direct sequencing. More than half of the mutations (60%, 95/159) were missense mutations; there were also 28 (18......%) frameshift or nonsense mutations, and 36 (23%) intronic or silent mutations. In coding region, the most common base change detected was C-->T transition (43/125; 34% of base changes in the coding region). G-->T transversions occurred at a frequency of 10% (12/125), which is less than reported in mutation...

  4. NPM1 mutations in therapy-related acute myeloid leukemia with uncharacteristic features

    DEFF Research Database (Denmark)

    Andersen, Morten Tolstrup; Andersen, Mette Klarskov; Christiansen, D.H.

    2008-01-01

    Frameshift mutations of the nucleophosmin gene (NPM1) were recently reported as a frequently occurring abnormality in patients with de novo acute myeloid leukemia (AML). To evaluate the frequency of NPM1 mutations in patients with therapy-related myelodysplasia (t-MDS) and therapy-related AML (t......-/-7, the most frequent abnormalities of t-MDS/t-AML, were not observed (P=0.002). This raises the question whether some of the cases presenting NPM1 mutations were in fact cases of de novo leukemia. The close association to class I mutations and the inverse association to class II mutations suggest...

  5. Upregulation of CFTR in patients with endometriosis and its involvement in NFκB-uPAR dependent cell migration.

    Science.gov (United States)

    Huang, Wenqing; Jin, Aihong; Zhang, Jieting; Wang, Chaoqun; Tsang, Lai Ling; Cai, Zhiming; Zhou, Xiaping; Chen, Hao; Chan, Hsiao Chang

    2017-09-15

    Endometriotic tissues exhibit high migration ability with the underlying mechanisms remain elusive. Our previous studies have demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR) acts as a tumor suppressor regulating cell migration. In the present study, we explored whether CFTR plays a role in the development of human endometriosis. We found that both mRNA and protein expression levels of CFTR and urokinase-type plasminogen activator receptor (uPAR) were significantly increased in ectopic endometrial tissues from patients with endometriosis compared to normal endometrial tissues from women without endometriosis and positively correlated. In human endometrial Ishikawa (ISK) cells, overexpression of CFTR stimulated cell migration with upregulated NFκB p65 and uPAR. Knockdown of CFTR inhibited cell migration. Furthermore, inhibition of NFκB with its inhibitors (curcumin or Bay) significantly reduced the expression of uPAR and cell migration in the CFTR-overexpressing ISK cells. Collectively, the present results suggest that the CFTR-NFκB-uPAR signaling may contribute to the progression of human endometriosis, and indicate potential targets for diagnosis and treatment.

  6. Benzopyrimido-pyrrolo-oxazine-dione (R)-BPO-27 Inhibits CFTR Chloride Channel Gating by Competition with ATP.

    Science.gov (United States)

    Kim, Yonjung; Anderson, Marc O; Park, Jinhong; Lee, Min Goo; Namkung, Wan; Verkman, A S

    2015-10-01

    We previously reported that benzopyrimido-pyrrolo-oxazinedione BPO-27 [6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid] inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with low nanomolar potency and reduces cystogenesis in a model of polycystic kidney disease. We used computational chemistry and patch-clamp to show that enantiomerically pure (R)-BPO-27 inhibits CFTR by competition with ATP, whereas (S)-BPO-27 is inactive. Docking computations using a homology model of CFTR structure suggested that (R)-BPO-27 binds near the canonical ATP binding site, and these findings were supported by molecular dynamics simulations showing a lower binding energy for the (R) versus (S) stereoisomers. Three additional lower-potency BPO-27 analogs were modeled in a similar fashion, with the binding energies predicted in the correct order. Whole-cell patch-clamp studies showed linear CFTR currents with a voltage-independent (R)-BPO-27 block mechanism. Single-channel recordings in inside-out patches showed reduced CFTR channel open probability and increased channel closed time by (R)-BPO-27 without altered unitary channel conductance. At a concentration of (R)-BPO-27 that inhibited CFTR chloride current by ∼50%, the EC50 for ATP activation of CFTR increased from 0.27 to 1.77 mM but was not changed by CFTRinh-172 [4-[[4-oxo-2-thioxo-3-[3-trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid], a thiazolidinone CFTR inhibitor that acts at a site distinct from the ATP binding site. Our results suggest that (R)-BPO-27 inhibition of CFTR involves competition with ATP. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  7. Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90.

    Directory of Open Access Journals (Sweden)

    Luiz Felipe M Prota

    Full Text Available Dexamethasone is widely used for pulmonary exacerbation in patients with cystic fibrosis, however, not much is known about the effects of glucocorticoids on the wild-type cystic fibrosis channel transmembrane regulator (CFTR. Our aim was to determine the effects of dexamethasone treatment on wild-type CFTR expression.Dose-response (1 nM to 10 µM and time course (3 to 48 h curves were generated for dexamethasone for mRNA expression in Calu-3 cells using a real-time PCR. Within 24 h, dexamethasone (10 nM showed a 0.3-fold decrease in CFTR mRNA expression, and a 3.2-fold increase in αENaC mRNA expression compared with control groups. Dexamethasone (10 nM induced a 1.97-fold increase in the total protein of wild-type CFTR, confirmed by inhibition by mifepristone. To access surface protein expression, biotinylation followed by Western blotting showed that dexamethasone treatment led to a 2.35-fold increase in the amount of CFTR in the cell surface compared with the untreated control groups. Once protein translation was inhibited with cycloheximide, dexamethasone could not increase the amount of CFTR protein. Protein stability was assessed by inhibition of protein synthesis with cycloheximide (50 µg/ml at different times in cells treated with dexamethasone and in untreated cells. Dexamethasone did not alter the degradation of wild-type CFTR. Assessment of the B band of CFTR within 15 min of metabolic pulse labeling showed a 1.5-fold increase in CFTR protein after treatment with dexamethasone for 24 h. Chaperone 90 (HSP90 binding to CFTR increased 1.55-fold after treatment with dexamethasone for 24 h, whereas chaperone 70 (HSP70 binding decreased 0.30 fold in an immunoprecipitation assay.Mature wild-type CFTR protein is regulated by dexamethasone post transcription, involving cotranslational mechanisms with HSP90 and HSP70, which enhances maturation and expression of wild-type CFTR.

  8. [Mutations of ACVRL1 gene in a pedigree with hereditary hemorrhagic telangiectasia].

    Science.gov (United States)

    Luo, Jie-wei; Chen, Hui; Yang, Liu-qing; Zhu, Ai-lan; Wu, Yan-an; Li, Jian-wei

    2008-06-01

    To identify the activin A receptor type II-like 1 gene (ACVRL1) mutations in a Chinese family with hereditary hemorrhagic telangiectasia (HHT2). The exons 3, 7 and 8 of ACVRL1 gene of the proband and her five family members were amplified by polymerase chain reaction (PCR), and the PCR products were sequenced. The proband had obvious telangiectasis of gastric mucosa, and small arteriovenous fistula in the right kidney. All the patients in the HHT2 family had iterative epistaxis or bleeding in other sites, and had telangiectasis of nasal mucosa, tunica mucosa oris and finger tips. ACVRL1 gene analysis confirmed that there is frameshift mutation caused by deletion of G145 in exon 3 in the 4 patients, but the mutation is absent in 2 members without HHT2. The HHT2 family is caused by a 145delG mutation of ACVRL1 gene, resulting in frameshift and a new stop codon at codon 53.

  9. Translational recoding as a feedback controller: systems approaches reveal polyamine-specific effects on the antizyme ribosomal frameshift

    Science.gov (United States)

    Rato, Claudia; Amirova, Svetlana R.; Bates, Declan G.; Stansfield, Ian; Wallace, Heather M.

    2011-01-01

    The antizyme protein, Oaz1, regulates synthesis of the polyamines putrescine, spermidine and spermine by controlling stability of the polyamine biosynthetic enzyme, ornithine decarboxylase. Antizyme mRNA translation depends upon a polyamine-stimulated +1 ribosomal frameshift, forming a complex negative feedback system in which the translational frameshifting event may be viewed in engineering terms as a feedback controller for intracellular polyamine concentrations. In this article, we present the first systems level study of the characteristics of this feedback controller, using an integrated experimental and modeling approach. Quantitative analysis of mutant yeast strains in which polyamine synthesis and interconversion were blocked revealed marked variations in frameshift responses to the different polyamines. Putrescine and spermine, but not spermidine, showed evidence of co-operative stimulation of frameshifting and the existence of multiple ribosome binding sites. Combinatorial polyamine treatments showed polyamines compete for binding to common ribosome sites. Using concepts from enzyme kinetics and control engineering, a mathematical model of the translational controller was developed to describe these complex ribosomal responses to combinatorial polyamine effects. Each one of a range of model predictions was successfully validated against experimental frameshift frequencies measured in S-adenosylmethionine-decarboxylase and antizyme mutants, as well as in the wild-type genetic background. PMID:21303766

  10. HNPCC: Six new pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Epplen Joerg T

    2004-06-01

    Full Text Available Abstract Background Hereditary non-polyposis colorectal cancer (HNPCC is an autosomal dominant disease with a high risk for colorectal and endometrial cancer caused by germline mutations in DNA mismatch-repair genes (MMR. HNPCC accounts for approximately 2 to 5% of all colorectal cancers. Here we present 6 novel mutations in the DNA mismatch-repair genes MLH1, MSH2 and MSH6. Methods Patients with clinical diagnosis of HNPCC were counselled. Tumor specimen were analysed for microsatellite instability and immunohistochemistry for MLH1, MSH2 and MSH6 protein was performed. If one of these proteins was not detectable in the tumor mutation analysis of the corresponding gene was carried out. Results We identified 6 frameshift mutations (2 in MLH1, 3 in MSH2, 1 in MSH6 resulting in a premature stop: two mutations in MLH1 (c.2198_2199insAACA [p.N733fsX745], c.2076_2077delTG [p.G693fsX702], three mutations in MSH2 (c.810_811delGT [p.C271fsX282], c.763_766delAGTGinsTT [p.F255fsX282], c.873_876delGACT [p.L292fsX298] and one mutation in MSH6 (c.1421_1422dupTG [p.C475fsX480]. All six tumors tested for microsatellite instability showed high levels of microsatellite instability (MSI-H. Conclusions HNPCC in families with MSH6 germline mutations may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations.

  11. A novel PCCB mutation in a Thai patient with propionic acidemia identified by exome sequencing.

    Science.gov (United States)

    Porntaveetus, Thantrira; Srichomthong, Chalurmpon; Suphapeetiporn, Kanya; Shotelersuk, Vorasuk

    2015-01-01

    Propionic acidemia (PA) is an inborn error of metabolism, caused by mutations in either the PCCA or PCCB gene, leading to mitochondrial accumulation of propionyl-CoA and its by-products. Here we report a 6-year-old Thai boy with PA who was born to consanguineous parents. Exome sequencing identified a novel homozygous frameshift insertion (c.379_380insA; p.T127NfsX160) in the PCCB gene, expanding its mutational spectrum.

  12. Graph-Theoretic Models of Mutations in the Nucleotide Binding Domain 1 of the Cystic Fibrosis Transmembrane Conductance Regulator

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    Debra J. Knisley

    2013-01-01

    Full Text Available Cystic fibrosis is one of the most common inherited diseases and is caused by a mutation in a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR. This protein serves as a chloride channel and regulates the viscosity of mucus lining the ducts of a number of organs. Although much has been learned about the consequences of mutations on the energy landscape and the resulting disrupted folding pathway of CFTR, a level of understanding needed to correct the misfolding has not been achieved. The most common mutations of CFTR are located in one of two nucleotide binding domains, namely, the nucleotide binding domain 1 (NBD1. We model NBD1 using a nested graph model. The vertices in the lowest layer each represent an atom in the structure of an amino acid residue, while the vertices in the mid layer each represent the residue. The vertices in the top layer each represent a subdomain of the nucleotide binding domain. We use this model to quantify the effects of a single point mutation on the protein domain. We compare the wildtype structure with eight of the most common mutations. The graph-theoretic model provides insight into how a single point mutation can have such profound structural consequences.

  13. Highest heterogeneity for cystic fibrosis: 36 mutations account for 75% of all CF chromosomes in Turkish patients.

    Science.gov (United States)

    Kilinç, Mehmet Okyay; Ninis, Vasiliki Ninidu; Dağli, Elif; Demirkol, Mübeccel; Ozkinay, Ferda; Arikan, Zeliha; Coğulu, Ozgür; Hüner, Gülden; Karakoç, Fazilet; Tolun, Aslihan

    2002-12-01

    We analyzed the CFTR locus in 83 Turkish cystic fibrosis patients to identify mutations, haplotypes, and the carrier frequency in the population. We detected 36 different mutations in 125 (75%) of the total 166 CF chromosomes. Seven novel mutations were identified: four missense (K68E, Q493P, E608G, and V1147I), two splice-site (406 -3T > C and 3849 +5G > A), and one deletion (CFTRdele17b,18). The data showed that the Turkish population has the highest genetic heterogeneity at the CFTR locus reported so far. The results of this thorough molecular analysis at the CFTR locus of a population not of European descent shows that CF is not uncommon in all such populations. The large number of mutations present, as well as the high heterogeneity in haplotypes associated with the mutations suggests that most of the mutations have persisted for a long time in the population. Consistently, the carrier frequency is assessed to be high, indicating that the disease in the population is ancient. Copyright 2002 Wiley-Liss, Inc.

  14. Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Hongbing; Franz, Carl J.; Wu, Guang; Renshaw, Hilary; Zhao, Guoyan [Departments of Molecular Microbiology and Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 (United States); Firth, Andrew E. [Department of Pathology, University of Cambridge, Cambridge CB2 1QP (United Kingdom); Wang, David, E-mail: davewang@borcim.wustl.edu [Departments of Molecular Microbiology and Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 (United States)

    2014-02-15

    Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated into Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha–delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses. - Highlights: • Orsay virus encodes a novel fusion protein by a ribosomal frameshifting mechanism. • Orsay capsid and fusion protein is translated from a non-canonical initiation site. • The fusion protein is likely incorporated into Orsay virions.

  15. The mutational spectrum in Waardenburg syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Read, A.P.; Tassabehji, M.; Liu, X.Z. [and others

    1994-09-01

    101 individuals or families with Waardenburg syndrome (WS) or related abnormalities have been screened for mutations in the PAX3 gene. PAX3 mutations were seen in 19 of 35 individuals or families with features of Type I Waardenburg syndrome. None of the 47 Type 2 WS families showed any PAX3 mutation, nor did any of 19 individuals with other neural crest syndromes or pigmentary disturbances. PAX3 mutations included substitutions of highly conserved amino acids, splice site mutations, nonsense mutations and frameshifting deletions or insertions. One patient (with Type 1 WS, mental retardation and growth retardation) had a chromosomal deletion of 7-8 Mb encompassing the PAX3 gene. Mutations were seen in each of exons 2-6, with a concentration in the 5{prime} part of the paired box (exon 2) and the 3{prime} part of the homeobox (exon 6). There was no evident relation between the molecular change and the clinical manifestations in mutation carriers. We conclude that PAX3 dosage effects very specifically produce dystopia canthorum, the distinguishing feature of Type 1 WS, and variably produce the other features of Type 1 WS depending on genetic background or chance events. Two of the Type 2 families showed linkage to markers from 3p14, the location of the MITF gene. MITF encodes a basic helix-loop-helix-zipper protein which is the homologue of the mouse microphthalmia gene product. It is likely that mutations in MITF cause some but not all Type 2 WS.

  16. Diagnosis of cystic fibrosis in the kindred of an infant with CFTR-related metabolic syndrome: importance of follow-up that includes monitoring sweat chloride concentrations over time.

    Science.gov (United States)

    Williams, Sophia N; Nussbaum, Eliezer; Chin, Terry W; Do, Paul C M; Singh, Kathryn E; Randhawa, Inderpal

    2014-03-01

    Newly implemented newborn screening (NBS) programs in California have resulted in a large subset of patients in whom at least two cystic fibrosis transmembrane conductance regulator (CFTR) mutations are identified, but subsequent sweat chloride analysis reveals normal or indeterminate values. These patients are diagnosed with CFTR-Related Metabolic Syndrome (CRMS). However, the natural progression and management of these patients are not clearly understood and frequently after the age of 1-year these patients are lost to follow-up with Cystic Fibrosis (CF) Centers. We present the first case of an infant who was referred to Miller Children's Hospital for a NBS positive for CF and subsequent discovery of identical mutations in six of his seven older brothers. Several siblings had positive sweat chloride results on repeat testing after the age of 3 years. We suggest the need for continued follow-up of CRMS in a CF center with diagnostic evaluation including repeat sweat chloride testing, beyond the currently recommended period. © 2013 Wiley Periodicals, Inc.

  17. DNA mutation motifs in the genes associated with inherited diseases.

    Directory of Open Access Journals (Sweden)

    Michal Růžička

    Full Text Available Mutations in human genes can be responsible for inherited genetic disorders and cancer. Mutations can arise due to environmental factors or spontaneously. It has been shown that certain DNA sequences are more prone to mutate. These sites are termed hotspots and exhibit a higher mutation frequency than expected by chance. In contrast, DNA sequences with lower mutation frequencies than expected by chance are termed coldspots. Mutation hotspots are usually derived from a mutation spectrum, which reflects particular population where an effect of a common ancestor plays a role. To detect coldspots/hotspots unaffected by population bias, we analysed the presence of germline mutations obtained from HGMD database in the 5-nucleotide segments repeatedly occurring in genes associated with common inherited disorders, in particular, the PAH, LDLR, CFTR, F8, and F9 genes. Statistically significant sequences (mutational motifs rarely associated with mutations (coldspots and frequently associated with mutations (hotspots exhibited characteristic sequence patterns, e.g. coldspots contained purine tract while hotspots showed alternating purine-pyrimidine bases, often with the presence of CpG dinucleotide. Using molecular dynamics simulations and free energy calculations, we analysed the global bending properties of two selected coldspots and two hotspots with a G/T mismatch. We observed that the coldspots were inherently more flexible than the hotspots. We assume that this property might be critical for effective mismatch repair as DNA with a mutation recognized by MutSα protein is noticeably bent.

  18. DNA mutation motifs in the genes associated with inherited diseases.

    Science.gov (United States)

    Růžička, Michal; Kulhánek, Petr; Radová, Lenka; Čechová, Andrea; Špačková, Naďa; Fajkusová, Lenka; Réblová, Kamila

    2017-01-01

    Mutations in human genes can be responsible for inherited genetic disorders and cancer. Mutations can arise due to environmental factors or spontaneously. It has been shown that certain DNA sequences are more prone to mutate. These sites are termed hotspots and exhibit a higher mutation frequency than expected by chance. In contrast, DNA sequences with lower mutation frequencies than expected by chance are termed coldspots. Mutation hotspots are usually derived from a mutation spectrum, which reflects particular population where an effect of a common ancestor plays a role. To detect coldspots/hotspots unaffected by population bias, we analysed the presence of germline mutations obtained from HGMD database in the 5-nucleotide segments repeatedly occurring in genes associated with common inherited disorders, in particular, the PAH, LDLR, CFTR, F8, and F9 genes. Statistically significant sequences (mutational motifs) rarely associated with mutations (coldspots) and frequently associated with mutations (hotspots) exhibited characteristic sequence patterns, e.g. coldspots contained purine tract while hotspots showed alternating purine-pyrimidine bases, often with the presence of CpG dinucleotide. Using molecular dynamics simulations and free energy calculations, we analysed the global bending properties of two selected coldspots and two hotspots with a G/T mismatch. We observed that the coldspots were inherently more flexible than the hotspots. We assume that this property might be critical for effective mismatch repair as DNA with a mutation recognized by MutSα protein is noticeably bent.

  19. ER-associated complexes (ERACs) containing aggregated cystic fibrosis transmembrane conductance regulator (CFTR) are degraded by autophagy

    OpenAIRE

    Fua, Lianwu; Sztula, Elizabeth

    2009-01-01

    The ubiquitin-proteasome pathway and autophagy are the two major mechanisms responsible for the clearance of cellular proteins. We have used the yeast Saccharomyces cerevisiae as a model system and the cystic fibrosis transmembrane conductance regulator (CFTR) as a model substrate to study the interactive function of these two pathways in the degradation of misfolded proteins. EGFP-tagged human CFTR was introduced into yeast and expressed under a copper-inducible promoter. The localization an...

  20. CFTR Influences Beta Cell Function and Insulin Secretion Through Non-Cell Autonomous Exocrine-Derived Factors.

    Science.gov (United States)

    Sun, Xingshen; Yi, Yaling; Xie, Weiliang; Liang, Bo; Winter, Michael C; He, Nan; Liu, Xiaoming; Luo, Meihui; Yang, Yu; Ode, Katie Larson; Uc, Aliye; Norris, Andrew W; Engelhardt, John F

    2017-10-01

    Although β-cell dysfunction in cystic fibrosis (CF) leads to diabetes, the mechanism by which the cystic fibrosis transmembrane conductance regulator (CFTR) channel influences islet insulin secretion remains debated. We investigated the CFTR-dependent islet-autonomous mechanisms affecting insulin secretion by using islets isolated from CFTR knockout ferrets. Total insulin content was lower in CF as compared with wild-type (WT) islets. Furthermore, glucose-stimulated insulin secretion (GSIS) was impaired in perifused neonatal CF islets, with reduced first, second, and amplifying phase secretion. Interestingly, CF islets compensated for reduced insulin content under static low-glucose conditions by secreting a larger fraction of islet insulin than WT islets, probably because of elevated SLC2A1 transcripts, increased basal inhibition of adenosine triphosphate-sensitive potassium channels (K-ATP), and elevated basal intracellular Ca2+. Interleukin (IL)-6 secretion by CF islets was higher relative to WT, and IL-6 treatment of WT ferret islets produced a CF-like phenotype with reduced islet insulin content and elevated percentage insulin secretion in low glucose. CF islets exhibited altered expression of INS, CELA3B, and several β-cell maturation and proliferation genes. Pharmacologic inhibition of CFTR reduced GSIS by WT ferret and human islets but similarly reduced insulin secretion and intracellular Ca2+ in CFTR knockout ferret islets, indicating that the mechanism of action is not through CFTR. Single-molecule fluorescent in situ hybridization, on isolated ferret and human islets and ferret pancreas, demonstrated that CFTR RNA colocalized within KRT7+ ductal cells but not endocrine cells. These results suggest that CFTR affects β-cell function via a paracrine mechanism involving proinflammatory factors secreted from islet-associated exocrine-derived cell types. Copyright © 2017 Endocrine Society.

  1. Glucocorticoids Distinctively Modulate the CFTR Channel with Possible Implications in Lung Development and Transition into Extrauterine Life.

    Science.gov (United States)

    Laube, Mandy; Bossmann, Miriam; Thome, Ulrich H

    2015-01-01

    During fetal development, the lung is filled with fluid that is secreted by an active Cl- transport promoting lung growth. The basolateral Na+,K+,2Cl- cotransporter (NKCC1) participates in Cl- secretion. The apical Cl- channels responsible for secretion are unknown but studies suggest an involvement of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is developmentally regulated with a high expression in early fetal development and a decline in late gestation. Perinatal lung transition is triggered by hormones that stimulate alveolar Na+ channels resulting in fluid absorption. Little is known on how hormones affect pulmonary Cl- channels. Since the rise of fetal cortisol levels correlates with the decrease in fetal CFTR expression, a causal relation may be assumed. The aim of this study was to analyze the influence of glucocorticoids on pulmonary Cl- channels. Alveolar cells from fetal and adult rats, A549 cells, bronchial Calu-3 and 16HBE14o- cells, and primary rat airway cells were studied with real-time quantitative PCR and Ussing chambers. In fetal and adult alveolar cells, glucocorticoids strongly reduced Cftr expression and channel activity, which was prevented by mifepristone. In bronchial and primary airway cells CFTR mRNA expression was also reduced, whereas channel activity was increased which was prevented by LY-294002 in Calu-3 cells. Therefore, glucocorticoids strongly reduce CFTR expression while their effect on CFTR activity depends on the physiological function of the cells. Another apical Cl- channel, anoctamin 1 showed a glucocorticoid-induced reduction of mRNA expression in alveolar cells and an increase in bronchial cells. Furthermore, voltage-gated chloride channel 5 and anoctamine 6 mRNA expression were increased in alveolar cells. NKCC1 expression was reduced by glucocorticoids in alveolar and bronchial cells alike. The results demonstrate that glucocorticoids differentially modulate pulmonary Cl- channels and are likely

  2. Evidence against the rescue of defective DeltaF508-CFTR cellular processing by curcumin in cell culture and mouse models.

    Science.gov (United States)

    Song, Yuanlin; Sonawane, N D; Salinas, Danieli; Qian, Liman; Pedemonte, Nicoletta; Galietta, Luis J V; Verkman, A S

    2004-09-24

    Curcumin, the yellow colored component of the spice turmeric, has been reported to rescue defective DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) cellular processing in homozygous mutant mice, restoring nasal potential differences and improving survival (Egan, M. E., Pearson, M., Weiner, S. A., Rajendran, V., Rubin, D., Glockner-Pagel, J., Canny, S., Du, K., Lukacs, G. L., and Caplan, M. J. (2004) Science 304, 600-602). Because of the implied potential use of curcumin or similar compounds in the therapy of cystic fibrosis caused by the DeltaF508 mutation, we tried to reproduce and extend the pre-clinical data of Egan et al. Fluorometric measurements of iodide influx in Fischer rat thyroid cells expressing DeltaF508-CFTR showed no effect of curcumin (1-40 microm) when added for up to 24 h prior to assay in cells grown at 37 degrees C. Controls, including 27 degrees C rescue and 4 mm phenylbutyrate at 37 degrees C, were strongly positive. Also, curcumin did not increase short circuit current in primary cultures of a human airway epithelium homozygous for DeltaF508-CFTR with a 27 degrees C rescue-positive control. Nasal potential differences in mice were measured in response to topical perfusion with serial solutions containing amiloride, low Cl-, and forskolin. Robust low Cl- and forskolin-induced hyperpolarization of 22 +/- 3 mV was found in wild type mice, with 2.1 +/- 0.4 mV hyperpolarization in DeltaF508 homozygous mutant mice. No significant increase in Cl-/forskolin hyperpolarization was seen in any of the 22 DeltaF508 mice studied using different curcumin preparations and administration regimens, including that used by Egan et al. Assay of serum curcumin by ethyl acetate extraction followed by liquid chromatography/mass spectrometry indicated a maximum serum concentration of 60 nm, well below that of 5-15 microm, where cellular effects by sarcoplasmic/endoplasmic reticulum calcium pump inhibition are proposed to occur. Our results do not

  3. Chlorogenic Acid Activates CFTR-Mediated Cl- Secretion in Mice and Humans: Therapeutic Implications for Chronic Rhinosinusitis.

    Science.gov (United States)

    Illing, Elisa A; Cho, Do-Yeon; Zhang, Shaoyan; Skinner, Daniel F; Dunlap, Quinn A; Sorscher, Eric J; Woodworth, Bradford A

    2015-08-01

    Salubrious effects of the green coffee bean are purportedly secondary to high concentrations of chlorogenic acid. Chlorogenic acid has a molecular structure similar to bioflavonoids that activate transepithelial Cl(-) transport in sinonasal epithelia. In contrast to flavonoids, the drug is freely soluble in water. The objective of this study is to evaluate the Cl(-) secretory capability of chlorogenic acid and its potential as a therapeutic activator of mucus clearance in sinus disease. Basic research. Laboratory. Chlorogenic acid was tested on primary murine nasal septal epithelial (MNSE) (CFTR(+/+) and transgenic CFTR(-/-)) and human sinonasal epithelial (HSNE) (CFTR(+/+) and F508del/F508del) cultures under pharmacologic conditions in Ussing chambers to evaluate effects on transepithelial Cl(-) transport. Cellular cyclic adenosine monophosphate (cAMP), phosphorylation of the CFTR regulatory domain (R-D), and CFTR mRNA transcription were also measured. Chlorogenic acid stimulated transepithelial Cl(-) secretion (change in short-circuit current [ΔISC = µA/cm(2)]) in MNSE (13.1 ± 0.9 vs 0.1 ± 0.1; P chlorogenic acid does not work through a PKA-dependent mechanism. Chlorogenic acid is a water-soluble agent that promotes CFTR-mediated Cl(-) transport in mouse and human sinonasal epithelium. Translating activators of mucociliary transport to clinical use provides a new therapeutic approach to sinus disease. Further in vivo evaluation is planned. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.

  4. Lack of CFTR in skeletal muscle predisposes to muscle wasting and diaphragm muscle pump failure in cystic fibrosis mice.

    Directory of Open Access Journals (Sweden)

    Maziar Divangahi

    2009-07-01

    Full Text Available Cystic fibrosis (CF patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR-deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr(-/- mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1 involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.

  5. Unusual long survival despite severe lung disease of a child with biallelic loss of function mutations in ABCA-3

    Directory of Open Access Journals (Sweden)

    P. El Boustany

    Full Text Available Homozygous or compound heterozygous for frameshift or nonsense mutations in the ATP–binding cassette transporter A3 (ABCA3 is associated with neonatal respiratory failure and death within the first year of life without lung transplantation. We report the case of a newborn baby girl who developed severe respiratory distress soon after birth. She was diagnosed with compound heterozygous frameshift mutation of the ABCA3 gene. Despite extensive treatment (intravenous corticosteroids pulse therapy, oral corticosteroids, azithromycin, and hydroxychloroquine, she developed chronic respiratory failure. As the parents refused cardio-pulmonary transplantation and couldn't resolve to an accompaniment of end of life, a tracheostomy was performed resulting in continuous mechanical ventilation. A neurodevelopmental delay and an overall muscular dystrophy were noted. At the age of 5 years, after 2 episodes of pneumothorax, the patient died from severe respiratory failure. To our knowledge, this was the first case of a child with compound heterozygous frameshift mutation who posed such an ethical dilemma with a patient surviving till the age of five years. Keywords: ABCA3 deficiency, Compound heterozygous frameshift mutation, Neonatal respiratory failure, Tracheostomy, Mechanical ventilation, Ethical dilemma

  6. Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

    Science.gov (United States)

    Vernon, Robert M; Chong, P Andrew; Lin, Hong; Yang, Zhengrong; Zhou, Qingxian; Aleksandrov, Andrei A; Dawson, Jennifer E; Riordan, John R; Brouillette, Christie G; Thibodeau, Patrick H; Forman-Kay, Julie D

    2017-08-25

    Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Second-hand cigarette smoke impairs bacterial phagocytosis in macrophages by modulating CFTR dependent lipid-rafts.

