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Sample records for splice donor mutation

  1. Prenatal diagnosis and a donor splice site mutation in fibrillin in a family with Marfan syndrome

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    Godfrey, M.; Vandemark, N.; Wang, M.; Han, J.; Rao, V.H. (Univ. of Nebraska Medical Center, Omaha (United States)); Velinov, M.; Tsipouras, P. (Univ. of Connecticut Health Sciences Center, Farmington (United States)); Wargowski, D.; Becker, J.; Robertson, W.; Droste, S. (Univ. of Wisconsin, Madison (United States))

    1993-08-01

    The Marfan syndrome, an autosomal dominant connective tissue disorder, is manifested by abnormalities in the cardiovascular, skeletal, and ocular systems. Recently, fibrillin, an elastic-associated microfibrillar glycoprotein, has been linked to the Marfan syndrome, and fibrillin mutations in affected individuals have been documented. In this study, genetic linkage analysis with fibrillin-specific markers was used to establish the prenatal diagnosis in an 11-wk-gestation fetus in a four-generation Marfan kindred. At birth, skeletal changes suggestive of the Marfan syndrome were observed. Reverse transcription-PCR amplification of the fibrillin gene mRNA detected a deletion of 123 bp in one allele in affected relatives. This deletion corresponds to an exon encoding an epidermal growth factor-like motif. Examination of genomic DNA showed a G[yields]C transversion at the +1 consensus donor splice site. 45 refs., 7 figs.

  2. A donor splice site mutation in CISD2 generates multiple truncated, non-functional isoforms in Wolfram syndrome type 2 patients.

    Science.gov (United States)

    Cattaneo, Monica; La Sala, Lucia; Rondinelli, Maurizio; Errichiello, Edoardo; Zuffardi, Orsetta; Puca, Annibale Alessandro; Genovese, Stefano; Ceriello, Antonio

    2017-12-13

    Mutations in the gene that encodes CDGSH iron sulfur domain 2 (CISD2) are causative of Wolfram syndrome type 2 (WFS2), a rare autosomal recessive neurodegenerative disorder mainly characterized by diabetes mellitus, optic atrophy, peptic ulcer bleeding and defective platelet aggregation. Four mutations in the CISD2 gene have been reported. Among these mutations, the homozygous c.103 + 1G > A substitution was identified in the donor splice site of intron 1 in two Italian sisters and was predicted to cause a exon 1 to be skipped. Here, we employed molecular assays to characterize the c.103 + 1G > A mutation using the patient's peripheral blood mononuclear cells (PBMCs). 5'-RACE coupled with RT-PCR were used to analyse the effect of the c.103 + 1G > A mutation on mRNA splicing. Western blot analysis was used to analyse the consequences of the CISD2 mutation on the encoded protein. We demonstrated that the c.103 + 1G > A mutation functionally impaired mRNA splicing, producing multiple splice variants characterized by the whole or partial absence of exon 1, which introduced amino acid changes and a premature stop. The affected mRNAs resulted in either predicted targets for nonsense mRNA decay (NMD) or non-functional isoforms. We concluded that the c.103 + 1G > A mutation resulted in the loss of functional CISD2 protein in the two Italian WFS2 patients.

  3. Mild recessive epidermolytic hyperkeratosis associated with a novel keratin 10 donor splice-site mutation in a family of Norfolk terrier dogs.

    Science.gov (United States)

    Credille, K M; Barnhart, K F; Minor, J S; Dunstan, R W

    2005-07-01

    Epidermolytic hyperkeratosis in humans is caused by dominant-negative mutations in suprabasal epidermal keratins 1 and 10. However, spontaneous keratin mutations have not been confirmed in a species other than human. To describe an autosomal recessive, mild, nonpalmar/plantar epidermolytic ichthyosis segregating in an extended pedigree of Norfolk terrier dogs due to a splice-site mutation in the gene encoding keratin 10 (KRT10). Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Genomic DNA samples and cDNA from skin RNA were sequenced and defined a mutation in KRT10. Consequences of the mutation were evaluated by assessing protein expression with immunohistochemistry and Western blotting and gene expression with real-time RT-PCR (reverse transcriptase-polymerase chain reaction). Adult dogs with the disease had generalized, pigmented hyperkeratosis with epidermal fragility. Light microscopic examination defined epidermolysis with hyperkeratosis; ultrastructural changes included a decrease in tonofilaments and abnormal filament aggregation in upper spinous and granular layer keratinocytes. Affected dogs were homozygous for a single base GT-->TT change in the consensus donor splice site of intron 5 in KRT10. Keratin 10 protein was not detected with immunoblotting in affected dogs. Heterozygous dogs were normal based on clinical and histological appearance and keratin 10 protein expression. The mutation caused activation of at least three cryptic or alternative splice sites. Use of the cryptic sites resulted in transcripts containing premature termination codons. One transcript could result in shortening of the proximal portion of the 2B domain before the stutter region. Quantitative real-time PCR indicated a significant decrease in KRT10 mRNA levels in affected dogs compared with wild-type dogs. This disease is the first confirmed spontaneous keratin mutation in a nonhuman species and is the first reported recessive form

  4. A novel point mutation (G[sup [minus]1] to T) in a 5[prime] splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker Muscular Dystrophy

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    Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko; Takeshima, Yasuhiro; Narita, Naoko; Wada, Hiroko; Yokoyama, Mitsuhiro; Nakamura, Hajime; Matsuo, Masafumi (Kobe Univ. School of Medicine (Japan))

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.

  5. Lethal autosomal recessive epidermolytic ichthyosis due to a novel donor splice-site mutation in KRT10.

    Science.gov (United States)

    Covaciu, C; Castori, M; De Luca, N; Ghirri, P; Nannipieri, A; Ragone, G; Zambruno, G; Castiglia, D

    2010-06-01

    Epidermolytic ichthyosis (EI; MIM 113800), previously named bullous congenital ichthyosiform erythroderma or epidermolytic hyperkeratosis, is a rare and clinically variable defect of cornification characterized by generalized erythema, erosions, scaling and easily breaking blisters that become less frequent later in life while hyperkeratosis increases. EI is caused by dominant mutations in either KRT1 or KRT10, encoding keratin 1 (K1) and keratin 10 (K10), respectively. Usually, mutations are missense substitutions into the highly conserved α-helical rod domains of the proteins. However, three inbred pedigrees in which EI is transmitted as a recessive trait due to KRT10 null mutations have been described. © 2010 The Authors. Journal Compilation © 2010 British Association of Dermatologists.

  6. Accumulation of GC donor splice signals in mammals

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    Koonin Eugene V

    2008-07-01

    Full Text Available Abstract The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signals. However, in a small fraction of donor sites, GT is replaced by GC. A substantial enrichment of GC in donor sites of alternatively spliced genes has been observed previously in human, nematode and Arabidopsis, suggesting that GC signals are important for regulation of alternative splicing. We used parsimony analysis to reconstruct evolution of donor splice sites and inferred 298 GT > GC conversion events compared to 40 GC > GT conversion events in primate and rodent genomes. Thus, there was substantive accumulation of GC donor splice sites during the evolution of mammals. Accumulation of GC sites might have been driven by selection for alternative splicing. Reviewers This article was reviewed by Jerzy Jurka and Anton Nekrutenko. For the full reviews, please go to the Reviewers' Reports section.

  7. HIV-1 splicing at the major splice donor site is restricted by RNA structure.

    Science.gov (United States)

    Mueller, Nancy; van Bel, Nikki; Berkhout, Ben; Das, Atze T

    2014-11-01

    The 5' leader region of the HIV-1 RNA contains the major 5' splice site (ss) that is used in the production of all spliced viral RNAs. This splice-donor (SD) region can fold a stem-loop structure. We demonstrate that whereas stabilization of this SD hairpin reduces splicing efficiency, destabilization increases splicing. Both stabilization and destabilization reduce viral fitness. These results demonstrate that the stability of the SD hairpin can modulate the level of splicing, most likely by controlling the accessibility of the 5'ss for the splicing machinery. The natural stability of the SD hairpin restricts splicing and this stability seems to be fine-tuned to reach the optimal balance between unspliced and spliced RNAs for efficient virus replication. The 5'ss region of different HIV-1 isolates and the related SIVmac239 can fold a similar structure. This evolutionary conservation supports the importance of this structure in viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. X-linked Alport syndrome associated with a synonymous p.Gly292Gly mutation alters the splicing donor site of the type IV collagen alpha chain 5 gene.

    Science.gov (United States)

    Fu, Xue Jun; Nozu, Kandai; Eguchi, Aya; Nozu, Yoshimi; Morisada, Naoya; Shono, Akemi; Taniguchi-Ikeda, Mariko; Shima, Yuko; Nakanishi, Koichi; Vorechovsky, Igor; Iijima, Kazumoto

    2016-10-01

    X-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the type IV collagen alpha chain 5 gene (COL4A5). Although many COL4A5 mutations have previously been identified, pathogenic synonymous mutations have not yet been described. A family with XLAS underwent mutational analyses of COL4A5 by PCR and direct sequencing, as well as transcript analysis of potential splice site mutations. In silico analysis was also conducted to predict the disruption of splicing factor binding sites. Immunohistochemistry (IHC) of kidney biopsies was used to detect α2 and α5 chain expression. We identified a hemizygous point mutation, c.876A>T, in exon 15 of COL4A5 in the proband and his brother, which is predicted to result in a synonymous amino acid change, p.(Gly292Gly). Transcript analysis showed that this mutation potentially altered splicing because it disrupted the splicing factor binding site. The kidney biopsy of the proband showed lamellation of the glomerular basement membrane (GBM), while IHC revealed negative α5(IV) staining in the GBM and Bowman's capsule, which is typical of XLAS. This is the first report of a synonymous COL4A5 substitution being responsible for XLAS. Our findings suggest that transcript analysis should be conducted for the future correct assessment of silent mutations.

  9. Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG).

    Science.gov (United States)

    Laine, C M; Chung, B D; Susic, M; Prescott, T; Semler, O; Fiskerstrand, T; D'Eufemia, P; Castori, M; Pekkinen, M; Sochett, E; Cole, W G; Netzer, C; Mäkitie, O

    2011-08-01

    Osteoporosis-pseudoglioma sydrome (OPPG) is an autosomal recessive disorder with early-onset severe osteoporosis and blindness, caused by biallelic loss-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Heterozygous carriers exhibit a milder bone phenotype. Only a few splice mutations in LRP5 have been published. We present clinical and genetic data for four patients with novel LRP5 mutations, three of which affect splicing. Patients were evaluated clinically and by radiography and bone densitometry. Genetic screening of LRP5 was performed on the basis of the clinical diagnosis of OPPG. Splice aberrances were confirmed by cDNA sequencing or exon trapping. The effect of one splice mutation on LRP5 protein function was studied. A novel splice-site mutation c.1584+4A>T abolished the donor splice site of exon 7 and activated a cryptic splice site, which led to an in-frame insertion of 21 amino acids (p.E528_V529ins21). Functional studies revealed severely impaired signal transduction presumably caused by defective intracellular transport of the mutated receptor. Exon trapping was used on two samples to confirm that splice-site mutations c.4112-2A>G and c.1015+1G>T caused splicing-out of exons 20 and 5, respectively. One patient carried a homozygous deletion of exon 4 causing the loss of exons 4 and 5, as demonstrated by cDNA analysis. Our results broaden the spectrum of mutations in LRP5 and provide the first functional data on splice aberrations.

  10. Splice Site Mutations in the ATP7A Gene

    DEFF Research Database (Denmark)

    Skjørringe, Tina; Tümer, Zeynep; Møller, Lisbeth Birk

    2011-01-01

    Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12...... mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation...... to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations...

  11. Systematic Analysis of Splice-Site-Creating Mutations in Cancer

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    Reyka G. Jayasinghe

    2018-04-01

    Full Text Available Summary: For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases. : Jayasinghe et al. identify nearly 2,000 splice-site-creating mutations (SCMs from over 8,000 tumor samples across 33 cancer types. They provide a more accurate interpretation of previously mis-annotated mutations, highlighting the importance of integrating data types to understand the functional and the clinical implications of splicing mutations in human disease. Keywords: splicing, RNA, mutations of clinical relevance

  12. Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites.

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    Straniero, Letizia; Rimoldi, Valeria; Soldà, Giulia; Mauri, Lucia; Manfredini, Emanuela; Andreucci, Elena; Bargiacchi, Sara; Penco, Silvana; Gesu, Giovanni P; Del Longo, Alessandra; Piozzi, Elena; Asselta, Rosanna; Primignani, Paola

    2015-09-01

    Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient.

  13. A functional alternative splicing mutation in AIRE gene causes autoimmune polyendocrine syndrome type 1.

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    Junyu Zhang

    Full Text Available Autoimmune polyendocrine syndrome type 1 (APS-1 is a rare autosomal recessive disease defined by the presence of two of the three conditions: mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease. Loss-of-function mutations of the autoimmune regulator (AIRE gene have been linked to APS-1. Here we report mutational analysis and functional characterization of an AIRE mutation in a consanguineous Chinese family with APS-1. All exons of the AIRE gene and adjacent exon-intron sequences were amplified by PCR and subsequently sequenced. We identified a homozygous missense AIRE mutation c.463G>A (p.Gly155Ser in two siblings with different clinical features of APS-1. In silico splice-site prediction and minigene analysis were carried out to study the potential pathological consequence. Minigene splicing analysis and subsequent cDNA sequencing revealed that the AIRE mutation potentially compromised the recognition of the splice donor of intron 3, causing alternative pre-mRNA splicing by intron 3 retention. Furthermore, the aberrant AIRE transcript was identified in a heterozygous carrier of the c.463G>A mutation. The aberrant intron 3-retaining transcript generated a truncated protein (p.G155fsX203 containing the first 154 AIRE amino acids and followed by 48 aberrant amino acids. Therefore, our study represents the first functional characterization of the alternatively spliced AIRE mutation that may explain the pathogenetic role in APS-1.

  14. Splice site mutations in the ATP7A gene.

    Directory of Open Access Journals (Sweden)

    Tina Skjørringe

    Full Text Available Menkes disease (MD is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12 mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations identified in 12 patients with milder phenotypes were predicted to have no significant effect on splicing, which concurs with the presence of wild-type transcript in 7 out of 9 patients investigated in vivo. Both the in silico predictions and the in vivo results support the hypothesis previously suggested by us and others, that the presence of some wild-type transcript is correlated to a milder phenotype.

  15. Splicing aberrations caused by constitutional RB1 gene mutations in ...

    Indian Academy of Sciences (India)

    Analysis of RB1 mRNA from blood leukocytes of patients with retinoblastoma identified the effects of mutations involving consensus splice site, exonic substitution and whole-exon deletions identified in genomic DNA of these patients. In addition, this study identified mutations in cases in which no mutations were detectable ...