    Directory of Open Access Journals (Sweden)

    Inzer Ni

    Full Text Available First/Second-hand cigarette-smoke (FHS/SHS exposure weakens immune defenses inducing chronic obstructive pulmonary disease (COPD but the underlying mechanisms are not fully understood. Hence, we evaluated if SHS induced changes in membrane/lipid-raft (m-/r-CFTR (cystic fibrosis transmembrane conductance regulator expression/activity is a potential mechanism for impaired bacterial phagocytosis in COPD.RAW264.7 murine macrophages were exposed to freshly prepared CS-extract (CSE containing culture media and/or Pseudomonas-aeruginosa-PA01-GFP for phagocytosis (fluorescence-microscopy, bacterial survival (colony-forming-units-CFU, and immunoblotting assays. The CFTR-expression/activity and lipid-rafts were modulated by transient-transfection or inhibitors/inducers. Next, mice were exposed to acute/sub-chronic-SHS or room-air (5-days/3-weeks and infected with PA01-GFP, followed by quantification of bacterial survival by CFU-assay.We investigated the effect of CSE treatment on RAW264.7 cells infected by PA01-GFP and observed that CSE treatment significantly (p<0.01 inhibits PA01-GFP phagocytosis as compared to the controls. We also verified this in murine model, exposed to acute/sub-chronic-SHS and found significant (p<0.05, p<0.02 increase in bacterial survival in the SHS-exposed lungs as compared to the room-air controls. Next, we examined the effect of impaired CFTR ion-channel-activity on PA01-GFP infection of RAW264.7 cells using CFTR172-inhibitor and found no significant change in phagocytosis. We also similarly evaluated the effect of a CFTR corrector-potentiator compound, VRT-532, and observed no significant rescue of CSE impaired PA01-GFP phagocytosis although it significantly (p<0.05 decreases CSE induced bacterial survival. Moreover, induction of CFTR expression in macrophages significantly (p<0.03 improves CSE impaired PA01-GFP phagocytosis as compared to the control. Next, we verified the link between m-/r-CFTR expression and phagocytosis

  8. Immunodeficiency associated with FCN3 mutation and ficolin-3 deficiency

    DEFF Research Database (Denmark)

    Munthe-Fog, Lea; Hummelshøj, Tina; Honoré, Christian

    2009-01-01

    Ficolin-3, encoded by the FCN3 gene and expressed in the lung and liver, is a recognition molecule in the lectin pathway of the complement system. Heterozygosity for an FCN3 frameshift mutation (rs28357092), leading to a distortion of the C-terminal end of the molecule, occurs in people without...... disease (allele frequency among whites, 0.01). We describe a patient with recurrent infections who was homozygous for this mutation, who had undetectable serum levels of ficolin-3, and who had a deficiency in ficolin-3-dependent complement activation....

  9. EFFECT OF ALLERGY AND INFLAMMATION ON EICOSANOID GENE EXPRESSION IN CFTR DEFICIENCY

    Science.gov (United States)

    Bickford, Justin S.; Mueller, Christian; Newsom, Kimberly J.; Barilovits, Sarah J.; Beachy, Dawn E.; Herlihy, John D.; Keeler, Benjamin; Flotte, Terence R.; Nick, Harry S.

    2012-01-01

    Background Allergic bronchopulmonary aspergillosis (ABPA) is a complicating factor in cystic fibrosis (CF), affecting 2–15% of patients. We hypothesized that sensitization/challenge of CFTR−/− mice with an Aspergillus fumigatus (Af) extract will affect eicosanoid pathway gene expression, impacting ABPA and CF. Methods FABP-hCFTR+/−-CFTR−/− mice were sensitized/challenged with an Af extract and gene expression of lung mRNA was evaluated for >40 genes, with correlative data in human CF (IB3.1) and CFTR-corrected (S9) bronchoepithelial cell lines. Results Pla2g4c, Pla2g2c, Pla2g2d and Pla2g5 were induced in response to Af in CFTR−/− mice. Interestingly, PLA2G2D was induced by LPS, IL-2, IL-6, IL-13, and Af only in CFTR-deficient human IB3.1 cells. Prostanoid gene expression was relatively constant, however, several 12/15-lipoxygenase genes were induced in response to Af. Numerous cytokines also caused differential expression of ALOX15 only in IB3.1 cells. Conclusions The distinct regulation of PLA2G4C, PLA2G2D and ALOX15 genes in Aspergillus sensitization and/or cystic fibrosis could provide new insights into diagnosis and treatment of ABPA and CF. PMID:22985691

  10. Stimulation of Intestinal Cl- Secretion Through CFTR by Caffeine Intake in Salt-Sensitive Hypertensive Rats

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    Xiao Wei

    2018-03-01

    Full Text Available Background/Aims: High salt consumption is a major risk factor for hypertension, and sodium homeostasis is regulated by both intestinal sodium absorption and urinary sodium excretion. Chronic caffeine intake has been reported to attenuate salt-sensitive hypertension by promoting urinary sodium excretion; however, its exact role in intestinal sodium absorption remains unknown. Here, we investigated whether and how chronic caffeine consumption antagonizes salt-sensitive hypertension by inhibiting intestinal sodium absorption. Methods: Dahl salt-sensitive rats were fed 8% NaCl chow and 0.1% caffeine in their drinking water for 15 days. The blood pressure and fecal sodium content were measured. The effect of caffeine on the movement of Cl- in enterocyte cells was determined with the Ussing chamber assay. Results: Rats that were treated with caffeine displayed significantly lower mean blood pressure and higher fecal sodium content than the controls. Consistent with these findings, caffeine intake decreased fluid absorption by the intestine in the fluid perfusion experiment. Further, the results from the Ussing chamber assay indicated that caffeine promoted Cl- secretion through enterocyte apical cystic fibrosis transmembrane conductance regulator (CFTR, and thus inhibited sodium absorption. Moreover, depletion of cAMP or inhibition of CFTR completely abolished the effect of caffeine on Cl- secretion. Conclusion: The results indicate that chronic caffeine consumption reduces sodium absorption by promoting CFTR-mediated Cl- secretion in the intestine, which contributes to the anti-hypertensive effect of caffeine in salt-sensitive rats.

  11. Longevity and plasticity of CFTR provide an argument for noncanonical SNP organization in hominid DNA.

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    Aubrey E Hill

    Full Text Available Like many other ancient genes, the cystic fibrosis transmembrane conductance regulator (CFTR has survived for hundreds of millions of years. In this report, we consider whether such prodigious longevity of an individual gene--as opposed to an entire genome or species--should be considered surprising in the face of eons of relentless DNA replication errors, mutagenesis, and other causes of sequence polymorphism. The conventions that modern human SNP patterns result either from purifying selection or random (neutral drift were not well supported, since extant models account rather poorly for the known plasticity and function (or the established SNP distributions found in a multitude of genes such as CFTR. Instead, our analysis can be taken as a polemic indicating that SNPs in CFTR and many other mammalian genes may have been generated--and continue to accrue--in a fundamentally more organized manner than would otherwise have been expected. The resulting viewpoint contradicts earlier claims of 'directional' or 'intelligent design-type' SNP formation, and has important implications regarding the pace of DNA adaptation, the genesis of conserved non-coding DNA, and the extent to which eukaryotic SNP formation should be viewed as adaptive.

  12. CFTR-dependent defect in alternatively-activated macrophages in cystic fibrosis.

    Science.gov (United States)

    Tarique, Abdullah A; Sly, Peter D; Holt, Patrick G; Bosco, Anthony; Ware, Robert S; Logan, Jayden; Bell, Scott C; Wainwright, Claire E; Fantino, Emmanuelle

    2017-07-01

    The role of the macrophages in cystic fibrosis (CF) lung disease has been poorly studied. We hypothesized that alternatively activated M2 macrophages are abnormal in CF lung disease. Blood samples were collected from adults (n=13) children (n=27) with CF on admission for acute pulmonary exacerbation and when clinically stable. Monocytes were differentiated into macrophages and polarized into classical (M1) and alternatively-activated (M2) phenotypes, function determined ex-vivo and compared with healthy controls. In the absence of functional cystic fibrosis trans-membrane conductance regulator (CFTR), either naturally in patients with CF or induced with CFTR inhibitors, monocyte-derived macrophages do not respond to IL-13/IL-4, fail to polarize into M2s associated with a post-transcriptional failure to produce and express IL-13Rα1 on the macrophage surface Polarization to the M1 phenotype was unaffected. CFTR-dependent imbalance of macrophage phenotypes and functions could contribute to the exaggerated inflammatory response seen in CF lung disease. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  13. Benign and Deleterious Cystic Fibrosis Transmembrane Conductance Regulator Mutations Identified by Sequencing in Positive Cystic Fibrosis Newborn Screen Children from California.

    Science.gov (United States)

    Salinas, Danieli B; Sosnay, Patrick R; Azen, Colleen; Young, Suzanne; Raraigh, Karen S; Keens, Thomas G; Kharrazi, Martin

    2016-01-01

    Of the 2007 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutations, 202 have been assigned disease liability. California's racially diverse population, along with CFTR sequencing as part of newborn screening model, provides the opportunity to examine the phenotypes of children with uncategorized mutations to help inform disease liability and penetrance. We conducted a retrospective cohort study based on children screened from 2007 to 2011 and followed for two to six years. Newborns that screened positive were divided into three genotype groups: those with two CF-causing mutations (CF-C); those with one mutation of varying clinic consequence (VCC); and those with one mutation of unknown disease liability (Unknown). Sweat chloride tests, pancreatic sufficiency status, and Pseudomonas aeruginosa colonization were compared. Children with two CF-causing mutations had a classical CF phenotype, while 5% of VCC (4/78) and 11% of Unknown (27/244) met diagnostic criteria of CF. Children carrying Unknown mutations 2215insG with D836Y, and T1036N had early and classical CF phenotype, while others carrying 1525-42G>A, L320V, L967S, R170H, and 296+28A>G had a benign clinical presentation, suggesting that these are non-CF causing. While most infants with VCC and Unknown CFTR mutations do not meet diagnostic criteria for CF, a small proportion do. These findings highlight the range of genotypes and phenotypes in the first few years of life following CF newborn screening when CFTR sequencing is performed.

  14. Benign and Deleterious Cystic Fibrosis Transmembrane Conductance Regulator Mutations Identified by Sequencing in Positive Cystic Fibrosis Newborn Screen Children from California.

    Directory of Open Access Journals (Sweden)

    Danieli B Salinas

    Full Text Available Of the 2007 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR mutations, 202 have been assigned disease liability. California's racially diverse population, along with CFTR sequencing as part of newborn screening model, provides the opportunity to examine the phenotypes of children with uncategorized mutations to help inform disease liability and penetrance.We conducted a retrospective cohort study based on children screened from 2007 to 2011 and followed for two to six years. Newborns that screened positive were divided into three genotype groups: those with two CF-causing mutations (CF-C; those with one mutation of varying clinic consequence (VCC; and those with one mutation of unknown disease liability (Unknown. Sweat chloride tests, pancreatic sufficiency status, and Pseudomonas aeruginosa colonization were compared.Children with two CF-causing mutations had a classical CF phenotype, while 5% of VCC (4/78 and 11% of Unknown (27/244 met diagnostic criteria of CF. Children carrying Unknown mutations 2215insG with D836Y, and T1036N had early and classical CF phenotype, while others carrying 1525-42G>A, L320V, L967S, R170H, and 296+28A>G had a benign clinical presentation, suggesting that these are non-CF causing.While most infants with VCC and Unknown CFTR mutations do not meet diagnostic criteria for CF, a small proportion do. These findings highlight the range of genotypes and phenotypes in the first few years of life following CF newborn screening when CFTR sequencing is performed.

  15. Airway Clearance Techniques (ACTs)

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    Full Text Available ... The Basics CF Mutations Video Series CFTR2 Personalized Medicine Types of CFTR Mutations DIAGNOSIS If you or ... The Basics CF Mutations Video Series CFTR2 Personalized Medicine Types of CFTR Mutations Researcher Resources Researchers, supported ...

  16. ΔF508 CFTR surface stability is regulated by DAB2 and CHIP-mediated ubiquitination in post-endocytic compartments.

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    Lianwu Fu

    Full Text Available The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2 and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2 on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR.

  17. Stimulation of wild-type, F508del- and G551D-CFTR chloride channels by non toxic modified pyrrolo[2,3-b]pyrazine derivatives

    Directory of Open Access Journals (Sweden)

    Luc eDannhoffer

    2011-08-01

    Full Text Available Cystic Fibrosis is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs due to abnormal function of Cystic Fibrosis Transmembrane conductance Regulator (CFTR protein. We recently identified a family of CFTR activators, which contains the hit: RP107 [7-n-butyl-6-(4-hydroxyphenyl[5H]-pyrrolo[2,3-b]pyrazine]. Here, we further evaluated the effect of the chemical modifications of the RP107-OH radical on CFTR activation. The replacement of the OH radical by a fluorine atom at position 2 (RP193 or 4 (RP185 significantly decreased the toxicity of the compounds without altering the ability to activate CFTR, especially for RP193. The non-toxic compound RP193 has no effect on cAMP production but stimulates the channel activity of wild-type CFTR in stably transfected CHO cells, in human bronchial epithelial NuLi-1 cells and in primary culture of human bronchial epithelial cells. Whole cell and single patch clamp recordings showed that RP193 induced a linear, time and voltage-independent current, which was fully inhibited by two different and selective CFTR inhibitors (CFTRinh-172 and GPinh-5a. Moreover, RP193 stimulates CFTR in temperature-rescued CuFi-1 (F508del/F508del human bronchial epithelial cells and in CHO cells stably expressing G551D-CFTR. This study shows that it is feasible to reduce cytotoxicity of chemical compounds without affecting their potency to activate CFTR and to rescue the class 2 F508del-CFTR and class 3 G551D-CFTR CF mutant activities.

  18. Using exome data to identify malignant hyperthermia susceptibility mutations.

    Science.gov (United States)

    Gonsalves, Stephen G; Ng, David; Johnston, Jennifer J; Teer, Jamie K; Stenson, Peter D; Cooper, David N; Mullikin, James C; Biesecker, Leslie G

    2013-11-01

    Malignant hyperthermia susceptibility (MHS) is a life-threatening, inherited disorder of muscle calcium metabolism, triggered by anesthetics and depolarizing muscle relaxants. An unselected cohort was screened for MHS mutations using exome sequencing. The aim of this study was to pilot a strategy for the RYR1 and CACNA1S genes. Exome sequencing was performed on 870 volunteers not ascertained for MHS. Variants in RYR1 and CACNA1S were annotated using an algorithm that filtered results based on mutation type, frequency, and information in mutation databases. Variants were scored on a six-point pathogenicity scale. Medical histories and pedigrees were reviewed for malignant hyperthermia and related disorders. The authors identified 70 RYR1 and 53 CACNA1S variants among 870 exomes. Sixty-three RYR1 and 41 CACNA1S variants passed the quality and frequency metrics but the authors excluded synonymous variants. In RYR1, the authors identified 65 missense mutations, one nonsense, two that affected splicing, and one non-frameshift indel. In CACNA1S, 48 missense, one frameshift deletion, one splicing, and one non-frameshift indel were identified. RYR1 variants predicted to be pathogenic for MHS were found in three participants without medical or family histories of MHS. Numerous variants, previously described as pathogenic in mutation databases, were reclassified by the authors as being of unknown pathogenicity. Exome sequencing can identify asymptomatic patients at risk for MHS, although the interpretation of exome variants can be challenging. The use of exome sequencing in unselected cohorts is an important tool to understand the prevalence and penetrance of MHS, a critical challenge for the field.

  19. Fitness is strongly influenced by rare mutations of large effect in a microbial mutation accumulation experiment.

    Science.gov (United States)

    Heilbron, Karl; Toll-Riera, Macarena; Kojadinovic, Mila; MacLean, R Craig

    2014-07-01

    Our understanding of the evolutionary consequences of mutation relies heavily on estimates of the rate and fitness effect of spontaneous mutations generated by mutation accumulation (MA) experiments. We performed a classic MA experiment in which frequent sampling of MA lines was combined with whole genome resequencing to develop a high-resolution picture of the effect of spontaneous mutations in a hypermutator (ΔmutS) strain of the bacterium Pseudomonas aeruginosa. After ∼644 generations of mutation accumulation, MA lines had accumulated an average of 118 mutations, and we found that average fitness across all lines decayed linearly over time. Detailed analyses of the dynamics of fitness change in individual lines revealed that a large fraction of the total decay in fitness (42.3%) was attributable to the fixation of rare, highly deleterious mutations (comprising only 0.5% of fixed mutations). Furthermore, we found that at least 0.64% of mutations were beneficial and probably fixed due to positive selection. The majority of mutations that fixed (82.4%) were base substitutions and we failed to find any signatures of selection on nonsynonymous or intergenic mutations. Short indels made up a much smaller fraction of the mutations that were fixed (17.4%), but we found evidence of strong selection against indels that caused frameshift mutations in coding regions. These results help to quantify the amount of natural selection present in microbial MA experiments and demonstrate that changes in fitness are strongly influenced by rare mutations of large effect. Copyright © 2014 by the Genetics Society of America.

  20. Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I.

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    Seung Bum Park

    Full Text Available Hepatitis C virus (HCV actively evades host interferon (IFN responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP and poly(IC. The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity.

  1. Chromatin remodeling mediated by the FOXA1/A2 transcription factors activates CFTR expression in intestinal epithelial cells.

    Science.gov (United States)

    Kerschner, Jenny L; Gosalia, Nehal; Leir, Shih-Hsing; Harris, Ann

    2014-04-01

    The forkhead box A transcription factors, FOXA1 and FOXA2, function as pioneer factors to open condensed chromatin and facilitate binding of other proteins. We showed previously that these factors are key components of a transcriptional network that drives enhancer function at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in intestinal epithelial cells. The CFTR promoter apparently lacks tissue-specific regulatory elements and expression of the gene is controlled by multiple cis-acting elements, which coordinate gene expression in different cell types. Here we show that concurrent depletion of FOXA1 and FOXA2 represses CFTR expression and alters the three-dimensional architecture of the active locus by diminishing interactions between the promoter and intronic cis-acting elements. Reduction of FOXA1/A2 also modifies the enrichment profile of the active enhancer marks H3K27ac and H3K4me2 across the CFTR locus and alters chromatin accessibility at individual cis-elements. Moreover, loss of FOXA1/A2 suppresses the recruitment of other members of the transcriptional network including HNF1 and CDX2, to multiple cis-elements. These data reveal a complex molecular mechanism underlying the role of FOXA1/A2 in achieving high levels of CFTR expression in intestinal epithelial cells.

  2. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

    Science.gov (United States)

    Stevers, Loes M; Lam, Chan V; Leysen, Seppe F R; Meijer, Femke A; van Scheppingen, Daphne S; de Vries, Rens M J M; Carlile, Graeme W; Milroy, Lech G; Thomas, David Y; Brunsveld, Luc; Ottmann, Christian

    2016-03-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics.

  3. A Crohn's disease-associated NOD2 mutation suppresses transcription of human IL10 by inhibiting activity of the nuclear ribonucleoprotein hnRNP-A1.

    NARCIS (Netherlands)

    Noguchi, E.; Homma, Y.; Kang, X.; Netea, M.G.; Ma, X.

    2009-01-01

    A common mutation in the gene encoding the cytoplasmic sensor Nod2, involving a frameshift insertion at nucleotide 3020 (3020insC), is strongly associated with Crohn's disease. How 3020insC contributes to this disease is a controversial issue. Clinical studies have identified defective production of

  4. Screening of 1331 Danish breast and/or ovarian cancer families identified 40 novel BRCA1 and BRCA2 mutations

    DEFF Research Database (Denmark)

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

    2011-01-01

    Germ-line mutations in the tumour suppressor genes BRCA1 and BRCA2 predispose to breast and ovarian cancer. Since 1999 we have performed mutational screening of breast and/or ovarian cancer patients in East Denmark. During this period we have identified 40 novel sequence variations in BRCA1...... and BRCA2 in high risk breast and/or ovarian cancer families. The mutations were detected via pre-screening using dHPLC or high-resolution melting and direct sequencing. We identified 16 variants in BRCA1, including 9 deleterious frame-shift mutations, 2 intronic variants, 4 missense mutations, and 1......, the presumed significance of the missense mutations was predicted in silico using the align GVGD algorithm. In conclusion, the mutation screening identified 40 novel variants in the BRCA1 and BRCA2 genes and thereby extends the knowledge of the BRCA1/BRCA2 mutation spectrum. Nineteen of the mutations were...

  5. Identification of a Danish breast/ovarian cancer family double heterozygote for BRCA1 and BRCA2 mutations

    DEFF Research Database (Denmark)

    Steffensen, Ane Y; Jønson, Lars; Ejlertsen, Bent

    2010-01-01

    Mutations in the two breast cancer susceptibility genes BRCA1 and BRCA2 are associated with increased risk of breast and ovarian cancer. Patients with mutations in both genes are rarely reported and often involve Ashkenazi founder mutations. Here we report the first identification of a Danish...... breast and ovarian cancer family heterozygote for mutations in the BRCA1 and BRCA2 genes. The BRCA1 nucleotide 5215G > A/c.5096G > A mutation results in the missense mutation Arg1699Gln, while the BRCA2 nucleotide 859 + 4A > G/c.631 + 4A > G is novel. Exon trapping experiments and reverse transcriptase...... (RT)-PCR analysis revealed that the BRCA2 mutation results in skipping of exon 7, thereby introducing a frameshift and a premature stop codon. We therefore classify the mutation as disease causing. Since the BRCA1 Arg1699Gln mutation is also suggested to be disease-causing, we consider this family...

  6. Manifestation of cystic fibrosis transmembrane regulator (CFTR) in hepatic ductal structures and renal tubules of female rats with experimental cholestasis of pregnancy.

    Science.gov (United States)

    Aleksandrova, M I; Kushnareva, N S; Smirnova, O V

    2014-03-01

    Studies by the immunohistochemical method with semiquantitative analysis of images showed that hyperprolactinemia stimulated CFTR protein manifestation in the bile ducts of female rats, which was clearly expressed in experimental cholestasis of pregnancy. The expression of CFTR in the renal tubules was reduced in hyperprolactinemia under conditions of normal liver function and in cholestasis of pregnancy. Significant positive correlations between CFTR, prolactin receptor, and multiple drug resistance protein 3 were detected in the bile ducts, but not in the renal tubules. Presumably, prolactin has a direct effect on CFTR expression in the bile ducts and indirect effect in the renal tubules. Changes in CFTR protein manifestation in the hepatic ductal structures and renal tubules in experimental pregnancy cholestasis could aggravate the disease.

  7. Crystallographic and single-particle analyses of native- and nucleotide-bound forms of the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

    Science.gov (United States)

    Awayn, N H; Rosenberg, M F; Kamis, A B; Aleksandrov, L A; Riordan, J R; Ford, R C

    2005-11-01

    Cystic fibrosis, one of the major human inherited diseases, is caused by defects in the CFTR (cystic fibrosis transmembrane conductance regulator), a cell-membrane protein. CFTR acts as a chloride channel which can be opened by ATP. Low-resolution structural studies of purified recombinant human CFTR are described in the present paper. Localization of the C-terminal decahistidine tag in CFTR was achieved by Ni2+-nitriloacetate nanogold labelling, followed by electron microscopy and single-particle analysis. The presence of the gold label appears to improve the single-particle-alignment procedure. Projection structures of CFTR from two-dimensional crystals analysed by electron crystallography displayed two alternative conformational states in the presence of nucleotide and nanogold, but only one form of the protein was observed in the quiescent (nucleotide-free) state.

  8. A novel cystic fibrosis gene mutation c.2490insT in a Palestinian patient: A case report and review of the literature

    Directory of Open Access Journals (Sweden)

    Hassan Chami

    2017-01-01

    Full Text Available We report the case of a 19-year-old male patient of Palestinian descent, who presented with a 1-year history of recurrent Pseudomonas aeruginosa respiratory infections, weight loss, chronic diarrhea, and a normal chloride sweat test. A panel for common cystic fibrosis transmembrane conductance regulator (CFTR gene mutations test was also negative. Cystic fibrosis (CF was still clinically suspected thus, full CFTR gene sequencing was performed, which revealed a homozygous unreported mutation c.2490insT (GenBank accession number: BankIt2019289 seq1 MF167456. Both parents were also found to be heterozygous for this mutation. This case highlights the importance of clinical evaluation and the need for extensive genetic investigation when dealing with a genetic disease with wide variability in a clinical presentation such as CF.

  9. A Novel Mutation in the EDAR Gene Causes Severe Autosomal Recessive Hypohidrotic Ectodermal Dysplasia

    DEFF Research Database (Denmark)

    Henningsen, Emil; Svendsen, Mathias Tiedemann; Lildballe, D. L.

    2014-01-01

    nasal discharge. The girl was the second born child of first-cousin immigrants from Northern Iraq. A novel homozygous mutation (c.84delC) in the EDAR gene was identified. This mutation most likely causes a frameshift in the protein product (p.S29fs*74). This results in abolition of all ectodysplasin......-mediated NF-kB signalling. This complete loss-of-function mutation likely accounts for the severe clinical abnormalities in ectodermal structures in the described patient. (C) 2014 Wiley Periodicals, Inc....

  10. Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

    DEFF Research Database (Denmark)

    Christiansen, L; Ged, C; Hombrados, I

    1999-01-01

    , confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...... to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual...

  11. Frequent somatic reversion of KRT1 mutations in ichthyosis with confetti.

    Science.gov (United States)

    Choate, Keith A; Lu, Yin; Zhou, Jing; Elias, Peter M; Zaidi, Samir; Paller, Amy S; Farhi, Anita; Nelson-Williams, Carol; Crumrine, Debra; Milstone, Leonard M; Lifton, Richard P

    2015-04-01

    Widespread reversion of genetic disease is rare; however, such events are particularly evident in some skin disorders in which normal clones develop on a background of affected skin. We previously demonstrated that mutations in keratin 10 (KRT10) cause ichthyosis with confetti (IWC), a severe dominant disorder that is characterized by progressive development of hundreds of normal skin spots via revertant mosaicism. Here, we report on a clinical and histological IWC subtype in which affected subjects have red, scaly skin at birth, experience worsening palmoplantar keratoderma in childhood, and develop hundreds of normal skin spots, beginning at around 20 years of age, that increase in size and number over time. We identified a causal de novo mutation in keratin 1 (KRT1). Similar to IWC-causing KRT10 mutations, this mutation in KRT1 resulted in a C-terminal frameshift, replacing 22 C-terminal amino acids with an alternate 30-residue peptide. Mutant KRT1 caused partial collapse of the cytoplasmic intermediate filament network and mislocalized to the nucleus. As with KRT10 mutations causing IWC, reversion of KRT1 mutations occurred via mitotic recombination. Because reversion is not observed with other disease-causing keratin mutations, the results of this study implicate KRT1 and KRT10 C-terminal frameshift mutations in the high frequency of revertant mosaicism in IWC.

  12. Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients

    International Nuclear Information System (INIS)

    Mihalatos, Markos; Fountzilas, George; Agnantis, Niki J; Nasioulas, Georgios; Apessos, Angela; Dauwerse, Hans; Velissariou, Voula; Psychias, Aristidis; Koliopanos, Alexander; Petropoulos, Konstantinos; Triantafillidis, John K; Danielidis, Ioannis

    2005-01-01

    Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions / deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation – dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription – Polymerase Chain Reaction). A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations

  13. Compound heterozygous ASPM mutations in Pakistani MCPH families

    DEFF Research Database (Denmark)

    Muhammad, Farooq; Mahmood Baig, Shahid; Hansen, Lars

    2009-01-01

    Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference (50% of all reported families. In spite of the high frequency of MCPH in Pakistan only one case of compound heterozygosity for mutations in ASPM has been reported yet. In this large MCPH study we...... confirmed compound heterozygosity in two and homozygous mutations in 20 families, respectively, showing that up to 10% of families with MCPH caused by ASPM are compound heterozygous. In total we identified 16 different nonsense or frameshift mutations of which 12 were novel thereby increasing the number...... of mutations in ASPM significantly from 35 to 47. We found no correlation between the severity of the condition and the site of truncation. We suggest that the high frequency of compound heterozygosity observed in this study is taken into consideration as part of future genetic testing and counseling...

  14. 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.

  15. A negative feedback modulator of antigen processing evolved from a frameshift in the cowpox virus genome.

    Directory of Open Access Journals (Sweden)

    Jiacheng Lin

    2014-12-01

    Full Text Available Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.

  16. Large-scale mass spectrometry-based analysis of Euplotes octocarinatus supports the high frequency of +1 programmed ribosomal frameshift

    OpenAIRE

    Wang, Ruanlin; Zhang, Zhiyun; Du, Jun; Fu, Yuejun; Liang, Aihua

    2016-01-01

    Programmed ribosomal frameshifting (PRF) is commonly used to express many viral and some cellular genes. We conducted a genome-wide investigation of +1 PRF in ciliate Euplotes octocarinatus through genome and transcriptome sequencing and our results demonstrated that approximately 11.4% of genes require +1 PRF to produce complete gene products. While nucleic acid-based evidence for candidate genes with +1 PRF is strong, only very limited information is available at protein levels to date. In ...

  17. Adenine Enrichment at the Fourth CDS Residue in Bacterial Genes Is Consistent with Error Proofing for +1 Frameshifts.

    Science.gov (United States)

    Abrahams, Liam; Hurst, Laurence D

    2017-12-01

    Beyond selection for optimal protein functioning, coding sequences (CDSs) are under selection at the RNA and DNA levels. Here, we identify a possible signature of "dual-coding," namely extensive adenine (A) enrichment at bacterial CDS fourth sites. In 99.07% of studied bacterial genomes, fourth site A use is greater than expected given genomic A-starting codon use. Arguing for nucleotide level selection, A-starting serine and arginine second codons are heavily utilized when compared with their non-A starting synonyms. Several models have the ability to explain some of this trend. In part, A-enrichment likely reduces 5' mRNA stability, promoting translation initiation. However T/U, which may also reduce stability, is avoided. Further, +1 frameshifts on the initiating ATG encode a stop codon (TGA) provided A is the fourth residue, acting either as a frameshift "catch and destroy" or a frameshift stop and adjust mechanism and hence implicated in translation initiation. Consistent with both, genomes lacking TGA stop codons exhibit weaker fourth site A-enrichment. Sequences lacking a Shine-Dalgarno sequence and those without upstream leader genes, that may be more error prone during initiation, have greater utilization of A, again suggesting a role in initiation. The frameshift correction model is consistent with the notion that many genomic features are error-mitigation factors and provides the first evidence for site-specific out of frame stop codon selection. We conjecture that the NTG universal start codon may have evolved as a consequence of TGA being a stop codon and the ability of NTGA to rapidly terminate or adjust a ribosome. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  18. Correction of chloride transport and mislocalization of CFTR protein by vardenafil in the gastrointestinal tract of cystic fibrosis mice.

    Directory of Open Access Journals (Sweden)

    Barbara Dhooghe

    Full Text Available Although lung disease is the major cause of mortality in cystic fibrosis (CF, gastrointestinal (GI manifestations are the first hallmarks in 15-20% of affected newborns presenting with meconium ileus, and remain major causes of morbidity throughout life. We have previously shown that cGMP-dependent phosphodiesterase type 5 (PDE5 inhibitors rescue defective CF Transmembrane conductance Regulator (CFTR-dependent chloride transport across the mouse CF nasal mucosa. Using F508del-CF mice, we examined the transrectal potential difference 1 hour after intraperitoneal injection of the PDE5 inhibitor vardenafil or saline to assess the amiloride-sensitive sodium transport and the chloride gradient and forskolin-dependent chloride transport across the GI tract. In the same conditions, we performed immunohistostaining studies in distal colon to investigate CFTR expression and localization. F508del-CF mice displayed increased sodium transport and reduced chloride transport compared to their wild-type littermates. Vardenafil, applied at a human therapeutic dose (0.14 mg/kg used to treat erectile dysfunction, increased chloride transport in F508del-CF mice. No effect on sodium transport was detected. In crypt colonocytes of wild-type mice, the immunofluorescence CFTR signal was mostly detected in the apical cell compartment. In F508del-CF mice, a 25% reduced signal was observed, located mostly in the subapical region. Vardenafil increased the peak of intensity of the fluorescence CFTR signal in F508del-CF mice and displaced it towards the apical cell compartment. Our findings point out the intestinal mucosa as a valuable tissue to study CFTR transport function and localization and to evaluate efficacy of therapeutic strategies in CF. From our data we conclude that vardenafil mediates potentiation of the CFTR chloride channel and corrects mislocalization of the mutant protein. The study provides compelling support for targeting the cGMP signaling pathway in CF

  19. A review on architecture of the gag-pol ribosomal frameshifting RNA in human immunodeficiency virus: a variability survey of virus genotypes.

    Science.gov (United States)

    Qiao, Qi; Yan, Yanhua; Guo, Jinmei; Du, Shuqiang; Zhang, Jiangtao; Jia, Ruyue; Ren, Haimin; Qiao, Yuanbiao; Li, Qingshan

    2017-06-01

    Programmed '-1' ribosomal frameshifting is necessary for expressing the pol gene overlapped from a gag of human immunodeficiency virus. A viral RNA structure that requires base pairing across the overlapping sequence region suggests a mechanism of regulating ribosome and helicase traffic during expression. To get precise roles of an element around the frameshift site, a review on architecture of the frameshifting RNA is performed in combination of reported information with augments of a representative set of 19 viral samples. In spite of a different length for the viral RNAs, a canonical comparison on the element sequence allocation is performed for viewing variability associations between virus genotypes. Additionally, recent and historical insights recognized in frameshifting regulation are looked back as for indel and single nucleotide polymorphism of RNA. As specially noted, structural changes at a frameshift site, the spacer sequence, and a three-helix junction element, as well as two Watson-Crick base pairs near a bulge and a C-G pair close a loop, are the most vital strategies for the virus frameshifting regulations. All of structural changes, which are dependent upon specific sequence variations, facilitate an elucidation about the RNA element conformation-dependent mechanism for frameshifting. These facts on disrupting base pair interactions also allow solving the problem of competition between ribosome and helicase on a same RNA template, common to single-stranded RNA viruses. In a broad perspective, each new insight of frameshifting regulation in the competition systems introduced by the RNA element construct changes will offer a compelling target for antiviral therapy.

  20. Detection of a novel silent deletion, a missense mutation and a nonsense mutation in TCOF1.

    Science.gov (United States)

    Fujioka, Hirotaka; Ariga, Tadashi; Horiuchi, Katsumi; Ishikiriyama, Satoshi; Oyama, Kimie; Otsu, Makoto; Kawashima, Kunihiro; Yamamoto, Yuhei; Sugihara, Tsuneki; Sakiyama, Yukio

    2008-12-01

    Treacher Collins syndrome (TCS) is a disorder of craniofacial development, that is caused by mutations in the TCOF1 gene. TCS is inherited as an autosomal dominant trait, and haploinsufficiency of the TCOF1 gene product treacle is proposed to be etiologically involved. Mutational analysis of the TCOF1 gene was done in 10 patients diagnosed with TCS using single-strand conformation polymorphism and direct sequencing. Among these 10 patients, a novel 9 bp deletion was found, together with a previously reported 2 bp deletion, a novel missense mutation and a novel nonsense mutation in three different families. Familial studies allowed judgment of whether these abnormal findings were responsible for the TCS phenotype, or not. The 9 bp deletion of three amino acids Lys-Glu-Lys (1378-1380), which was located in the nuclear localization domain of treacle, seemed not essential for the treacle function. In contrast, the novel mutation of Ala26Val is considered to affect the LisH domain, an important domain of treacle. All of the mutations thus far detected in exon 5 have resulted in frameshift, but a nonsense mutation was detected (Lys159Stop). The information obtained in the present study provides additional insights into the functional domains of treacle.

  1. Heterozygous mutations in the LDL receptor-related protein 5 (LRP5) gene are associated with primary osteoporosis in children.

    Science.gov (United States)

    Hartikka, Heini; Mäkitie, Outi; Männikkö, Minna; Doria, Andrea S; Daneman, Alan; Cole, William G; Ala-Kokko, Leena; Sochett, Etienne B

    2005-05-01

    Three of 20 patients with juvenile osteoporosis were found to have a heterozygous mutation in the LRP5 gene. No mutations were found in the type I collagen genes. Mutations in the other family members with similar bone phenotype confirmed that LRP5 has a role in both juvenile and adult osteoporosis. The gene encoding the low-density lipoprotein receptor-related protein 5 (LRP5) gene has recently been shown to affect bone mass accrual during growth and to be involved in osteoporosis-pseudoglioma syndrome and a high bone mass phenotype. Mutations in the type I collagen genes (COL1A1 and COL1A2) are known to cause osteogenesis imperfecta, characterized by increased bone fragility. Here we analyzed COL1A1, COL1A2, and LRP5 for mutations in 20 pediatric patients with primary osteoporosis characterized by low BMD, recurrent fractures, and absent extraskeletal manifestations. No mutations were detected in the type I collagen genes, but two missense mutations (A29T and R1036Q) and one frameshift mutation (C913fs) were found in the LRP5 gene in three of the patients. The frameshift mutation was also seen in the proband's father and brother, who both were found to have significant osteoporosis. R1036Q was observed in the proband's mother and two brothers, who all had osteoporosis. These results indicate that heterozygous mutations in the LRP5 gene can cause osteoporosis in both children and adults.

  2. A novel TSC2 mutation in a Chinese family with tuberous sclerosis ...

    Indian Academy of Sciences (India)

    Supplementary data, J. Genet. 93, 169-172. Figure 1. Amino acid changes caused by the deletion c.2690delT. The mutation (p.Phe897SerfsX947) resulted in a frameshift and a run of 50 missense amino acids followed by a premature stop codon (TGA), resulting in a truncation protein of 946 amino acids. Journal of Genetics ...

  3. Two novel NIPBL gene mutations in Chinese patients with Cornelia de Lange syndrome.

    Science.gov (United States)

    Mei, Libin; Liang, Desheng; Huang, Yanru; Pan, Qian; Wu, Lingqian

    2015-01-25

    Cornelia de Lange syndrome (CdLS) is a dominantly inherited developmental disorder characterized by distinctive facial features, mental retardation, and upper limb defects, with the involvement of multiple organs and systems. To date, mutations have been identified in five genes responsible for CdLS: NIPBL, SMC1A, SMC3, RAD21, and HDAC8. Here, we present a clinical and molecular characterization of five unrelated Chinese patients whose clinical presentation is consistent with that of CdLS. There were no chromosomal abnormalities in the five children. In three patients, DNA sequencing revealed a previously reported frameshift mutation c.2479delA (p.Arg827GlyfsX20), and two novel mutations including a heterozygous mutation c.6272 G>T (p.Cys2091Phe) and a frameshift mutation c.1672delA (p.Thr558LeufsX7) in NIPBL. For the remaining patients, large deletions and/or duplications within the NIPBL gene were excluded as playing a role in the pathogenesis, by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. These findings broaden the mutation spectrum of NIPBL and further our understanding of the diverse and variable effects of NIPBL mutations on CdLS. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Juvenil polypose-syndrom og hereditær hæmoragisk telangiektasi hos en patient med SMAD4-mutation

    DEFF Research Database (Denmark)

    Jelsig, Anne Marie; Tørring, Pernille Mathiesen; Wikman, Friedrik

    2014-01-01

    Germ line mutations in SMAD4 can cause both juvenile polyposis syndrome and hereditary haemorrhagic telangiectasia syndrome. In this case we present a 37-year-old man with a frameshift mutation in SMAD4. The patient had multiple polyps in the gastrointestinal tract and was diagnosed with colon ca...... cancer at the age of 21 and gastro-oesophageal junction cancer at the age of 37. Furthermore the patient had telangiectasias and recurrent epistaxis....

  5. Development of rAAV2-CFTR: History of the First rAAV Vector Product to be Used in Humans.

    Science.gov (United States)

    Loring, Heather S; ElMallah, Mai K; Flotte, Terence R

    2016-04-01

    The first human gene therapy trials using recombinant adeno-associated virus (rAAV) vectors were performed in cystic fibrosis (CF) patients. Over 100 CF patients were enrolled in 5 separate trials of rAAV2-CFTR administration via nasal, endobronchial, maxillary sinus, and aerosol delivery. Recombinant AAV vectors were designed to deliver the CF transmembrane regulator (CFTR) gene and correct the basic CFTR defect by restoring chloride transport and reverting the upregulation of proinflammatory cytokines. However, vector DNA expression was limited in duration because of the low incidence of integration and natural airway epithelium turnover. In addition, repeated administration of AAV-CFTR vector resulted in a humoral immune response that prevented effective gene transfer from subsequent doses of vector. AAV serotype 2 was used in human trials before the comparison with other serotypes and determination that serotypes 1 and 5 not only possess higher tropism for the airway epithelium, but also are capable of bypassing the binding and trafficking processes-both were important hindrances to the effectiveness of rAAV2. Although rAAV-CFTR gene therapy does not appear likely to supplant newer small-molecule CFTR modulators in the near future, early work with rAAV-CFTR provided an important foundation for later use of rAAV in humans.

  6. The zebrafish Kupffer's vesicle as a model system for the molecular mechanisms by which the lack of Polycystin-2 leads to stimulation of CFTR

    Directory of Open Access Journals (Sweden)

    Mónica Roxo-Rosa

    2015-11-01

    Full Text Available In autosomal dominant polycystic kidney disease (ADPKD, cyst inflation and continuous enlargement are associated with marked transepithelial ion and fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR. Indeed, the inhibition or degradation of CFTR prevents the fluid accumulation within cysts. The in vivo mechanisms by which the lack of Polycystin-2 leads to CFTR stimulation are an outstanding challenge in ADPKD research and may bring important biomarkers for the disease. However, hampering their study, the available ADPKD in vitro cellular models lack the three-dimensional architecture of renal cysts and the ADPKD mouse models offer limited access for live-imaging experiments in embryonic kidneys. Here, we tested the zebrafish Kupffer's vesicle (KV as an alternative model-organ. KV is a fluid-filled vesicular organ, lined by epithelial cells that express both CFTR and Polycystin-2 endogenously, being each of them easily knocked-down. Our data on the intracellular distribution of Polycystin-2 support its involvement in the KV fluid-flow induced Ca2+-signalling. Mirroring kidney cysts, the KV lumen inflation is dependent on CFTR activity and, as we clearly show, the knockdown of Polycystin-2 results in larger KV lumens through overstimulation of CFTR. In conclusion, we propose the zebrafish KV as a model organ to study the renal cyst inflation. Favouring its use, KV volume can be easily determined by in vivo imaging offering a live readout for screening compounds and genes that may prevent cyst enlargement through CFTR inhibition.

  7. CFTR in cystic fibrosis and cholera: from membrane transport to clinical practice.

    Science.gov (United States)

    Goodman, Barbara E; Percy, William H

    2005-06-01

    We have used a brief analysis of transport via cystic fibrosis (CF) transmembrane conductance regulators (CFTRs) in various organ systems to highlight the importance of basic membrane transport processes across epithelial cells for first-year medical students in physiology. Because CFTRs are involved in transport both physiologically and pathologically in various systems, we have used this clinical correlation to analyze how a defective gene leading to defective transport proteins can be directly involved in the symptoms of cholera and CF. This article is a "Staying Current" approach to transport via CFTRs including numerous helpful references with further information for a teaching faculty member. The article follows our normal presentation which begins with a discussion of the involvement of CFTR transport in the intestine and how cholera affects intestinal transport, extends to CFTR transport in various organ systems in CF, and concludes with the logic behind many of the treatments that improve CF. Student learning objectives are included to assist in assessment of student understanding of the basic concepts.

  8. Novel and recurrent BRCA1/BRCA2 mutations in early onset and familial breast and ovarian cancer detected in the Program of Genetic Counseling in Cancer of Valencian Community (eastern Spain). Relationship of family phenotypes with mutation prevalence.

    Science.gov (United States)

    de Juan Jiménez, Inmaculada; García Casado, Zaida; Palanca Suela, Sarai; Esteban Cardeñosa, Eva; López Guerrero, José Antonio; Segura Huerta, Ángel; Chirivella González, Isabel; Sánchez Heras, Ana Beatriz; Juan Fita, Ma José; Tena García, Isabel; Guillen Ponce, Carmen; Martínez de Dueñas, Eduardo; Romero Noguera, Ignacio; Salas Trejo, Dolores; Goicoechea Sáez, Mercedes; Bolufer Gilabert, Pascual

    2013-12-01

    During the first 6 years of the Program of Genetic Counselling in Cancer of Valencia (eastern Spain), 310 mutations (155 in BRCA1 and 155 in BRCA2) in 1,763 hereditary breast (BC) and ovarian cancer (OC) families were identified. Of the mutations found 105 were distinct (53 in BRCA1 and 52 in BRCA2), eight new and 37 recurrent. Two of the novel mutations were frame-shift placed in exons 2 and 11 of BRCA1 and the remaining six were placed in BRCA2; four frame-shift (three in exon 11 and one in exon 23), one deletion of the entire exon 19 and one in the intervening sequence of exon 22. The BRCA1 mutations with higher recurrence were c.66_68delAG, c.5123C > A, c.1961delA, c.3770_3771delAG and c.5152+5G > A that covered 45.2% of mutations of this gene. The age of onset of BCs of c.68_69delAG mutation carriers occurs later than for the other recurrent mutations of this gene (45 vs. 37 years; p = 0.008). The BRCA2 mutations with higher recurrence were c.9026_9030delATCAT, c.3264insT and c.8978_8991del14 which represented 43.2% of all mutations in this gene, being the most recurrent mutation by far c.9026_9030delATCAT that represents 21.3% of BRCA2 mutations and 10.6% of all mutations. Probands with family histories of BC and OC, or OC and/or BC in at least two first degree relatives, were the more likely to have BRCA1/BRCA2 mutations (35.2% of the total mutations). And that most BRCA1mutations (73.19% mutations) occurred in probands with early-onset BC or with family history of OC.

  9. Restoration of CFTR Activity in Ducts Rescues Acinar Cell Function and Reduces Inflammation in Pancreatic and Salivary Glands of Mice.

    Science.gov (United States)

    Zeng, Mei; Szymczak, Mitchell; Ahuja, Malini; Zheng, Changyu; Yin, Hongen; Swaim, William; Chiorini, John A; Bridges, Robert J; Muallem, Shmuel

    2017-10-01

    Sjögren's syndrome and autoimmune pancreatitis are disorders with decreased function of salivary, lacrimal glands, and the exocrine pancreas. Nonobese diabetic/ShiLTJ mice and mice transduced with the cytokine BMP6 develop Sjögren's syndrome and chronic pancreatitis and MRL/Mp mice are models of autoimmune pancreatitis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a ductal Cl -  channel essential for ductal fluid and HCO 3 - secretion. We used these models to ask the following questions: is CFTR expression altered in these diseases, does correction of CFTR correct gland function, and most notably, does correcting ductal function correct acinar function? We treated the mice models with the CFTR corrector C18 and the potentiator VX770. Glandular, ductal, and acinar cells damage, infiltration, immune cells and function were measured in vivo and in isolated duct/acini. In the disease models, CFTR expression is markedly reduced. The salivary glands and pancreas are inflamed with increased fibrosis and tissue damage. Treatment with VX770 and, in particular, C18 restored salivation, rescued CFTR expression and localization, and nearly eliminated the inflammation and tissue damage. Transgenic overexpression of CFTR exclusively in the duct had similar effects. Most notably, the markedly reduced acinar cell Ca 2+ signaling, Orai1, inositol triphosphate receptors, Aquaporin 5 expression, and fluid secretion were restored by rescuing ductal CFTR. Our findings reveal that correcting ductal function is sufficient to rescue acinar cell function and suggests that CFTR correctors are strong candidates for the treatment of Sjögren's syndrome and pancreatitis. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

  10. The FOXA2 transcription factor is frequently somatically mutated in uterine carcinosarcomas and carcinomas.

    Science.gov (United States)

    Le Gallo, Matthieu; Rudd, Meghan L; Urick, Mary Ellen; Hansen, Nancy F; Merino, Maria J; Mutch, David G; Goodfellow, Paul J; Mullikin, James C; Bell, Daphne W

    2018-01-01

    Uterine carcinosarcomas (UCSs) are a rare but clinically aggressive form of cancer. They are biphasic tumors consisting of both epithelial and sarcomatous components. The majority of uterine carcinosarcomas are clonal, with the carcinomatous cells undergoing metaplasia to give rise to the sarcomatous component. The objective of the current study was to identify novel somatically mutated genes in UCSs. We whole exome sequenced paired tumor and nontumor DNAs from 14 UCSs and orthogonally validated 464 somatic variants using Sanger sequencing. Fifteen genes that were somatically mutated in at least 2 tumor exomes were Sanger sequenced in another 39 primary UCSs. Overall, among 53 UCSs in the current study, the most frequently mutated of these 15 genes were tumor protein p53 (TP53) (75.5%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (34.0%), protein phosphatase 2, regulatory subunit A, alpha (PPP2R1A) (18.9%), F-box and WD repeat domain containing 7 (FBXW7) (18.9%), chromodomain helicase DNA binding protein 4 (CHD4) (17.0%), and forkhead box A2 (FOXA2) (15.1%). FOXA2 has not previously been implicated in UCSs and was predominated by frameshift and nonsense mutations. One UCS with a FOXA2 frameshift mutation expressed truncated FOXA2 protein by immunoblotting. Sequencing of FOXA2 in 160 primary endometrial carcinomas revealed somatic mutations in 5.7% of serous, 22.7% of clear cell, 9% of endometrioid, and 11.1% of mixed endometrial carcinomas, the majority of which were frameshift mutations. Collectively, the findings of the current study provide compelling genetic evidence that FOXA2 is a pathogenic driver gene in the etiology of primary uterine cancers, including UCSs. Cancer 2018;124:65-73. © 2017 American Cancer Society. © 2017 American Cancer Society.

  11. Mutation analysis of the gene involved in adrenoleukodystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Oost, B.A. van; Ligtenberg, M.J.L. [Univ. Hospital Nijmegen (Netherlands); Kemp, S.; Bolhuis, P.A. [Academic Medical Center, Amsterdam (Netherlands)

    1994-09-01

    A gene responsible for the X-linked genetic disorder adrenoleukodystrophy (ALD) that is characterized by demyelination of the nervous system and adrenocortical insufficiency has been identified by positional cloning. The gene encodes an ATP-binding transporter which is located in the peroxisomal membrane. Deficiency of the gene leads to accumulation of unsaturated very long chain fatty acids due to impaired peroxisomal {beta}-oxidation. A systematic analysis of the open reading frame of the ALD gene unraveled the mutations in 28 different families using reverse transcriptase-PCR followed by direct sequencing. No entire gene deletions or drastic promoter mutations have been detected. Only in one family did the mutation involved multiple exons. The remaining mutations were subtle alterations leading to missense (about 50%) or nonsense mutations, frameshifts or splice acceptor site defects. In one patient a single codon was missing. Mutations affecting a single amino acid were concentrated in the region between the third and fourth putative membrane spanning fragments and in the ATP-binding domain. This overview of mutations aids in the determination of structural and functional important regions and facilitates the screening for mutations in other ALD patients. The detection of mutations in virtually all ALD families tested indicates that the isolated gene is the only gene responsible for ALD located in Xq28.

  12. Cystic fibrosis and the role of gastrointestinal outcome measures in the new era of therapeutic CFTR modulation

    NARCIS (Netherlands)

    Bodewes, Frank A J A; Verkade, Henkjan J; Taminiau, Jan A J M; Borowitz, Drucy; Wilschanski, Michael

    With the development of new drugs that directly affect CFTR protein function, clinical trials are being designed or initiated for a growing number of patients with cystic fibrosis. The currently available and accepted clinical endpoints, FEV1 and BMI, have limitations. The aim of this report is to

  13. Association of sweat chloride concentration at time of diagnosis and CFTR genotype with mortality and cystic fibrosis phenotype.

    Science.gov (United States)

    McKone, Edward F; Velentgas, Priscilla; Swenson, Anna J; Goss, Christopher H

    2015-09-01

    The extent to which sweat chloride concentration predicts survival and clinical phenotype independently of CFTR genotype in cystic fibrosis is not well understood. We analyzed the US Cystic Fibrosis Foundation Patient Registry data using Cox regression to examine the relationship between sweat chloride concentration (Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  14. Mutation analysis in Norwegian families with hereditary hemorrhagic telangiectasia: founder mutations in ACVRL1.

    Science.gov (United States)

    Heimdal, K; Dalhus, B; Rødningen, O K; Kroken, M; Eiklid, K; Dheyauldeen, S; Røysland, T; Andersen, R; Kulseth, M A

    2016-02-01

    Hereditary hemorrhagic telangiectasia (HHT, Osler-Weber-Rendu disease) is an autosomal dominant inherited disease defined by the presence of epistaxis and mucocutaneous telangiectasias and arteriovenous malformations (AVMs) in internal organs. In most families (~85%), HHT is caused by mutations in the ENG (HHT1) or the ACVRL1 (HHT2) genes. Here, we report the results of genetic testing of 113 Norwegian families with suspected or definite HHT. Variants in ENG and ACVRL1 were found in 105 families (42 ENG, 63 ACVRL1), including six novel variants of uncertain pathogenic significance. Mutation types were similar to previous reports with more missense variants in ACVRL1 and more nonsense, frameshift and splice-site mutations in ENG. Thirty-two variants were novel in this study. The preponderance of ACVRL1 mutations was due to founder mutations, specifically, c.830C>A (p.Thr277Lys), which was found in 24 families from the same geographical area of Norway. We discuss the importance of founder mutations and present a thorough evaluation of missense and splice-site variants. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Clusters of Cl- channels in CFTR-expressing Sf9 cells switch spontaneously between slow and fast gating modes

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Price, E. M.; Gabriel, S. E.

    1996-01-01

    The Sf9 insect Spodoptora frugiperda cell line was used for heterologous expression of the cloned human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, or the cloned ß-galactosidase gene, using the baculovirus Autographa califonica as the infection vector. Using application...... of the patch-clamp technique, evidence for functional expression of CFTR was obtained according to the following three criteria. Firstly, whole-cell currents recorded 2 days after infection with CFTR revealed a statistically significant increase of membrane conductance, ˜25 times above that of mock...

  16. Mutation detection and prenatal diagnosis of XLHED pedigree

    Directory of Open Access Journals (Sweden)

    Yao Lin

    2017-08-01

    Full Text Available Background The phenotypic characters of X -linked Hypohidrotic Ectodermal Dysplasia (XLHED are the dysplasia of epithelial- and mesenchymal-derived organs. Ectodysplasin (EDA is the causative gene of XLHED. Methods The current study reported a large Chinese XLHED pedigree. The genomic DNA of adult and fetus was extracted from peripheral blood and shed chorion cell respectively. The nucleotide variation in EDA gene was screened through direct sequencing the coding sequence. The methylation state of EDA gene’s promoter was evaluated by pyrosequencing. Results This Chinese XLHED family had two male patients and three carriers. All of them were with a novel EDA frameshift mutation. The mutation, c.172-173insGG, which leads to an immediate premature stop codon in exon one caused severe structural changes of EDA. Prenatal diagnosis suggested that the fetus was a female carrier. The follow-up observation of this child indicated that she had mild hypodontia of deciduous teeth at age six. The methylation level of EDA gene’s promoter was not related to carriers’ phenotype changes in this family. Discussion We reported a new frameshift mutation of EDA gene in a Chinese family. Prenatal diagnosis can help to predict the disease status of the fetus.

  17. ENG mutational mosaicism in a family with hereditary hemorrhagic telangiectasia

    DEFF Research Database (Denmark)

    Tørring, Pernille M; Kjeldsen, Anette D; Ousager, Lilian Bomme

    2018-01-01

    BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disorder caused by mutations in ENG, ACVRL1, or SMAD4. Around 90% of HHT patients present with a heterozygous pathogenic genetic variation. Almost all cases of HHT have a family history. Very few cases are de...... novo or mosaicism. We describe a case with mutational mosaicism that would not be observed in the clinical routine when using Sanger sequencing or a NGS read coverage below app. 100. METHODS: DNA was extracted from peripheral blood leukocytes, and buccal swabs. The coding region, exon-intron boundaries......, and the flanking sequences of the genes were sequenced by NGS. RESULTS: The proband had clinical HHT fulfilling the Curaçao criteria and genetic testing identified a frameshift mutation in ENG. The mother of the proband, also with clinical HHT, was found negative when analyzing DNA from blood for the familial...

  18. Correlation between connexin 32 gene mutations and clinical phenotype in X-linked dominant Charcot-Marie-Tooth neuropathy

    Energy Technology Data Exchange (ETDEWEB)

    Ionasescu, V.; Ionasescu, R.; Searby, C. [Univ. of Iowa Hospitals and Clinics, Iowa City, IA (United States)

    1996-06-14

    We studied the relationship between the genotype and clinical phenotype in 27 families with dominant X-linked Charcot-Marie-Tooth (CMTX1) neuropathy. Twenty-two families showed mutations in the coding region of the connexin32 (cx32) gene. The mutations include four nonsense mutations, eight missense mutations, two medium size deletions, and one insertion. Most missense mutations showed a mild clinical phenotype (five out of eight), whereas all nonsense mutations, the larger of the two deletions, and the insertion that produced frameshifts showed severe phenotypes. Five CMTX1 families with mild clinical phenotype showed no point mutations of the cx32 gene coding region. Three of these families showed positive genetic linkage with the markers of the Xq13.1 region. The genetic linkage of the remaining two families could not be evaluated because of their small size. 25 refs., 1 fig., 1 tab.

  19. [X-linked adrenoleukodystrophy ABCD1 gene mutation analysis in China].

    Science.gov (United States)

    Pan, Hong; Xiong, Hui; Zhang, Yue-hua; Wu, Ye; Bao, Xin-hua; Jiang, Yu-wu; Wu, Xi-ru

    2004-02-01

    To investigate mutations of ABCD1 gene in X- linked adrenoleukodystrophy (ALD) patients in China. Polymerase chain reaction and DNA direct sequencing were employed to analyze the 10 exons of ABCD1 gene in 25 ALD patients. Seventeen mutations in different exons (except exons 4, 9 and 10) were identified in 18 of 25 patients. Most of the mutations were missense mutations, including R182P, G266R, H283D, S404P, N509I, R518G, L520Q, Q556R, S606L and R617C, four (H283D, S40 4P, N509I, R518G) of 10 missense mutations were novel. Also identified were 3 nonsense mutations (W132X, W242X, W595X), 1 dinucleotides deletion mutation (1414 del AG) resulting in frameshift, and 1 base pair deletion at splice acceptor site (IVS5-6 del C). Two synonymous mutations (L516L and V349V) appeared simultaneously in 2 unrelated patients, and no other mutations could be found with them in all 10 exons screened. There were no hot spot mutations in ABCD1 gene in China. Mutations in gene were found over 70% of patients with ALD in China. The ABCD1 gene mutations identified revealed no obvious correlation between the type of mutation and phenotype.

  20. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.