  16. Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II.

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    Rimoldi, Valeria; Straniero, Letizia; Asselta, Rosanna; Mauri, Lucia; Manfredini, Emanuela; Penco, Silvana; Gesu, Giovanni P; Del Longo, Alessandra; Piozzi, Elena; Soldà, Giulia; Primignani, Paola

    2014-03-01

    Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type II (OCA2) is one of the four commonly-recognized forms of albinism, and is determined by mutation in the OCA2 gene. In the present study, we investigated the molecular basis of OCA2 in two siblings and one unrelated patient. The mutational screening of the OCA2 gene identified two hitherto-unknown putative splicing mutations. The first one (c.1503+5G>A), identified in an Italian proband and her affected sibling, lies in the consensus sequence of the donor splice site of OCA2 intron 14 (IVS14+5G>A), in compound heterozygosity with a frameshift mutation, c.1450_1451insCTGCCCTGACA, which is predicted to determine the premature termination of the polypeptide chain (p.I484Tfs*19). In-silico prediction of the effect of the IVS14+5G>A mutation on splicing showed a score reduction for the mutant splice site and indicated the possible activation of a newly-created deep-intronic acceptor splice site. The second mutation is a synonymous transition (c.2139G>A, p.K713K) involving the last nucleotide of exon 20. This mutation was found in a young African albino patient in compound heterozygosity with a previously-reported OCA2 missense mutation (p.T404M). In-silico analysis predicted that the mutant c.2139G>A allele would result in the abolition of the splice donor site. The effects on splicing of these two novel mutations were investigated using an in-vitro hybrid-minigene approach that led to the demonstration of the causal role of the two mutations and to the identification of aberrant transcript variants. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Insertion of a T next to the donor splice site of intron 1 causes aberrantly spliced mRNA in a case of infantile GM1-gangliosidosis.

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    Morrone, A; Morreau, H; Zhou, X Y; Zammarchi, E; Kleijer, W J; Galjaard, H; d'Azzo, A

    1994-01-01

    The lysosomal storage disorders GM1-gangliosidosis and Morquio B syndrome are caused by a complete or partial deficiency of acid beta-galactosidase. Here, we have characterized the mutation segregating in a family with two siblings affected by the severe infantile form of GM1-gangliosidosis. In total mRNA preparations derived from the patients' fibroblasts at least two aberrantly spliced beta-galactosidase transcripts (1 and 2) have been identified. Both transcripts contain a 20 nucleotide (nt) insertion derived from the 5' end of intron 1 of the beta-galactosidase gene. Furthermore, in transcript 2 sequences encoded by exon II are deleted during the splicing process. Comparison of the 20-nt insertion with wild-type intronic sequences indicated that in the genomic DNA of the patients an extra T nucleotide is present immediately downstream of the conserved GT splice donor dinucleotide of intron 1. Both patients are homozygous for the T nucleotide insertion. We propose that this single base insertion is the mutation responsible for aberrant splicing of beta-galactosidase pre-mRNA, giving rise to transcripts that cannot encode a normal protein.

  18. A novel splicing mutation in the V2 vasopressin receptor

    DEFF Research Database (Denmark)

    Kamperis, Konstantinos; Siggaard, C; Herlin, Troels

    2000-01-01

    as clinical investigations comprising a fluid deprivation test and a 1-deamino-8-D-arginine-vasopressin (dDAVP) infusion test in the study subject and his mother. We found a highly unusual, novel, de novo 1447A-->C point mutation (gDNA), involving the invariable splice acceptor of the second intron...... of the gene in both the affected male (hemizygous) and his mother (heterozygous). This mutation is likely to cause aberrant splicing of the terminal intron of the gene, leading to a non-functional AVP receptor. The clinical studies were consistent with such a hypothesis, as the affected subject had a severe...

  19. Splicing aberrations caused by constitutional RB1 gene mutations in ...

    Indian Academy of Sciences (India)

    . [Parsam VL, Ali MJ, Honavar SG, Vemuganti GK and Kannabiran C 2011 Splicing aberrations caused by constitutional RB1 gene mutations in retinoblastoma. J. Biosci. 36 281–287] DOI 10.1007/s12038-011-9062-9. 1. Introduction.

  20. Splicing aberrations caused by constitutional RB1 gene mutations in ...

    Indian Academy of Sciences (India)

    Analysis of RB1 mRNA from blood leukocytes of patients with retinoblastoma identified the effects of mutations involving consensus splice site, .... bilateral Rb. Genomic DNA analysis from peripheral blood was as described by Parsam .... the patterns are not always the same in different studies (Klutz et al. 2002; Taylor et al.

  1. A BRCA2 mutation incorrectly mapped in the original BRCA2 reference sequence, is a common West Danish founder mutation disrupting mRNA splicing

    DEFF Research Database (Denmark)

    Thomassen, Mads; Pedersen, Inge Søkilde; Vogel, Ida

    2011-01-01

    Inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 predispose carriers to breast and ovarian cancer. The authors have identified a mutation in BRCA2, 7845+1G>A (c.7617+1G>A), not previously regarded as deleterious because of incorrect mapping of the splice junction in the originally...... published genomic reference sequence. This reference sequence is generally used in many laboratories and it maps the mutation 16 base pairs inside intron 15. However, according to the recent reference sequences the mutation is located in the consensus donor splice sequence. By reverse transcriptase analysis......, loss of exon 15 in the final transcript interrupting the open reading frame was demonstrated. Furthermore, the mutation segregates with a cancer phenotype in 18 Danish families. By genetic analysis of more than 3,500 Danish breast/ovarian cancer risk families, the mutation was identified as the most...

  2. G to A substitution in 5{prime} donor splice site of introns 18 and 48 of COL1A1 gene of type I collagen results in different splicing alternatives in osteogenesis imperfecta type I cell strains

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    Willing, M.; Deschenes, S. [Univ. of Iowa, Iowa City, IA (United States)

    1994-09-01

    We have identified a G to A substitution in the 5{prime} donor splice site of intron 18 of one COL1A1 allele in two unrelated families with osteogenesis imperfecta (OI) type I. A third OI type I family has a G to A substitution at the identical position in intron 48 of one COL1A1 allele. Both mutations abolish normal splicing and lead to reduced steady-state levels of mRNA from the mutant COL1A1 allele. The intron 18 mutation leads to both exon 18 skipping in the mRNA and to utilization of a single alternative splice site near the 3{prime} end of exon 18. The latter results in deletion of the last 8 nucleotides of exon 18 from the mRNA, a shift in the translational reading-frame, and the creation of a premature termination codon in exon 19. Of the potential alternative 5{prime} splice sites in exon 18 and intron 18, the one utilized has a surrounding nucleotide sequence which most closely resembles that of the natural splice site. Although a G to A mutation was detected at the identical position in intron 48 of one COL1A1 allele in another OI type I family, nine complex alternative splicing patterns were identified by sequence analysis of cDNA clones derived from fibroblast mRNA from this cell strain. All result in partial or complete skipping of exon 48, with in-frame deletions of portions of exons 47 and/or 49. The different patterns of RNA splicing were not explained by their sequence homology with naturally occuring 5{prime} splice sites, but rather by recombination between highly homologous exon sequences, suggesting that we may not have identified the major splicing alternative(s) in this cell strain. Both G to A mutations result in decreased production of type I collagen, the common biochemical correlate of OI type I.

  3. A novel 5' ATRX mutation with splicing consequences in acquired alpha thalassemia-myelodysplastic syndrome.

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    Nelson, Maria E; Thurmes, Paul J; Hoyer, James D; Steensma, David P

    2005-11-01

    Acquired alpha thalassemia (hemoglobin H (HbH) disease) is a rare complication of neoplastic chronic myeloid disorders, especially myelodysplastic syndrome. Acquired HbH has recently been associated with mutations in an X-linked gene, ATRX, previously linked to inherited ATR-X syndrome (alpha thalassemia-retardation-X linked). A Swiss man with chronic myelomonocytic leukemia complicated by various autoimmune disorders and by strikingly microcytic, hypochromic anemia was analyzed for the presence of acquired HbH. After HbH detection, we sought an underlying genetic cause. We used denaturing high-performance liquid chromatography to screen for an ATRX mutation, and measured ATRX expression by reverse transcriptase polymerase chain reaction. The patient had 50% HbH-containing cells on supravital staining. Marrow karyotype and the alpha globin cluster were normal. A clonally-restricted ATRX point mutation was detected in the conserved splice donor motif in intron 4 (IVS 4 +2 T-->C). Plasmid vector cloning of patient ATRX cDNA demonstrated both exon 4 skipping and partial intron retention with activation of a cryptic splice site, both outcomes resulting in frameshifts with premature stop codon generation in exon 5 and near-decimation of ATRX expression in myeloid cells. Normal exon 6 alternative splicing was retained. Intronic ATRX mutations with splicing consequences, uncommon in inherited ATR-X syndrome because of their devastating effect on expression of functional protein, should be routinely sought when undertaking molecular analysis of acquired HbH disease. Detection of an acquired ATRX mutation can help support clonality in karyotypically normal ambiguous myeloid disorders with HbH.

  4. Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

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    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

  5. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

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    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.

  6. Clinical presentation and molecular identification of four uncommon alpha globin variants in Thailand. Initiation codon mutation of α2-globin Gene (HBA2:c.1delA), donor splice site mutation of α1-globin gene (IVSI-1, HBA1:c.95 + 1G>A), hemoglobin Queens Park/Chao Pra Ya (HBA1:c.98T>A) and hemoglobin Westmead (HBA2:c.369C>G).

    Science.gov (United States)

    Viprakasit, Vip; Ekwattanakit, Supachai; Chalaow, Nipon; Riolueang, Suchada; Wijit, Sirirat; Tanyut, Porntep; Chat-Uthai, Nunthawut; Tachavanich, Kalaya

    2014-01-01

    Alpha thalassemia is the most common genetic disease in the world with the prevalence of carriers ranging from 5-50% in several populations. Coinheritance of two defective α-globin genes usually gives rise to a symptomatic condition, hemoglobin (Hb) H disease. Previously, it has been suggested from several studies in different populations that nondeletional Hb H disease (--/α(T)α or --/αα(T)) is generally more severe than the deletional type (--/-α). In this report, we describe four rare nondeletional α-thalassemia mutations in Thai individuals, including initiation codon mutation (HBA2:c.1delA), donor splice site mutation (IVSI-1, HBA1:c.95 + 1G>A), Hb Queens Park (HBA1:c.98T>A) [α32(B13)Met>Lys], and Hb Westmead (HBA2:c.369C>G) [α122(H5)His>Gln]. Interactions of the first three mutations with the α(0)-thalassemia resulted in nondeletional Hb H disease; however, their clinical presentations were rather mild and some were detected accidentally. This suggests that a genotype-phenotype correlation of α-thalassemia syndrome might be more heterogeneous and so the type of mutation does not simply imply the prediction of the resulting phenotype. Our data will be of use in future genetic counseling of such conditions that are increasingly identified thanks to the improvement of molecular analysis in routine laboratories. © 2013 S. Karger AG, Basel.

  7. Novel aberrant splicings caused by a splice site mutation (IVS1a+5g>a) in F7 gene.

    Science.gov (United States)

    Ding, Qiulan; Wu, Wenman; Fu, Qihua; Wang, Xuefeng; Hu, Yiqun; Wang, Hongli; Wang, Zhenyi

    2005-06-01

    Low FVII coagulant activity (FVII:C 8.2%) and antigen level (FVII:Ag 34.1%) in a 46-year-old Chinese male led to a diagnosis of coagulation factor VII (FVII) deficiency. Compound heterozygous mutations were identified in his F 7 gene:a G to A transition in the 5' donor splice site of intron 1a (IVS1a+5g>a) and a T to G transition at the nucleotide position 10961 in exon 8, resulting in a His to Gln substitution at amino acid residue 348. An analysis of ectopic transcripts of F7 in the leukocytes of the patient reveals that the mutation (IVS1a+5g>a) is associated with two novel aberrant patterns of splicing. The predominant alternative transcript removes exon 2, but retains intron 3, which shifts the reading frame and predicts a premature translation termination at the nucleotide positions 2-4 in intron 3. The minor alternative transcript skips both exon 2 and exon 3 (FVII Delta 2, 3), leading to an in-frame deletion of the propeptide and gamma-carboxylated glutamic acid (Gla) domains of mature FVII protein. In vitro expression studies of the alternative transcript FVII Delta 2,3 by transient transfection of HEK 293 cells with PcDNA 3.1(-) expression vector showed that although the mutant protein could be secreted, no pro-coagulation activity was detected. The coexistence of the two abnormal transcripts and a heterozygous mutation His348Gln, explained the patient's phenotype.