    Science.gov (United States)

    Nangalia, J; Massie, C E; Baxter, E J; Nice, F L; Gundem, G; Wedge, D C; Avezov, E; Li, J; Kollmann, K; Kent, D G; Aziz, A; Godfrey, A L; Hinton, J; Martincorena, I; Van Loo, P; Jones, A V; Guglielmelli, P; Tarpey, P; Harding, H P; Fitzpatrick, J D; Goudie, C T; Ortmann, C A; Loughran, S J; Raine, K; Jones, D R; Butler, A P; Teague, J W; O'Meara, S; McLaren, S; Bianchi, M; Silber, Y; Dimitropoulou, D; Bloxham, D; Mudie, L; Maddison, M; Robinson, B; Keohane, C; Maclean, C; Hill, K; Orchard, K; Tauro, S; Du, M-Q; Greaves, M; Bowen, D; Huntly, B J P; Harrison, C N; Cross, N C P; Ron, D; Vannucchi, A M; Papaemmanuil, E; Campbell, P J; Green, A R

    2013-12-19

    Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay

  1. Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer (SG); (Columbia); (JHU)

    2010-07-19

    Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

  2. NF kappaB expression increases and CFTR and MUC1 expression decreases in the endometrium of infertile patients with hydrosalpinx: a comparative study

    Directory of Open Access Journals (Sweden)

    Song Yong

    2012-10-01

    Full Text Available Abstract Background Hydrosalpinx are associated with infertility, due to reduced rates of implantation and increased abortion rates. The aims of this study were to investigate the expression of cystic fibrosis transmembrane conductance regulator (CFTR, nuclear factor kappa B (NF KappaB and mucin-1 (MUC-1, and analyze the correlation between the expression of CFTR and NF KappaB or MUC1, in the endometrium of infertile women with and without hydrosalpinx. Methods Thirty-one infertile women with laparoscopy-confirmed unilateral or bilateral hydrosalpinx and 20 infertile women without hydrosalpinx or pelvic inflammatory disease (control group were recruited. Endometrial biopsy samples were collected and the expression of CFTR, NF KappaB and MUC1 were analyzed using immunohistochemistry and quantitative real-time PCR. Results CFTR, NF KappaB and MUC1 mRNA and protein expression tended to increase in the secretory phase compared to the proliferative phase in both groups; however, these differences were not significantly different. The endometrium of infertile patients with hydrosalpinx had significantly higher NF KappaB mRNA and protein expression, and significantly lower CFTR and MUC1 mRNA and protein expression, compared to control infertile patients. A positive correlation was observed between CFTR and MUC1 mRNA expression (r = 0.65, P CFTR mRNA and NF KappaB mRNA expression (r = −0.59, P Conclusions Increased NF KappaB expression and decreased CFTR and MUC1 expression in the endometrium of infertile patients with hydrosalpinx reinforce the involvement of a molecular mechanism in the regulation of endometrial receptivity.

  3. Singlet oxygen-induced mutations in M13 lacZ phage DNA.

    OpenAIRE

    Decuyper-Debergh, D; Piette, J; Van de Vorst, A

    1987-01-01

    The mutagenic consequences of damages to M13 mp19 RF DNA produced by singlet oxygen have been determined in a forward mutational system capable of detecting all classes of mutagenic events. When the damaged M13 mp19 RF DNA is used to transfect competent E. coli JM105 cells, a 16.6-fold increase in mutation frequency is observed at 5% survivors when measured as a loss of alpha-complementation. The enhanced mutagenicity is largely due to single-nucleotide substitutions, frameshift events and do...

  4. Mismatch repair gene mutation spectrum in the Swedish Lynch syndrome population.

    Science.gov (United States)

    Lagerstedt-Robinson, Kristina; Rohlin, Anna; Aravidis, Christos; Melin, Beatrice; Nordling, Margareta; Stenmark-Askmalm, Marie; Lindblom, Annika; Nilbert, Mef

    2016-11-01

    Lynch syndrome caused by constitutional mismatch‑repair defects is one of the most common hereditary cancer syndromes with a high risk for colorectal, endometrial, ovarian and urothelial cancer. Lynch syndrome is caused by mutations in the mismatch repair (MMR) genes i.e., MLH1, MSH2, MSH6 and PMS2. After 20 years of genetic counseling and genetic testing for Lynch syndrome, we have compiled the mutation spectrum in Sweden with the aim to provide a population-based perspective on the contribution from the different MMR genes, the various types of mutations and the influence from founder mutations. Mutation data were collected on a national basis from all laboratories involved in genetic testing. Mutation analyses were performed using mainly Sanger sequencing and multiplex ligation-dependent probe amplification. A total of 201 unique disease-predisposing MMR gene mutations were identified in 369 Lynch syndrome families. These mutations affected MLH1 in 40%, MSH2 in 36%, MSH6 in 18% and PMS2 in 6% of the families. A large variety of mutations were identified with splice site mutations being the most common mutation type in MLH1 and frameshift mutations predominating in MSH2 and MSH6. Large deletions of one or several exons accounted for 21% of the mutations in MLH1 and MSH2 and 22% in PMS2, but were rare (4%) in MSH6. In 66% of the Lynch syndrome families the variants identified were private and the effect from founder mutations was limited and predominantly related to a Finnish founder mutation that accounted for 15% of the families with mutations in MLH1. In conclusion, the Swedish Lynch syndrome mutation spectrum is diverse with private MMR gene mutations in two-thirds of the families, has a significant contribution from internationally recognized mutations and a limited effect from founder mutations.

  5. Novel missense MTTP gene mutations causing abetalipoproteinemia.

    Science.gov (United States)

    Miller, Sharon A; Burnett, John R; Leonis, Mike A; McKnight, C James; van Bockxmeer, Frank M; Hooper, Amanda J

    2014-10-01

    The microsomal triglyceride transfer protein (MTTP) plays a critical role in the formation of hepatic very low density lipoprotein. Abetalipoproteinemia (ABL) is a rare, naturally occurring extreme form of MTTP inhibition, which is characterized by the virtual absence of apolipoprotein (apo) B-containing lipoproteins in blood. The goal of this study was to examine the effect that four novel MTTP missense mutations had on protein interactions, expression and lipid-transfer activity, and to determine which mutations were responsible for the ABL phenotype observed in two patients. In two patients with ABL, we identified in MTTP a novel frameshift mutation (K35Ffs*37), and four novel missense mutations, namely, G264R, Y528H, R540C, and N649S. When transiently expressed in COS-7 cells, all missense MTTP mutations interacted with apoB17, apoB48, and protein disulfide isomerase. Mutations Y528H and R540C, however, displayed negligible levels of MTTP activity and N649S displayed a partial reduction relative to the wild-type MTTP. In contrast, G264R retained full lipid-transfer activity. These studies indicate that missense mutations Y528H, R540C, and N649S appear to cause ABL by reducing MTTP activity rather than by reducing binding of MTTP with protein disulfide isomerase or apoB. The region of MTTP containing amino acids 528 and 540 constitutes a critical domain for its lipid-transfer activity. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells

    Directory of Open Access Journals (Sweden)

    Annalucia Carbone

    2018-01-01

    Full Text Available We previously found that human amniotic mesenchymal stem cells (hAMSCs in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ- mediated intercellular communication (GJIC in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43, a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C, and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

  7. Nocturnal frontal lobe epilepsy caused by a mutation in the GATOR1 complex gene NPRL3.

    Science.gov (United States)

    Korenke, Georg-Christoph; Eggert, Marlene; Thiele, Holger; Nürnberg, Peter; Sander, Thomas; Steinlein, Ortrud K

    2016-03-01

    Mutations in NPRL3, one of three genes that encode proteins of the mTORC1-regulating GATOR1 complex, have recently been reported to cause cortical dysplasia with focal epilepsy. We have now analyzed a multiplex epilepsy family by whole exome sequencing and identified a frameshift mutation (NM_001077350.2; c.1522delG; p.E508Rfs*46) within exon 13 of NPRL3. This truncating mutation causes an epilepsy phenotype characterized by early childhood onset of mainly nocturnal frontal lobe epilepsy. The penetrance in our family was low (three affected out of six mutation carriers), compared to families with either ion channel- or DEPDC5-associated familial nocturnal frontal lobe epilepsy. The absence of apparent structural brain abnormalities suggests that mutations in NPRL3 are not necessarily associated with focal cortical dysplasia but might be able to cause epilepsy by different, yet unknown pathomechanisms. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

  8. 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.

  9. Loss-of-function mutations of STXBP1 in patients with epileptic encephalopathy.

    Science.gov (United States)

    Yamamoto, Toshiyuki; Shimojima, Keiko; Yano, Tamami; Ueda, Yuki; Takayama, Rumiko; Ikeda, Hiroko; Imai, Katsumi

    2016-03-01

    Epileptic encephalopathy, which commences during early infancy, is a severe epileptic syndrome that manifests as age-dependent seizures and severe developmental delay. The syntaxin-binding protein 1 gene (STXBP1) is one of the genes responsible for epileptic encephalopathy. We conducted a cohort study to analyze STXBP1 in 42 patients with epileptic encephalopathy. We identified four novel mutations: two splicing mutations, a frameshift mutation, and a nonsense mutation. All of these mutations were predicted to cause loss-of-function. This result suggests loss-of-function is a common mechanism underlying STXBP1-related epileptic encephalopathy. The four patients showed epileptic features consistent with STXBP1-related epileptic encephalopathy, but showed variable radiological findings, including brain volume loss and myelination delay. Copyright © 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  10. Cystic fibrosis transmembrane conductance regulator mutations and polymorphisms associated with congenital bilateral absence of vas deferens in a restricted group of patients from North Africa.

    Science.gov (United States)

    Boudaya, M; Fredj, S Hadj; Haj, R Bel; Khrouf, M; Bouker, A; Halouani, L; Messaoud, T

    2012-01-01

    Congenital bilateral absence of vas deferens (CBAVD) is responsible for 2-6% of male infertility. It occurs in 95% of men with cystic fibrosis. This malformation is present in patients with a sterile obstructive azoospermia but without clinical evidence of cystic fibrosis. Molecular study of the cystic fibrosis transmembrane conductance regulator (CFTR) gene responsible for cystic fibrosis could show the relationship between this disease and bilateral absence of vas deferens. The study involved 20 male patients aged between 28-40 years, referred with suspected cystic fibrosis and in whom bilateral absence of vas deferens was confirmed by cyto-biochemical analyses and urogenital ultrasound. Molecular study of the CFTR gene was based on several techniques: DHPLC, DGGE and direct sequencing. Thirteen patients had CFTR mutations: F508del, G542X, W1282X, E1104X, 711+1G → T, V201M (TG) m and IVS8-5T. These mutations were associated with polymorphisms: M470V and D1270N. Seven cases presented only polymorphisms. The different mutations found in this study were associated with polymorphisms which decrease the severity of the disease and delay its onset. Thus, bilateral agenesis of the vas deferens is classed as a form of cystic fibrosis with only genital expression.

  11. TP53 mutation spectrum in smokers and never smoking lung cancer patients

    Directory of Open Access Journals (Sweden)

    Ann Rita Halvorsen

    2016-05-01

    Full Text Available AbstractBackground: TP53 mutations are among the most common mutations found in lung cancers, identified as an independent prognostic factor in many types of cancers. The purpose of this study was to investigate the frequency and prognostic impact of TP53 mutations in never-smokers and in different histological subtypes of lung cancer.Methods: We analysed tumour tissue from 394 non-small cell carcinomas including adenocarcinomas (n=229, squamous cell carcinomas (n=112, large cell carcinomas (n=30 and others (n=23 for mutations in TP53 by the use of Sanger sequencing (n=394 and next generation sequencing (n=100. Results: TP53 mutations were identified in 47.2% of the samples, with the highest frequency (65% of mutations among squamous cell carcinomas. Among never-smokers, 36% carried a TP53 mutation, identified as a significant independent negative prognostic factor in this subgroup. For large cell carcinomas, a significantly prolonged progression free survival was found for those carrying a TP53 mutation. In addition, the frequency of frameshift mutations was doubled in squamous cell carcinomas (20.3% compared to adenocarcinomas (9.1%.Conclusion: TP53 mutation patterns differ between the histological subgroups of lung cancers, as also influenced by smoking history. This indicates that the histological subtypes in lung cancer are genetically different, and that smoking-induced TP53 mutations may have a different biological impact than TP53 mutations occurring in never-smokers.

  12. Novel GABRG2 mutations cause familial febrile seizures

    Science.gov (United States)

    Boillot, Morgane; Morin-Brureau, Mélanie; Picard, Fabienne; Weckhuysen, Sarah; Lambrecq, Virginie; Minetti, Carlo; Striano, Pasquale; Zara, Federico; Iacomino, Michele; Ishida, Saeko; An-Gourfinkel, Isabelle; Daniau, Mailys; Hardies, Katia; Baulac, Michel; Dulac, Olivier; Leguern, Eric; Nabbout, Rima

    2015-01-01

    Objective: To identify the genetic cause in a large family with febrile seizures (FS) and temporal lobe epilepsy (TLE) and subsequently search for additional mutations in a cohort of 107 families with FS, with or without epilepsy. Methods: The cohort consisted of 1 large family with FS and TLE, 64 smaller French families recruited through a national French campaign, and 43 Italian families. Molecular analyses consisted of whole-exome sequencing and mutational screening. Results: Exome sequencing revealed a p.Glu402fs*3 mutation in the γ2 subunit of the GABAA receptor gene (GABRG2) in the large family with FS and TLE. Three additional nonsense and frameshift GABRG2 mutations (p.Arg136*, p.Val462fs*33, and p.Pro59fs*12), 1 missense mutation (p.Met199Val), and 1 exonic deletion were subsequently identified in 5 families of the follow-up cohort. Conclusions: We report GABRG2 mutations in 5.6% (6/108) of families with FS, with or without associated epilepsy. This study provides evidence that GABRG2 mutations are linked to the FS phenotype, rather than epilepsy, and that loss-of-function of GABAA receptor γ2 subunit is the probable underlying pathogenic mechanism. PMID:27066572

  13. X-ray-induced mutations in Escherichia coli K-12 strains with altered DNA polymerase I activities

    International Nuclear Information System (INIS)

    Nagata, Yuki; Kawata, Masakado; Komura, Jun-ichiro; Ono, Tetsuya; Yamamoto, Kazuo

    2003-01-01

    Spectra of ionizing radiation mutagenesis were determined by sequencing X-ray-induced endogenous tonB gene mutations in Escherichia coli polA strains. We used two polA alleles, the polA1 mutation, defective for Klenow domain, and the polA107 mutation, defective for flap domain. We demonstrated that irradiation of 75 and 50 Gy X-rays could induce 3.8- and 2.6-fold more of tonB mutation in polA1 and polA107 strains, respectively, than spontaneous level. The radiation induced spectrum of 51 tonB mutations in polA1 and 51 in polA107 indicated that minus frameshift, A:T→T:A transversion and G:C→T:A transversion were the types of mutations increased. Previously, we have reported essentially the same X-ray-induced tonB mutation spectra in the wild-type strain. These results indicate that (1) X-rays can induce minus frameshift, A:T→T:A transversion and G:C→T:A transversion in E. coli and (2) presence or absence of polymerase I (PolI) of E. coli does not have any effects on the process of X-ray mutagenesis

  14. Novel COL4A1 mutations cause cerebral small vessel disease by haploinsufficiency.

    Science.gov (United States)

    Lemmens, Robin; Maugeri, Alessandra; Niessen, Hans W M; Goris, An; Tousseyn, Thomas; Demaerel, Philippe; Corveleyn, Anniek; Robberecht, Wim; van der Knaap, Marjo S; Thijs, Vincent N; Zwijnenburg, Petra J G

    2013-01-15

    Mutations in COL4A1 have been identified in families with hereditary small vessel disease of the brain presumably due to a dominant-negative mechanism. Here, we report on two novel mutations in COL4A1 in two families with porencephaly, intracerebral hemorrhage and severe white matter disease caused by haploinsufficiency. Two families with various clinical presentations of cerebral microangiopathy and autosomal dominant inheritance were examined. Clinical, neuroradiological and genetic investigations were performed. Electron microscopy of the skin was also performed. In one of the families, sequence analysis revealed a one base deletion, c.2085del, leading to a frameshift and a premature stopcodon, p.(Gly696fs). In the other family, a splice site mutation was identified, c.2194-1G>A, which most likely leads to skipping of an exon with a frameshift and premature termination as a result. In fibroblasts of affected individuals from both the families, nonsense-mediated decay (NMD) of the mutant COL4A1 messenger RNAs (mRNAs) and a clear reduction of COL4A1 protein expression were demonstrated, indicating haploinsufficiency of COL4A1. Moreover, thickening of the capillary basement membrane in the skin was documented, similar to reports in patients with COL4A1 missense mutations. These findings suggest haploinsufficiency, a different mechanism from the commonly assumed dominant-negative effect, for COL4A1 mutations as a cause of (antenatal) intracerebral hemorrhage and white matter disease.

  15. Two novel RAD21 mutations in patients with mild Cornelia de Lange syndrome-like presentation and report of the first familial case.

    Science.gov (United States)

    Minor, Agata; Shinawi, Marwan; Hogue, Jacob S; Vineyard, Marisa; Hamlin, Damara R; Tan, Christopher; Donato, Kirsten; Wysinger, Latrice; Botes, Shaun; Das, Soma; Del Gaudio, Daniela

    2014-03-10

    Cornelia de Lange syndrome (CdLS) is a developmental disorder characterized by limb reduction defects, characteristic facial features and impaired cognitive development. Mutations in the NIPBL gene predominate; however, mutations in other cohesin complex genes have also been implicated, particularly in atypical and mild CdLS cases. Missense mutations and whole gene deletions in RAD21 have been identified in children with growth retardation, minor skeletal anomalies and facial features that overlap findings in individuals with CdLS. We report the first intragenic deletion and frameshift mutations identified in RAD21 in two patients presenting with atypical CdLS. One patient had an in-frame deletion of exon 13, while the second patient had a c.592_593dup frameshift mutation. The first patient presented with developmental delay, hypospadias, inguinal hernia and dysmorphic features while, the second patient presented with developmental delay, characteristic facial features, hirsutism, and hand and feet anomalies, with the first patient being milder than the second. The in-frame deletion mutation was found to be inherited from the mother who had a history of melanoma and other unspecified medical problems. This study expands the spectrum of RAD21 mutations and emphasizes the clinical utility of performing RAD21 mutation analysis in patients presenting with atypical forms of CdLS. Moreover, the variability of clinical presentation within families and low penetrance of mutations as well as the significance of performing molecular genetic testing in mildly affected patients are discussed. Published by Elsevier B.V.

  16. Screening for SH3TC2 gene mutations in a series of demyelinating recessive Charcot-Marie-Tooth disease (CMT4).

    Science.gov (United States)

    Piscosquito, Giuseppe; Saveri, Paola; Magri, Stefania; Ciano, Claudia; Gandioli, Claudia; Morbin, Michela; Bella, Daniela D; Moroni, Isabella; Taroni, Franco; Pareyson, Davide

    2016-09-01

    Charcot-Marie-Tooth disease type 4C (CMT4C) is an autosomal recessive (AR) demyelinating neuropathy associated to SH3TC2 mutations, characterized by early onset, spine deformities, and cranial nerve involvement. We screened 43 CMT4 patients (36 index cases) with AR inheritance, demyelinating nerve conductions, and negative testing for PMP22 duplication, GJB1 and MPZ mutations, for SH3TC2 mutations. Twelve patients (11 index cases) had CMT4C as they carried homozygous or compound heterozygous mutations in SH3TC2. We found six mutations: three nonsense (p.R1109*, p.R954*, p.Q892*), one splice site (c.805+2T>C), one synonymous variant (p.K93K) predicting altered splicing, and one frameshift (p.F491Lfs*32) mutation. The splice site and the frameshift mutations are novel. Mean onset age was 7 years (range: 1-14). Neuropathy was moderate-to-severe. Scoliosis was present in 11 patients (severe in 4), and cranial nerve deficits in 9 (hearing loss in 7). Scoliosis and cranial nerve involvement are frequent features of this CMT4 subtype, and their presence should prompt the clinician to look for SH3TC2 gene mutations. In our series of undiagnosed CMT4 patients, SH3TC2 mutation frequency is 30%, confirming that CMT4C may be the most common AR-CMT type. © 2016 Peripheral Nerve Society.

  17. Expanding the Clinical and Genetic Spectrum of KRT1, KRT2 and KRT10 Mutations in Keratinopathic Ichthyosis.

    Science.gov (United States)

    Hotz, Alrun; Oji, Vinzenz; Bourrat, Emmanuelle; Jonca, Nathalie; Mazereeuw-Hautier, Juliette; Betz, Regina C; Blume-Peytavi, Ulrike; Stieler, Karola; Morice-Picard, Fanny; Schönbuchner, Ines; Markus, Susanne; Schlipf, Nina; Fischer, Judith

    2016-05-01

    Twenty-six families with keratinopathic ichthyoses (epidermolytic ichthyosis, superficial epidermolytic ichthyosis or congenital reticular ichthyosiform erythroderma) were studied. Epidermolytic ichthyosis is caused by mutations in the genes KRT1 or KRT10, mutations in the gene KRT2 lead to superficial epidermolytic ichthyosis, and congenital reticular ichthyosiform erythroderma is caused by frameshift mutations in the genes KRT10 or KRT1, which lead to the phenomenon of revertant mosaicism. In this study mutations were found in KRT1, KRT2 and KRT10, including 8 mutations that are novel pathogenic variants. We report here the first case of a patient with congenital reticular ichthyosiform erythroderma carrying a mutation in KRT10 that does not lead to an arginine-rich reading frame. Novel clinical features found in patients with congenital reticular ichthyosiform erythroderma are described, such as mental retardation, spasticity, facial dysmorphisms, symblepharon and malposition of the 4th toe.

  18. Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic fibrosis adults homozygous for F508DEL-CFTR.

    Science.gov (United States)

    Donaldson, Scott H; Solomon, George M; Zeitlin, Pamela L; Flume, Patrick A; Casey, Alicia; McCoy, Karen; Zemanick, Edith T; Mandagere, Arun; Troha, Janice M; Shoemaker, Steven A; Chmiel, James F; Taylor-Cousar, Jennifer L

    2017-05-01

    Cavosonstat (N91115), an orally bioavailable inhibitor of S-nitrosoglutathione reductase, promotes cystic fibrosis transmembrane conductance regulator (CFTR) maturation and plasma membrane stability, with a mechanism of action complementary to CFTR correctors and potentiators. A Phase I program evaluated pharmacokinetics, drug-drug interactions and safety of cavosonstat in healthy and cystic fibrosis (CF) subjects homozygous for F508del-CFTR. Exploratory outcomes included changes in sweat chloride in CF subjects. Cavosonstat was rapidly absorbed and demonstrated linear and predictable pharmacokinetics. Exposure was unaffected by a high-fat meal or rifampin-mediated effects on drug metabolism and transport. Cavosonstat was well tolerated, with no dose-limiting toxicities or significant safety findings. At the highest dose, significant reductions from baseline in sweat chloride were observed (-4.1mmol/L; P=0.032) at day 28. The favorable safety and clinical profile warrant further study of cavosonstat in CF. ClinicalTrials.gov Numbers: NCT02275936, NCT02013388, NCT02500667. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  19. Cystic fibrosis and the role of gastrointestinal outcome measures in the new era of therapeutic CFTR modulation.

    Science.gov (United States)

    Bodewes, Frank A J A; Verkade, Henkjan J; Taminiau, Jan A J M; Borowitz, Drucy; Wilschanski, Michael

    2015-03-01

    With the development of new drugs that directly affect CFTR protein function, clinical trials are being designed or initiated for a growing number of patients with cystic fibrosis. The currently available and accepted clinical endpoints, FEV1 and BMI, have limitations. The aim of this report is to draw attention to the need and the ample possibilities for the development and validation of relevant gastrointestinal clinical endpoints for scientific evaluation of CFTR modulation treatment, particularly in young children and infants. The gastrointestinal tract offers very good opportunities to measure CFTR protein function and systematically evaluate CF related clinical outcomes based on the principal clinical gastrointestinal manifestations of CF: intestinal pH, intestinal transit time, intestinal bile salt malabsorption, intestinal inflammation, exocrine pancreatic function and intestinal fat malabsorption. We present a descriptive analysis of a variety of gastrointestinal outcome measures for clinical relevance, reliability, validity, responsiveness to interventions, feasibility in particular in young children and the availability of reference values. Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  20. Beta-thalassaemia mutations in northern India (Delhi).

    Science.gov (United States)

    Madan, N; Sharma, S; Rusia, U; Sen, S; Sood, S K

    1998-03-01

    The present study was undertaken to define beta-thalassaemia mutations prevalent in northern India (Delhi). Forty six children of beta-thalassaemia major and their families were investigated. DNA was extracted from leucocytes and screened for mutations prevalent in the Indian population. These mutations included 619bp deletion, IVS 1-1 (G-T), IVS 1-5 (G-C), frameshift mutations FS 8/9 (+G), FS 41/42 (-CTTT), Codon 16(-C), Codon 15 (G-A), codon 30 (G-C), IVS 1-110 (G-A) and -88 (C-T). 619 bp deletion mutation was detected directly by amplification of DNA by PCR followed by agarose gel electrophoresis. Other mutations were studied by DNA amplification and dot blot hybridization using synthetic normal and mutant oligonucleotide probes labelled at 5' end with gamma-32 P-ATP. Five mutations accounted for all the chromosomes in 46 patients. 619 bp deletion mutation was found to be the commonest mutation (34.8%) followed by IVS 1-5 (G-C) in 22.8 per cent, IVS 1-1 (G-T) in 19.6 per cent, FS 8/9 (+G) in 13 per cent and FS 41/42 (-CTTT) in 9.8 per cent. Nineteen (41.3%) patients were homozygous and 27 (58.7%) double heterozygous for different beta-thalassaemia mutations. This observation of limited number of mutations is significant and will be useful in planning strategies for prenatal diagnosis of beta-thalassaemia in northern India.

  1. Role of mutation in Pseudomonas aeruginosa biofilm development.

    Directory of Open Access Journals (Sweden)

    Tim C R Conibear

    2009-07-01

    Full Text Available The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor

  2. Frameshifted beta-amyloid precursor protein (APP(+1)) is a secretory protein, and the level of APP(+1) in cerebrospinal fluid is linked to Alzheimer pathology

    NARCIS (Netherlands)

    Hol, Elly M.; van Dijk, Renske; Gerez, Lisya; Sluijs, Jacqueline A.; Hobo, Barbara; Tonk, Martijn T.; de Haan, Annett; Kamphorst, Wouter; Fischer, David F.; Benne, Rob; van Leeuwen, Fred W.

    2003-01-01

    Molecular misreading of the beta-amyloid precursor protein (APP) gene generates mRNA with dinucleotide deletions in GAGAG motifs. The resulting truncated and partly frameshifted APP protein ( APP(+1)) accumulates in the dystrophic neurites and the neurofibrillary tangles in the cortex and

  3. Funciones de los canales iónicos CFTR y ENAC en la fibrosis quística

    Directory of Open Access Journals (Sweden)

    Alejandra G. Palma

    2014-04-01

    Full Text Available La fibrosis quística se debe a la ausencia o defecto del canal transmembrana regulador de la fibrosis quística (CFTR, un canal de cloruro codificado en el gen cftr que juega un papel clave en la homeostasis del agua e iones. El CFTR es activado por el AMPc y se localiza en las membranas apicales y basolaterales de las vías aéreas, intestino y glándulas exocrinas. Una de sus funciones primarias en los pulmones es mantener la capa de líquido superficial a través de su función de canal y regular el canal epitelial de sodio sensible al amiloride (ENaC. Se han identificado más de 1900 mutaciones en el gen cftr. La enfermedad se caracteriza por secreciones viscosas en las glándulas exocrinas y por niveles elevados de cloruro de sodio en el sudor. En la fibrosis quística el CFTR no funciona y el ENaC está desregulado; el resultado es un aumento en la reabsorción de sodio y agua con la formación de un líquido viscoso. En las glándulas sudoríparas tanto el Na+ como el Cl- se retienen en el lumen causando una pérdida de electrolitos durante la sudoración y el NaCl se elimina al sudor. Así, los niveles elevados de NaCl son la base del test del sudor inducido por pilocarpina, un método de diagnóstico para la enfermedad. En esta revisión se discuten los movimientos de Cl- y Na+ en las glándulas sudoríparas y pulmón así como el papel del ENaC en la patogénesis de la enfermedad.

  4. Solenopsis invicta virus 3: mapping of structural proteins, ribosomal frameshifting, and similarities to Acyrthosiphon pisum virus and Kelp fly virus.

    Directory of Open Access Journals (Sweden)

    Steven M Valles

    Full Text Available Solenopsis invicta virus 3 (SINV-3 is a positive-sense single-stranded RNA virus that infects the red imported fire ant, Solenopsis invicta. We show that the second open reading frame (ORF of the dicistronic genome is expressed via a frameshifting mechanism and that the sequences encoding the structural proteins map to both ORF2 and the 3' end of ORF1, downstream of the sequence that encodes the RNA-dependent RNA polymerase. The genome organization and structural protein expression strategy resemble those of Acyrthosiphon pisum virus (APV, an aphid virus. The capsid protein that is encoded by the 3' end of ORF1 in SINV-3 and APV is predicted to have a jelly-roll fold similar to the capsid proteins of picornaviruses and caliciviruses. The capsid-extension protein that is produced by frameshifting, includes the jelly-roll fold domain encoded by ORF1 as its N-terminus, while the C-terminus encoded by the 5' half of ORF2 has no clear homology with other viral structural proteins. A third protein, encoded by the 3' half of ORF2, is associated with purified virions at sub-stoichiometric ratios. Although the structural proteins can be translated from the genomic RNA, we show that SINV-3 also produces a subgenomic RNA encoding the structural proteins. Circumstantial evidence suggests that APV may also produce such a subgenomic RNA. Both SINV-3 and APV are unclassified picorna-like viruses distantly related to members of the order Picornavirales and the family Caliciviridae. Within this grouping, features of the genome organization and capsid domain structure of SINV-3 and APV appear more similar to caliciviruses, perhaps suggesting the basis for a "Calicivirales" order.

  5. The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, R.J.; Bobrow, M.; Roberts, R.G. [St. Thomas`s Hospitals, London (United Kingdom)

    1995-08-01

    The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frameshifting deletions in the dystrophin gene that are detectable by multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be mutation in the ORF. We believe that reverse-transcription-PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion. 29 refs., 2 figs., 3 tabs.

  6. A novel heterozygous SOX2 mutation causing congenital bilateral anophthalmia, hypogonadotropic hypogonadism and growth hormone deficiency.

    Science.gov (United States)

    Macchiaroli, Annamaria; Kelberman, Daniel; Auriemma, Renata Simona; Drury, Suzanne; Islam, Lily; Giangiobbe, Sara; Ironi, Gabriele; Lench, Nicholas; Sowden, Jane C; Colao, Annamaria; Pivonello, Rosario; Cavallo, Luciano; Gasperi, Maurizio; Faienza, Maria Felicia

    2014-01-25

    Heterozygous de novo mutations in SOX2 have been reported in approximately 10-20% of patients with unilateral or bilateral anophthalmia or microphthalmia. An additional phenotype of hypopituitarism, with anterior pituitary hypoplasia and hypogonadotropic hypogonadism, has been reported in patients carrying SOX2 alterations. We report a novel heterozygous mutation in the SOX2 gene in a male affected with congenital bilateral anophthalmia, hypogonadotrophic hypogonadism and growth hormone deficiency. The mutation we describe is a cytosine deletion in position 905 (c905delC) which causes frameshift and an aberrant C-terminal domain. Our report highlights the fact that subjects affected with eye anomalies and harboring SOX2 mutations are at high risk for gonadotropin deficiency, which has important implications for their clinical management. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Variability in Chromatin Architecture and Associated DNA Repair at Genomic Positions Containing Somatic Mutations.

    Science.gov (United States)

    Lim, Byungho; Mun, Jihyeob; Kim, Yong Sung; Kim, Seon-Young

    2017-06-01

    Dynamic chromatin structures result in differential chemical reactivity to mutational processes throughout the genome. To identify chromatin features responsible for mutagenesis, we compared chromatin architecture around single-nucleotide variants (SNV), insertion/deletions (indels), and their context-matched, nonmutated positions. We found epigenetic differences between genomic regions containing missense SNVs and those containing frameshift indels across multiple cancer types. Levels of active histone marks were higher around frameshift indels than around missense SNV, whereas repressive histone marks exhibited the reverse trend. Accumulation of repressive histone marks and nucleosomes distinguished mutated positions (both SNV and indels) from the context-matched, nonmutated positions, whereas active marks were associated with substitution- and cancer type-specific mutagenesis. We also explained mutagenesis based on genome maintenance mechanisms, including nucleotide excision repair (NER), mismatch repair (MMR), and DNA polymerase epsilon (POLE). Regional NER variation correlated strongly with chromatin features; NER machineries exhibited shifted or depleted binding around SNV, resulting in decreased NER at mutation positions, especially at sites of recurrent mutations. MMR-deficient tumors selectively acquired SNV in regions with high active histone marks, especially H3K36me3, whereas POLE-deficient tumors selectively acquired indels and SNV in regions with low active histone marks. These findings demonstrate the importance of fine-scaled chromatin structures and associated DNA repair mechanisms in mutagenesis. Cancer Res; 77(11); 2822-33. ©2017 AACR . ©2017 American Association for Cancer Research.

  8. [Relation between gene mutations and pancreatic exocrine function in patients with cystic fibrosis].

    Science.gov (United States)

    Radivojević, D; Guć-Sćekić, M; Djurisić, M; Lalić, T; Minić, P; Kanavakis, E

    2001-01-01

    Cystic fibrosis (CF), is the most common autosomal-recessive disease in Caucasians, with an incidence of approximately 1:2500 live births and a carrier frequency of approximately 4-5%. Causes of the disease are mutations in the CF gene which is located on chromosome 7 (region 7q31). Although a single mutation, a deletion of phenylalanine at position 508 (DF508) in exon 10, accounts for almost 70% of all CF chromosomes, over 900 other mutations have been identified in this large gene. CF gene encodes a membrane protein, which functions as aion channel- CFTR (cystic fibrosis transmembrane regulator protein). The exocrine pancreas is a gland that secretes water, enzymes and electrolytes into the intestinal lumen. These enzymes are needed for the normal digestion of food, and their reduced secretion in cystic fibrosis will cause malabsortion and malnutrition in CF patients. Pancreatic dysfunction in CF begins in uteri. Most patients with CF typically present insufficient pancreatic exocrine function (PI phenotype) and 10-15% of CF patients are pancreatic sufficient (PS phenotype). It has been shown elsewhere that the pancreatic function status in CF could be correlated to mutations in the CFTR gene. To determine the relation between genotype and pancreatic status, we analyzed 32 CF patients in whom both CF gene mutant alleles were identified (Table 1). Patients included in this study attended the Paediatric Department of Mother and Child Health Institute in Belgrade. The diagnosis was based on typical clinical manifestations and high levels of sweat chloride concentration (higher than 60 mmol/L). Of the 32 patients studied, only one (3.12%) was PS and the rest (96.88%) had PI phenotype. For each CF genotype the number of patients who were PI or PS is given in Table 1. The most striking observation was that all given genotypes correlated with either PI or PS, but not with both. On the basis of both preceding hypotheses and our present data (Table 2 and Table 3), it was

  9. Feasibility of placebo-controlled trial designs for new CFTR modulator evaluation.

    Science.gov (United States)

    VanDevanter, Donald R; Mayer-Hamblett, Nicole; Boyle, Michael

    2017-07-01

    New CFTR modulators are in development that sponsors anticipate will be comparable or superior to approved modulators. Testing these agents for efficacy will require either placebo-controlled or active-comparator trials. We surveyed US CF physicians and their patients eligible to receive approved modulators or their families for willingness to participate in placebo-controlled modulator trials of varying duration. Interest in placebo-controlled trials of short duration (2-4weeks) was greatest, with few respondents, particularly among patient respondents, willing to consider 6month studies. Patients/families with access to approved modulators were consistently less interested in placebo-controlled modulator trials of any duration. Sample size and interpretability advantages of placebo-controlled trials outweigh alternative active-comparator trials, but must consider physician and patient thresholds for forgoing treatment with approved modulators. Enrollment will be most feasible for short-duration trials and those conducted among populations without access to approved modulators. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  10. NPHS1 gene mutations confirm congenital nephrotic syndrome in four Brazilian cases: A novel mutation is described.

    Science.gov (United States)

    Guaragna, Mara S; Cleto, Thaís Lira; Souza, Marcela Lopes; Lutaif, Anna Cristina G B; de Castro, Luiz Cláudio Gonçalves; Penido, Maria Goretti Moreira Guimarães; Maciel-Guerra, Andréa T; Belangero, Vera M S; Guerra-Junior, Gil; De Mello, Maricilda P

    2016-09-01

    Autosomal recessive mutations in NPHS1 gene are a common cause of congenital nephrotic syndrome (CNS). The disorder is characterized by massive proteinuria that manifests in utero or in the neonatal period during the first 3 months of life. NPHS1 encodes nephrin, a member of the immunoglobulin family of cell adhesion molecules and the main protein expressed at the renal slit diaphragm. Currently, there are approximately 250 mutations described in the NPHS1 gene distributed among all nephrin domains. The main objective of this study was to perform the analysis of the NPHS1 gene in patients with congenital nephrotic syndrome in order to determine the molecular cause of the disease. Direct sequencing of NPHS1 gene in four children was performed. Each patient was heterozygous for two pathogenic mutations disclosing the molecular cause of the disease in 100% of the cases. We identified six different mutations, consisting of one in-frame deletion, one frameshift, and four missense substitutions. The p.Val736Met mutation that is described here for the first time was considered pathogenic by different mutation predictive algorithms. Regardless of the type of mutation, three patients had a bad outcome and died Despite the small size of the cohort, this study contributed to the increasing number of deleterious mutations in the NPHS1 gene by describing a new mutation. Also, since we identified NPHS1 pathogenic mutations as the cause of the disease in all cases analyzed, it might be a frequent cause of CNS in the South Eastern region of Brazil, although the analysis of a larger sample is required to obtain more indicative epidemiological data. © 2015 Asian Pacific Society of Nephrology.

  11. Mutation of RET proto-oncogene in Hirschsprung’s disease and intestinal neuronal dysplasia

    Science.gov (United States)

    Tou, Jin-Fa; Li, Min-Ju; Guan, Tao; Li, Ji-Cheng; Zhu, Xiong-Kai; Feng, Zhi-Gang

    2006-01-01

    AIM: To investigate the genetic relationship between Hirschsprung’s disease (HD) and intestinal neuronal dysplasia (IND) in Chinese population. METHODS: Peripheral blood samples were obtained from 30 HD patients, 20 IND patients, 18 HD/IND combined patients and 20 normal individuals as control. Genomic DNA was extracted according to standard procedure. Exons 11,13,15,17 of RET proto-oncogene were amplified by polymerase chain reaction (PCR). The mutations of RET proto-oncogene were analyzed by single strand conformational polymorphism (SSCP) and sequencing of the positive amplified products was performed. RESULTS: Eight germline sequence variants were detected. In HD patients, 2 missense mutations in exon 11 at nucleotide 15165 G→A (G667S), 2 frameshift mutations in exon 13 at nucleotide 18974 (18974insG), 1 missense mutation in exon 13 at nucleotide 18919 A→G (K756E) and 1 silent mutation in exon 15 at nucleotide 20692 G→A(Q916Q) were detected. In HD/IND combined patients, 1 missense mutation in exon 11 at nucleotide 15165 G→A and 1 silent mutation in exon 13 at nucleotide 18888 T→G (L745L) were detected. No mutation was found in IND patients and controls. CONCLUSION: Mutation of RET proto-oncogene is involved in the etiopathogenesis of HD. The frequency of RET proto-oncogene mutation is quite different between IND and HD in Chinese population. IND is a distinct clinical entity genetically different from HD. PMID:16534860

  12. Mutations in the gene for X-linked adrenoleukodystrophy in patients with different clinical phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Braun, A.; Ambach, H.; Kammerer, S.; Rolinski, B.; Roscher, A.; Rabl, W. [Univ. of Munich (Germany); Stoeckler, S. [Univ. of Graz (Germany); Gaertner, J. [Univ. of Duesseldorf (Germany); Zierz, S. [Univ. of Bonn (Germany)

    1995-04-01

    Recently, the gene for the most common peroxisomal disorder, X-linked adrenoleukodystrophy (X-ALD), has been described encoding a peroxisomal membrane transporter protein. We analyzed the entire protein-coding sequence of this gene by reverse-transcription PCR, SSCP, and DNA sequencing in five patients with different clinical expressions were cerebral childhood ALD, adrenomyecloneuropathy (AMN), and {open_quotes}Addison disease only{close_quotes} (AD) phenotype. In the three patients exhibiting the classical picture of severe childhood ALD we identified in the 5{prime} portion of the X-ALD gene a 38-bp deletion that causes a frameshift mutation, a 3-bp deletion leading to a deletion of an amino acid in the ATP-binding domain of the ALD protein, and a missense mutation. In the patient with the clinical phenotype of AMN, a nonsense mutation in codon 212, along with a second site mutation at codon 178, was observed. Analysis of the patient with the ADO phenotype revealed a further missense mutation at a highly conserved position in the ALDP/PMP70 comparison. The disruptive nature of two mutations (i.e., the frameshift and the nonsense mutation) in patients with biochemically proved childhood ALD and AMN further strongly supports the hypothesis that alterations in this gene play a crucial role in the pathogenesis of X-ALD. Since the current biochemical techniques for X-ALD carrier detection in affected families lack sufficient reliability, our procedure described for systematic mutation scanning is also capable of improving genetic counseling and prenatal diagnosis. 19 refs., 6 figs., 3 tabs.

  13. Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension. Retarded secretion and proteasomal degradation.

    Science.gov (United States)

    Komaru, Keiichi; Ishida, Yoko; Amaya, Yoshihiro; Goseki-Sone, Masae; Orimo, Hideo; Oda, Kimimitsu

    2005-04-01

    In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency. In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation/translocation system and COS-1 cells transiently expressing this mutant protein. We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by approximately 12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome. SDS/PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms a disulfide-bonded high-molecular-mass aggregate. Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C-terminal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers. Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT).

  14. [Suspected pathogenic mutation identified in two cases with oculocutaneous albinism].

    Science.gov (United States)

    He, Jiangmei; Zheng, Meiling; Zhang, Guilin; Hua, Ailing

    2015-08-01

    To detect potential mutations in genes related with non-syndromic oculocutaneous albinism I-IV and ocular albinism type I in two couples who had given births to children with albinism. All exons of the non-syndromic albinism related genes TYR, OCA2, TYRP-1, MITF, SLC45A2 and GPR143 were subjected to deep sequencing. The results were verified with Sanger sequencing. For the two female carriers, the coding region of the TYR gene was found to harbor a frameshift mutation c.925_926insC, which was also suspected to have been pathogenic. In one of the male partners, a nonsense mutations c.832C>T was found, which was also known to be pathogenic. Another male partner was found to harbor a TYR gene mutation c.346C>T, which was also known to be a pathogenic nonsense mutation. The coding region of the TYR gene c.925_926insC (p.Thr309ThrfsX9) probably underlies the OCA1 disease phenotype.

  15. Mitochondrial Mutation Rate, Spectrum and Heteroplasmy in Caenorhabditis elegans Spontaneous Mutation Accumulation Lines of Differing Population Size.

    Science.gov (United States)

    Konrad, Anke; Thompson, Owen; Waterston, Robert H; Moerman, Donald G; Keightley, Peter D; Bergthorsson, Ulfar; Katju, Vaishali

    2017-06-01

    Mitochondrial genomes of metazoans, given their elevated rates of evolution, have served as pivotal markers for phylogeographic studies and recent phylogenetic events. In order to determine the dynamics of spontaneous mitochondrial mutations in small populations in the absence and presence of selection, we evolved mutation accumulation (MA) lines of Caenorhabditis elegans in parallel over 409 consecutive generations at three varying population sizes of N = 1, 10, and 100 hermaphrodites. The N =1 populations should have a minimal influence of natural selection to provide the spontaneous mutation rate and the expected rate of neutral evolution, whereas larger population sizes should experience increasing intensity of selection. New mutations were identified by Illumina paired-end sequencing of 86 mtDNA genomes across 35 experimental lines and compared with published genomes of natural isolates. The spontaneous mitochondrial mutation rate was estimated at 1.05 × 10-7/site/generation. A strong G/C→A/T mutational bias was observed in both the MA lines and the natural isolates. This suggests that the low G + C content at synonymous sites is the product of mutation bias rather than selection as previously proposed. The mitochondrial effective population size per worm generation was estimated to be 62. Although it was previously concluded that heteroplasmy was rare in C. elegans, the vast majority of mutations in this study were heteroplasmic despite an experimental regime exceeding 400 generations. The frequencies of frameshift and nonsynonymous mutations were negatively correlated with population size, which suggests their deleterious effects on fitness and a potent role for selection in their eradication. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. A novel silent deletion, an insertion mutation and a nonsense mutation in the TCOF1 gene found in two Chinese cases of Treacher Collins syndrome.

    Science.gov (United States)

    Wang, Yan; Yin, Xiao-Juan; Han, Tao; Peng, Wei; Wu, Hong-Lin; Liu, Xin; Feng, Zhi-Chun

    2014-12-01

    Treacher Collins syndrome (TCS) is the most common and well-known craniofacial disorder caused by mutations in the genes involved in pre-rRNA transcription, which include the TCOF1 gene. This study explored the role of TCOF1 mutations in Chinese patients with TCS. Mutational analysis of the TCOF1 gene was performed in three patients using polymerase chain reaction and direct sequencing. Among these three patients, two additional TCOF1 variations, a novel 18 bp deletion and a novel 1 bp insertion mutation, were found in patient 1, together with a novel nonsense mutation (p.Ser476X) and a previously reported 4 bp deletion (c.1872_1875delTGAG) in other patients. Pedigree analysis allowed for prediction of the character of the mutation, which was either pathological or not. The 18 bp deletion of six amino acids, Ser-Asp-Ser-Glu-Glu-Glu (798*803), which was located in the CKII phosphorylation site of treacle, seemed relatively benign for TCS. By contrast, another novel mutation of c.1072_1073insC (p.Gln358ProfsX23) was a frameshift mutation and expected to result in a premature stop codon. This study provides insights into the functional domain of treacle and illustrates the importance of clinical and family TCS screening for the interpretation of novel sequence alterations.

  17. Ornithine decarboxylase antizyme finder (OAF: Fast and reliable detection of antizymes with frameshifts in mRNAs

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    Atkins John F

    2008-04-01

    Full Text Available Abstract Background Ornithine decarboxylase antizymes are proteins which negatively regulate cellular polyamine levels via their affects on polyamine synthesis and cellular uptake. In virtually all organisms from yeast to mammals, antizymes are encoded by two partially overlapping open reading frames (ORFs. A +1 frameshift between frames is required for the synthesis of antizyme. Ribosomes change translation phase at the end of the first ORF in response to stimulatory signals embedded in mRNA. Since standard sequence analysis pipelines are currently unable to recognise sites of programmed ribosomal frameshifting, proper detection of full length antizyme coding sequences (CDS requires conscientious manual evaluation by a human expert. The rapid growth of sequence information demands less laborious and more cost efficient solutions for this problem. This manuscript describes a rapid and accurate computer tool for antizyme CDS detection that requires minimal human involvement. Results We have developed a computer tool, OAF (ODC antizyme finder for identifying antizyme encoding sequences in spliced or intronless nucleic acid sequenes. OAF utilizes a combination of profile hidden Markov models (HMM built separately for the products of each open reading frame constituting the entire antizyme coding sequence. Profile HMMs are based on a set of 218 manually assembled antizyme sequences. To distinguish between antizyme paralogs and orthologs from major phyla, antizyme sequences were clustered into twelve groups and specific combinations of profile HMMs were designed for each group. OAF has been tested on the current version of dbEST, where it identified over six thousand Expressed Sequence Tags (EST sequences encoding antizyme proteins (over two thousand antizyme CDS in these ESTs are non redundant. Conclusion OAF performs well on raw EST sequences and mRNA sequences derived from genomic annotations. OAF will be used for the future updates of the RECODE

  18. Loss of function JAK1 mutations occur at high frequency in cancers with microsatellite instability and are suggestive of immune evasion.

    Science.gov (United States)

    Albacker, Lee A; Wu, Jeremy; Smith, Peter; Warmuth, Markus; Stephens, Philip J; Zhu, Ping; Yu, Lihua; Chmielecki, Juliann

    2017-01-01

    Immune evasion is a well-recognized hallmark of cancer and recent studies with immunotherapy agents have suggested that tumors with increased numbers of neoantigens elicit greater immune responses. We hypothesized that the immune system presents a common selective pressure on high mutation burden tumors and therefore immune evasion mutations would be enriched in high mutation burden tumors. The JAK family of kinases is required for the signaling of a host of immune modulators in tumor, stromal, and immune cells. Therefore, we analyzed alterations in this family for the hypothesized signature of an immune evasion mutation. Here, we searched a database of 61,704 unique solid tumors for alterations in the JAK family kinases (JAK1/2/3, TYK2). We used The Cancer Genome Atlas and Cancer Cell Line Encyclopedia data to confirm and extend our findings by analyzing gene expression patterns. Recurrent frameshift mutations in JAK1 were associated with high mutation burden and microsatellite instability. These mutations occurred in multiple tumor types including endometrial, colorectal, stomach, and prostate carcinomas. Analyzing gene expression signatures in endometrial and stomach adenocarcinomas revealed that tumors with a JAK1 frameshift exhibited reduced expression of interferon response signatures and multiple anti-tumor immune signatures. Importantly, endometrial cancer cell lines exhibited similar gene expression changes that were expected to be tumor cell intrinsic (e.g. interferon response) but not those expected to be tumor cell extrinsic (e.g. NK cells). From these data, we derive two primary conclusions: 1) JAK1 frameshifts are loss of function alterations that represent a potential pan-cancer adaptation to immune responses against tumors with microsatellite instability; 2) The mechanism by which JAK1 loss of function contributes to tumor immune evasion is likely associated with loss of the JAK1-mediated interferon response.

  19. GNRHR biallelic and digenic mutations in patients with normosmic congenital hypogonadotropic hypogonadism

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    Catarina I Gonçalves

    2017-07-01

    Full Text Available Objective: Normosmic congenital hypogonadotropic hypogonadism (nCHH is a rare disorder characterised by lack of pubertal development and infertility, due to deficient production, secretion or action of gonadotropin-releasing hormone (GnRH and, unlike Kallmann syndrome, is associated with a normal sense of smell. Mutations in the GNRHR gene cause autosomal recessive nCHH. The aim of this study was to determine the prevalence of GNRHR mutations in a group of 40 patients with nCHH. Design: Cross-sectional study of 40 unrelated patients with nCHH. Methods: Patients were screened for mutations in the GNRHR gene by DNA sequencing. Results: GNRHR mutations were identified in five of 40 patients studied. Four patients had biallelic mutations (including a novel frameshift deletion p.Phe313Metfs*3, in two families in agreement with autosomal recessive inheritance. One patient had a heterozygous GNRHR mutation associated with a heterozygous PROKR2 mutation, thus suggesting a possible role of synergistic heterozygosity in the pathogenesis of the disorder. Conclusions: This study further expands the spectrum of known genetic defects associated with nCHH. Although GNRHR mutations are usually biallelic and inherited in an autosomal recessive manner, the presence of a monoallelic mutation in a patient should raise the possibility of a digenic/oligogenic cause of nCHH.

  20. Identification of four novel mutations of the WFS1 gene in Iranian Wolfram syndrome pedigrees.

    Science.gov (United States)

    Ghahraman, Martha; Abbaszadegan, Mohammad Reza; Vakili, Rahim; Hosseini, Sousan; Fardi Golyan, Fatemeh; Ghaemi, Nosrat; Forghanifard, Mohammad Mahdi

    2016-12-01

    Wolfram syndrome is a rare neurodegenerative disorder with an autosomal recessive pattern of inheritance characterized by various clinical manifestations. The related gene, WFS1, encodes a transmembrane glycoprotein, named wolframin. Genetic analyses demonstrated that mutations in this gene are associated with WS type 1. Our aim in this study was to sequence WFS1 coding region in Iranian Wolfram syndrome pedigrees. Genomic DNA was extracted from peripheral blood of 12 WS patients and their healthy parents. Exons 2-8 and the exon-intron junctions of WFS1 were sequenced. DNA sequences were compared to the reference using Sequencher software. Molecular analysis of WFS1 revealed six different mutations. Four novel and two previously reported mutations were identified. One novel mutation, c.1379_1381del, is predicted to produce an aberrant protein. A second novel mutation, c.1384G > T, encodes a truncated protein. Novel mutation, c.1097-1107dup (11 bp), causes a frameshift which results in a premature stop codon. We screened for the novel missense mutation, c.1010C > T, in 100 control alleles. This mutation was not found in any of the healthy controls. Our study increased the spectrum of WFS1 mutations and supported the role of WFS1 in susceptibility to WS. We hope that these findings open new horizons to future molecular investigations which may help to prevent and treat this devastating disease.

  1. Assaying Mutations Associated With Gene Conversion Repair of a Double-Strand Break.

    Science.gov (United States)

    Dwivedi, Gajendrahar; Haber, James E

    2018-01-01

    DNA double-strand break (DSB) is a cytotoxic lesion and needs to be repaired immediately. There are several metabolic pathways evolved to repair a DSB. Gene conversion is one of the least error-prone pathway for repair of a DNA DSB. Despite this there is nearly 1000-fold increase in mutation rate associated with gene conversion. Not only higher mutation rate is associated with gene conversion but also there is a very distinct mutation profile compared to spontaneous mutation events. Gene conversion is characterized by the presence of very high frameshift mutation events and other complex mutations that are not present during regular DNA replication. Another DNA DSB repair pathway widely studied is "break-induced replication" (BIR). BIR has been shown to be highly mutagenic in nature. BIR may lead to chromosomal rearrangement and has potential to cause cluster mutations with serious disease implications. In this chapter, the design of assay systems to study various mutation types and experimental procedures to measure specific mutation frequency associated with gene conversion are discussed. © 2018 Elsevier Inc. All rights reserved.

  2. Identification of Two Novel LAMA2 Mutations in a Chinese Patient with Congenital Muscular Dystrophy.

    Science.gov (United States)

    Zhou, Jing; Tan, Jianxin; Ma, Dingyuan; Zhang, Jingjing; Cheng, Jian; Luo, Chunyu; Liu, Gang; Wang, Yuguo; Xu, Zhengfeng

    2018-01-01

    Merosin-deficient CMD type 1A (MDC1A), caused by mutations of laminin subunit alpha 2 (LAMA2), is a predominant subtype of congenital muscular dystrophy (CMD). Herein, we described a Chinese patient with MDC1A who was admitted to hospital 17 days after birth because of marasmus and feeding difficulties. Mutations were identified by targeted capture and next generation sequencing (NGS) and further confirmed by Sanger sequencing. Paternity was confirmed by short tandem repeat analysis. Physical examination showed malnutrition, poor suck and appendicular hypotonia. Her serum CK levels were 2483 and 1962 U/L at 2 and 4 months of age, respectively. Brain magnetic resonance imaging performed at 1 month of age presented hyperintensity on T2-weighted images, T1-weighted images in parietal and occipital lobes, and diffusion-weighted image (DWI) as well as hypointensity on fluid attenuated inversion recovery (FLAIR) image; however, the cerebellum and corpus arenaceum were normal. At 7 months of age, delayed developmental milestones were observed, and she failed to turn her body over and raise her head up. A point mutation (c.1782+2T > G) and a frameshift duplication (c.8217dupT) in the LAMA2 gene were identified by targeted capture and NGS and further confirmed by Sanger sequencing. Moreover, genotyping with multiple short tandem repeat markers confirmed paternity to demonstrate that the point mutation is de novo . The frameshift duplication (c.8217dupT), inherited from her mother, was predicted to cause a substitution of Pro (P) to Ser (S) at the 2740th amino-acid residue and generate a prematurely truncated protein. The in silico analysis suggests that the mutation (c.1782+2T > G) may lead to aberrant splicing of LAMA2. Our case further confirms the heterogeneous clinical spectrum of MDC1A and presents two novel LAMA2 mutations to expand the mutation spectrum of MDC1A.

  3. Identification of Two Novel LAMA2 Mutations in a Chinese Patient with Congenital Muscular Dystrophy

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    Jing Zhou

    2018-02-01

    Full Text Available Merosin-deficient CMD type 1A (MDC1A, caused by mutations of laminin subunit alpha 2 (LAMA2, is a predominant subtype of congenital muscular dystrophy (CMD. Herein, we described a Chinese patient with MDC1A who was admitted to hospital 17 days after birth because of marasmus and feeding difficulties. Mutations were identified by targeted capture and next generation sequencing (NGS and further confirmed by Sanger sequencing. Paternity was confirmed by short tandem repeat analysis. Physical examination showed malnutrition, poor suck and appendicular hypotonia. Her serum CK levels were 2483 and 1962 U/L at 2 and 4 months of age, respectively. Brain magnetic resonance imaging performed at 1 month of age presented hyperintensity on T2-weighted images, T1-weighted images in parietal and occipital lobes, and diffusion-weighted image (DWI as well as hypointensity on fluid attenuated inversion recovery (FLAIR image; however, the cerebellum and corpus arenaceum were normal. At 7 months of age, delayed developmental milestones were observed, and she failed to turn her body over and raise her head up. A point mutation (c.1782+2T > G and a frameshift duplication (c.8217dupT in the LAMA2 gene were identified by targeted capture and NGS and further confirmed by Sanger sequencing. Moreover, genotyping with multiple short tandem repeat markers confirmed paternity to demonstrate that the point mutation is de novo. The frameshift duplication (c.8217dupT, inherited from her mother, was predicted to cause a substitution of Pro (P to Ser (S at the 2740th amino-acid residue and generate a prematurely truncated protein. The in silico analysis suggests that the mutation (c.1782+2T > G may lead to aberrant splicing of LAMA2. Our case further confirms the heterogeneous clinical spectrum of MDC1A and presents two novel LAMA2 mutations to expand the mutation spectrum of MDC1A.

  4. Novel insertion mutation in a non-Jewish Caucasian type 1 Gaucher disease patient

    Energy Technology Data Exchange (ETDEWEB)

    Choy, F.Y.M.; Humphries, M.L. [Univ. of Victoria, British Columbia (Canada); Ferreira, P. [Univ. of Alberta, Edmonton (Canada)

    1997-01-20

    Gaucher disease is the most prevalent lysosomal storage disorder. It is autosomal recessive, resulting in lysosomal glucocerebrosidase deficiency. Three clinical forms of Gaucher disease have been described: type 1 (nonneuronopathic), type 2 (acute neuronopathic), and type 3 (subacute neuronopathic). We performed PCR-thermal cycle sequence analysis of glucocerebrosidase genomic DNA and identified a novel mutation in a non-Jewish type 1 Gaucher disease patient. It is a C insertion in exon 3 at cDNA nucleotide position 122 and genomic nucleotide position 1626. This mutation causes a frameshift and, subsequently, four of the five codons immediately downstream of the insertion were changed while the sixth was converted to a stop codon, resulting in premature termination of protein translation. The 122CC insertion abolishes a Cac81 restriction endonuclease cleavage site, allowing a convenient and reliable method for detection using RFLP analysis of PCR-amplified glucocerebrosidase genomic DNA. The mutation in the other Gaucher allele was found to be an A{r_arrow}G substitution at glucocerebrosidase cDNA nucleotide position 1226 that so far has only been reported among type 1 Gaucher disease patients. Since mutation 122CC causes a frameshift and early termination of protein translation, it most likely results in a meaningless transcript and subsequently no residual glucocerebrosidase enzyme activity. We speculate that mutation 122CC may result in a worse prognosis than mutations associated with partial activity. When present in the homozygous form, it could be a lethal allele similar to what has been postulated for the other known insertion mutation, 84GG. Our patient, who is a compound heterozygote 122CC/1226G, has moderately severe type 1 Gaucher disease. Her clinical response to Ceredase{reg_sign} therapy that began 31 months ago has been favorable, though incomplete. 30 refs., 3 figs., 2 tabs.