  8. Somatic Mutational Landscape of Splicing Factor Genes and Their Functional Consequences across 33 Cancer Types

    Directory of Open Access Journals (Sweden)

    Michael Seiler

    2018-04-01

    Full Text Available Summary: Hotspot mutations in splicing factor genes have been recently reported at high frequency in hematological malignancies, suggesting the importance of RNA splicing in cancer. We analyzed whole-exome sequencing data across 33 tumor types in The Cancer Genome Atlas (TCGA, and we identified 119 splicing factor genes with significant non-silent mutation patterns, including mutation over-representation, recurrent loss of function (tumor suppressor-like, or hotspot mutation profile (oncogene-like. Furthermore, RNA sequencing analysis revealed altered splicing events associated with selected splicing factor mutations. In addition, we were able to identify common gene pathway profiles associated with the presence of these mutations. Our analysis suggests that somatic alteration of genes involved in the RNA-splicing process is common in cancer and may represent an underappreciated hallmark of tumorigenesis. : Seiler et al. report that 119 splicing factor genes carry putative driver mutations over 33 tumor types in TCGA. The most common mutations appear to be mutually exclusive and are associated with lineage-independent altered splicing. Samples with these mutations show deregulation of cell-autonomous pathways and immune infiltration. Keywords: splicing, SF3B1, U2AF1, SRSF2, RBM10, FUBP1, cancer, mutation

  9. Characterization of a splicing mutation in group A xeroderma pigmentosum

    International Nuclear Information System (INIS)

    Satokata, Ichiro; Tanaka, Kiyoji; Miura, Naoyuki; Miyamoto, Iwai; Okada, Yoshio; Satoh, Yoshiaki; Kondo, Seiji

    1990-01-01

    The molecular basis of group A xeroderma pigmentosum (WP) was investigated by comparison of the nucleotide sequences of multiple clones of the XP group A complementing gene (XPAC) from a patient with group A XP with that of a normal gene. The clones showed a G → C substitution at the 3' splice acceptor site of intron 3, which altered the obligatory AG acceptor dinucleotide to AC. Nucleotide sequencing of cDNAs amplified by the polymerase chain reaction revealed that this single base substitution abolishes the canonical 3' splice site, thus creating two abnormally spliced mRNA forms. The larger form is identical with normal mRNA except for a dinucleotide deletion at the 5' end of exon 4. This deletion results in a frameshift with premature translation termination in exon 4. The smaller form has a deletion of the entire exon 3 and the dinucleotide at the 5' end of exon 4. The result of a transfection study provided additional evidence that this single base substitution is the disease-causing mutation. This single base substitution creates a new cleavage site for the restriction nuclease AlwNI. Analysis of AlwNI restriction fragment length polymorphism showed a high frequency of this mutation in Japanese patients with group A XP: 16 of 21 unrelated Japanese patients were homozygous and 4 were heterozygous for this mutation. However, 11 Caucasians and 2 Blacks with group A XP did not have this mutant allele. The polymorphic AlwNI restriction fragments are concluded to be useful for diagnosis of group A XP in Japanese subjects, including prenatal cases and carriers

  10. SQSTM1 splice site mutation in distal myopathy with rimmed vacuoles.

    Science.gov (United States)

    Bucelli, Robert C; Arhzaouy, Khalid; Pestronk, Alan; Pittman, Sara K; Rojas, Luisa; Sue, Carolyn M; Evilä, Anni; Hackman, Peter; Udd, Bjarne; Harms, Matthew B; Weihl, Conrad C

    2015-08-25

    To identify the genetic etiology and characterize the clinicopathologic features of a novel distal myopathy. We performed whole-exome sequencing on a family with an autosomal dominant distal myopathy and targeted exome sequencing in 1 patient with sporadic distal myopathy, both with rimmed vacuolar pathology. We also evaluated the pathogenicity of identified mutations using immunohistochemistry, Western blot analysis, and expression studies. Sequencing identified a likely pathogenic c.1165+1 G>A splice donor variant in SQSTM1 in the affected members of 1 family and in an unrelated patient with sporadic distal myopathy. Affected patients had late-onset distal lower extremity weakness, myopathic features on EMG, and muscle pathology demonstrating rimmed vacuoles with both TAR DNA-binding protein 43 and SQSTM1 inclusions. The c.1165+1 G>A SQSTM1 variant results in the expression of 2 alternatively spliced SQSTM1 proteins: 1 lacking the C-terminal PEST2 domain and another lacking the C-terminal ubiquitin-associated (UBA) domain, both of which have distinct patterns of cellular and skeletal muscle localization. SQSTM1 is an autophagic adaptor that shuttles aggregated and ubiquitinated proteins to the autophagosome for degradation via its C-terminal UBA domain. Similar to mutations in VCP, dominantly inherited mutations in SQSTM1 are now associated with rimmed vacuolar myopathy, Paget disease of bone, amyotrophic lateral sclerosis, and frontotemporal dementia. Our data further suggest a pathogenic connection between the disparate phenotypes. © 2015 American Academy of Neurology.

  11. Recurrent Hyperparathyroidism Due to a Novel CDC73 Splice Mutation.

    Science.gov (United States)

    Hattangady, Namita Ganesh; Wilson, Tremika Le-Shan; Miller, Barbra Sue; Lerario, Antonio Marcondes; Giordano, Thomas James; Choksi, Palak; Else, Tobias

    2017-08-01

    The recognition of hereditary causes of primary hyperparathyroidism (pHPT) is important because clinical care and surveillance differ significantly between sporadic and hereditary pHPT. In addition, the increasing number of genetic tests poses a challenge to classify mutations as benign or pathogenic. Functional work-up of variants remains a mainstay to provide evidence for pathogenicity. We describe a 52-year-old male patient with recurrent pHPT since age 35 years. Despite several neck surgeries with complete parathyroidectomy, he experienced persistent pHPT, necessitating repeated surgery for a forearm autotransplant, which finally resulted in unmeasurable parathyroid hormone (PTH) levels. Genetic testing revealed a new CDC73 variant (c.238-8G>A [IVS2-8G>A]), initially classified as a variant of uncertain significance. Parathyroid tissue from the initial surgeries showed loss of heterozygosity. Using an RT-PCR approach, we show that the mutation leads to the use of a cryptic splice site in peripheral mononuclear cells. In addition, a minigene approach confirms the use of the cryptic splice site in a heterologous cell system. The novel c.238-8G>A CDC73 variant activates a cryptic splice site, and the functional data provided justify the classification as a likely pathogenic variant. Our results underscore the importance of functional work-up for variant classification in the absence of other available data, such as presence in disease-specific databases, other syndromic clinical findings, or family history. In addition, the presented case exemplifies the importance to consider a hereditary condition in young patients with pHPT, particularly those with multi-gland involvement. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

  12. Functional Characterization of NIPBL Physiological Splice Variants and Eight Splicing Mutations in Patients with Cornelia de Lange Syndrome

    Directory of Open Access Journals (Sweden)

    María E. Teresa-Rodrigo

    2014-06-01

    Full Text Available Cornelia de Lange syndrome (CdLS is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21 or functionally associated factors (NIPBL, HDAC8 of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B’. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.

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

    Science.gov (United States)

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

    2014-12-01

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

  14. TCOF1 mutation database: novel mutation in the alternatively spliced exon 6A and update in mutation nomenclature.

    Science.gov (United States)

    Splendore, Alessandra; Fanganiello, Roberto D; Masotti, Cibele; Morganti, Lucas S C; Passos-Bueno, M Rita

    2005-05-01

    Recently, a novel exon was described in TCOF1 that, although alternatively spliced, is included in the major protein isoform. In addition, most published mutations in this gene do not conform to current mutation nomenclature guidelines. Given these observations, we developed an online database of TCOF1 mutations in which all the reported mutations are renamed according to standard recommendations and in reference to the genomic and novel cDNA reference sequences (www.genoma.ib.usp.br/TCOF1_database). We also report in this work: 1) results of the first screening for large deletions in TCOF1 by Southern blot in patients without mutation detected by direct sequencing; 2) the identification of the first pathogenic mutation in the newly described exon 6A; and 3) statistical analysis of pathogenic mutations and polymorphism distribution throughout the gene.

  15. Features of 5'-splice-site efficiency derived from disease-causing mutations and comparative genomics

    DEFF Research Database (Denmark)

    Roca, Xavier; Olson, Andrew J; Rao, Atmakuri R

    2008-01-01

    Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonuria, can be caused by 5'-splice-site (5'ss) mutations that are not predicted to disrupt splicing, according to position weight matrices. By using comparative genomics, we identify pairwise dependencies...

  16. Deletion of a splice donor site ablates expression of the following exon and produces an unphosphorylated RB protein unable to bind SV40 T antigen.

    Science.gov (United States)

    Shew, J Y; Chen, P L; Bookstein, R; Lee, E Y; Lee, W H

    1990-01-01

    Studies of mutated retinoblastoma (RB) proteins in human tumor cells potentially reveal regions of the normal RB gene product that are required for its cancer suppression function. We here characterize a mutated RB protein of Mr 104,000 (p104) from a primary small-cell lung carcinoma. Unlike normal RB protein (pp110RB), p104 was unphosphorylated and unable to bind T antigen of SV40 both in vivo and in vitro. On the other hand, nuclear localization and DNA binding activity were preserved in the mutated protein. p104 was immunoprecipitable with four separate polyclonal antibodies recognizing different epitopes of the RB polypeptide, suggesting the presence of most exons in their correct reading frame. Following reverse transcription and in vitro amplification, RB mRNA from this tumor was shown to lack nucleotides encoded by exon 16. Analysis of genomic DNA from this tumor showed that exon 16 and its flanking splice donor and acceptor sequences were present and entirely normal; however, a 43-base pair (bp) region containing the splice donor site of intron 15 was deleted instead. Exon 15 was joined directly to exon 17 during mRNA processing via a cryptic splice donor site; exon 16 was presumably skipped because the preceding mutated intron was of insufficient length (less than 80 bp) for normal RB mRNA processing. These results demonstrate that loss of a single small exon disrupts several important biochemical properties of RB protein. In addition, sequence features of the 43-bp depletion suggest involvement of a novel deletional mechanism.

  17. Mutational analyses on X-linked adrenoleukodystrophy reveal a novel cryptic splicing and three missense mutations in the ABCD1 gene.

    Science.gov (United States)

    Hung, Kun-Long; Wang, Jinn-Shyan; Keng, Wee Teik; Chen, Hui-Ju; Liang, Jao-Shwann; Ngu, Lock Hock; Lu, Jyh-Feng

    2013-09-01

    X-linked adrenoleukodystrophy is caused by a defective peroxisomal membrane transporter, ABCD1, responsible for transporting very-long-chain fatty acid substrate into peroxisomes for degradation. The main biochemical defect, which is also one of the major diagnostic hallmarks, of X-linked adrenoleukodystrophy is the accumulation of saturated very-long-chain fatty acids in all tissues and body fluids. Direct and reverse-transcribed polymerase chain reactions followed by DNA sequencing-based mutational analyses were performed on one Taiwanese and three Malaysian X-linked adrenoleukodystrophy families. A novel splicing donor site mutation (c.1272+1g>a) was identified in a Taiwanese X-linked adrenoleukodystrophy patient, resulting in a deletion of 121 bp and a premature stop codon (p.Val425fs*92) in messenger-RNA transcript. This deletion is caused by the activation of a cryptic splicing donor site in exon 4 of the ABCD1 gene, which is consistent with the prediction by several online algorithms. In addition, three previously described missense mutations (c.965T>C, c.1978C>T, and c.2006A>G), leading to aberrant ABCD1 of p.Leu322Pro, p.Arg660Trp, and p.His669Arg, were also identified in Malaysian probands. This is the first report to unveil unequivocally that cryptic splicing-induced aberrant messenger-RNA carrying an internal frameshift deletion results from an intronic mutation in the ABCD1 gene. Furthermore, a polymorphism in intron 9 (c.1992-32c/t; refSNP: rs4898368) of the ABCD1 gene was commonly observed in both Taiwanese and Malaysian populations. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. A missense mutation (Q279R) in the fumarylacetoacetate hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation.

    Science.gov (United States)

    Dreumont, N; Poudrier, J A; Bergeron, A; Levy, H L; Baklouti, F; Tanguay, R M

    2001-01-01

    Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was found in tumor regions while a healthy region contained enzyme-expressing nodules. Analysis of DNA from a FAH expressing region showed that the expression of the protein was due to correction of the Q279R mutation. RT-PCR was used to assess if Q279R RNA was produced in the liver cells and in fibroblasts from the patient. Normal mRNA was found in the liver region where the mutation had reverted while splicing intermediates were found in non-expressing regions suggesting that the Q279R mutation acted as a splicing mutation in vivo. Sequence of transcripts showed skipping of exon 8 alone or together with exon 9. Using minigenes in transfection assays, the Q279R mutation was shown to induce skipping of exon 9 when placed in a constitutive splicing environment. These data suggest that the putative missense mutation Q279R in the FAH gene acts as a splicing mutation in vivo. Moreover FAH expression can be partially restored in certain liver cells as a result of a reversion of the Q279R mutation and expansion of the corrected cells.

  19. Novel mutations in EVC cause aberrant splicing in Ellis-van Creveld syndrome.

    Science.gov (United States)

    Shi, Lisong; Luo, Chunyan; Ahmed, Mairaj K; Attaie, Ali B; Ye, Xiaoqian

    2016-04-01

    Ellis-van Creveld syndrome (EvC) is a rare autosomal recessive disorder characterized by disproportionate chondrodysplasia, postaxial polydactyly, nail dystrophy, dental abnormalities and in a proportion of patients, congenital cardiac malformations. Weyers acrofacial dysostosis (Weyers) is another dominantly inherited disorder allelic to EvC syndrome but with milder phenotypes. Both disorders can result from loss-of-function mutations in either EVC or EVC2 gene, and phenotypes associated with the two gene mutations are clinically indistinguishable. We present here a clinical and molecular analysis of a Chinese family manifested specific features of EvC syndrome. Sequencing of both EVC and EVC2 identified two novel heterozygous splice site mutations c.384+5G>C in intron 3 and c.1465-1G>A in intron 10 in EVC, which were inherited from mother and father, respectively. In vitro minigene expression assay, RT-PCR and sequencing analysis demonstrated that c.384+5G>C mutation abolished normal splice site and created a new cryptic acceptor site within exon 4, whereas c.1465-1G>A mutation affected consensus splice junction site and resulted in full exon 11 skipping. These two aberrant pre-mRNA splicing processes both produced in-frame abnormal transcripts that possibly led to abolishment of important functional domains. To our knowledge, this is the first report of EVC mutations that cause EvC syndrome in Chinese population. Our data revealed that EVC splice site mutations altered splicing pattern and helped elucidate the pathogenesis of EvC syndrome.

  20. Depletion of somatic mutations in splicing-associated sequences in cancer genomes.

    Science.gov (United States)

    Hurst, Laurence D; Batada, Nizar N

    2017-11-07

    An important goal of cancer genomics is to identify systematically cancer-causing mutations. A common approach is to identify sites with high ratios of non-synonymous to synonymous mutations; however, if synonymous mutations are under purifying selection, this methodology leads to identification of false-positive mutations. Here, using synonymous somatic mutations (SSMs) identified in over 4000 tumours across 15 different cancer types, we sought to test this assumption by focusing on coding regions required for splicing. Exon flanks, which are enriched for sequences required for splicing fidelity, have ~ 17% lower SSM density compared to exonic cores, even after excluding canonical splice sites. While it is impossible to eliminate a mutation bias of unknown cause, multiple lines of evidence support a purifying selection model above a mutational bias explanation. The flank/core difference is not explained by skewed nucleotide content, replication timing, nucleosome occupancy or deficiency in mismatch repair. The depletion is not seen in tumour suppressors, consistent with their role in positive tumour selection, but is otherwise observed in cancer-associated and non-cancer genes, both essential and non-essential. Consistent with a role in splicing modulation, exonic splice enhancers have a lower SSM density before and after controlling for nucleotide composition; moreover, flanks at the 5' end of the exons have significantly lower SSM density than at the 3' end. These results suggest that the observable mutational spectrum of cancer genomes is not simply a product of various mutational processes and positive selection, but might also be shaped by negative selection.

  1. Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events.

    Science.gov (United States)

    Liu, Jinfeng; Lee, William; Jiang, Zhaoshi; Chen, Zhongqiang; Jhunjhunwala, Suchit; Haverty, Peter M; Gnad, Florian; Guan, Yinghui; Gilbert, Houston N; Stinson, Jeremy; Klijn, Christiaan; Guillory, Joseph; Bhatt, Deepali; Vartanian, Steffan; Walter, Kimberly; Chan, Jocelyn; Holcomb, Thomas; Dijkgraaf, Peter; Johnson, Stephanie; Koeman, Julie; Minna, John D; Gazdar, Adi F; Stern, Howard M; Hoeflich, Klaus P; Wu, Thomas D; Settleman, Jeff; de Sauvage, Frederic J; Gentleman, Robert C; Neve, Richard M; Stokoe, David; Modrusan, Zora; Seshagiri, Somasekar; Shames, David S; Zhang, Zemin

    2012-12-01

    Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology.

  2. Recurrent disruption of the Imu splice donor site in t(14;18) positive lymphomas: a potential molecular basis for aberrant downstream class switch recombination.

    Science.gov (United States)

    Ruminy, Philippe; Jardin, Fabrice; Penther, Dominique; Picquenot, Jean-Michel; Parmentier, Françoise; Buchonnet, Gérard; Bertrand, Philippe; Tilly, Hervé; Bastard, Christian

    2007-08-01

    t(14;18) positive lymphomas are mature germinal center B-cell neoplasms. In agreement with this cellular origin, most have somatically mutated immunoglobulin variable genes and the IGH@ locus has almost always been reorganized by class switch recombination (CSR). However, contrasting with normal B-cells, a majority of cases still express an IgM while the constant genes are normally rearranged only on the non-productive allele. Concurrently, aberrant intra-allelic junctions involving downstream switch regions, with a lack of engagement of the switch mu (Smu), often accumulate on the functional alleles, suggesting some recurrent CSR perturbation during the onset of the disease. To clarify these surprising observations, we addressed the accessibility of the Smu to the CSR machinery in a large series of patients by characterizing the mutations that are expected to accumulate at this place upon CSR activation. Our data indicate that the Smu is mutated in a large majority of cases, often on both alleles, indicating that these cells usually reach a differentiation stage where CSR is activated and where this region remains accessible. Interestingly, we also identified a significant cluster of mutations at the splicing donor site of the first exon of the Smu germline transcripts, on the functional allele. This location suggests a possible relation with CSR perturbations in lymphoma and the clustering points to a probable mechanism of selection. In conclusion, our data suggest that an acquired mutation at the splicing donor site of the Smu transcripts may participate in the selection of lymphoma cells and play a significant role during the onset of the disease.

  3. Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT.

    Science.gov (United States)

    Sposito, Teresa; Preza, Elisavet; Mahoney, Colin J; Setó-Salvia, Núria; Ryan, Natalie S; Morris, Huw R; Arber, Charles; Devine, Michael J; Houlden, Henry; Warner, Thomas T; Bushell, Trevor J; Zagnoni, Michele; Kunath, Tilo; Livesey, Frederick J; Fox, Nick C; Rossor, Martin N; Hardy, John; Wray, Selina

    2015-09-15

    The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC-neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing. © The Author 2015. Published by Oxford University Press.

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

    Science.gov (United States)

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

    2016-10-13

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

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  6. A Missense Mutation (Q279R) in the Fumarylacetoacetate Hydrolase Gene, Responsible for Hereditary Tyrosinemia, Acts as a Splicing Mutation

    OpenAIRE

    Dreumont, Natacha; Poudrier, Jacques A; Bergeron, Anne; Baklouti, Faouzi; Tanguay, Robert M; Levy, Harvey Louis

    2001-01-01

    Abstract Background Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was...

  7. Analysis of 30 putative BRCA1 splicing mutations in hereditary breast and ovarian cancer families identifies exonic splice site mutations that escape in silico prediction.

    Directory of Open Access Journals (Sweden)

    Barbara Wappenschmidt

    Full Text Available Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis.

  8. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

    DEFF Research Database (Denmark)

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S

    2008-01-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian...... DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent...... cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor...

  9. A novel RNA-splicing mutation in TRAPPC2 gene causing X-linked ...

    Indian Academy of Sciences (India)

    large Chinese family with SEDT and identified a novel RNA- splicing mutation in the TRAPPC2 gene. Materials and methods. Patients. The proband of this family is 38-year-old man who sought medical attention because of chronic pain in weight-bearing joints. The proband's height is 135 cm and his arm span is 155 cm.

  10. Ab initio prediction of mutation-induced cryptic splice-site activation and exon skipping

    Czech Academy of Sciences Publication Activity Database

    Divina, Petr; Kvitkovicova, Andrea; Buratti, E.; Vorechovsky, I.

    2009-01-01

    Roč. 17, č. 6 (2009), s. 759-765 ISSN 1018-4813 Institutional research plan: CEZ:AV0Z50520514 Keywords : mutation * cryptic splice site * exon skipping Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.564, year: 2009

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    to be compound heterozygotes for mutations in the IR gene. The maternal allele was alternatively spliced in exon 17 due to a point mutation in the -1 donor splice site of the exon. The abnormal skipping of exon 17 shifts the amino acid reading frame and leads to a truncated IR, missing the entire tyrosine kinase......The insulin receptor (IR) in two brothers with a rare syndrome of congenital muscle fiber type disproportion myopathy (CFTDM) associated with diabetes and severe insulin resistance was studied. By direct sequencing of Epstein-Barr virus-transformed lymphocytes both patients were found...

  12. Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5' splice site

    DEFF Research Database (Denmark)

    Martínez-Pizarro, Ainhoa; Dembic, Maja; Pérez, Belén

    2018-01-01

    Phenylketonuria (PKU), one of the most common inherited diseases of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Recently, PAH exon 11 was identified as a vulnerable exon due to a weak 3' splice site, with different exonic mutations affecting exon 11...

  13. Splice, insertion-deletion and nonsense mutations that perturb the phenylalanine hydroxylase transcript cause phenylketonuria in India.

    Science.gov (United States)

    Bashyam, Murali D; Chaudhary, Ajay K; Kiran, Manjari; Nagarajaram, Hampapathalu A; Devi, Radha Rama; Ranganath, Prajnya; Dalal, Ashwin; Bashyam, Leena; Gupta, Neerja; Kabra, Madhulika; Muranjan, Mamta; Puri, Ratna D; Verma, Ishwar C; Nampoothiri, Sheela; Kadandale, Jayarama S

    2014-03-01

    Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutational inactivation of the phenylalanine hydroxylase (PAH) gene. Missense mutations are the most common PAH mutation type detected in PKU patients worldwide. We performed PAH mutation analysis in 27 suspected Indian PKU families (including 7 from our previous study) followed by structure and function analysis of specific missense and splice/insertion-deletion/nonsense mutations, respectively. Of the 27 families, disease-causing mutations were detected in 25. A total of 20 different mutations were identified of which 7 "unique" mutations accounted for 13 of 25 mutation positive families. The unique mutations detected exclusively in Indian PKU patients included three recurrent mutations detected in three families each. The 20 mutations included only 5 missense mutations in addition to 5 splice, 4 each nonsense and insertion-deletion mutations, a silent variant in coding region and a 3'UTR mutation. One deletion and two nonsense mutations were characterized to confirm significant reduction in mutant transcript levels possibly through activation of nonsense mediated decay. All missense mutations affected conserved amino acid residues and sequence and structure analysis suggested significant perturbations in the enzyme activity of respective mutant proteins. This is probably the first report of identification of a significantly low proportion of missense PAH mutations from PKU families and together with the presence of a high proportion of splice, insertion-deletion, and nonsense mutations, points to a unique PAH mutation profile in Indian PKU patients. © 2013 Wiley Periodicals, Inc.

  14. A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal.

    Science.gov (United States)

    Mueller, Nancy; Das, Atze T; Berkhout, Ben

    2016-07-21

    RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5' splice site (5'ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5'ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5'ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations.

  15. Identification of a novel splice-site mutation in MIP in a Chinese congenital cataract family.

    Science.gov (United States)

    Jiang, Jin; Jin, Chongfei; Wang, Wei; Tang, Xiajing; Shentu, Xingchao; Wu, Renyi; Wang, Yao; Xia, Kun; Yao, Ke

    2009-01-01

    To map the locus and identify the gene causing autosomal dominant congenital cataract (ADCC) with "snail-like" phenotype in a large Chinese family. Clinical and ophthalmologic examinations were conducted on family members and documented by slit lamp photography. Linkage analysis was performed with an initial 41 microsatellite markers, then 3 additional markers flanking the major intrinsic protein (MIP) gene. Mutations were screened by DNA sequencing and verified by restriction fragment length polymorphism (RFLP) analysis. Significant two-point LOD scores were obtained at 5 markers flanking MIP with the highest 3.08 (theta=0.00) at marker D12S1632. Mutation screening of MIP identified a heterozygous G>A transition at the acceptor splice site of intron 3 (IVS3 -1 G>A), abolishing a BstSF I restriction site in one allele of all the affected individuals. We identified a novel splice-site mutation (IVS3 -1 G>A in MIP) in a Chinese ADCC family. To our knowledge, this is the first report on an acceptor splice-site mutation in human genes associated with ADCC.

  16. Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Pleiss

    2007-04-01

    Full Text Available Appropriate expression of most eukaryotic genes requires the removal of introns from their pre-messenger RNAs (pre-mRNAs, a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.

  17. A spontaneous Fatp4/Scl27a4 splice site mutation in a new murine model for congenital ichthyosis.

    Science.gov (United States)

    Tao, Jianning; Koster, Maranke I; Harrison, Wilbur; Moran, Jennifer L; Beier, David R; Roop, Dennis R; Overbeek, Paul A

    2012-01-01

    Congenital ichthyoses are life-threatening conditions in humans. We describe here the identification and molecular characterization of a novel recessive mutation in mice that results in newborn lethality with severe congenital lamellar ichthyosis. Mutant newborns have a taut, shiny, non-expandable epidermis that resembles cornified manifestations of autosomal-recessive congenital ichthyosis in humans. The skin is stretched so tightly that the newborn mice are immobilized. The genetic defect was mapped to a region near the proximal end of chromosome 2 by SNP analysis, suggesting Fatp4/Slc27a4 as a candidate gene. FATP4 mutations in humans cause ichthyosis prematurity syndrome (IPS), and mutations of Fatp4 in mice have previously been found to cause a phenotype that resembles human congenital ichthyoses. Characterization of the Fatp4 cDNA revealed a fusion of exon 8 to exon 10, with deletion of exon 9. Genomic sequencing identified an A to T mutation in the splice donor sequence at the 3'-end of exon 9. Loss of exon 9 results in a frame shift mutation upstream from the conserved very long-chain acyl-CoA synthase (VLACS) domain. Histological studies revealed that the mutant mice have defects in keratinocyte differentiation, along with hyperproliferation of the stratum basale of the epidermis, a hyperkeratotic stratum corneum, and reduced numbers of secondary hair follicles. Since Fatp4 protein is present primarily at the stratum granulosum and the stratum spinosum, the hyperproliferation and the alterations in hair follicle induction suggest that very long chain fatty acids, in addition to being required for normal cornification, may influence signals from the stratum corneum to the basal cells that help to orchestrate normal skin differentiation.

  18. Congenital lipoid adrenal hyperplasia caused by a novel splicing mutation in the gene for the steroidogenic acute regulatory protein.

    Science.gov (United States)

    González, Alexis A; Reyes, M Loreto; Carvajal, Cristian A; Tobar, Jaime A; Mosso, Lorena M; Baquedano, Paulina; Solar, Antonieta; Venegas, Alejandro; Fardella, Carlos E

    2004-02-01

    Steroidogenic acute regulatory protein (StAR) plays a crucial role in the transport of cholesterol from the cytoplasm to the inner mitochondrial membrane, facilitating its conversion to pregnenolone by cytochrome P450scc. Its essential role in steroidogenesis was demonstrated after observing that StAR gene mutations gave rise to a potentially lethal disease named congenital lipoid adrenal hyperplasia, in which virtually no steroids are produced. We report here a 2-month-old female patient, karyotype 46XY, who presented with growth failure, convulsions, dehydration, hypoglycemia, hyponatremia, hypotension, and severe hyperpigmentation suggestive of adrenal insufficiency. Serum cortisol, 17OH-progesterone, dehydroepiandrosterone sulfate, testosterone, 17OH-pregnenolone, and aldosterone levels were undetectable in the presence of high ACTH and plasma renin activity levels. Immunohistochemical analysis of testis tissues revealed the absence of StAR protein. Molecular analysis of StAR gene demonstrated a homozygous G to T mutation within the splice donor site of exon 1 (IVS1 + 1G>T). Her parents and one brother were heterozygous for this mutation. In vitro analysis of the mutation was performed in COS cells transfected with minigenes coding regions spanning exon-intron 1 to 3 carrying the mutant and the wild-type sequences. RT-PCR analyses of the mutant gene showed an abnormal mRNA transcript of 2430 bp (normal size 433 bp). Sequence analysis of the mutant mRNA demonstrated the retention of intron 1. Immunolocalization of the StAR minigene product detected the peptide in the mitochondria of COS cells transfected with the wild-type minigene but not in those transfected with the mutant minigene. We conclude that this mutation gives rise to a truncated StAR protein, which lacks an important N-terminal region and the entire lipid transfer domain.

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

    Directory of Open Access Journals (Sweden)

    Omar Soukarieh

    2016-01-01

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

  20. Clinical and genetic studies in a family with a new splice-site mutation in the choroideremia gene.

    Science.gov (United States)

    Contestabile, Maria T; Piane, Maria; Cascone, Nikhil C; Pasquale, Nadia; Ciarnella, Angela; Recupero, Santi M; Chessa, Luciana

    2014-01-01

    To describe the clinical and molecular findings of an Italian family with a new mutation in the choroideremia (CHM) gene. We performed a comprehensive ophthalmologic examination, fundus photography, macular optical coherence tomography, perimetry, electroretinography, and fluorescein angiography in an Italian family. The clinical diagnosis was supported by western blot analysis of lymphoblastoid cell lines from patients with CHM and carriers, using a monoclonal antibody against the 415 C-terminal amino acids of Rab escort protein-1 (REP-1). Sequencing of the CHM gene was undertaken on genomic DNA from affected men and carriers; the RNA transcript was analyzed with reverse transcriptase-PCR. The affected men showed a variability in the rate of visual change and in the degree of clinical and functional ophthalmologic involvement, mainly age-related, while the women displayed aspecific areas of chorioretinal degeneration. Western blot did not show a detectable amount of normal REP-1 protein in affected men who were hemizygous for a novel mutation, c.819+2T>A at the donor splicing site of intron 6 of the CHM gene; the mutation was confirmed in heterozygosity in the carriers. Western blot of the REP-1 protein confirmed the clinical diagnosis, and molecular analysis showed the new in-frame mutation, c.819+2T>A, leading to loss of function of the REP-1 protein. These results emphasize the value of a diagnostic approach that correlates genetic and ophthalmologic data for identifying carriers in families with CHM. An early diagnosis might be crucial for genetic counseling of this type of progressive and still untreatable disease.