  5. Secuenciación del gen CFTR en un grupo de pacientes chilenos con fibrosis quística

    OpenAIRE

    Lay-Son, Guillermo; Vásquez, Marcos; Puga, Alonso; Manque, Patricio; Repetto, Gabriela

    2014-01-01

    La fibrosis quística (FQ) es un trastorno autosómico recesivo causado por mutaciones en el gen CFTR, en el cual se han identificado más de 1.900 mutaciones diferentes. En Chile, el panel diagnóstico con las 36 mutaciones más comunes permite una tasa de detección cercana al 50% de los alelos, mientras que en caucásicos la tasa es casi de 90%. El objetivo fue ampliar la capacidad de detección mutacional en los pacientes chilenos y buscar mutaciones que pudieran ser recurrentes a nivel local....

  6. WDR62 is associated with the spindle pole and is mutated in human microcephaly

    OpenAIRE

    Nicholas, Adeline K; Khurshid, Maryam; Désir, Julie; Carvalho, Ofélia P; Cox, James J; Thornton, Gemma; Kausar, Rizwana; Ansar, Muhammad; Ahmad, Wasim; Verloes, Alain; Passemard, Sandrine; Misson, Jean-Paul; Lindsay, Susan; Gergely, Fanni; Dobyns, William B

    2010-01-01

    Autosomal recessive primary microcephaly (MCPH) is a disorder of neurodevelopment resulting in a small brain1,2. We identified WDR62 as the second most common cause of MCPH after finding homozygous missense and frame-shifting mutations in seven MCPH families. In human cell lines, we found that WDR62 is a spindle pole protein, as are ASPM and STIL, the MCPH7 and MCHP7 proteins3–5. Mutant WDR62 proteins failed to localize to the mitotic spindle pole. In human and mouse embryonic brain, we found...

  7. Novel FKBP10 Mutation in a Patient with Osteogenesis Imperfecta Type XI.

    Science.gov (United States)

    Seyedhassani, Seyed Mohammad; Hashemi-Gorji, Feyzollah; Yavari, Mahdieh; Harazi, Fahimeh; Yassaee, Vahid Reza

    2016-01-01

    Osteogenesis imperfecta (OI) is a set of clinically and genetically heterogeneous disorders with autosomal dominant, recessive and X-linked inheritance patterns. The aim of this study was to describe a novel genetic abnormality in a case of OI type XI with mild joint contractures, kyphoscoliosis, muscular atrophy, progressively deforming and multiple bone fractures in a consanguineous Iranian family. Based on the phenotype, investigation of two candidate genes, CRTAP (OI type VII) and FKBP10 (OI type XI) detected a novel homozygous frameshift mutation in the FKBP10 gene. This finding can be useful in accurate genetic counseling and prioritization of molecular analysis of OI in Iranian patients.

  8. Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast.

    Science.gov (United States)

    Belew, Ashton T; Advani, Vivek M; Dinman, Jonathan D

    2011-04-01

    Although first discovered in viruses, previous studies have identified operational -1 ribosomal frameshifting (-1 RF) signals in eukaryotic genomic sequences, and suggested a role in mRNA stability. Here, four yeast -1 RF signals are shown to promote significant mRNA destabilization through the nonsense mediated mRNA decay pathway (NMD), and genetic evidence is presented suggesting that they may also operate through the no-go decay pathway (NGD) as well. Yeast EST2 mRNA is highly unstable and contains up to five -1 RF signals. Ablation of the -1 RF signals or of NMD stabilizes this mRNA, and changes in -1 RF efficiency have opposing effects on the steady-state abundance of the EST2 mRNA. These results demonstrate that endogenous -1 RF signals function as mRNA destabilizing elements through at least two molecular pathways in yeast. Consistent with current evolutionary theory, phylogenetic analyses suggest that -1 RF signals are rapidly evolving cis-acting regulatory elements. Identification of high confidence -1 RF signals in ∼10% of genes in all eukaryotic genomes surveyed suggests that -1 RF is a broadly used post-transcriptional regulator of gene expression.

  9. Highly conserved RNA pseudoknots at the gag-pol junction of HIV-1 suggest a novel mechanism of −1 ribosomal frameshifting

    Science.gov (United States)

    Huang, Xiaolan; Yang, Yang; Wang, Guan; Cheng, Qiang; Du, Zhihua

    2014-01-01

    −1 programmed ribosomal frameshifting (PRF) is utilized by many viruses to synthesize their enzymatic (Pol) and structural (Gag) proteins at a defined ratio. For efficient −1 PRF, two cis-acting elements are required: a heptanucleotide frameshift site and a downstream stimulator such as a pseudoknot. We have analyzed the gag-pol junction sequences from 4254 HIV-1 strains. Approximately ninety-five percent of the sequences can form four pseudoknots PK1–PK4 (∼97% contain PK1, PK3, and PK4), covering ∼72 nt including the frameshift site. Some pseudoknots are mutually excluded due to sequence overlap. PK1 and PK3 arrange tandemly. Their stems form a quasi-continuous helix of ∼22 bp. We propose a novel mechanism for possible roles of these pseudoknots. Multiple alternative structures may exist at the gag-pol junction. In most strains, the PK1–PK3 tandem pseudoknots may dominate the structurally heterogeneous pool of RNA due to their greater overall stability. The tandem pseudoknots may function as a breaking system to slow down the ribosome. The ribosome unwinds PK1 and stem 1 of PK3 before it can reach the frameshift site. Then, PK4 can form rapidly because the intact stem 2 of PK3 makes up a large part of the stem 1 of PK4. The newly formed PK4 jams the entrance of the mRNA tunnel. The process then proceeds as in a typical case of −1 PRF. This mechanism incorporates several exquisite new features while still being consistent with the current paradigm of pseudoknot-dependent −1 PRF. PMID:24671765

  10. Novel SOX9 Gene Mutation in Campomelic Dysplasia with Autosomal Sex Reversal

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    Hui-Pin Hsiao

    2006-01-01

    Full Text Available Campomelic dysplasia (CD; OMIM #114290 is an autosomal dominant, frequently lethal dysplasia syndrome whose primary features include angular bowing and shortening of the limbs, and sex reversal in the majority of affected XY individuals. Most CD cases have heterozygous de novo mutations in the coding region of the transcription factor gene SOX9 (SRY-related high-mobility group [HMG] box 9 in chromosome 17q. Here, we report a novel mutation of SOX9 in a female neonate with CD with autosomal sex reversal. Respiratory distress and cyanosis were noted at birth, and endotracheal intubation with mechanical ventilation was performed due to respiratory failure. The presenting phenotypes included dysmorphic face with macrocephaly prominent forehead, low nasal bridge, cleft palate and micrognathia. Skeletal deformities characteristic of CD were observed, including narrow thoracic cage, hypoplastic scapulae, scoliosis and short limbs with anterolateral femoral and tibial bowing. The karyotype was 46,XY despite female external genitalia. SOX9 gene analysis revealed frameshift mutation (at nucleotide position 1095G →AT in the open reading frame, resulting in a frameshift with 211 new amino acids.

  11. Novel mutation in forkhead box G1 (FOXG1) gene in an Indian patient with Rett syndrome.

    Science.gov (United States)

    Das, Dhanjit Kumar; Jadhav, Vaishali; Ghattargi, Vikas C; Udani, Vrajesh

    2014-03-15

    Rett syndrome (RTT) is a severe neurodevelopmental disorder characterized by the progressive loss of intellectual functioning, fine and gross motor skills and communicative abilities, deceleration of head growth, and the development of stereotypic hand movements, occurring after a period of normal development. The classic form of RTT involves mutation in MECP2 while the involvement of CDKL5 and FOXG1 genes has been identified in atypical RTT phenotype. FOXG1 gene encodes for a fork-head box protein G1, a transcription factor acting primarily as transcriptional repressor through DNA binding in the embryonic telencephalon as well as a number of other neurodevelopmental processes. In this report we have described the molecular analysis of FOXG1 gene in Indian patients with Rett syndrome. FOXG1 gene mutation analysis was done in a cohort of 34 MECP2/CDKL5 mutation negative RTT patients. We have identified a novel mutation (p. D263VfsX190) in FOXG1 gene in a patient with congenital variant of Rett syndrome. This mutation resulted into a frameshift, thereby causing an alteration in the reading frames of the entire coding sequence downstream of the mutation. The start position of the frameshift (Asp263) and amino acid towards the carboxyl terminal end of the protein was found to be well conserved across species using multiple sequence alignment. Since the mutation is located at forkhead binding domain, the resultant mutation disrupts the secondary structure of the protein making it non-functional. This is the first report from India showing mutation in FOXG1 gene in Rett syndrome. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Isolated hypogonadotropic hypogonadism with SOX2 mutation and anophthalmia/microphthalmia in offspring

    Science.gov (United States)

    Stark, Zornitza; Storen, Rebecca; Bennetts, Bruce; Savarirayan, Ravi; Jamieson, Robyn V

    2011-01-01

    Isolated hypogonadotropic hypogonadism (IHH) is a genetically heterogeneous condition in which patients frequently require assisted reproduction to achieve fertility. In patients with IHH who are otherwise well, no particular increased risk of congenital anomalies in the resultant offspring has been highlighted. Heterozygous mutations in SOX2 are the commonest single-gene cause of anophthalmia/microphthalmia (A/M) and sometimes result in pituitary abnormalities. We report a family with a novel frameshift mutation in the SOX2 transactivation domain, p.Gly280AlafsX91, resulting in bilateral anophthalmia and subtle endocrinological abnormalities in a male sibling, and unilateral microphthalmia in a female sibling. The mutation is present in their mother who has IHH, but has no eye disorders or other anomalies. She underwent assisted reproduction to achieve fertility. This report has important implications for the evaluation of patients with IHH, particularly in the setting of planned infertility treatment. PMID:21326281

  13. TBC1D24 Mutations in a Sibship with Multifocal Polymyoclonus

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    Adeline Ngoh

    2017-04-01

    Full Text Available Background: Advances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus.Case Report: A family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in TBC1D24, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G>A (p.Glu157Lys, and a novel frameshift mutation c.545del (p.Thr182Serfs*6.Discussion: We propose that TBC1D24-related diseases should be in the differential diagnosis for children with polymyoclonus. 

  14. Kinetics of gene and chromosome mutations induced by UV-C in yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Koltovaya, N.; Kokoreva, A.; Senchenko, D.; Shvaneva, N.; Zhuchkina, N.

    2017-01-01

    The systematic study of the kinetics of UV-induced gene and structural mutations in eukaryotic cells was carried out on the basis of model yeast S. cerevisiae. A variety of genetic assays (all types of base pair substitutions, frameshifts, forward mutations canl, chromosomal and plasmid rearrangements) in haploid strains were used. Yeast cells were treated by UV-C light of fluence of energy up to 200 J/m 2 . The kinetics of the induced gene and structural mutations is represented by a linear-quadratic and exponential functions. The slope of curves in log-log plots was not constant, had the value 2-4 and depended on the interval of doses. It was suggested that it is the superposition and dynamics of different pathways form the mutagenic responses of eukaryotic cells to UV-C light that cause the high-order curves. [ru

  15. Normosmic congenital hypogonadotropic hypogonadism due to TAC3/TACR3 mutations: characterization of neuroendocrine phenotypes and novel mutations.

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    Bruno Francou

    Full Text Available CONTEXT: TAC3/TACR3 mutations have been reported in normosmic congenital hypogonadotropic hypogonadism (nCHH (OMIM #146110. In the absence of animal models, studies of human neuroendocrine phenotypes associated with neurokinin B and NK3R receptor dysfunction can help to decipher the pathophysiology of this signaling pathway. OBJECTIVE: To evaluate the prevalence of TAC3/TACR3 mutations, characterize novel TACR3 mutations and to analyze neuroendocrine profiles in nCHH caused by deleterious TAC3/TACR3 biallelic mutations. RESULTS: From a cohort of 352 CHH, we selected 173 nCHH patients and identified nine patients carrying TAC3 or TACR3 variants (5.2%. We describe here 7 of these TACR3 variants (1 frameshift and 2 nonsense deleterious mutations and 4 missense variants found in 5 subjects. Modeling and functional studies of the latter demonstrated the deleterious consequence of one missense mutation (Tyr267Asn probably caused by the misfolding of the mutated NK3R protein. We found a statistically significant (p<0.0001 higher mean FSH/LH ratio in 11 nCHH patients with TAC3/TACR3 biallelic mutations than in 47 nCHH patients with either biallelic mutations in KISS1R, GNRHR, or with no identified mutations and than in 50 Kallmann patients with mutations in KAL1, FGFR1 or PROK2/PROKR2. Three patients with TAC3/TACR3 biallelic mutations had an apulsatile LH profile but low-frequency alpha-subunit pulses. Pulsatile GnRH administration increased alpha-subunit pulsatile frequency and reduced the FSH/LH ratio. CONCLUSION: The gonadotropin axis dysfunction associated with nCHH due to TAC3/TACR3 mutations is related to a low GnRH pulsatile frequency leading to a low frequency of alpha-subunit pulses and to an elevated FSH/LH ratio. This ratio might be useful for pre-screening nCHH patients for TAC3/TACR3 mutations.

  16. Novel BRCA1 and BRCA2 pathogenic mutations in Slovene hereditary breast and ovarian cancer families.

    Science.gov (United States)

    Novaković, Srdjan; Milatović, Maša; Cerkovnik, Petra; Stegel, Vida; Krajc, Mateja; Hočevar, Marko; Zgajnar, Janez; Vakselj, Aleš

    2012-11-01

    The estimated proportion of hereditary breast and ovarian cancers among all breast and ovarian cancer cases is 5-10%. According to the literature, inherited mutations in the BRCA1 and BRCA2 tumour-suppressor genes, account for the majority of hereditary breast and ovarian cancer cases. The aim of this report is to present novel mutations that have not yet been described in the literature and pathogenic BRCA1 and BRCA2 mutations which have been detected in HBOC families for the first time in the last three years. In the period between January 2009 and December 2011, 559 individuals from 379 families affected with breast and/or ovarian cancer were screened for mutations in the BRCA1 and BRCA2 genes. Three novel mutations were detected: one in BRCA1 - c.1193C>A (p.Ser398*) and two in BRCA2 - c.5101C>T (p.Gln1701*) and c.5433_5436delGGAA (p.Glu1811Aspfs*3). These novel mutations are located in the exons 11 of BRCA1 or BRCA2 and encode truncated proteins. Two of them are nonsense while one is a frameshift mutation. Also, 11 previously known pathogenic mutations were detected for the first time in the HBOC families studied here (three in BRCA1 and eight in BRCA2). All, except one cause premature formation of stop codons leading to truncation of the respective BRCA1 or BRCA2 proteins.

  17. Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

    Science.gov (United States)

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  18. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    Directory of Open Access Journals (Sweden)

    Jonàs Juan-Mateu

    Full Text Available DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements.

  19. Contribution of CFTR to Alveolar Fluid Clearance by Lipoxin A4 via PI3K/Akt Pathway in LPS-Induced Acute Lung Injury

    Directory of Open Access Journals (Sweden)

    Yi Yang

    2013-01-01

    Full Text Available The lipoxins are the first proresolution mediators to be recognized and described as the endogenous “braking signals” for inflammation. We evaluated the anti-inflammatory and proresolution bioactions of lipoxin A4 in our lipopolysaccharide (LPS-induced lung injury model. We demonstrated that lipoxin A4 significantly improved histology of rat lungs and inhibited IL-6 and TNF-α in LPS-induced lung injury. In addition, lipoxin A4 increased alveolar fluid clearance (AFC and the effect of lipoxin A4 on AFC was abolished by CFTRinh-172 (a specific inhibitor of CFTR. Moreover, lipoxin A4 could increase cystic fibrosis transmembrane conductance regulator (CFTR protein expression in vitro and in vivo. In rat primary alveolar type II (ATII cells, LPS decreased CFTR protein expression via activation of PI3K/Akt, and lipoxin A4 suppressed LPS-stimulated phosphorylation of Akt. These results showed that lipoxin A4 enhanced CFTR protein expression and increased AFC via PI3K/Akt pathway. Thus, lipoxin A4 may provide a potential therapeutic approach for acute lung injury.

  20. A forward mutation assay using ampicillin-resistance in Escherichia coli designed for investigating the mutagenicity of biological samples.

    Science.gov (United States)

    Bosworth, D; Crofton-Sleigh, C; Venitt, S

    1987-11-01

    The development of a bacterial mutation assay using forward mutation to ampicillin-resistance is described. It is a technically simple assay using Escherichia coli D494uvrB transformed with a multi-copy mutator plasmid pGW1700. Mutation is detected by an increase in the frequency of ampicillin-resistant colonies following treatment of bacteria with the test material during logarithmic growth. The determination of viable counts allows a correction factor to be applied to compensate for the effects of sample-induced growth enhancement or toxicity on the bacterial population. The assay has been tested with a range of reference mutagens. It is particularly sensitive to base-pair substitution mutagens, detecting these at doses equal to or less than those detected in the Salmonella/microsome assay or the SOS Chromotest. The assay also detects frameshift mutagens but with lower sensitivity than the Salmonella/microsome assay.

  1. A novel splicing mutation in COL1A1 gene caused type I osteogenesis imperfecta in a Chinese family.

    Science.gov (United States)

    Peng, Hao; Zhang, Yuhui; Long, Zhigao; Zhao, Ding; Guo, Zhenxin; Xue, Jinjie; Xie, Zhiguo; Xiong, Zhimin; Xu, Xiaojuan; Su, Wei; Wang, Bing; Xia, Kun; Hu, Zhengmao

    2012-07-10

    Osteogenesis imperfect (OI) is a heritable connective tissue disorder with bone fragility as a cardinal manifestation, accompanied by short stature, dentinogenesis imperfecta, hyperlaxity of ligaments and skin, blue sclerae and hearing loss. Dominant form of OI is caused by mutations in the type I procollagen genes, COL1A1/A2. Here we identified a novel splicing mutation c.3207+1G>A (GenBank ID: JQ236861) in the COL1A1 gene that caused type I OI in a Chinese family. RNA splicing analysis proved that this mutation created a new splicing site at c.3200, and then led to frameshift. This result further enriched the mutation spectrum of type I procollagen genes. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Naturally occurred frame-shift mutations in the tvb receptor gene are responsible for decreased susceptibility to subgroups B, D, and E avian leukosis virus infection in chicken

    Science.gov (United States)

    The group of highly related avian leukosis viruses (ALVs) in chickens were thought to have evolved from a common retroviral ancestor into six subgroups, A to E and J. These ALV subgroups use diverse cellular proteins encoded by four genetic loci in chickens as receptors to gain entry into host cells...

  3. In vivo mutations in human blood cells: Biomarkers for molecular epidemiology

    Energy Technology Data Exchange (ETDEWEB)

    Albertini, R.J.; Branda, R.F.; O' Neill, J.P. (Univ. of Vermont, Burlington (United States)); Nicklas, J.A.; Fuscoe, J.C. (Environmental Health Research and Testing, Inc., Research Triangle Park, NC (United States)); Skopek, T.R. (Univ. of North Carolina, Chapel Hill (United States))

    1993-03-01

    Mutations arising in vivo in recorder genes of human blood cells provide biomarkers for molecular epidemiology by serving as surrogates for cancer-causing genetic changes. Current markers include mutations of the glycophorin-A (GPA) or hemoglobin (Hb) genes, measured in red blood cells, or mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) or HLA genes, measured in T-lymphocytes. Mean mutant frequencies (variant frequencies) for normal young adults are approximately: Hb (4 [times] 10[sup [minus]8]) < hprt (5 [times] 10[sup [minus]6]) = GPA (10 [times] 10[sup [minus]6]) < HLA (30 [times] 10[sup [minus]6]). Mutagen-exposed individuals show decided elevations. Molecular mutational spectra are also being defined. For the hprt marker system, about 15% of background mutations are gross structural alterations of the hprt gene (e.g., deletions); the remainder are point mutations (e.g., base substitutions or frameshifts). Ionizing radiations result in dose-related increases in total gene deletions. Large deletions may encompass several megabases as shown by co-deletions of linked markers. Possible hprt spectra for defining radiation and chemical exposures are being sought. In addition to their responsiveness to environmental mutagens/carcinogens, three additional findings suggest that the in vivo recorder mutations are relevant in vivo surrogates for cancer mutations. First, a large fraction of GPA and HLA mutations show exchanges due to homologous recombination, an important mutational event in cancer. Second, hprt mutations arise preferentially in dividing T-cells, which can accumulate additional mutations in the same clone, reminiscent of the multiple hits required in the evolution of malignancy. Finally, fetal hprt mutations frequently have characteristic deletions of hprt exons 2 and 3, which appear to be mediated by the VDJ recombinase that rearranges the T-cell receptor genes during thymic ontogeny. 60 refs., 3 tabs.

  4. Prognostic impact of mismatch repair genes germline defects in colorectal cancer patients: are all mutations equal?

    Science.gov (United States)

    Maccaroni, Elena; Bracci, Raffaella; Giampieri, Riccardo; Bianchi, Francesca; Belvederesi, Laura; Brugiati, Cristiana; Pagliaretta, Silvia; Del Prete, Michela; Scartozzi, Mario; Cascinu, Stefano

    2015-01-01

    Background Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, caused by germline mutations in MisMatch Repair (MMR) genes, particularly in MLH1, MSH2 and MSH6. Patients with LS seem to have a more favourable prognosis than those with sporadic CRC, although the prognostic impact of different mutation types is unknown. Aim of our study is to compare survival outcomes of different types of MMR mutations in patients with LS-related CRC. Methods 302 CRC patients were prospectively selected on the basis of Amsterdam or Revised Bethesda criteria to undergo genetic testing: direct sequencing of DNA and MLPA were used to examine the entire MLH1, MSH2 and MSH6 coding sequence. Patients were classified as mutation-positive or negative according to the genetic testing result. Results A deleterious MMR mutation was found in 38/302 patients. Median overall survival (OS) was significantly higher in mutation-positive vs mutation-negative patients (102.6 vs 77.7 months, HR:0.63, 95%CI:0.46–0.89, p = 0.0083). Different types of mutation were significantly related with OS: missense or splicing-site mutations were associated with better OS compared with rearrangement, frameshift or non-sense mutations (132.5 vs 82.5 months, HR:0.46, 95%CI:0.16–0.82, p = 0.0153). Conclusions Our study confirms improved OS for LS-patients compared with mutation-negative CRC patients. In addition, not all mutations could be considered equal: the better prognosis in CRC patients with MMR pathogenic missense or splicing site mutation could be due to different functional activity of the encoded MMR protein. This matter should be investigated by use of functional assays in the future. PMID:26485756

  5. Molecular characterization of 21 X-ALD Portuguese families: identification of eight novel mutations in the ABCD1 gene.

    Science.gov (United States)

    Guimarães, Carla P; Lemos, Manuela; Sá-Miranda, Clara; Azevedo, Jorge E

    2002-05-01

    X-linked adrenoleukodystrophy (X-ALD) is the most common inherited peroxisomal disorder. The gene associated with X-ALD, ABCD1, encodes a peroxisomal ATP-binding cassette half-transporter. In this study, we describe the molecular characterization of 21 affected Portuguese families. The complete coding region of the ABCD1 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) or genomic PCR. After conformation-sensitive gel electrophoresis analysis, fragments with a conformational heteroduplex pattern were sequenced. Using this strategy, we have identified 14 missense mutations, two nonsense mutations, two splicing site defects, and three small deletions, two of them resulting in frameshifts. Eight of the genetic alterations characterized in this study are novel. The levels of the ABCD1 transcript as well as the levels of ALDP in cultured skin fibroblasts of male probands were also determined in most cases. The levels of the ABCD1 transcript in one patient (corresponding to a nonsense mutation) were below the detection limit of Northern-blotting analysis. ALDP was found at normal levels in only three patients, absent in five (corresponding to a double missense, two nonsense, and two frameshift mutations), and decreased in all the others.

  6. Impact of two myostatin (MSTN mutations on weight gain and lamb carcass classification in Norwegian White Sheep (Ovis aries

    Directory of Open Access Journals (Sweden)

    Blichfeldt Thor

    2010-01-01

    Full Text Available Abstract Background Our aim was to estimate the effect of two myostatin (MSTN mutations in Norwegian White Sheep, one of which is close to fixation in the Texel breed. Methods The impact of two known MSTN mutations was examined in a field experiment with Norwegian White Sheep. The joint effect of the two MSTN mutations on live weight gain and weaning weight was studied on 644 lambs. Carcass weight gain from birth to slaughter, carcass weight, carcass conformation and carcass fat classes were calculated in a subset of 508 lambs. All analyses were carried out with a univariate linear animal model. Results The most significant impact of both mutations was on conformation and fat classes. The largest difference between the genotype groups was between the wild type for both mutations and the homozygotes for the c.960delG mutation. Compared to the wild types, these mutants obtained a conformation score 5.1 classes higher and a fat score 3.0 classes lower, both on a 15-point scale. Conclusions Both mutations reduced fatness and increased muscle mass, although the effect of the frameshift mutation (c.960delG was more important as compared to the 3'-UTR mutation (c.2360G>A. Lambs homozygous for the c.960delG mutation grew more slowly than those with other MSTN genotypes, but had the least fat and the largest muscle mass. Only c.960delG showed dominance effects.

  7. Common mutations in the phosphofructokinase-M gene in Ashkenazi Jewish patients with glycogenesis VII - and their population frequency

    Energy Technology Data Exchange (ETDEWEB)

    Sherman, J.B.; Raben, N.; Nicastri, C.; Adams, E.M.; Plotz, P.H. (National Institutes of Health, Bethesda, MD (United States)); Argov, Z. (Hebrew Univ., Jerusalem (Israel)); Nakajima, Hiromu (Osaka Univ. (Japan)); Eng, C.M.; Cowan, T.M. (Univ. of Maryland School of Medicine, Baltimore, MD (United States))

    1994-08-01

    Phosphofructokinase (PFK) catalyzes the rate-limiting step of glycolysis. Deficiency of the muscle enzyme is manifested by exercise intolerance and a compensated hemolytic anemia. Case reports of this autosomal recessive disease suggest a predominance in Ashkenazi Jews in the United States. The authors have explored the genetic basis for this illness in nine affected families and surveyed the normal Ashkenazi population for the mutations found. Genomic DNA was amplified using PCR, and denaturing gradient-gel electrophoresis. The polymorphic exons were sequenced or digested with restriction enzymes. A previously described splicing mutation, [Delta]5, accounted for 11 (61%) of 18 abnormal alleles in the nine families. A single base deletion leading to a frameshift mutation in exon 22 ([Delta]C-22) was found in six of seven alleles. A third mutation, resulting in a nonconservative amino acid substitution in exon 4, accounted for the remaining allele. Thus, three mutations could account for an illness in this group, and two mutations could account for 17 of 18 alleles. In screening 250 normal Ashkenazi individuals for all three mutations, they found only one [Delta]5 allele. Clinical data revealed no correlation between the particular mutations and symptoms, but male patients were more symptomatic than females, and only males had frank hemolysis and hyperuricemia. Because PFK deficiency in Ashkenazi Jews is caused by a limited number of mutations, screening genomic DNA from peripheral blood for the described mutations in this population should enable rapid diagnosis without muscle biopsy. 41 refs., 4 figs., 2 tabs.

  8. Nucleotide sequence of Zygosaccharomyces bailii virus Z: Evidence for +1 programmed ribosomal frameshifting and for assignment to family Amalgaviridae.

    Science.gov (United States)

    Depierreux, Delphine; Vong, Minh; Nibert, Max L

    2016-06-02

    Zygosaccharomyces bailii virus Z (ZbV-Z) is a monosegmented dsRNA virus that infects the yeast Zygosaccharomyces bailii and remains unclassified to date despite its discovery >20years ago. The previously reported nucleotide sequence of ZbV-Z (GenBank AF224490) encompasses two nonoverlapping long ORFs: upstream ORF1 encoding the putative coat protein and downstream ORF2 encoding the RNA-dependent RNA polymerase (RdRp). The lack of overlap between these ORFs raises the question of how the downstream ORF is translated. After examining the previous sequence of ZbV-Z, we predicted that it contains at least one sequencing error to explain the nonoverlapping ORFs, and hence we redetermined the nucleotide sequence of ZbV-Z, derived from the same isolate of Z. bailii as previously studied, to address this prediction. The key finding from our new sequence, which includes several insertions, deletions, and substitutions relative to the previous one, is that ORF2 in fact overlaps ORF1 in the +1 frame. Moreover, a proposed sequence motif for +1 programmed ribosomal frameshifting, previously noted in influenza A viruses, plant amalgaviruses, and others, is also present in the newly identified ORF1-ORF2 overlap region of ZbV-Z. Phylogenetic analyses provided evidence that ZbV-Z represents a distinct taxon most closely related to plant amalgaviruses (genus Amalgavirus, family Amalgaviridae). We conclude that ZbV-Z is the prototype of a new species, which we propose to assign as type species of a new genus of monosegmented dsRNA mycoviruses in family Amalgaviridae. Comparisons involving other unclassified mycoviruses with RdRps apparently related to those of plant amalgaviruses, and having either mono- or bisegmented dsRNA genomes, are also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Founding BRCA1 mutations in hereditary breast and ovarian cancer in southern Sweden

    Energy Technology Data Exchange (ETDEWEB)

    Johannsson, O.; Hakansson, S.; Johannson, U. [Univ. Hospital, Lund (Sweden)] [and others

    1996-03-01

    Nine different germ-line mutations in the BRCA1 breast and ovarian cancer susceptibility gene were identified in 15 of 47 kindreds from southern Sweden, by use of SSCP and heteroduplex analysis of all exons and flanking intron region and by a protein-truncation test for exon 11, followed by direct sequencing. All but one of the mutations are predicted to give rise to premature translation termination and include seven frameshift insertions or deletions, a nonsense mutation, and a splice acceptor site mutation. The remaining mutation is a missense mutation (Cys61Gly) in the zinc-binding motif. Four novel Swedish founding mutations were identified: the nucleotide 2595 deletion A was found in five families, the C 1806 T nonsense mutation in three families, the 3166 insertion TGAGA in three families, and the nucleotide 1201 deletion 11 in two families. Analysis of the intragenic polymorphism D17S855 supports common origins of the mutations. Eleven of the 15 kindreds manifesting BRCA1 mutations were breast-ovarian cancer families, several of them with a predominant ovarian cancer phenotype. The set of 32 families in which no BRCA1 alterations were detected included 1 breast-ovarian cancer kindred manifesting clear linkage to the BRCA1 region and loss of the wild-type chromosome in associated tumors. Other tumor types found in BRCA1 mutation/haplotype carriers included prostatic, pancreas, skin, and lung cancer, a malignant melanoma, an oligodendroglioma, and a carcinosarcoma. In all, 12 of 16 kindreds manifesting BRCA1 mutation or linkage contained ovarian cancer, as compared with only 6 of the remaining 31 families (P < .001). The present study confirms the involvement of BRCA1 in disease predisposition for a subset of hereditary breast cancer families often characterized by ovarian cancers. 28 refs., 3 figs., 4 tabs.