  1. A novel AVPR2 splice site mutation leads to partial X-linked nephrogenic diabetes insipidus in two brothers.

    Science.gov (United States)

    Schernthaner-Reiter, Marie Helene; Adams, David; Trivellin, Giampaolo; Ramnitz, Mary Scott; Raygada, Margarita; Golas, Gretchen; Faucz, Fabio R; Nilsson, Ola; Nella, Aikaterini A; Dileepan, Kavitha; Lodish, Maya; Lee, Paul; Tifft, Cynthia; Markello, Thomas; Gahl, William; Stratakis, Constantine A

    2016-05-01

    X-linked nephrogenic diabetes insipidus (NDI, OMIM#304800) is caused by mutations in the arginine vasopressin (AVP, OMIM*192340) receptor type 2 (AVPR2, OMIM*300538) gene. A 20-month-old boy and his 8-year-old brother presented with polyuria, polydipsia, and failure to thrive. Both boys demonstrated partial DDAVP (1-desamino-8-D AVP or desmopressin) responses; thus, NDI diagnosis was delayed. While routine sequencing of AVPR2 showed a potential splice site variant, it was not until exome sequencing confirmed the AVPR2 splice site variant and did not reveal any more likely candidates that the patients' diagnosis was made and proper treatment was instituted. Both patients were hemizygous for two AVPR2 variants predicted in silico to affect AVPR2 messenger RNA (mRNA) splicing. A minigene assay revealed that the novel AVPR2 c.276A>G mutation creates a novel splice acceptor site leading to 5' truncation of AVPR2 exon 2 in HEK293 human kidney cells. Both patients have been treated with high-dose DDAVP with a remarkable improvement of their symptoms and accelerated linear growth and weight gain. We present here a unique case of partial X-linked NDI due to an AVPR2 splice site mutation; patients with diabetes insipidus of unknown etiology may harbor splice site mutations that are initially underestimated in their pathogenicity on sequence analysis. • X-linked nephrogenic diabetes insipidus is caused by AVPR2 mutations, and disease severity can vary depending on the functional effect of the mutation. What is New: • We demonstrate here that a splice site mutation in AVPR2 leads to partial X-linked NDI in two brothers. • Treatment with high-dose DDAVP led to improvement of polyuria and polydipsia, weight gain, and growth.

  2. BAP1 missense mutation c.2054 A>T (p.E685V completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line.

    Directory of Open Access Journals (Sweden)

    Arianne Morrison

    Full Text Available BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela¬noma, malignant pleural mesothelioma (MPM, clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val, identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3' end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1 a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5' splice site (GU, which resulted in the deletion of 4 base pairs and presumably protein truncation; 2 a variety of alternative splicing products that led to retention of different introns: introns 14-16; introns 15-16; intron 14 and intron 16; 3 partial intron 14 and 15 retentions caused by activation of alternative 3' splice acceptor sites (AG in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing through creation of a novel 5' splice site and activation of cryptic splice sites. These data support the conclusion that BAP1 c.2054 A&T (p.E685V variant is a pathogenic mutation and contributes to MPM through disruption of normal splicing.

  3. 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.

  4. A short in-frame deletion in NTRK1 tyrosine kinase domain caused by a novel splice site mutation in a patient with congenital insensitivity to pain with anhidrosis

    Directory of Open Access Journals (Sweden)

    Arístegui Javier

    2011-06-01

    Full Text Available Abstract Background Congenital insensitivity to pain with anhidrosis (CIPA is a rare autosomal recessive genetic disease characterized by the lack of reaction to noxious stimuli and anhidrosis. It is caused by mutations in the NTRK1 gene, which encodes the high affinity tyrosine kinase receptor I for Neurotrophic Growth Factor (NGF. Case Presentation We present the case of a female patient diagnosed with CIPA at the age of 8 months. The patient is currently 6 years old and her psychomotor development conforms to her age (RMN, SPECT and psychological study are in the range of normality. PCR amplification of DNA, followed by direct sequencing, was used to investigate the presence of NTRK1 gene mutations. Reverse transcriptase (RT-PCR amplification of RNA, followed by cloning and sequencing of isolated RT-PCR products was used to characterize the effect of the mutations on NTRK1 mRNA splicing. The clinical diagnosis of CIPA was confirmed by the detection of two splice-site mutations in NTRK1, revealing that the patient was a compound heterozygote at this gene. One of these alterations, c.574+1G>A, is located at the splice donor site of intron 5. We also found a second mutation, c.2206-2 A>G, not previously reported in the literature, which is located at the splice acceptor site of intron 16. Each parent was confirmed to be a carrier for one of the mutations by DNA sequencing analysis. It has been proposed that the c.574+1G>A mutation would cause exon 5 skipping during NTRK1 mRNA splicing. We could confirm this prediction and, more importantly, we provide evidence that the novel c.2206-2A>G mutation also disrupts normal NTRK1 splicing, leading to the use of an alternative splice acceptor site within exon 17. As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain. Conclusions We present the first description of a CIPA-associated NTRK1 mutation

  5. Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

    International Nuclear Information System (INIS)

    Akeson, A.L.; Wiginton, D.A.; States, C.J.; Perme, C.M.; Dusing, M.R.; Hutton, J.J.

    1987-01-01

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency

  6. Developmental splicing deregulation in leukodystrophies related to EIF2B mutations.

    Directory of Open Access Journals (Sweden)

    Aurélia Huyghe

    Full Text Available Leukodystrophies (LD are rare inherited disorders that primarily affect the white matter (WM of the central nervous system. The large heterogeneity of LD results from the diversity of the genetically determined defects that interfere with glial cells functions. Astrocytes have been identified as the primary target of LD with cystic myelin breakdown including those related to mutations in the ubiquitous translation initiation factor eIF2B. EIF2B is involved in global protein synthesis and its regulation under normal and stress conditions. Little is known about how eIF2B mutations have a major effect on WM. We performed a transcriptomic analysis using fibroblasts of 10 eIF2B-mutated patients with a severe phenotype and 10 age matched patients with other types of LD in comparison to control fibroblasts. ANOVA was used to identify genes that were statistically significantly differentially expressed at basal state and after ER-stress. The pattern of differentially expressed genes between basal state and ER-stress did not differ significantly among each of the three conditions. However, 70 genes were specifically differentially expressed in eIF2B-mutated fibroblasts whatever the stress conditions tested compared to controls, 96% being under-expressed. Most of these genes were involved in mRNA regulation and mitochondrial metabolism. The 13 most representative genes, including genes belonging to the Heterogeneous Nuclear Ribonucleoprotein (HNRNP family, described as regulators of splicing events and stability of mRNA, were dysregulated during the development of eIF2B-mutated brains. HNRNPH1, F and C mRNA were over-expressed in foetus but under-expressed in children and adult brains. The abnormal regulation of HNRNP expression in the brain of eIF2B-mutated patients was concomitant with splicing dysregulation of the main genes involved in glial maturation such as PLP1 for oligodendrocytes and GFAP in astrocytes. These findings demonstrate a developmental

  7. Urogenital and caudal dysgenesis in adrenocortical dysplasia (acd) mice is caused by a splicing mutation in a novel telomeric regulator.

    Science.gov (United States)

    Keegan, Catherine E; Hutz, Janna E; Else, Tobias; Adamska, Maja; Shah, Sonalee P; Kent, Amy E; Howes, John M; Beamer, Wesley G; Hammer, Gary D

    2005-01-01

    Adrenocortical dysplasia (acd) is a spontaneous autosomal recessive mouse mutant with developmental defects in organs derived from the urogenital ridge. In surviving adult mutants, adrenocortical dysplasia and hypofunction are predominant features. Adults are infertile due to lack of mature germ cells, and 50% develop hydronephrosis due to ureteral hyperplasia. We report the identification of a splice donor mutation in a novel gene, which is the mouse ortholog of a newly discovered telomeric regulator. This gene (Acd) has recently been characterized as a novel component of the TRF1 protein complex that controls telomere elongation by telomerase. Characterization of Acd transcripts in mutant animals reveals two abnormal transcripts, consistent with a splicing defect. Expression of a wild-type Acd transgene in acd mutants rescues the observed phenotype. Most mutants die within 1-2 days of life on the original genetic background. Analysis of these mutant embryos reveals variable, yet striking defects in caudal specification, limb patterning and axial skeleton formation. In the tail bud, reduced expression of Wnt3a and Dll1 correlates with phenotypic severity of caudal regression. In the limbs, expression of Fgf8 is expanded in the dorsal-ventral axis of the apical ectodermal ridge and shortened in the anterior-posterior axis, consistent with the observed loss of anterior digits in older embryos. The axial skeleton of mutant embryos shows abnormal vertebral fusions in cervical, lumbar and caudal regions. This is the first report to show that a telomeric regulator is required for proper urogenital ridge differentiation, axial skeleton specification and limb patterning in mice.

  8. Splice site mutations in mismatch repair genes and risk of cancer in the general population

    DEFF Research Database (Denmark)

    Thomsen, Mette; Nordestgaard, Børge G; Tybjærg-Hansen, Anne

    2013-01-01

    We tested the hypothesis that splice site variations in MSH2 and MLH1 are associated with increased risk of hereditary non-polyposis colorectal cancer (HNPCC) and of cancer in general in the general population. In a cohort of 154 HNPCC patients with sequenced MSH2 and MLH1, we identified four pos......-related cancers and of all cancers combined in the general population. These findings are novel and important in the counseling of HNPCC patients and their relatives.......We tested the hypothesis that splice site variations in MSH2 and MLH1 are associated with increased risk of hereditary non-polyposis colorectal cancer (HNPCC) and of cancer in general in the general population. In a cohort of 154 HNPCC patients with sequenced MSH2 and MLH1, we identified four...... possible splice-site mutations, which we subsequently genotyped in more than 9,000 individuals from the general population. Allele frequencies in the general population were 0 % for 942+3A>T in MSH2, 0.05 % for 307-19A>G, 0.005 % for 1,667+(2-8)del(taaatca);ins(attt), and 4.4 % for 1039-8T>A in MLH1. Odds...

  9. Pancreatic α-cell hyperplasia and hyperglucagonemia due to a glucagon receptor splice mutation

    Directory of Open Access Journals (Sweden)

    Etienne Larger

    2016-11-01

    Full Text Available Glucagon stimulates hepatic glucose production by activating specific glucagon receptors in the liver, which in turn increase hepatic glycogenolysis as well as gluconeogenesis and ureagenesis from amino acids. Conversely, glucagon secretion is regulated by concentrations of glucose and amino acids. Disruption of glucagon signaling in rodents results in grossly elevated circulating glucagon levels but no hypoglycemia. Here, we describe a patient carrying a homozygous G to A substitution in the invariant AG dinucleotide found in a 3′ mRNA splice junction of the glucagon receptor gene. Loss of the splice site acceptor consensus sequence results in the deletion of 70 nucleotides encoded by exon 9, which introduces a frame shift and an early termination signal in the receptor mRNA sequence. The mutated receptor neither bound 125I-labeled glucagon nor induced cAMP production upon stimulation with up to 1 μM glucagon. Despite the mutation, the only obvious pathophysiological trait was hyperglucagonemia, hyperaminoacidemia and massive hyperplasia of the pancreatic α-cells assessed by histology. Our case supports the notion of a hepato–pancreatic feedback system, which upon disruption leads to hyperglucagonemia and α-cell hyperplasia, as well as elevated plasma amino acid levels. Together with the glucagon-induced hypoaminoacidemia in glucagonoma patients, our case supports recent suggestions that amino acids may provide the feedback link between the liver and the pancreatic α-cells.

  10. A pan-cancer analysis of transcriptome changes associated with somatic mutations in U2AF1 reveals commonly altered splicing events.

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    Angela N Brooks

    Full Text Available Although recurrent somatic mutations in the splicing factor U2AF1 (also known as U2AF35 have been identified in multiple cancer types, the effects of these mutations on the cancer transcriptome have yet to be fully elucidated. Here, we identified splicing alterations associated with U2AF1 mutations across distinct cancers using DNA and RNA sequencing data from The Cancer Genome Atlas (TCGA. Using RNA-Seq data from 182 lung adenocarcinomas and 167 acute myeloid leukemias (AML, in which U2AF1 is somatically mutated in 3-4% of cases, we identified 131 and 369 splicing alterations, respectively, that were significantly associated with U2AF1 mutation. Of these, 30 splicing alterations were statistically significant in both lung adenocarcinoma and AML, including three genes in the Cancer Gene Census, CTNNB1, CHCHD7, and PICALM. Cell line experiments expressing U2AF1 S34F in HeLa cells and in 293T cells provide further support that these altered splicing events are caused by U2AF1 mutation. Consistent with the function of U2AF1 in 3' splice site recognition, we found that S34F/Y mutations cause preferences for CAG over UAG 3' splice site sequences. This report demonstrates consistent effects of U2AF1 mutation on splicing in distinct cancer cell types.

  11. Seemingly neutral polymorphic variants may confer immunity to splicing-inactivating mutations: a synonymous SNP in exon 5 of MCAD protects from deleterious mutations in a flanking exonic splicing enhancer

    DEFF Research Database (Denmark)

    Nielsen, Karsten Bork; Sørensen, Suzette; Cartegni, Luca

    2007-01-01

    The idea that point mutations in exons may affect splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vit...

  12. A novel splice mutation in the TP53 gene associated with Leydig cell tumor and primitive neuroectodermal tumor

    DEFF Research Database (Denmark)

    Stecher, Chalotte Willemann; Grønbaek, Kirsten; Hasle, Henrik

    2008-01-01

    A 20-month-old boy presented with precocious puberty due to a Leydig cell tumor, and at the age of 6 years with a primitive neuroectodermal brain-tumor (PNET). A novel splice site mutation of the TP53-gene, likely to be associated with a nonfunctional protein, was found in the proband, his father...

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

    Science.gov (United States)

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

    2017-01-01

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

  14. A Novel SLC27A4 Splice Acceptor Site Mutation in Great Danes with Ichthyosis.

    Science.gov (United States)

    Metzger, Julia; Wöhlke, Anne; Mischke, Reinhard; Hoffmann, Annalena; Hewicker-Trautwein, Marion; Küch, Eva-Maria; Naim, Hassan Y; Distl, Ottmar

    2015-01-01

    Ichthyoses are a group of various different types of hereditary disorders affecting skin cornification. They are characterized by hyperkeratoses of different severity levels and are associated with a dry and scaling skin. Genome-wide association analysis of nine affected and 13 unaffected Great Danes revealed a genome-wide significant peak on chromosome 9 at 57-58 Mb in the region of SLC27A4. Sequence analysis of genomic DNA of SLC27A4 revealed the non-synonymous SNV SLC27A4:g.8684G>A in perfect association with ichthyosis-affection in Great Danes. The mutant transcript of SLC27A4 showed an in-frame loss of 54 base pairs in exon 8 probably induced by a new splice acceptor site motif created by the mutated A- allele of the SNV. Genotyping 413 controls from 35 different breeds of dogs and seven wolves revealed that this mutation could not be found in other populations except in Great Danes. Affected dogs revealed high amounts of mutant transcript but only low levels of the wild type transcript. Targeted analyses of SLC27A4 protein from skin tissues of three affected and two unaffected Great Danes indicated a markedly reduced or not detectable wild type and truncated protein levels in affected dogs but a high expression of wild type SLC27A4 protein in unaffected controls. Our data provide evidence of a new splice acceptor site creating SNV that results in a reduction or loss of intact SLC27A4 protein and probably explains the severe skin phenotype in Great Danes. Genetic testing will allow selective breeding to prevent ichthyosis-affected puppies in the future.