  10. GJB2 mutation spectrum in 2063 Chinese patients with nonsyndromic hearing impairment

    Directory of Open Access Journals (Sweden)

    Tang Liang

    2009-04-01

    Full Text Available Abstract Background Mutations in GJB2 are the most common molecular defects responsible for autosomal recessive nonsyndromic hearing impairment (NSHI. The mutation spectra of this gene vary among different ethnic groups. Methods In order to understand the spectrum and frequency of GJB2 mutations in the Chinese population, the coding region of the GJB2 gene from 2063 unrelated patients with NSHI was PCR amplified and sequenced. Results A total of 23 pathogenic mutations were identified. Among them, five (p.W3X, c.99delT, c.155_c.158delTCTG, c.512_c.513insAACG, and p.Y152X are novel. Three hundred and seven patients carry two confirmed pathogenic mutations, including 178 homozygotes and 129 compound heterozygotes. One hundred twenty five patients carry only one mutant allele. Thus, GJB2 mutations account for 17.9% of the mutant alleles in 2063 NSHI patients. Overall, 92.6% (684/739 of the pathogenic mutations are frame-shift truncation or nonsense mutations. The four prevalent mutations; c.235delC, c.299_c.300delAT, c.176_c.191del16, and c.35delG, account for 88.0% of all mutantalleles identified. The frequency of GJB2 mutations (alleles varies from 4% to 30.4% among different regions of China. It also varies among different sub-ethnic groups. Conclusion In some regions of China, testing of the three most common mutations can identify at least one GJB2 mutant allele in all patients. In other regions such as Tibet, the three most common mutations account for only 16% the GJB2 mutant alleles. Thus, in this region, sequencing of GJB2 would be recommended. In addition, the etiology of more than 80% of the mutant alleles for NSHI in China remains to be identified. Analysis of other NSHI related genes will be necessary.

  11. Mutations in the hedgehog pathway genes SMO and PTCH1 in human gastric tumors.

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    Xi-De Wang

    Full Text Available The causal role of the hedgehog pathway in cancer has been best documented in basal cell carcinoma of the skin. To assess potential DNA alterations of the hedgehog pathway in gastric cancer, we sequenced SMO and PTCH1 genes in a set of 39 gastric tumors. Tumors were classified by histology based on the Lauren classification and Sanger sequencing was performed to obtain full length coding sequences. Genomic instability was evident in these tumors as a number of silent or missense mutations were found. In addition to those that are potential germline polymorphisms, we found three SMO missense mutations, and one PTCH1 frameshift mutation that are novel and have not been documented in basal cell carcinoma. Mutations were found in both intestinal and diffuse type gastric tumors as well as in tumors that exhibit both intestinal and diffuse features. mRNA expression of hedgehog pathway genes was also examined and their levels do not indicate unequivocal higher pathway activity in tumors with mutations than those without. In summary, SMO and/or PTCH1 mutations are present at low frequency in different histologic subtypes of gastric tumors and these do not appear to be driver mutations.

  12. Identities and frequencies of mutations of the otoferlin gene (OTOF) causing DFNB9 deafness in Pakistan

    Science.gov (United States)

    Choi, BY; Ahmed, ZM; Riazuddin, S; Bhinder, MA; Shahzad, M; Husnain, T; Riazuddin, S; Griffith, AJ; Friedman, TB

    2012-01-01

    Mutations in OTOF, encoding otoferlin, cause non-syndromic recessive hearing loss. The goal of our study was to define the identities and frequencies of OTOF mutations in a model population. We screened a cohort of 557 large consanguineous Pakistani families segregating recessive, severe-to-profound, prelingual-onset deafness for linkage to DFNB9. There were 13 families segregating deafness consistent with linkage to markers for DFNB9. We analyzed the genomic nucleotide sequence of OTOF and detected probable pathogenic sequence variants among all 13 families. These include the previously reported nonsense mutation p.R708X and 10 novel variants: 3 nonsense mutations (p.R425X, p.W536X, and p.Y1603X), 1 frameshift (c.1103_1104delinsC), 1 single amino acid deletion (p.E766del) and 5 missense substitutions of conserved residues (p.L573R, p.A1090E, p.E1733K, p.R1856Q and p.R1939W). OTOF mutations thus account for deafness in 13 (2.3%) of 557 Pakistani families. This overall prevalence is similar, but the mutation spectrum is different from those for Western populations. In addition, we demonstrate the existence of an alternative splice isoform of OTOF expressed in the human cochlea. This isoform must be required for human hearing because it encodes a unique alternative C-terminus affected by some DFNB9 mutations. PMID:19250381

  13. A review on non-syndromic tooth agenesis associated with PAX9 mutations

    Directory of Open Access Journals (Sweden)

    Nurul Hasyiqin Fauzi

    2018-02-01

    Full Text Available Summary: Tooth agenesis in the reduction of tooth number which includes hypodontia, oligodontia and anodontia is caused by disturbances and gene mutations that occur during odontogenesis. To date, several genetic mutations that unlock the causes of non-syndromic tooth agenesis are being discovered; these have been associated with certain illnesses because tooth development involves the interaction of several genes for tooth epithelium and mesenchyme odontogenesis. Mutation of candidate genes PAX9 and MSX1 have been identified as the main causes of hypodontia and oligodontia; meanwhile, AXIN2 mutation is associated with anodontia. Previous study using animal models reported that PAX9-deficient knockout mice exhibit missing molars due to an arrest of tooth development at the bud stage. PAX9 frameshift, missense and nonsense mutations are reported to be responsible; however, the most severe condition showed by the phenotype is caused by haploinsufficiency. This suggests that PAX9 is dosage-sensitive. Understanding the mechanism of genetic mutations will benefit clinicians and human geneticists in future alternative treatment investigations. Keywords: Hypodontia, Oligodontia, Mutation, PAX9

  14. Mutations in epilepsy and intellectual disability genes in patients with features of Rett syndrome.

    Science.gov (United States)

    Olson, Heather E; Tambunan, Dimira; LaCoursiere, Christopher; Goldenberg, Marti; Pinsky, Rebecca; Martin, Emilie; Ho, Eugenia; Khwaja, Omar; Kaufmann, Walter E; Poduri, Annapurna

    2015-09-01

    Rett syndrome and neurodevelopmental disorders with features overlapping this syndrome frequently remain unexplained in patients without clinically identified MECP2 mutations. We recruited a cohort of 11 patients with features of Rett syndrome and negative initial clinical testing for mutations in MECP2. We analyzed their phenotypes to determine whether patients met formal criteria for Rett syndrome, reviewed repeat clinical genetic testing, and performed exome sequencing of the probands. Using 2010 diagnostic criteria, three patients had classical Rett syndrome, including two for whom repeat MECP2 gene testing had identified mutations. In a patient with neonatal onset epilepsy with atypical Rett syndrome, we identified a frameshift deletion in STXBP1. Among seven patients with features of Rett syndrome not fulfilling formal diagnostic criteria, four had suspected pathogenic mutations, one each in MECP2, FOXG1, SCN8A, and IQSEC2. MECP2 mutations are highly correlated with classical Rett syndrome. Genes associated with atypical Rett syndrome, epilepsy, or intellectual disability should be considered in patients with features overlapping with Rett syndrome and negative MECP2 testing. While most of the identified mutations were apparently de novo, the SCN8A variant was inherited from an unaffected parent mosaic for the mutation, which is important to note for counseling regarding recurrence risks. © 2015 Wiley Periodicals, Inc.

  15. EIF2S3 Mutations Associated with Severe X-Linked Intellectual Disability Syndrome MEHMO.

    Science.gov (United States)

    Skopkova, Martina; Hennig, Friederike; Shin, Byung-Sik; Turner, Clesson E; Stanikova, Daniela; Brennerova, Katarina; Stanik, Juraj; Fischer, Ute; Henden, Lyndal; Müller, Ulrich; Steinberger, Daniela; Leshinsky-Silver, Esther; Bottani, Armand; Kurdiova, Timea; Ukropec, Jozef; Nyitrayova, Olga; Kolnikova, Miriam; Klimes, Iwar; Borck, Guntram; Bahlo, Melanie; Haas, Stefan A; Kim, Joo-Ran; Lotspeich-Cole, Leda E; Gasperikova, Daniela; Dever, Thomas E; Kalscheuer, Vera M

    2017-04-01

    Impairment of translation initiation and its regulation within the integrated stress response (ISR) and related unfolded-protein response has been identified as a cause of several multisystemic syndromes. Here, we link MEHMO syndrome, whose genetic etiology was unknown, to this group of disorders. MEHMO is a rare X-linked syndrome characterized by profound intellectual disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. We have identified a C-terminal frameshift mutation (Ile465Serfs) in the EIF2S3 gene in three families with MEHMO syndrome and a novel maternally inherited missense EIF2S3 variant (c.324T>A; p.Ser108Arg) in another male patient with less severe clinical symptoms. The EIF2S3 gene encodes the γ subunit of eukaryotic translation initiation factor 2 (eIF2), crucial for initiation of protein synthesis and regulation of the ISR. Studies in patient fibroblasts confirm increased ISR activation due to the Ile465Serfs mutation and functional assays in yeast demonstrate that the Ile465Serfs mutation impairs eIF2γ function to a greater extent than tested missense mutations, consistent with the more severe clinical phenotype of the Ile465Serfs male mutation carriers. Thus, we propose that more severe EIF2S3 mutations cause the full MEHMO phenotype, while less deleterious mutations cause a milder form of the syndrome with only a subset of the symptoms. © 2017 WILEY PERIODICALS, INC.

  16. KIT Mutation and Loss of 14q May Be Sufficient for the Development of Clinically Symptomatic Very Low-Risk GIST.

    Directory of Open Access Journals (Sweden)

    Olaf Karl Klinke

    Full Text Available The aim of this study was to determine the minimal set of genetic alterations required for the development of a very low risk clinically symptomatic gastro-intestinal stromal tumour within the stomach wall. We studied the genome of a very low-risk gastric gastro-intestinal stromal tumour by whole-genome sequencing, comparative genomic hybridisation and methylation profiling. The studied tumour harboured two typical genomic lesions: loss of the long arm of chromosome 14 and an activating mutation in exon 11 of KIT. Besides these genetic lesions, only two point mutations that may affect tumour progression were identified: A frame-shift deletion in RNF146 and a missense mutation in a zinc finger of ZNF407. Whilst the frameshift deletion in RNF146 seemed to be restricted to this particular tumour, a similar yet germline mutation in ZNF407 was found in a panel of 52 gastro-intestinal stromal tumours from different anatomical sites and different categories. Germline polymorphisms in the mitotic checkpoint proteins Aurora kinase A and BUB1 kinase B may have furthered tumour growth. The epigenetic profile of the tumour matches that of other KIT-mutant tumours. We have identified mutations in three genes and loss of the long arm of chromosome 14 as the so far minimal set of genetic abnormalities sufficient for the development of a very low risk clinically symptomatic gastric stromal tumour.

  17. Pancreatic Agenesis due to Compound Heterozygosity for a Novel Enhancer and Truncating Mutation in the PTF1A Gene.

    Science.gov (United States)

    Gabbay, Monica; Ellard, Sian; De Franco, Elisa; Moisés, Regina S

    2017-09-01

    Neonatal diabetes, defined as the onset of diabetes within the first six months of life, is very rarely caused by pancreatic agenesis. Homozygous truncating mutations in the PTF1A gene, which encodes a transcriptional factor, have been reported in patients with pancreatic and cerebellar agenesis, whilst mutations located in a distal pancreatic-specific enhancer cause isolated pancreatic agenesis. We report an infant, born to healthy non-consanguineous parents, with neonatal diabetes due to pancreatic agenesis. Initial genetic investigation included sequencing of KCNJ11, ABCC8 and INS genes, but no mutations were found. Following this, 22 neonatal diabetes associated genes were analyzed by a next generation sequencing assay. We found compound heterozygous mutations in the PTF1A gene: A frameshift mutation in exon 1 (c.437_462 del, p.Ala146Glyfs*116) and a mutation affecting a highly conserved nucleotide within the distal pancreatic enhancer (g.23508442A>G). Both mutations were confirmed by Sanger sequencing. Isolated pancreatic agenesis resulting from compound heterozygosity for truncating and enhancer mutations in the PTF1A gene has not been previously reported. This report broadens the spectrum of mutations causing pancreatic agenesis.

  18. Renoguanylin stimulates apical CFTR translocation and decreases HCO3-secretion through PKA activity in the Gulf toadfish (Opsanus beta).

    Science.gov (United States)

    Ruhr, Ilan M; Schauer, Kevin L; Takei, Yoshio; Grosell, Martin

    2018-03-26

    The guanylin peptides - guanylin, uroguanylin and renoguanylin (RGN) - are endogenously produced hormones in teleost fish enterocytes that are activators of guanylyl cyclase-C (GC-C) and are potent modulators of intestinal physiology, particularly in seawater teleosts. Most notably, they reverse normal net ion-absorbing mechanisms that are vital to water absorption, an important process for seawater teleost survival. The role of guanylin-peptide stimulation of the intestine remains unclear, but it is hypothesized to facilitate the removal of solids from the intestine by providing fluid to enable their removal by peristalsis. The present study used one member of this group of peptides - RGN - to provide evidence for the prominent role that protein kinase A (PKA) plays in mediating the effects of guanylin-peptide stimulation in the posterior intestine of the Gulf toadfish ( Opsanus beta ). Protein kinase G was found to not mediate the intracellular effects of RGN, despite previous evidence showing that GC-C activation leads to higher cyclic guanosine monophosphate formation. RGN reversed the absorptive short-circuit current and increased conductance in the Gulf toadfish intestine. These effects are correlated to increased trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl - channel to the apical membrane, which is negated by PKA inhibition. Moreover, RGN decreased HCO 3 - secretion, likely by limiting apical HCO 3 - /Cl - exchange (possibly by reducing SLC26a6 activity), a reduction that was enhanced by PKA inhibition. RGN seems to alter PKA activity in the posterior intestine to recruit CFTR to the apical membrane and reduce HCO 3 - secretion. © 2018. Published by The Company of Biologists Ltd.

  19. The CFTR Met 470 allele is associated with lower birth rates in fertile men from a population isolate.

    Directory of Open Access Journals (Sweden)

    Gülüm Kosova

    2010-06-01

    Full Text Available Although little is known about the role of the cystic fibrosis transmembrane regulator (CFTR gene in reproductive physiology, numerous variants in this gene have been implicated in etiology of male infertility due to congenital bilateral absence of the vas deferens (CBAVD. Here, we studied the fertility effects of three CBAVD-associated CFTR polymorphisms, the (TGm and polyT repeat polymorphisms in intron 8 and Met470Val in exon 10, in healthy men of European descent. Homozygosity for the Met470 allele was associated with lower birth rates, defined as the number of births per year of marriage (P = 0.0029. The Met470Val locus explained 4.36% of the phenotypic variance in birth rate, and men homozygous for the Met470 allele had 0.56 fewer children on average compared to Val470 carrier men. The derived Val470 allele occurs at high frequencies in non-African populations (allele frequency = 0.51 in HapMap CEU, whereas it is very rare in African population (Fst = 0.43 between HapMap CEU and YRI. In addition, haplotypes bearing Val470 show a lack of genetic diversity and are thus longer than haplotypes bearing Met470 (measured by an integrated haplotype score [iHS] of -1.93 in HapMap CEU. The fraction of SNPs in the HapMap Phase2 data set with more extreme Fst and iHS measures is 0.003, consistent with a selective sweep outside of Africa. The fertility advantage conferred by Val470 relative to Met470 may provide a selective mechanism for these population genetic observations.

  20. Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis.

    Directory of Open Access Journals (Sweden)

    Ayad A A Amer

    Full Text Available Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia

  1. p53 mutation in carcinomas arising in ovarian cystic teratomas.

    Science.gov (United States)

    Fujii, T; Oguni, S; Kikuchi, M; Kanai, N; Saito, K

    1995-09-01

    Carcinomas arising in mature cystic teratomas of the ovaries from nine women were examined for the presence of p53 mutations. The nine tumors comprised six squamous cell carcinomas, one squamous cell carcinoma in situ, one undifferentiated small cell carcinoma, and one mucoepidermoid carcinoma. Abnormal nuclear accumulation of the p53 protein was observed in four of the tumors. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue blocks and subjected to polymerase chain reaction (PCR) for specific amplification of the p53 gene exons 5-8, followed by direct chemiluminescence sequencing analysis. A frameshift mutation in exon 8 (codon 278, CCT > del T; stop at codon 344) was detected in one poorly differentiated squamous cell carcinoma. The samples were also evaluated for the possible association of 'benign' and 'malignant' types of human papillomavirus (HPV) by PCR using universal primer sets. None of the samples contained detectable HPV genome. These data suggest that p53 mutations are relatively uncommon in secondary carcinomas developing in ovarian dermoid cysts, although the number of samples studied was admittedly small.

  2. Potentiators (specific therapies for class III and IV mutations) for cystic fibrosis.

    Science.gov (United States)

    Patel, Sanjay; Sinha, Ian P; Dwan, Kerry; Echevarria, Carlos; Schechter, Michael; Southern, Kevin W

    2015-03-26

    Cystic fibrosis is the most common inherited life-shortening illness in Caucasians and caused by a mutation in the gene that codes for the cystic fibrosis transmembrane regulator protein (CFTR), which functions as a salt transporter. This mutation most notably affects the airways of people with cystic fibrosis. Excess salt absorption by defective CFTR dehydrates the airway lining and leads to defective mucociliary clearance. Consequent accumulation of thick, sticky mucus makes the airway prone to chronic infection and progressive inflammation; respiratory failure often ensues. Additionally, abnormalities with CFTR lead to systemic complications like malnutrition, diabetes and subfertility.Since the discovery of the causative gene, our understanding of the structure and function of CFTR and the impact of different mutations has increased and allowed pharmaceutical companies to design new mutation-specific therapies targeting the underlying molecular defect. Therapies targeting mutation classes III and IV (CFTR potentiators) aim to normalise airway surface liquid and help re-establish mucociliary clearance, which then has a beneficial impact on the chronic infection and inflammation that characterizes lung disease in people with cystic fibrosis. These therapies may also affect other mutations. To evaluate the effects of CFTR potentiators on clinically important outcomes in children and adults with cystic fibrosis. We searched the Cochrane Cystic Fibrosis Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched the reference lists of relevant articles and reviews. Last search: 05 March 2015.We searched the EU Clinical Trials Register, clinicaltrials.gov (US Clinical Trials Register) and the International Clinical Trials Registry Platform (ICTRP). Last search of clinical trial registries: 06 February 2014. Randomised controlled trials of parallel design comparing CFTR potentiators to

  3. Compound heterozygous mutations (p.T561M and c.2422delT) in the TPO gene associated with congenital hypothyroidism.

    Science.gov (United States)

    Ma, Shao-Gang; Zheng, Xiao; Qiu, Ya-Li; Guo, Man-Li; Shao, Xiao-Juan

    2016-05-01

    The objective of the study was to determine the genetic basis of goitrous congenital hypothyroidism (GCH) in Chinese siblings. The proband and her younger brother with GCH were enrolled for molecular analysis of the dual oxidase 2 (DUOX2), dual oxidase maturation factor 2 (DUOXA2), and thyroid peroxidase (TPO) genes. Mutation screening was performed by Sanger sequencing the fragments amplified from genomic DNA. The detected mutations were verified among the close relatives of the patients and 105 controls. All participants underwent clinical examination and laboratory tests. Analysis of the TPO gene revealed two heterozygous mutations, the frameshift mutation c.2422delT in the exon14 of the TPO gene, that has been reported previously, and a novel missense mutation c.1682C>T (p.T561M) in the exon10 of the TPO gene. Nine family members of the patients were enrolled for mutation screening. The patients' parents and grandfathers harbored a single heterozygous mutation. The germline mutations from this family were consistent with an autosomal recessive inheritance pattern. No mutations in the DUOXA2 and DUOX2 genes were observed. The inactivating mutations (c.2422delT and p.T561M) in the TPO gene were identified in the Chinese siblings with GCH. The compound heterozygous mutations can cause GCH.

  4. Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease.

    Science.gov (United States)

    Casari, G; De Fusco, M; Ciarmatori, S; Zeviani, M; Mora, M; Fernandez, P; De Michele, G; Filla, A; Cocozza, S; Marconi, R; Dürr, A; Fontaine, B; Ballabio, A

    1998-06-12

    Hereditary spastic paraplegia (HSP) is characterized by progressive weakness and spasticity of the lower limbs due to degeneration of corticospinal axons. We found that patients from a chromosome 16q24.3-linked HSP family are homozygous for a 9.5 kb deletion involving a gene encoding a novel protein, named Paraplegin. Two additional Paraplegin mutations, both resulting in a frameshift, were found in a complicated and in a pure form of HSP. Paraplegin is highly homologous to the yeast mitochondrial ATPases, AFG3, RCA1, and YME1, which have both proteolytic and chaperon-like activities at the inner mitochondrial membrane. Immunofluorescence analysis and import experiments showed that Paraplegin localizes to mitochondria. Analysis of muscle biopsies from two patients carrying Paraplegin mutations showed typical signs of mitochondrial OXPHOS defects, thus suggesting a mechanism for neurodegeneration in HSP-type disorders.

  5. A novel SERPINA1 mutation causing serum alpha(1-antitrypsin deficiency.

    Directory of Open Access Journals (Sweden)

    Darren N Saunders

    Full Text Available Mutations in the SERPINA1 gene can cause deficiency in the circulating serine protease inhibitor α(1-Antitrypsin (α(1AT. α(1AT deficiency is the major contributor to pulmonary emphysema and liver disease in persons of European ancestry, with a prevalence of 1 in 2500 in the USA. We present the discovery and characterization of a novel SERPINA1 mutant from an asymptomatic Middle Eastern male with circulating α(1AT deficiency. This 49 base pair deletion mutation (T379Δ, originally mistyped by IEF, causes a frame-shift replacement of the last sixteen α(1AT residues and adds an extra twenty-four residues. Functional analysis showed that the mutant protein is not secreted and prone to intracellular aggregation.

  6. Wiskott-Aldrich Syndrome With Normal-Sized Platelets in an Eighteen-Month-Old Boy: A Rare Mutation

    Directory of Open Access Journals (Sweden)

    Jayitri Mazumdar

    2015-07-01

    Full Text Available Introduction: Wiskott-Aldrich syndrome (WAS is an X-linked recessive disorder characterized by thrombocytopenia, eczema, and recurrent infections. The disease is usually associated with small defective platelets. Case Presentation: We described an 18-month-old boy who presented with lower gastrointestinal bleeding, eczema, and recurrent infections. There was pancytopenia with normal-sized platelets. In addition, the CD4 count was significantly low and serum IgA and IgE levels were increased. The diagnosis of WAS was confirmed by detecting a mutation of WAS gene, which was due to a deletion mutation resulting in frameshift (c.177DelT. Conclusions: Usually microplatelets with mean platelet volume of 4-5 fL are seen in WAS, but in this case, the patient had normal-sized platelets with a rare mutation of WAS gene. Therefore, high index of clinical suspicion is needed to diagnose WAS.

  7. Identification of a novel BRCA1 nucleotide 4803delCC/c.4684delCC mutation and a nucleotide 249T>A/c.130T>A (p.Cys44Ser) mutation in two Greenlandic Inuit families

    DEFF Research Database (Denmark)

    Hansen, Thomas van Overeem; Jønson, Lars; Albrechtsen, Anders

    2010-01-01

    Germ-line mutations in the tumour suppressor proteins BRCA1 and BRCA2 predispose to breast and ovarian cancer. We have recently identified a Greenlandic Inuit BRCA1 nucleotide 234T>G/c.115T>G (p.Cys39Gly) founder mutation, which at that time was the only disease-causing BRCA1/BRCA2 mutation...... identified in this population. Here, we describe the identification of a novel disease-causing BRCA1 nucleotide 4803delCC/c.4684delCC mutation in a Greenlandic Inuit with ovarian cancer. The mutation introduces a frameshift and a premature stop at codon 1572. We have also identified a BRCA1 nucleotide 249T......>A/c.130T>A (p.Cys44Ser) mutation in another Greenlandic individual with ovarian cancer. This patient share a 1-2 Mb genomic fragment, containing the BRCA1 gene, with four Danish families harbouring the same mutation, suggesting that the 249T>A/c.130T>A (p.Cys44Ser) mutation originates from a Danish...

  8. Detección molecular de tres grandes deleciones del gen CFTR en pacientes fibroquísticos de la provincia de Buenos Aires

    OpenAIRE

    Echeverría Llumipanta, Inés Catalina

    2011-01-01

    La fibrosis quística (FQ) es la enfermedad hereditaria autosómica recesiva más frecuente en la población mundial. La enfermedad fibroquística es la expresión de diferentes mutaciones en el gen que codifica la proteína reguladora de la conductancia transmembrana de la fibrosis quística (CFTR), responsable del flujo de iones cloruro a través de la membrana celular del tejido epitelial. El gen CFTR posee acción pleiotrópica que le confiere a la fibrosis quística la característica de ser multisis...

  9. Purinergic regulation of CFTR and Ca2+ -activated Cl- channels and K+ channels in human pancreatic duct epithelium

    DEFF Research Database (Denmark)

    Wang, Jing; Haanes, Kristian A; Novak, Ivana

    2013-01-01

    dependent on intracellular Ca(2+). Apically applied ATP/UTP stimulated CF transmembrane conductance regulator (CFTR) and Ca(2+)-activated Cl(-) (CaCC) channels, which were inhibited by CFTRinh-172 and niflumic acid, respectively. The basolaterally applied ATP stimulated CFTR. In CFPAC-1 cells, which have.......1). The apical effects of ATP/UTP were greatly potentiated by the IK channel opener DC-EBIO. Determination of RNA and protein levels revealed that Capan-1 cells have high expression of TMEM16A (ANO1), a likely CaCC candidate. We conclude that in human pancreatic duct cells ATP/UTP regulates via purinergic......Purinergic agonists have been considered for the treatment of respiratory epithelia in cystic fibrosis (CF) patients. The pancreas, one of the most seriously affected organs in CF, expresses various purinergic receptors. Studies on the rodent pancreas show that purinergic signaling regulates...

  10. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

    Science.gov (United States)

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease.

  11. Germline PMS2 mutation screened by mismatch repair protein immunohistochemistry of colorectal cancer in Japan.

    Science.gov (United States)

    Sugano, Kokichi; Nakajima, Takeshi; Sekine, Shigeki; Taniguchi, Hirokazu; Saito, Shinya; Takahashi, Masahiro; Ushiama, Mineko; Sakamoto, Hiromi; Yoshida, Teruhiko

    2016-11-01

    Germline PMS2 gene mutations were detected by RT-PCR/direct sequencing of total RNA extracted from puromycin-treated peripheral blood lymphocytes (PBL) and multiplex ligation-dependent probe amplification (MLPA) analyses of Japanese patients with colorectal cancer (CRC) fulfilling either the revised Bethesda Guidelines or being an age at disease onset of younger than 70 years, and screened by mismatch repair protein immunohistochemistry of formalin-fixed paraffin embedded sections. Of the 501 subjects examined, 7 (1.40%) showed the downregulated expression of the PMS2 protein alone and were referred to the genetic counseling clinic. Germline PMS2 mutations were detected in 6 (85.7%), including 3 nonsense and 1 frameshift mutations by RT-PCR/direct sequencing and 2 genomic deletions by MLPA. No mutations were identified in the other MMR genes (i.e. MSH2, MLH1 and MSH6). The prevalence of the downregulated expression of the PMS2 protein alone was 1.40% among the subjects examined and IHC results predicted the presence of PMS2 germline mutations. RT-PCR from puromycin-treated PBL and MLPA may be employed as the first screening step to detect PMS2 mutations without pseudogene interference, followed by the long-range PCR/nested PCR validation using genomic DNA. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  12. Different inactivating mutations of the mineralocorticoid receptor in fourteen families affected by type I pseudohypoaldosteronism.

    Science.gov (United States)

    Sartorato, Paola; Lapeyraque, Anne-Laure; Armanini, Decio; Kuhnle, Ursula; Khaldi, Yasmina; Salomon, Rémi; Abadie, Véronique; Di Battista, Eliana; Naselli, Arturo; Racine, Alain; Bosio, Maurizio; Caprio, Massimiliano; Poulet-Young, Véronique; Chabrolle, Jean-Pierre; Niaudet, Patrick; De Gennes, Christiane; Lecornec, Marie-Hélène; Poisson, Elodie; Fusco, Anna Maria; Loli, Paola; Lombès, Marc; Zennaro, Maria-Christina

    2003-06-01

    We have analyzed the human mineralocorticoid receptor (hMR) gene in 14 families with autosomal dominant or sporadic pseudohypoaldosteronism (PHA1), a rare form of mineralocorticoid resistance characterized by neonatal renal salt wasting and failure to thrive. Six heterozygous mutations were detected. Two frameshift mutations in exon 2 (insT1354, del8bp537) and one nonsense mutation in exon 4 (C2157A, Cys645stop) generate truncated proteins due to premature stop codons. Three missense mutations (G633R, Q776R, L979P) differently affect hMR function. The DNA binding domain mutant R633 exhibits reduced maximal transactivation, although its binding characteristics and ED(50) of transactivation are comparable with wild-type hMR. Ligand binding domain mutants R776 and P979 present reduced or absent aldosterone binding, respectively, which is associated with reduced or absent ligand-dependent transactivation capacity. Finally, P979 possesses a transdominant negative effect on wild-type hMR activity, whereas mutations G633R and Q776R probably result in haploinsufficiency in PHA1 patients. We conclude that hMR mutations are a common feature of autosomal dominant PHA1, being found in 70% of our familial cases. Their absence in some families underscores the importance of an extensive investigation of the hMR gene and the role of precise diagnostic procedures to allow for identification of other genes potentially involved in the disease.