  15. A Novel SLC27A4 Splice Acceptor Site Mutation in Great Danes with Ichthyosis.

    Directory of Open Access Journals (Sweden)

    Julia Metzger

    Full Text Available Ichthyoses are a group of various different types of hereditary disorders affecting skin cornification. They are characterized by hyperkeratoses of different severity levels and are associated with a dry and scaling skin. Genome-wide association analysis of nine affected and 13 unaffected Great Danes revealed a genome-wide significant peak on chromosome 9 at 57-58 Mb in the region of SLC27A4. Sequence analysis of genomic DNA of SLC27A4 revealed the non-synonymous SNV SLC27A4:g.8684G>A in perfect association with ichthyosis-affection in Great Danes. The mutant transcript of SLC27A4 showed an in-frame loss of 54 base pairs in exon 8 probably induced by a new splice acceptor site motif created by the mutated A- allele of the SNV. Genotyping 413 controls from 35 different breeds of dogs and seven wolves revealed that this mutation could not be found in other populations except in Great Danes. Affected dogs revealed high amounts of mutant transcript but only low levels of the wild type transcript. Targeted analyses of SLC27A4 protein from skin tissues of three affected and two unaffected Great Danes indicated a markedly reduced or not detectable wild type and truncated protein levels in affected dogs but a high expression of wild type SLC27A4 protein in unaffected controls. Our data provide evidence of a new splice acceptor site creating SNV that results in a reduction or loss of intact SLC27A4 protein and probably explains the severe skin phenotype in Great Danes. Genetic testing will allow selective breeding to prevent ichthyosis-affected puppies in the future.

  16. Transcriptome sequencing reveals potential mechanism of cryptic 3' splice site selection in SF3B1-mutated cancers.

    Directory of Open Access Journals (Sweden)

    Christopher DeBoever

    2015-03-01

    Full Text Available Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3' splice sites (3'SSs are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3' SSs and propose that cryptic 3'SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3'SSs are present at low frequency (<10% relative to nearby canonical 3'SSs, we identified ten genes that preferred out-of-frame cryptic 3'SSs. We show that cancers with mutations in the SF3B1 HEAT 5-9 repeats use cryptic 3'SSs downstream of the branch point and provide both a mechanistic model consistent with published experimental data and affected targets that will guide further research into the oncogenic effects of SF3B1 mutation.

  17. Early-onset encephalopathy with epilepsy associated with a novel splice site mutation in SMC1A.

    Science.gov (United States)

    Lebrun, Nicolas; Lebon, Sébastien; Jeannet, Pierre-Yves; Jacquemont, Sébastien; Billuart, Pierre; Bienvenu, Thierry

    2015-12-01

    We report on the clinical and molecular characterization of a female patient with early-onset epileptic encephalopathy, who was found to carry a de novo novel splice site mutation in SMC1A. This girl shared some morphologic and anthropometric traits described in patients with clinical diagnosis of Cornelia de Lange syndrome and with SMC1A mutation but also has severe encephalopathy with early-onset epilepsy. In addition, she had midline hand stereotypies and scoliosis leading to the misdiagnosis of a Rett overlap syndrome. Molecular studies found a novel de novo splice site mutation (c.1911 + 1G > T) in SMC1A. This novel splice mutation was associated with an aberrantly processed mRNA that included intron 11 of the gene. Moreover, quantitative approach by RT-PCR showed a severe reduction of the SMC1A transcript suggesting that this aberrant transcript may be unstable and degraded. Taken together, our data suggest that the phenotype may be due to a loss-of-function of SMC1A in this patient. Our findings suggest that loss-of-function mutations of SMC1A may be associated with early-onset encephalopathy with epilepsy. © 2015 Wiley Periodicals, Inc.

  18. Mutations in SNRPB, encoding components of the core splicing machinery, cause cerebro-costo-mandibular syndrome.

    Science.gov (United States)

    Bacrot, Séverine; Doyard, Mathilde; Huber, Céline; Alibeu, Olivier; Feldhahn, Niklas; Lehalle, Daphné; Lacombe, Didier; Marlin, Sandrine; Nitschke, Patrick; Petit, Florence; Vazquez, Marie-Paule; Munnich, Arnold; Cormier-Daire, Valérie

    2015-02-01

    Cerebro-costo-mandibular syndrome (CCMS) is a developmental disorder characterized by the association of Pierre Robin sequence and posterior rib defects. Exome sequencing and Sanger sequencing in five unrelated CCMS patients revealed five heterozygous variants in the small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) gene. This gene includes three transcripts, namely transcripts 1 and 2, encoding components of the core spliceosomal machinery (SmB' and SmB) and transcript 3 undergoing nonsense-mediated mRNA decay. All variants were located in the premature termination codon (PTC)-introducing alternative exon of transcript 3. Quantitative RT-PCR analysis revealed a significant increase in transcript 3 levels in leukocytes of CCMS individuals compared to controls. We conclude that CCMS is due to heterozygous mutations in SNRPB, enhancing inclusion of a SNRPB PTC-introducing alternative exon, and show that this developmental disease is caused by defects in the splicing machinery. Our finding confirms the report of SNRPB mutations in CCMS patients by Lynch et al. (2014) and further extends the clinical and molecular observations. © 2014 WILEY PERIODICALS, INC.

  19. An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements.

    Science.gov (United States)

    Disset, A; Bourgeois, C F; Benmalek, N; Claustres, M; Stevenin, J; Tuffery-Giraud, Sylvie

    2006-03-15

    A nonsense mutation c.4250T>A (p.Leu1417X) in the dystrophin gene of a patient with an intermediate phenotype of muscular dystrophy induces partial in-frame skipping of exon 31. On the basis of UV cross-linking assays and pull-down analysis, we present evidence that the skipping of this exon is because of the creation of an exonic splicing silencer, which acts as a highly specific binding site (UAGACA) for a known repressor protein, hnRNP A1. Recombinant hnRNP A1 represses exon inclusion both in vitro and in vivo upon transient transfection of C2C12 cells with Duchenne muscular dystrophy (DMD) minigenes carrying the c.4250T>A mutation. Furthermore, we identified a downstream splicing enhancer in the central region of exon 31. This region functions as a Tra2beta-dependent exonic splicing enhancer (ESE) in vitro when inserted into a heterologous splicing reporter, and deletion of the ESE showed that incorporation of exon 31 depends on the Tra2beta-dependent enhancer both in the wild-type and mutant context. We conclude that dystrophin exon 31 contains juxtaposed sequence motifs that collaborate to regulate exon usage. This is the first elucidation of the molecular mechanism leading to exon skipping in the dystrophin gene and allowing the occurrence of a milder phenotype than the expected DMD phenotype. The knowledge of which cis-acting sequence within an exon is important for its definition will be essential for the alternative gene therapy approaches based on modulation of splicing to bypass DMD-causing mutations in the endogenous dystrophin gene.

  20. A splice site mutation in laminin-α2 results in a severe muscular dystrophy and growth abnormalities in zebrafish.

    Directory of Open Access Journals (Sweden)

    Vandana A Gupta

    Full Text Available Congenital muscular dystrophy (CMD is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2. Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501, exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

  1. A family with attenuated familial adenomatous polyposis due to a mutation in the alternatively spliced region of APC exon 9.

    Science.gov (United States)

    Young, J; Simms, L A; Tarish, J; Buttenshaw, R; Knight, N; Anderson, G J; Bell, A; Leggett, B

    1998-01-01

    A family is presented with attenuated familial adenomatous polyposis of variable phenotype. The clinical features range from sparse right-sided polyposis and cancer in the proximal colon at the age of 34 to pan-colonic polyposis and cancer at the age of 68. Rectal sparing is common to all affected members. Heteroduplex analysis detected bands of altered mobility in exon 9 of the APC gene in all affected family members. Subsequently, a frameshift mutation was found in the alternatively spliced region of exon 9 at codon 398 which resulted in a stop signal 4 codons downstream. Alternatively spliced transcripts that delete the mutation were readily amplified from normal colonic mucosa and therefore create a mechanism for the attenuated phenotype seen in this family.

  2. Cryptic splice activation but not exon skipping is observed in minigene assays of dystrophin c.9361+1G>A mutation identified by NGS.

    Science.gov (United States)

    Niba, Emma Tabe Eko; Nishida, Atsushi; Tran, Van Khanh; Vu, Dung Chi; Matsumoto, Masaaki; Awano, Hiroyuki; Lee, Tomoko; Takeshima, Yasuhiro; Nishio, Hisahide; Matsuo, Masafumi

    2017-04-01

    Next-generation sequencing (NGS) discloses nucleotide changes in the genome. Mutations at splicing regulatory elements are expected to cause splicing errors, such as exon skipping, cryptic splice site activation, partial exon loss or intron retention. In dystrophinopathy patients, prediction of splicing outcomes is essential to determine the phenotype: either severe Duchenne or mild Becker muscular dystrophy, based on the reading frame rule. In a Vietnamese patient, NGS identified a c.9361+1G>A mutation in the dystrophin gene and an additional DNA variation of A>G at +117 bases in intron 64. To ascertain the consequences of these DNA changes on dystrophin splicing, minigene constructs were prepared inserting dystrophin exon 64 plus various lengths of intron 64. Exon 64 skipping was observed in the minigene construct with 160 nucleotide (nt) of intron 64 sequence with both c.9361+1A and +117G. In contrast, minigene constructs with larger flanking intronic domains resulted in cryptic splice site activation rather than exon skipping. Meanwhile, the cryptic splice site activation was induced even in +117G when intron 64 was elongated to 272 nt and longer. It was expected that cryptic splice site activation is an in vivo splicing outcome.

  3. A Novel Splicing Mutation of KIT Results in Piebaldism and Auburn Hair Color in a Chinese Family

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    Yong-jia Yang

    2013-01-01

    Full Text Available Piebaldism is a rare autosomal dominant disorder of melanocyte development, which is mostly caused by KIT gene. The key characteristics of piebaldism include localized poliosis, congenital leukoderma, and other variable manifestations. The previous study has illustrated that the homogeneous MC1R (a gene which is associated with the hair color variant (p.I120T coordinating with KIT mutation may lead to auburn hair color and piebaldism. In this study, we have investigated a Chinese family with piebaldism and auburn hair color; the mutation screening of KIT and MC1R genes identified that only a splicing mutation (c. 2484+1G>A of KIT gene cosegregated with the auburn hair color and piebaldism. The data of this study and others suggests that the KIT mutation may causes of the auburn hair color in the piebaldism patients.

  4. IVS8+1 DelG, a Novel Splice Site Mutation Causing DFNA5 Deafness in a Chinese Family.

    Science.gov (United States)

    Li-Yang, Mei-Na; Shen, Xiao-Fei; Wei, Qin-Jun; Yao, Jun; Lu, Ya-Jie; Cao, Xin; Xing, Guang-Qian

    2015-09-20

    Nonsyndromic hearing loss (NSHL) is highly heterogeneous, in which more than 90 causative genes have currently been identified. DFNA5 is one of the deafness genes that known to cause autosomal dominant NSHL. Until date, only five DFNA5 mutations have been described in eight families worldwide. In this study, we reported the identification of a novel pathogenic mutation causing DFNA5 deafness in a five-generation Chinese family. After detailed clinical evaluations of this family, the genomic DNA of three affected individuals was selected for targeted exome sequencing of 101 known deafness genes, as well as mitochondrial DNA and microRNA regions. Co-segregation analysis between the hearing loss and the candidate variant was confirmed in available family members by direct polymerase chain reaction (PCR)-Sanger sequencing. Real-time PCR (RT-PCR) was performed to investigate the potential effect of the pathogenic mutation on messenger RNA splicing. Clinical evaluations revealed a similar deafness phenotype in this family to that of previously reported DFNA5 families with autosomal dominant, late-onset hearing loss. Molecular analysis identified a novel splice site mutation in DFNA5 intron 8 (IVS8+1 delG). The mutation segregated with the hearing loss of the family and was absent in 120 unrelated control DNA samples of Chinese origin. RT-PCR showed skipping of exon 8 in the mutant transcript. We identified a novel DFNA5 mutation IVS8+1 delG in a Chinese family which led to skipping of exon 8. This is the sixth DFNA5 mutation relates to hearing loss and the second one in DFNA5 intron 8. Our findings provide further support to the hypothesis that the DFNA5-associated hearing loss represents a mechanism of gain-of-function.

  5. A splice acceptor mutation in C. elegans daf-19/Rfx disrupts functional specialization of male-specific ciliated neurons but does not affect ciliogenesis.

    Science.gov (United States)

    Wells, Kristen L; Rowneki, Mazhgan; Killian, Darrell J

    2015-04-01

    RFX transcription factors are master regulators of ciliogenesis in diverse animal species. The sole Caenorhabditis elegans RFX homolog, DAF-19, plays at least two roles in the formation of functional cilia. The DAF-19(C) isoform is required for ciliogenesis and the DAF-19(M) isoform is required for the functional specialization of a subset of male-specific ciliated neurons called PKD neurons. Here we report the identification of a novel mutation, daf-19(sm129), which disrupts the functional specification of PKD neurons and thus suggests that daf-19m activity is compromised. However, ciliogenesis is not disrupted in daf-19(sm129) mutants suggesting that daf-19c activity is retained. The sm129 mutation disrupts a splice acceptor site adjacent to an exon common to the daf-19c and daf-19m isoforms resulting in aberrant splicing in a proportion of transcripts. While aberrant splicing of daf-19c to upstream cryptic sites results in in-frame and functional products, a large proportion of daf-19m mRNAs include the entire upstream intron, which introduces a frameshift and stop codons. At least 15% of disease-causing mutations affect splicing of the gene bearing the mutation, thus it is important to understand the consequences of splice site mutations on gene function. However, predicting the effects of a splice site mutation remains difficult and experimental determination is still required. Using daf-19(sm129) as a model, our results suggest that this problem is exacerbated when a splice acceptor mutation is used by multiple isoforms of the same gene because the effects on each isoform can be dramatically different. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene.