  13. Genotype-phenotype correlation in a large population of muscular dystrophy patients with LAMA2 mutations.

    LENUS (Irish Health Repository)

    Geranmayeh, Fatemeh

    2010-04-01

    Merosin deficient congenital muscular dystrophy 1A (MDC1A) results from mutations in the LAMA2 gene. We report 51 patients with MDC1A and examine the relationship between degree of merosin expression, genotype and clinical features. Thirty-three patients had absence of merosin and 13 showed some residual merosin. Compared to the residual merosin group, patients with absent merosin had an earlier presentation (<7days) (P=0.0073), were more likely to lack independent ambulation (P=0.0215), or require enteral feeding (P=0.0099) and ventilatory support (P=0.0354). We identified 33 novel LAMA2 mutations; these were distributed throughout the gene in patients with absent merosin, with minor clusters in exon 27, 14, 25 and 26 (55% of mutations). Patients with residual merosin often carried at least one splice site mutation and less frequently frameshift mutations. This large study identified novel LAMA2 mutations and highlights the role of immunohistochemical studies for merosin status in predicting clinical severity of MDC1A.

  14. [Analysis of UQCRB gene mutation in a child with mitochondrial complex III deficiency].

    Science.gov (United States)

    Zhang, Ting; Hong, Fang; Qian, Guling; Tong, Fan; Zhou, Xuelian; Huang, Xiaolei; Yang, Rulai; Huang, Xinwen

    2017-06-10

    To delineate the clinical, biochemical and genetic mutational characteristics of a child with mitochondrial complex III deficiency. Clinical information and results of auxiliary examination of the patient were analyzed. Next-generation sequencing of the mitochondrial genome and related nuclear genes was carried out. Suspected mutation was confirmed in both parents with Sanger sequencing. Heterozygous deletion was mapped with chromosomal microarray analysis and confirmed with real-time PCR. The patient presented with vomiting, polypnea, fever, metabolic acidosis, hyperlactatemia, hypoglycemia, dysfunction of coagulation and immune system, in addition with increased lactate dehydrogenase and creatine kinase isoenzyme. Elevation of blood alanine and acylcarnitines as well as urinary ketotic dicarboxylic acid were also noted. The patient also presented development delay, mental retardation and hypotonia. Sequence analysis revealed two mutations in the nuclear gene UQCRB, which included a previously reported frameshift mutation c.306_309delAAAA(p.Arg105Lysfs*22) and a novel large deletion encompassing the entire UQCRB gene. The clinical, biochemical and gene mutation characteristics of a child with mitochondrial complex III deficiency caused by mutations of the UQCRB gene have been delineated.

  15. One gene, two proteins: coordinated production of a copper chaperone by differential transcript formation and translational frameshifting in Escherichia coli.

    Science.gov (United States)

    Drees, Steffen L; Klinkert, Birgit; Helling, Stefan; Beyer, Dominik F; Marcus, Katrin; Narberhaus, Franz; Lübben, Mathias

    2017-11-01

    Programmed ribosomal frameshifting (PRF) is a translational anomaly causing the ribosome to shift into an alternative reading frame. PRFs are common in viral genomes, using a single nucleotide sequence to code for two proteins in overlapping frames. In bacteria and eukaryota, PRFs are less frequent. We report on a PRF in the copper detoxification system of Escherichia coli where a metallochaperone is generated out of the first 69 amino acids and a C-terminal out-of-frame glycine of the gene copA. copA besides codes for the P 1B -ATPase CopA, a membrane-integral protein and principal interaction target of the chaperone. To enhance the production of the frameshift-generated cytosolic copper binding protein a truncated transcript is produced from the monocistronic copA gene. This shorter transcript is essential for producing sufficient amounts of the chaperone to support the membrane pump. The findings close the gap in our understanding of the molecular physiology of cytoplasmic copper transport in E. coli, revealing that a chaperone-like entity is required for full functionality of the P 1B -ATPase copper pump. We, moreover, demonstrate that the primary transcriptional response to copper results in formation of the small transcript and concurrently, the metallochaperone plays a key role in resistance against copper shock. © 2017 John Wiley & Sons Ltd.

  16. Relationship between repair processes and mutation induction in bacteria. [UV radiation; methyl methanesulfonate; N-methyl-N/sup 1/-nitro-N-nitrosoguanidine; N-methyl-N-nitrosourea; N-ethyl-N-nitrosourea; ethyl methanesulfanate; N-ethyl-nitrosoguanidine; 4-nitroquinoline 1-oxide

    Energy Technology Data Exchange (ETDEWEB)

    Kimball, R F

    1979-01-01

    The main repair and replication-associated processes that can influence the induction of mutations by various mutagens in bacteria are reviewed. These include both constitutive and induced, error-free and error-prone systems. The mutation yield from a treatment with a mutagen can be markedly affected by which of these systems is operating in a given bacterial species or strain. The effect of these systems on mutation induction by ultraviolet light, monofunctional alkylating agents, base analogues, and frameshift mutagens is discussed in some detail. The bearing of these studies on the practical problems of estimating hazards is briefly considered. 79 references.

  17. X-Linked Agammagobulinemia in a Large Series of North African Patients: Frequency, Clinical Features and Novel BTK Mutations.

    Science.gov (United States)

    Aadam, Zahra; Kechout, Nadia; Barakat, Abdelhamid; Chan, Koon-Wing; Ben-Ali, Meriem; Ben-Mustapha, Imen; Zidi, Fethi; Ailal, Fatima; Attal, Nabila; Doudou, Fatouma; Abbadi, Mohamed-Cherif; Kaddache, Chawki; Smati, Leila; Touri, Nabila; Chemli, Jalel; Gargah, Tahar; Brini, Ines; Bakhchane, Amina; Charoute, Hicham; Jeddane, Leila; El Atiqi, Sara; El Hafidi, Naïma; Hida, Mustapha; Saile, Rachid; Alj, Hanane Salih; Boukari, Rachida; Bejaoui, Mohamed; Najib, Jilali; Barbouche, Mohamed-Ridha; Lau, Yu-Lung; Mellouli, Fethi; Bousfiha, Ahmed Aziz

    2016-04-01

    X-linked agammagobulinemia (XLA) is a primary immunodeficiency caused by Bruton's tyrosine kinase (BTK) gene defect. XLA patients have absent or reduced number of peripheral B cells and a profound deficiency in all immunoglobulin isotypes. This multicenter study reports the clinical, immunological and molecular features of Bruton's disease in 40 North African male patients. Fifty male out of 63 (male and female) patients diagnosed with serum agammaglobulinemia and non detectable to less than 2% peripheral B cells were enrolled. The search for BTK gene mutations was performed for all of them by genomic DNA amplification and Sanger sequencing. We identified 33 different mutations in the BTK gene in 40 patients including 12 missense mutations, 6 nonsense mutations, 6 splice-site mutations, 5 frameshift, 2 large deletions, one complex mutation and one in-frame deletion. Seventeen of these mutations are novel. This large series shows a lower frequency of XLA among male patients from North Africa with agammaglobulinemia and absent to low B cells compared with other international studies (63.5% vs. 85%). No strong evidence for genotype-phenotype correlation was observed. This study adds to other reports from highly consanguineous North African populations, showing lower frequency of X-linked forms as compared to AR forms of the same primary immunodeficiency. Furthermore, a large number of novel BTK mutations were identified and could further help identify carriers for genetic counseling.

  18. Functional analysis of the novel TBX5 c.1333delC mutation resulting in an extended TBX5 protein

    Directory of Open Access Journals (Sweden)

    Ekman-Joelsson Britt-Marie

    2008-10-01

    Full Text Available Abstract Background Autosomal dominant Holt-Oram syndrome (HOS is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS. Methods The functional impact of this novel mutation was assessed by investigating the intracellular localization of the resulting TBX5 protein and its ability to activate the expression of its downstream target ANF. Results The deletion of the cytosine is the first TBX5 frameshift mutation predicted to result in an elongated TBX5 protein with 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter. Conclusion The mutation c.1333delC does not locate within functional domains, but impairs the activation of the downstream target. This suggests that misfolding of the protein prevents its biological function.

  19. A novel de novo COL1A1 mutation in a Thai boy with osteogenesis imperfecta born to consanguineous parents

    Directory of Open Access Journals (Sweden)

    Siraprapa Tongkobpetch

    2017-09-01

    Full Text Available Abstract Osteogenesis imperfecta (OI is genetically heterogeneous. Mutations in COL1A1 and COL1A2 are responsible for at least 90% of the cases, which are transmitted in an autosomal dominant manner or are de novo events. We identified a Thai boy with OI whose parents were first cousins. Because the proband was the product of a consanguineous marriage, we hypothesized that he might be homozygous for a mutation in a known gene causing a recessive form of OI. Using whole exome sequencing (WES, we did not find any pathogenic mutations in any known gene responsible for an autosomal recessive form of OI. Instead, we identified a COL1A1 frameshift mutation, c.1290delG (p.Gly431Valfs*110 in heterozygosis. By Sanger sequencing, the mutation was confirmed in the proband, and not detected in his parents, indicating that it was a de novo mutation. These findings had implication for genetic counseling. In conclusion, we expanded the mutational spectrum of COL1A1 and provided another example of a de novo pathogenic mutation in heterozygosis in a patient born to consanguineous parents.

  20. Histamine 1 receptor-Gβγ-cAMP/PKA-CFTR pathway mediates the histamine-induced resetting of the suprachiasmatic circadian clock.

    Science.gov (United States)

    Kim, Yoon Sik; Kim, Young-Beom; Kim, Woong Bin; Lee, Seung Won; Oh, Seog Bae; Han, Hee-Chul; Lee, C Justin; Colwell, Christopher S; Kim, Yang In

    2016-05-06

    Recent evidence indicates that histamine, acting on histamine 1 receptor (H1R), resets the circadian clock in the mouse suprachiasmatic nucleus (SCN) by increasing intracellular Ca(2+) concentration ([Ca(2+)]i) through the activation of CaV1.3 L-type Ca(2+) channels and Ca(2+)-induced Ca(2+) release from ryanodine receptor-mediated internal stores. In the current study, we explored the underlying mechanisms with various techniques including Ca(2+)- and Cl(-)-imaging and extracellular single-unit recording. Our hypothesis was that histamine causes Cl(-) efflux through cystic fibrosis transmembrane conductance regulator (CFTR) to elicit membrane depolarization needed for the activation of CaV1.3 Ca(2+) channels in SCN neurons. We found that histamine elicited Cl(-) efflux and increased [Ca(2+)]i in dissociated mouse SCN cells. Both of these events were suppressed by bumetanide [Na(+)-K(+)-2Cl(-) cotransporter isotype 1 (NKCC1) blocker], CFTRinh-172 (CFTR inhibitor), gallein (Gβγ protein inhibitor) and H89 [protein kinase A (PKA) inhibitor]. By itself, H1R activation with 2-pyridylethylamine increased the level of cAMP in the SCN and this regulation was prevented by gallein. Finally, histamine-evoked phase shifts of the circadian neural activity rhythm in the mouse SCN slice were blocked by bumetanide, CFTRinh-172, gallein or H89 and were not observed in NKCC1 or CFTR KO mice. Taken together, these results indicate that histamine recruits the H1R-Gβγ-cAMP/PKA pathway in the SCN neurons to activate CaV1.3 channels through CFTR-mediated Cl(-) efflux and ultimately to phase-shift the circadian clock. This pathway and NKCC1 may well be potential targets for agents designed to treat problems resulting from the disturbance of the circadian system.

  1. Mutations in Danish patients with long QT syndrome and the identification of a large founder family with p.F29L in KCNH2

    DEFF Research Database (Denmark)

    Christiansen, Michael; Hedley, Paula L; Theilade, Juliane

    2014-01-01

    in KCNQ1, 28 in KCNH2, 9 in SCN5A, 3 in KCNE1 and 2 in KCNE2. Twenty-six of these have only been described in the Danish population and 18 are novel. One double heterozygote (1.4% of families) was found. A founder mutation, p.F29L in KCNH2, was identified in 5 "unrelated" families. Disease association......, in 31.2% of cases, was based on the type of mutation identified (nonsense, insertion/deletion, frameshift or splice-site). Functional data was available for 22.7% of the missense mutations. None of the mutations were found in 364 Danish alleles and only three, all functionally characterised, were...

  2. Somatic USP8 Gene Mutations Are a Common Cause of Pediatric Cushing Disease.

    Science.gov (United States)

    Faucz, Fabio R; Tirosh, Amit; Tatsi, Christina; Berthon, Annabel; Hernández-Ramírez, Laura C; Settas, Nikolaos; Angelousi, Anna; Correa, Ricardo; Papadakis, Georgios Z; Chittiboina, Prashant; Quezado, Martha; Pankratz, Nathan; Lane, John; Dimopoulos, Aggeliki; Mills, James L; Lodish, Maya; Stratakis, Constantine A

    2017-08-01

    Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have been recently identified as the most common genetic alteration in patients with Cushing disease (CD). However, the frequency of these mutations in the pediatric population has not been extensively assessed. We investigated the status of the USP8 gene at the somatic level in a cohort of pediatric patients with corticotroph adenomas. The USP8 gene was fully sequenced in both germline and tumor DNA samples from 42 pediatric patients with CD. Clinical, biochemical, and imaging data were compared between patients with and without somatic USP8 mutations. Five different USP8 mutations (three missense, one frameshift, and one in-frame deletion) were identified in 13 patients (31%), all of them located in exon 14 at the previously described mutational hotspot, affecting the 14-3-3 binding motif of the protein. Patients with somatic mutations were older at disease presentation [mean 5.1 ± 2.1 standard deviation (SD) vs 13.1 ± 3.6 years, P = 0.03]. Levels of urinary free cortisol, midnight serum cortisol, and adrenocorticotropic hormone, as well as tumor size and frequency of invasion of the cavernous sinus, were not significantly different between the two groups. However, patients harboring somatic USP8 mutations had a higher likelihood of recurrence compared with patients without mutations (46.2% vs 10.3%, P = 0.009). Somatic USP8 gene mutations are a common cause of pediatric CD. Patients harboring a somatic mutation had a higher likelihood of tumor recurrence, highlighting the potential importance of this molecular defect for the disease prognosis and the development of targeted therapeutic options. Copyright © 2017 Endocrine Society

  3. DMD Mutations in 576 Dystrophinopathy Families: A Step Forward in Genotype-Phenotype Correlations.

    Science.gov (United States)

    Juan-Mateu, Jonas; Gonzalez-Quereda, Lidia; Rodriguez, Maria Jose; Baena, Manel; Verdura, Edgard; Nascimento, Andres; Ortez, Carlos; Baiget, Montserrat; Gallano, Pia

    2015-01-01

    Recent advances in molecular therapies for Duchenne muscular dystrophy (DMD) require precise genetic diagnosis because most therapeutic strategies are mutation-specific. To understand more about the genotype-phenotype correlations of the DMD gene we performed a comprehensive analysis of the DMD mutational spectrum in a large series of families. Here we provide the clinical, pathological and genetic features of 576 dystrophinopathy patients. DMD gene analysis was performed using the MLPA technique and whole gene sequencing in blood DNA and muscle cDNA. The impact of the DNA variants on mRNA splicing and protein functionality was evaluated by in silico analysis using computational algorithms. DMD mutations were detected in 576 unrelated dystrophinopathy families by combining the analysis of exonic copies and the analysis of small mutations. We found that 471 of these mutations were large intragenic rearrangements. Of these, 406 (70.5%) were exonic deletions, 64 (11.1%) were exonic duplications, and one was a deletion/duplication complex rearrangement (0.2%). Small mutations were identified in 105 cases (18.2%), most being nonsense/frameshift types (75.2%). Mutations in splice sites, however, were relatively frequent (20%). In total, 276 mutations were identified, 85 of which have not been previously described. The diagnostic algorithm used proved to be accurate for the molecular diagnosis of dystrophinopathies. The reading frame rule was fulfilled in 90.4% of DMD patients and in 82.4% of Becker muscular dystrophy patients (BMD), with significant differences between the mutation types. We found that 58% of DMD patients would be included in single exon-exon skipping trials, 63% from strategies directed against multiexon-skipping exons 45 to 55, and 14% from PTC therapy. A detailed analysis of missense mutations provided valuable information about their impact on the protein structure.

  4. Mutational analysis of Btk, the defective gene in X-linked agammaglobulinemia

    Energy Technology Data Exchange (ETDEWEB)

    Conley, M.E.; Fitch-Hilgenberg, M.E.; Rohrer, J. [St. Jude Children`s Research Hospital, Memphis, TN (United States)

    1994-09-01

    Recent studies have shown that X-linked agammaglobulinemia (XLA), a disorder of B cell development, is due to mutations in an scr-like cytoplasmic tyrosine kinase, Btk. Thus far, mutations in this gene have been identified by sequencing of cDNA. To permit the detection of mutations in genomic DNA, we determined the structure of Btk and identified 19 exons in 37 kb of DNA. PCR primers were designed to amplify each exon with its splice sites. Two overlapping PCR products were employed for exons longer than 230 base pairs. Single strand conformation polymorphism (SSCP) analysis was used to screen genomic DNA from 30 unrelated families presumed to carry a mutation in Btk. It was possible to amplify DNA in every reaction from every patient. None of the DNA samples demonstrated more than one aberrant SSCP pattern. Twenty three mutations were detected in 25 families. Seven point mutations resulting in amino acid substitutions were seen. An additional 7 base pair substitutions gave rise to premature stop codons. Two splice defects were noted. Small insertions or deletions, all resulting in frameshifts and premature stop codons were seen in eight patients. One patient had an A to G transition in the ATG start codon. Two mutations, both at CpG dinucleotides, were seen in more than one family. Haplotype analysis, using CA repeats closely linked to Btk, demonstrated that the mutations in these families arose independently. We conclude from these studies that the mutations in Btk in patients with XLA are highly variable. Large deletions are uncommon, although small 1 to 4 bp insertions or deletions constitute as many as one third of the mutations. Further analysis of patients with amino acid substitutions will permit structure/function correlations.

  5. DMD Mutations in 576 Dystrophinopathy Families: A Step Forward in Genotype-Phenotype Correlations.

    Directory of Open Access Journals (Sweden)

    Jonas Juan-Mateu

    Full Text Available Recent advances in molecular therapies for Duchenne muscular dystrophy (DMD require precise genetic diagnosis because most therapeutic strategies are mutation-specific. To understand more about the genotype-phenotype correlations of the DMD gene we performed a comprehensive analysis of the DMD mutational spectrum in a large series of families. Here we provide the clinical, pathological and genetic features of 576 dystrophinopathy patients. DMD gene analysis was performed using the MLPA technique and whole gene sequencing in blood DNA and muscle cDNA. The impact of the DNA variants on mRNA splicing and protein functionality was evaluated by in silico analysis using computational algorithms. DMD mutations were detected in 576 unrelated dystrophinopathy families by combining the analysis of exonic copies and the analysis of small mutations. We found that 471 of these mutations were large intragenic rearrangements. Of these, 406 (70.5% were exonic deletions, 64 (11.1% were exonic duplications, and one was a deletion/duplication complex rearrangement (0.2%. Small mutations were identified in 105 cases (18.2%, most being nonsense/frameshift types (75.2%. Mutations in splice sites, however, were relatively frequent (20%. In total, 276 mutations were identified, 85 of which have not been previously described. The diagnostic algorithm used proved to be accurate for the molecular diagnosis of dystrophinopathies. The reading frame rule was fulfilled in 90.4% of DMD patients and in 82.4% of Becker muscular dystrophy patients (BMD, with significant differences between the mutation types. We found that 58% of DMD patients would be included in single exon-exon skipping trials, 63% from strategies directed against multiexon-skipping exons 45 to 55, and 14% from PTC therapy. A detailed analysis of missense mutations provided valuable information about their impact on the protein structure.

  6. Identification of novel mutations in Chinese Hans with autosomal dominant polycystic kidney disease

    Directory of Open Access Journals (Sweden)

    Yu Chaowen

    2011-12-01

    Full Text Available Abstract Background Autosomal dominant polycystic kidney disease (ADPKD is the most common inherited renal disease with an incidence of 1 in 400 to 1000. The disease is genetically heterogeneous, with two genes identified: PKD1 (16p13.3 and PKD2 (4q21. Molecular diagnosis of the disease in at-risk individuals is complicated due to the structural complexity of PKD1 gene and the high diversity of the mutations. This study is the first systematic ADPKD mutation analysis of both PKD1 and PKD2 genes in Chinese patients using denaturing high-performance liquid chromatography (DHPLC. Methods Both PKD1 and PKD2 genes were mutation screened in each proband from 65 families using DHPLC followed by DNA sequencing. Novel variations found in the probands were checked in their family members available and 100 unrelated normal controls. Then the pathogenic potential of the variations of unknown significance was examined by evolutionary comparison, effects of amino acid substitutions on protein structure, and effects of splice site alterations using online mutation prediction resources. Results A total of 92 variations were identified, including 27 reported previously. Definitely pathogenic mutations (ten frameshift, ten nonsense, two splicing defects and one duplication were identified in 28 families, and probably pathogenic mutations were found in an additional six families, giving a total detection level of 52.3% (34/65. About 69% (20/29 of the mutations are first reported with a recurrent mutation rate of 31%. Conclusions Mutation study of PKD1 and PKD2 genes in Chinese Hans with ADPKD may contribute to a better understanding of the genetic diversity between different ethnic groups and enrich the mutation database. Besides, evaluating the pathogenic potential of novel variations should also facilitate the clinical diagnosis and genetic counseling of the disease.

  7. Multi-layered mutation in hedgehog-related genes in Gorlin syndrome may affect the phenotype.

    Directory of Open Access Journals (Sweden)

    Shoko Onodera

    Full Text Available Gorlin syndrome is a genetic disorder of autosomal dominant inheritance that predisposes the affected individual to a variety of disorders that are attributed largely to heterozygous germline patched1 (PTCH1 mutations. PTCH1 is a hedgehog (Hh receptor as well as a repressor, mutation of which leads to constitutive activation of Hh pathway. Hh pathway encompasses a wide variety of cellular signaling cascades, which involve several molecules; however, no associated genotype-phenotype correlations have been reported. Recently, mutations in Suppressor of fused homolog (SUFU or PTCH2 were reported in patients with Gorlin syndrome. These facts suggest that multi-layered mutations in Hh pathway may contribute to the development of Gorlin syndrome. We demonstrated multiple mutations of Hh-related genes in addition to PTCH1, which possibly act in an additive or multiplicative manner and lead to Gorlin syndrome. High-throughput sequencing was performed to analyze exome sequences in four unrelated Gorlin syndrome patient genomes. Mutations in PTCH1 gene were detected in all four patients. Specific nucleotide variations or frameshift variations of PTCH1 were identified along with the inferred amino acid changes in all patients. We further filtered 84 different genes which are closely related to Hh signaling. Fifty three of these had enough coverage of over ×30. The sequencing results were filtered and compared to reduce the number of sequence variants identified in each of the affected individuals. We discovered three genes, PTCH2, BOC, and WNT9b, with mutations with a predicted functional impact assessed by MutationTaster2 or PolyPhen-2 (Polymorphism Phenotyping v2 analysis. It is noticeable that PTCH2 and BOC are Hh receptor molecules. No significant mutations were observed in SUFU. Multi-layered mutations in Hh pathway may change the activation level of the Hh signals, which may explain the wide phenotypic variability of Gorlin syndrome.

  8. Multiple endocrine neoplasia type 1: analysis of germline MEN1 mutations in the Italian multicenter MEN1 patient database.

    Science.gov (United States)

    Marini, Francesca; Giusti, Francesca; Fossi, Caterina; Cioppi, Federica; Cianferotti, Luisella; Masi, Laura; Boaretto, Francesca; Zovato, Stefania; Cetani, Filomena; Colao, Annamaria; Davì, Maria Vittoria; Faggiano, Antongiulio; Fanciulli, Giuseppe; Ferolla, Piero; Ferone, Diego; Loli, Paola; Mantero, Franco; Marcocci, Claudio; Opocher, Giuseppe; Beck-Peccoz, Paolo; Persani, Luca; Scillitani, Alfredo; Guizzardi, Fabiana; Spada, Anna; Tomassetti, Paola; Tonelli, Francesco; Brandi, Maria Luisa

    2018-03-01

    Multiple endocrine neoplasia type 1 (MEN1) is caused by germline inactivating mutations of the MEN1 gene. Currently, no direct genotype-phenotype correlation is identified. We aim to analyze MEN1 mutation site and features, and possible correlations between the mutation type and/or the affected menin functional domain and clinical presentation in patients from the Italian multicenter MEN1 database, one of the largest worldwide MEN1 mutation series published to date. The study included the analysis of MEN1 mutation profile in 410 MEN1 patients [370 familial cases from 123 different pedigrees (48 still asymptomatic at the time of this study) and 40 single cases]. We identified 99 different mutations: 41 frameshift [small intra-exon deletions (28) or insertions (13)], 13 nonsense, 26 missense and 11 splicing site mutations, 4 in-frame small deletions, and 4 intragenic large deletions spanning more than one exon. One family had two different inactivating MEN1 mutations on the same allele. Gastro-entero-pancreatic tumors resulted more frequent in patients with a nonsense mutation, and thoracic neuroendocrine tumors in individuals bearing a splicing-site mutation. Our data regarding mutation type frequency and distribution are in accordance with previously published data: MEN1 mutations are scattered through the entire coding region, and truncating mutations are the most common in MEN1 syndrome. A specific direct correlation between MEN1 genotype and clinical phenotype was not found in all our families, and wide intra-familial clinical variability and variable disease penetrance were both confirmed, suggesting a role for modifying, still undetermined, factors, explaining the variable MEN1 tumorigenesis.

  9. Spectrum of SMPD1 mutations in Asian-Indian patients with acid sphingomyelinase (ASM)-deficient Niemann-Pick disease.

    Science.gov (United States)

    Ranganath, Prajnya; Matta, Divya; Bhavani, Gandham SriLakshmi; Wangnekar, Savita; Jain, Jamal Mohammed Nurul; Verma, Ishwar C; Kabra, Madhulika; Puri, Ratna Dua; Danda, Sumita; Gupta, Neerja; Girisha, Katta M; Sankar, Vaikom H; Patil, Siddaramappa J; Ramadevi, Akella Radha; Bhat, Meenakshi; Gowrishankar, Kalpana; Mandal, Kausik; Aggarwal, Shagun; Tamhankar, Parag Mohan; Tilak, Preetha; Phadke, Shubha R; Dalal, Ashwin

    2016-10-01

    Acid sphingomyelinase (ASM)-deficient Niemann-Pick disease is an autosomal recessive lysosomal storage disorder caused by biallelic mutations in the SMPD1 gene. To date, around 185 mutations have been reported in patients with ASM-deficient NPD world-wide, but the mutation spectrum of this disease in India has not yet been reported. The aim of this study was to ascertain the mutation profile in Indian patients with ASM-deficient NPD. We sequenced SMPD1 in 60 unrelated families affected with ASM-deficient NPD. A total of 45 distinct pathogenic sequence variants were found, of which 14 were known and 31 were novel. The variants included 30 missense, 4 nonsense, and 9 frameshift (7 single base deletions and 2 single base insertions) mutations, 1 indel, and 1 intronic duplication. The pathogenicity of the novel mutations was inferred with the help of the mutation prediction software MutationTaster, SIFT, Polyphen-2, PROVEAN, and HANSA. The effects of the identified sequence variants on the protein structure were studied using the structure modeled with the help of the SWISS-MODEL workspace program. The p. (Arg542*) (c.1624C>T) mutation was the most commonly identified mutation, found in 22% (26 out of 120) of the alleles tested, but haplotype analysis for this mutation did not identify a founder effect for the Indian population. To the best of our knowledge, this is the largest study on mutation analysis of patients with ASM-deficient Niemann-Pick disease reported in literature and also the first study on the SMPD1 gene mutation spectrum in India. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. CtIP Mutations Cause Seckel and Jawad Syndromes.

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    Per Qvist

    2011-10-01

    Full Text Available Seckel syndrome is a recessively inherited dwarfism disorder characterized by microcephaly and a unique head profile. Genetically, it constitutes a heterogeneous condition, with several loci mapped (SCKL1-5 but only three disease genes identified: the ATR, CENPJ, and CEP152 genes that control cellular responses to DNA damage. We previously mapped a Seckel syndrome locus to chromosome 18p11.31-q11.2 (SCKL2. Here, we report two mutations in the CtIP (RBBP8 gene within this locus that result in expression of C-terminally truncated forms of CtIP. We propose that these mutations are the molecular cause of the disease observed in the previously described SCKL2 family and in an additional unrelated family diagnosed with a similar form of congenital microcephaly termed Jawad syndrome. While an exonic frameshift mutation was found in the Jawad family, the SCKL2 family carries a splicing mutation that yields a dominant-negative form of CtIP. Further characterization of cell lines derived from the SCKL2 family revealed defective DNA damage induced formation of single-stranded DNA, a critical co-factor for ATR activation. Accordingly, SCKL2 cells present a lowered apoptopic threshold and hypersensitivity to DNA damage. Notably, over-expression of a comparable truncated CtIP variant in non-Seckel cells recapitulates SCKL2 cellular phenotypes in a dose-dependent manner. This work thus identifies CtIP as a disease gene for Seckel and Jawad syndromes and defines a new type of genetic disease mechanism in which a dominant negative mutation yields a recessively inherited disorder.

  11. Rescue of defective ATP8B1 trafficking by CFTR correctors as a therapeutic strategy for familial intrahepatic cholestasis

    NARCIS (Netherlands)

    van der Woerd, Wendy L.; Wichers, Catharina G. K.; Vestergaard, Anna L.; Andersen, Jens Peter; Paulusma, Coen C.; Houwen, Roderick H. J.; van de Graaf, Stan F. J.

    Background & Aims ATP8B1 deficiency is an autosomal recessive liver disease characterized by intrahep