    Science.gov (United States)

    Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; Van Kuilenburg, André B P

    2016-01-12

    Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

  7. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS Gene

    Directory of Open Access Journals (Sweden)

    Yoko Nakajima

    2016-01-01

    Full Text Available Dihydropyrimidinase (DHP deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

  8. Identification of a 5' splice site mutation in the RPGR gene in a family with X-linked retinitis pigmentosa (RP3)

    NARCIS (Netherlands)

    Dry, K. L.; Manson, F. D.; Lennon, A.; Bergen, A. A.; van Dorp, D. B.; Wright, A. F.

    1999-01-01

    We have identified a novel RPGR gene mutation in a large Dutch family with X-linked retinitis pigmentosa (RP3). In affected members, a G-->T transversion was found at position +1 of the 5' splice site of intron 5 of the RPGR (retinitis pigmentosa GTPase regulator) gene. Analysis of this mutation at

  9. Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II

    Directory of Open Access Journals (Sweden)

    Liliana Matos

    2015-12-01

    The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in “Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II” Matos et al. (2015 [1].

  10. Successful living donor kidney transplantation in a patient with prothrombin gene mutation: Case report and literature review

    Directory of Open Access Journals (Sweden)

    Edward Shen

    2014-01-01

    Full Text Available We present a patient with known prothrombin gene mutation and a history of prior vascular events, who underwent living donor kidney transplantation. Given the presumed elevated risk of complication from known prothrombin mutation, clinical management was directed towards optimizing living donor allograft function.

  11. Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.

    Directory of Open Access Journals (Sweden)

    Yesenia Ríos

    2011-01-01

    Full Text Available Loss of retinoblastoma (Rb tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39 mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130, and cdkn1a (p21 expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

  12. Dwarfism with joint laxity in Friesian horses is associated with a splice site mutation in B4GALT7.

    Science.gov (United States)

    Leegwater, Peter A; Vos-Loohuis, Manon; Ducro, Bart J; Boegheim, Iris J; van Steenbeek, Frank G; Nijman, Isaac J; Monroe, Glen R; Bastiaansen, John W M; Dibbits, Bert W; van de Goor, Leanne H; Hellinga, Ids; Back, Willem; Schurink, Anouk

    2016-10-28

    Inbreeding and population bottlenecks in the ancestry of Friesian horses has led to health issues such as dwarfism. The limbs of dwarfs are short and the ribs are protruding inwards at the costochondral junction, while the head and back appear normal. A striking feature of the condition is the flexor tendon laxity that leads to hyperextension of the fetlock joints. The growth plates of dwarfs display disorganized and thickened chondrocyte columns. The aim of this study was to identify the gene defect that causes the recessively inherited trait in Friesian horses to understand the disease process at the molecular level. We have localized the genetic cause of the dwarfism phenotype by a genome wide approach to a 3 Mb region on the p-arm of equine chromosome 14. The DNA of two dwarfs and one control Friesian horse was sequenced completely and we identified the missense mutation ECA14:g.4535550C > T that cosegregated with the phenotype in all Friesians analyzed. The mutation leads to the amino acid substitution p.(Arg17Lys) of xylosylprotein beta 1,4-galactosyltransferase 7 encoded by B4GALT7. The protein is one of the enzymes that synthesize the tetrasaccharide linker between protein and glycosaminoglycan moieties of proteoglycans of the extracellular matrix. The mutation not only affects a conserved arginine codon but also the last nucleotide of the first exon of the gene and we show that it impedes splicing of the primary transcript in cultured fibroblasts from a heterozygous horse. As a result, the level of B4GALT7 mRNA in fibroblasts from a dwarf is only 2 % compared to normal levels. Mutations in B4GALT7 in humans are associated with Ehlers-Danlos syndrome progeroid type 1 and Larsen of Reunion Island syndrome. Growth retardation and ligamentous laxity are common manifestations of these syndromes. We suggest that the identified mutation of equine B4GALT7 leads to the typical dwarfism phenotype in Friesian horses due to deficient splicing of transcripts of

  13. Comparative in vitro and in silico analyses of variants in splicing regions of BRCA1 and BRCA2 genes and characterization of novel pathogenic mutations.

    Science.gov (United States)

    Colombo, Mara; De Vecchi, Giovanna; Caleca, Laura; Foglia, Claudia; Ripamonti, Carla B; Ficarazzi, Filomena; Barile, Monica; Varesco, Liliana; Peissel, Bernard; Manoukian, Siranoush; Radice, Paolo

    2013-01-01

    Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for

  14. Deep intronic mis-splicing mutation in JAK3 gene underlies T-B+NK- severe combined immunodeficiency phenotype.

    Science.gov (United States)

    Stepensky, Polina; Keller, Baerbel; Shamriz, Oded; NaserEddin, Adeeb; Rumman, Nisreen; Weintraub, Michael; Warnatz, Klaus; Elpeleg, Orly; Barak, Yaacov

    2016-02-01

    Severe combined immune deficiency (SCID) is a group of genetically heterogeneous diseases caused by an early block in T cell differentiation and present with life threatening infections, often within the first year of life. Janus kinase (JAK)3 gene mutations have been found to cause autosomal recessive T-B+ SCID phenotype. In this study we describe three patients with a novel deep intronic mis-splicing mutation in JAK3 as a cause of T-B+NK- SCID highlighting the need for careful evaluation of intronic regulatory elements of known genes associated with clearly defined clinical phenotypes. We present the cases and discuss the current literature. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. A novel BTK gene mutation creates a de-novo splice site in an X-linked agammaglobulinemia patient.

    Science.gov (United States)

    Chear, Chai Teng; Ripen, Adiratna Mat; Mohamed, Sharifah Adlena Syed; Dhaliwal, Jasbir Singh

    2015-04-15

    Bruton's tyrosine kinase (BTK), encoded by the BTK gene, is a cytoplasmic protein critical in B cell development. Mutations in the BTK gene cause X-linked agammaglobulinemia (XLA), a primary immunodeficiency with characteristically low or absent B cells and antibodies. This report describes a five year-old boy who presented with otitis externa, arthritis, reduced immunoglobulins and no B cells. Flow cytometry showed undetectable monocyte BTK expression. Sequencing revealed a novel mutation at exon 13 of the BTK gene which created a de novo splice site with a proximal 5 nucleotide loss resulting in a truncated BTK protein. The patient still suffered from ear infection despite intravenous immunoglobulin replacement therapy. In this study, mosaicism was seen only in the mother's genomic DNA. These results suggest that a combination of flow cytometry and BTK gene analysis is important for XLA diagnosis and carrier screening. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. An Analysis of the Sensitivity of Proteogenomic Mapping of Somatic Mutations and Novel Splicing Events in Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Ruggles, Kelly V.; Tang, Zuojian; Wang, Xuya; Grover, Himanshu; Askenazi, Manor; Teubl, Jennifer; Cao, Song; McLellan, Michael D.; Clauser, Karl R.; Tabb, David L.; Mertins, Philipp; Slebos, Robbert; Erdmann-Gilmore, Petra; Li, Shunqiang; Gunawardena, Harsha P.; Xie, Ling; Liu, Tao; Zhou, Jian-Ying; Sun, Shisheng; Hoadley, Katherine A.; Perou, Charles M.; Chen, Xian; Davies, Sherri R.; Maher, Christopher A.; Kinsinger, Christopher R.; Rodland, Karen D.; Zhang, Hui; Zhang, Zhen; Ding, Li; Townsend, R. Reid; Rodriguez, Henry; Chan, Daniel; Smith, Richard D.; Liebler, Daniel C.; Carr, Steven A.; Payne, Samuel; Ellis, Matthew J.; Fenyő, David

    2015-12-02

    Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations and splice variants identified in cancer cells are translated. Herein we therefore describe a proteogenomic data integration tool (QUILTS) and illustrate its application to whole genome, transcriptome and global MS peptide sequence datasets generated from a pair of luminal and basal-like breast cancer patient derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS process replicates. Despite over thirty sample replicates, only about 10% of all SNV (somatic and germline) were detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNV without a detectable mRNA transcript were also observed demonstrating the transcriptome coverage was also incomplete (~80%). In contrast to germ-line variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than the luminal tumor raising the possibility of differential translation or protein degradation effects. In conclusion, the QUILTS program integrates DNA, RNA and peptide sequencing to assess the degree to which somatic mutations are translated and therefore biologically active. By identifying gaps in sequence coverage QUILTS benchmarks current technology and assesses progress towards whole cancer proteome and transcriptome analysis.

  17. The rare nonsense mutation in p53 triggers alternative splicing to produce a protein capable of inducing apoptosis.

    Directory of Open Access Journals (Sweden)

    Evgeny M Makarov

    Full Text Available P53 protein is more frequently mutated in human tumours compared with the other proteins. While the majority of the p53 mutations, especially within its DNA-binding domain, lead to the loss of the wild-type function, there are accumulating data demonstrating that the p53 mutants gain tumour promoting activities; the latter triggers a revitalised interest in functional analysis of the p53 mutants. A systematic screening for p53 mutations in surgical materials from patients with glioma revealed a 378C>G mutation that creates a stop codon at the position of amino acid residue 126. The mutation eliminates the recognition site for the restriction endonuclease Sca I that allowed us to carry out RFLP analysis of DNA extracted from the clinical samples and suggests that this mutation is more frequent than is documented in the p53 databases. Both the ECV-304 and EJ cell lines, that probably originate from the bladder carcinoma T24 cell line, were confirmed to contain the homozygous 378C>G mutation but were shown to produce the p53 protein of expected full-length size detected by Western blotting. We provide evidence that the 378C>G mutation generates an alternative 3' splice site (ss which is more often used instead of the authentic upstream 3' ss, driving the production of mRNA encoding the protein with the single amino acid deletion (p53ΔY126. Using endogenous expression, we demonstrated that the p53ΔY126 protein is nearly as active as the wild type protein in inducing the p21/Waf1 expression and apoptosis.

  18. Ehlers-Danlos syndrome with lethal cardiac valvular dystrophy in males carrying a novel splice mutation in FLNA.

    Science.gov (United States)

    Ritelli, Marco; Morlino, Silvia; Giacopuzzi, Edoardo; Carini, Giulia; Cinquina, Valeria; Chiarelli, Nicola; Majore, Silvia; Colombi, Marina; Castori, Marco

    2017-01-01

    Filamin A is an X-linked, ubiquitous actin-binding protein whose mutations are associated to multiple disorders with limited genotype-phenotype correlations. While gain-of-function mutations cause various bone dysplasias, loss-of-function variants are the most common cause of periventricular nodular heterotopias with variable soft connective tissue involvement, as well as X-linked cardiac valvular dystrophy (XCVD). The term "Ehlers-Danlos syndrome (EDS) with periventricular heterotopias" has been used in females with neurological, cardiovascular, integument and joint manifestations, but this nosology is still a matter of debate. We report the clinical and molecular update of an Italian family with an X-linked recessive soft connective tissue disorder and which was described, in 1975, as the first example of EDS type V of the Berlin nosology. The cutaneous phenotype of the index patient was close to classical EDS and all males died for a lethal cardiac valvular dystrophy. Whole exome sequencing identified the novel c.1829-1G>C splice variation in FLNA in two affected cousins. The nucleotide change was predicted to abolish the canonical splice acceptor site of exon 13 and to activate a cryptic acceptor site 15 bp downstream, leading to in frame deletion of five amino acid residues (p.Phe611_Gly615del). The predicted in frame deletion clusters with all the mutations previously identified in XCVD and falls within the N-terminus rod 1 domain of filamin A. Our findings expand the male-specific phenotype of FLNA mutations that now includes classical-like EDS with lethal cardiac valvular dystrophy, and offer further insights for the genotype-phenotype correlations within this spectrum. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing-defective genes.

    Directory of Open Access Journals (Sweden)

    Andrea B Eberle

    Full Text Available BACKGROUND: Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human beta-globin gene with mutated splice sites in intron 2 (mut beta-globin. The transcripts encoded by the mut beta-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut beta-globin transcripts are much lower than those of wild type (wt ss-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt beta-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut beta-globin transcripts are processed at the 3', but the mut beta-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut beta-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut beta-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut beta-globin gene shows reduced levels of H3K4me3. CONCLUSIONS/SIGNIFICANCE: Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The

  20. A five' splice-region G → C mutation in exon 1 of the human β-globin gene inhibits pre-mRNA splicing: A mechanism for β+-thalassemia

    International Nuclear Information System (INIS)

    Vidaud, M.; Vidaud, D.; Amselem, S.; Rosa, J.; Goossens, M.; Gattoni, R.; Stevenin, J.; Chibani, J.

    1989-01-01

    The authors have characterized a Mediterranean β-thalassemia allele containing a sequence change at codon 30 that alters both β-globin pre-mRNA splicing and the structure of the homoglobin product. Presumably, this G → C transversion at position -1 of intron 1 reduces severely the utilization of the normal 5' splice site since the level of the Arg → Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position -1 of the CAG/GTAAGT consensus sequence had been isolated previously. They investigated the role of this nucleotide in the constitution of an active 5' splice site by studying the splicing of the pre-mRNA in cell-free extracts. They demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Their results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5

  1. Novel FGFR1 mutations in Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism: evidence for the involvement of an alternatively spliced isoform.

    Science.gov (United States)

    Gonçalves, Catarina; Bastos, Margarida; Pignatelli, Duarte; Borges, Teresa; Aragüés, José M; Fonseca, Fernando; Pereira, Bernardo D; Socorro, Sílvia; Lemos, Manuel C

    2015-11-01

    To determine the prevalence of fibroblast growth factor receptor 1 (FGFR1) mutations and their predicted functional consequences in patients with idiopathic hypogonadotropic hypogonadism (IHH). Cross-sectional study. Multicentric. Fifty unrelated patients with IHH (21 with Kallmann syndrome and 29 with normosmic IHH). None. Patients were screened for mutations in FGFR1. The functional consequences of mutations were predicted by in silico structural and conservation analysis. Heterozygous FGFR1 mutations were identified in six (12%) kindreds. These consisted of frameshift mutations (p.Pro33-Alafs*17 and p.Tyr654*) and missense mutations in the signal peptide (p.Trp4Cys), in the D1 extracellular domain (p.Ser96Cys) and in the cytoplasmic tyrosine kinase domain (p.Met719Val). A missense mutation was identified in the alternatively spliced exon 8A (p.Ala353Thr) that exclusively affects the D3 extracellular domain of FGFR1 isoform IIIb. Structure-based and sequence-based prediction methods and the absence of these variants in 200 normal controls were all consistent with a critical role for the mutations in the activity of the receptor. Oligogenic inheritance (FGFR1/CHD7/PROKR2) was found in one patient. Two FGFR1 isoforms, IIIb and IIIc, result from alternative splicing of exons 8A and 8B, respectively. Loss-of-function of isoform IIIc is a cause of IHH, whereas isoform IIIb is thought to be redundant. Ours is the first report of normosmic IHH associated with a mutation in the alternatively spliced exon 8A and suggests that this disorder can be caused by defects in either of the two alternatively spliced FGFR1 isoforms. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  2. Whole exome sequencing identifies a novel splice-site mutation in ADAMTS17 in an Indian family with Weill-Marchesani syndrome.

    Science.gov (United States)

    Shah, Mohd Hussain; Bhat, Vishwanath; Shetty, Jyoti S; Kumar, Arun

    2014-01-01

    Weill-Marchesani syndrome (WMS) is a rare connective tissue disorder, characterized by short stature, microspherophakic lens, and stubby hands and feet (brachydactyly). WMS is caused by mutations in the FBN1, ADAMTS10, and LTBP2 genes. Mutations in the LTBP2 and ADAMTS17 genes cause a WMS-like syndrome, in which the affected individuals show major features of WMS but do not display brachydactyly and joint stiffness. The main purpose of our study was to determine the genetic cause of WMS in an Indian family. Whole exome sequencing (WES) was used to identify the genetic cause of WMS in the family. The cosegregation of the mutation was determined with Sanger sequencing. Reverse transcription (RT)-PCR analysis was used to assess the effect of a splice-site mutation on splicing of the ADAMTS17 transcript. The WES analysis identified a homozygous novel splice-site mutation c.873+1G>T in a known WMS-like syndrome gene, ADAMTS17, in the family. RT-PCR analysis in the patient showed that exon 5 was skipped, which resulted in the deletion of 28 amino acids in the ADAMTS17 protein. The mutation in the WMS-like syndrome gene ADAMTS17 also causes WMS in an Indian family. The present study will be helpful in genetic diagnosis of this family and increases the number of mutations of this gene to six.

  3. Retinitis Pigmentosa Mutations of SNRNP200 Enhance Cryptic Splice-Site Recognition

    Czech Academy of Sciences Publication Activity Database

    Cvačková, Zuzana; Matějů, Daniel; Staněk, David

    2014-01-01

    Roč. 35, č. 3 (2014), s. 308-317 ISSN 1059-7794 R&D Projects: GA ČR GPP301/12/P425; GA ČR GAP302/11/1910; GA AV ČR KAN200520801 Institutional support: RVO:68378050 Keywords : Retinitis pigmentosa * pre-mRNA splicing * fidelity Subject RIV: EB - Gene tics ; Molecular Biology Impact factor: 5.144, year: 2014

  4. Retinitis Pigmentosa Mutations of SNRNP200 Enhance Cryptic Splice-Site Recognition

    Czech Academy of Sciences Publication Activity Database

    Cvačková, Zuzana; Matějů, Daniel; Staněk, David

    2014-01-01

    Roč. 35, č. 3 (2014), s. 308-317 ISSN 1059-7794 R&D Projects: GA ČR GPP301/12/P425; GA ČR GAP302/11/1910; GA AV ČR KAN200520801 Institutional support: RVO:68378050 Keywords : Retinitis pigmentosa * pre-mRNA splicing * fidelity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.144, year: 2014

  5. Regulation of HIV-1 splicing

    NARCIS (Netherlands)

    Müller, N.

    2016-01-01

    Human immunodeficiency virus type-1 (HIV-1) produces a single primary RNA transcript. The full-length transcript functions as RNA genome that is packaged into virions and as mRNA for translation of the Gag and Pol proteins. HIV-1 RNA contains several splice donor (5’splice site; 5’ss) and splice

  6. Clinical, in silico, and experimental evidence for pathogenicity of two novel splice site mutations in the SH3TC2 gene

    Czech Academy of Sciences Publication Activity Database

    Laššuthová, P.; Gregor, Martin; Sarnová, Lenka; Machalová, Eliška; Sedláček, Radislav; Seeman, P.

    2012-01-01

    Roč. 26, 3-4 (2012), s. 413-420 ISSN 0167-7063 R&D Projects: GA ČR GAP303/10/2044 Institutional support: RVO:68378050 Keywords : exon trapping * peripheral neuropathy * SH3TC2 gene * splice site mutation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.159, year: 2012

  7. Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals.

    Directory of Open Access Journals (Sweden)

    Keren Borensztajn

    2006-09-01

    Full Text Available Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5' splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7 a 37-bp VNTR minisatellite whose first element spans the exon7-IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5' pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5' splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5' splice site, rather than repressing the 3' following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5' pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5' splice site follows a scanning-type mechanism, precluding competition with other functional 5' pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5' pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type-independent 5'-3'-oriented scanning process for accurate recognition of the authentic 5' splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5' splice site selection.

  8. Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus

    DEFF Research Database (Denmark)

    Sørensen, Annette Balle; Lund, Anders H; Kunder, Sandra

    2007-01-01

    to be associated with specific tumor diagnoses or individual viral mutants. CONCLUSION: We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential......) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms......BACKGROUND: Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine...

  9. Multipotent genetic suppression of retrotransposon-induced mutations by Nxf1 through fine-tuning of alternative splicing.

    Directory of Open Access Journals (Sweden)

    Dorothy Concepcion

    2009-05-01

    Full Text Available Cellular gene expression machinery has coevolved with molecular parasites, such as viruses and transposons, which rely on host cells for their expression and reproduction. We previously reported that a wild-derived allele of mouse Nxf1 (Tap, a key component of the host mRNA nuclear export machinery, suppresses two endogenous retrovirus-induced mutations and shows suggestive evidence of positive selection. Here we show that Nxf1(CAST suppresses a specific and frequent class of intracisternal A particle (IAP-induced mutations, including Ap3d1(mh2J, a model for Hermansky-Pudlak syndrome, and Atcay(hes, an orthologous gene model for Cayman ataxia, among others. The molecular phenotype of suppression includes approximately two-fold increase in the level of correctly-spliced mRNA and a decrease in mutant-specific, alternatively-processed RNA accumulating from the inserted allele. Insertional mutations involving ETn and LINE elements are not suppressed, demonstrating a high degree of specificity to this suppression mechanism. These results implicate Nxf1 in some instances of pre-mRNA processing, demonstrate the useful range of Nxf1(CAST alleles for manipulating existing mouse models of disease, and specifically imply a low functional threshold for therapeutic benefit in Cayman ataxia.

  10. Splicing mutation in the ATR-X gene can lead to a dysmorphic mental retardation phenotype without {alpha}-thalassemia

    Energy Technology Data Exchange (ETDEWEB)

    Villard, L.; Lossi, A.M.; Fontes, M. [and others

    1996-03-01

    We have previously reported the isolation of a gene from Xq13 that codes for a putative regulator of transcription (XNP) and has now been shown to be the gene involved in the X-linked {alpha}-thalassemia with mental retardation (ATR-X) syndrome. The widespread expression and numerous domains present in the putative protein suggest that this gene could be involved in other phenotypes. The predominant expression of the gene in the developing brain, as well as its association with neuron differentiation, indicates that mutations of this gene might result in a mental retardation (MR) phenotype. In this paper we present a family with a splice junction mutation in XNP that results in the skipping of an exon and in the introduction of a stop codon in the middle of the XNP-coding sequence. Only the abnormal transcript is expressed in two first cousins presenting the classic ATR-X phenotype (with {alpha}-thalassemia and HbH inclusions). In a distant cousin presenting a similar dysmorphic MR phenotype but not having thalassemia, {approximately}30% of the XNP transcripts are normal. These data demonstrate that the mode of action of the XNP gene product on globin expression is distinct from its mode of action in brain development and facial morphogenesis and suggest that other dysmorphic mental retardation phenotypes, such as Juberg-Marsidi or some sporadic cases of Coffin-Lowry, could be due to mutations in XNP. 20 refs., 5 figs., 2 tabs.

  11. Splice mutations preserve myophosphorylase activity that ameliorates the phenotype in McArdle disease

    DEFF Research Database (Denmark)

    Vissing, John; Duno, Morten; Schwartz, Marianne

    2009-01-01

    Over 100 mutations in the myophosphorylase gene, which cause McArdle disease, are known. All these mutations have resulted in a complete block of muscle glycogenolysis, and accordingly, no genotype-phenotype correlation has been identified in this condition. We evaluated physiologic and genetic f...

  12. Becker muscular dystrophy due to an intronic splicing mutation inducing a dual dystrophin transcript.

    Science.gov (United States)

    Todeschini, Alice; Gualandi, Francesca; Trabanelli, Cecilia; Armaroli, Annarita; Ravani, Anna; Fanin, Marina; Rota, Silvia; Bello, Luca; Ferlini, Alessandra; Pegoraro, Elena; Padovani, Alessandro; Filosto, Massimiliano

    2016-10-01

    We describe a 29-year-old patient who complained of left thigh muscle weakness since he was 23 and of moderate proximal weakness of both lower limbs with difficulty in climbing stairs and running since he was 27. Mild weakness of iliopsoas and quadriceps muscles and muscle atrophy of both the distal forearm and thigh were observed upon clinical examination. He harboured a novel c.1150-3C>G substitution in the DMD gene, affecting the intron 10 acceptor splice site and causing exon 11 skipping and an out-of-frame transcript. However, protein of normal molecular weight but in reduced amounts was observed on Western Blot analysis. Reverse transcription analysis on muscle RNA showed production, via alternative splicing, of a transcript missing exon 11 as well as a low abundant full-length transcript which is enough to avoid the severe Duchenne phenotype. Our study showed that a reduced amount of full length dystrophin leads to a mild form of Becker muscular dystrophy. These results confirm earlier findings that low amounts of dystrophin can be associated with a milder phenotype, which is promising for therapies aiming at dystrophin restoration. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. New splicing mutation in the choline kinase beta (CHKB) gene causing a muscular dystrophy detected by whole-exome sequencing.

    Science.gov (United States)

    Oliveira, Jorge; Negrão, Luís; Fineza, Isabel; Taipa, Ricardo; Melo-Pires, Manuel; Fortuna, Ana Maria; Gonçalves, Ana Rita; Froufe, Hugo; Egas, Conceição; Santos, Rosário; Sousa, Mário

    2015-06-01

    Muscular dystrophies (MDs) are a group of hereditary muscle disorders that include two particularly heterogeneous subgroups: limb-girdle MD and congenital MD, linked to 52 different genes (seven common to both subgroups). Massive parallel sequencing technology may avoid the usual stepwise gene-by-gene analysis. We report the whole-exome sequencing (WES) analysis of a patient with childhood-onset progressive MD, also presenting mental retardation and dilated cardiomyopathy. Conventional sequencing had excluded eight candidate genes. WES of the trio (patient and parents) was performed using the ion proton sequencing system. Data analysis resorted to filtering steps using the GEMINI software revealed a novel silent variant in the choline kinase beta (CHKB) gene. Inspection of sequence alignments ultimately identified the causal variant (CHKB:c.1031+3G>C). This splice site mutation was confirmed using Sanger sequencing and its effect was further evaluated with gene expression analysis. On reassessment of the muscle biopsy, typical abnormal mitochondrial oxidative changes were observed. Mutations in CHKB have been shown to cause phosphatidylcholine deficiency in myofibers, causing a rare form of CMD (only 21 patients reported). Notwithstanding interpretative difficulties that need to be overcome before the integration of WES in the diagnostic workflow, this work corroborates its utility in solving cases from highly heterogeneous groups of diseases, in which conventional diagnostic approaches fail to provide a definitive diagnosis.

  14. A polymorphism in the splice donor site of ZNF419 results in the novel renal cell carcinoma-associated minor histocompatibility antigen ZAPHIR.

    Directory of Open Access Journals (Sweden)

    Kelly Broen

    Full Text Available Nonmyeloablative allogeneic stem cell transplantation (SCT can induce remission in patients with renal cell carcinoma (RCC, but this graft-versus-tumor (GVT effect is often accompanied by graft-versus-host disease (GVHD. Here, we evaluated minor histocompatibility antigen (MiHA-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI. One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT.

  15. Is it time to revisit contraindications to organ donation from donors with a JAK-2 mutation? Safe use of a liver allograft from a donor with essential thrombocythaemia.

    Science.gov (United States)

    Haldar, Debashis; Chen, Frederick; Byron, Jane; Elsharkawy, Ahmed Mohamed; Perera, M Thamara Pr

    2015-07-01

    Transplantation can cure end-stage liver disease and hepatocellular carcinoma. However, the balance of organ demand and provision is heavily tipped to the detriment of patients. Patients awaiting transplantation rely on the greater use of marginal donors that may carry a risk to the recipient. UK authorities have decreed donor haematological malignancy an absolute contraindication. The authors describe the first report of a patient being safely transplanted with a liver from a donor who suffered from JAK2 V617F mutation-driven essential thrombocythaemia to a patient with a critical burden of hepatocellular carcinoma. A year after transplantation, the patient has neither evidence of acquisition of the donor's pathology, nor evidence of carcinoma recurrence. The case highlights the responsibility of the recipient team to maximize the use of organs by expert risk assessment. Dissemination of experience should inform future decisions, benefit patients and bolster utility in an era of growing waiting-list mortality. © 2015 Steunstichting ESOT.

  16. A cryptic BAP1 splice mutation in a family with uveal and cutaneous melanoma, and paraganglioma

    DEFF Research Database (Denmark)

    Wadt, K.; Choi, J.; Chung, J.Y.

    2012-01-01

    Inactivating germ line BRCA1-associated protein-1 (BAP1) mutations have recently been reported in families with uveal or cutaneous malignant melanoma (UMM, CMM), mesothelioma, and meningioma. Although apparently predisposing to a wide range of tumors, the exact tumor spectrum associated with germ...

  17. Splicing mutation in Sbf1 causes nonsyndromic male infertility in the rat

    Czech Academy of Sciences Publication Activity Database

    Liška, F.; Chylíková, B.; Janků, M.; Šeda, Ondřej; Vernerová, Z.; Pravenec, Michal; Křen, Vladimír

    2016-01-01

    Roč. 152, č. 3 (2016), s. 215-223 ISSN 1470-1626 R&D Projects: GA CR(CZ) GA16-06548S Institutional support: RVO:67985823 ; RVO:68378050 Keywords : Sbf1 (SET binding factor 1) gene mutation * male infertility * spontaneously hypertensive rat Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.100, year: 2016

  18. Novel exonic mutation inducing aberrant splicing in the IL10RA gene and resulting in infantile-onset inflammatory bowel disease: a case report.

    Science.gov (United States)

    Yanagi, Tadahiro; Mizuochi, Tatsuki; Takaki, Yugo; Eda, Keisuke; Mitsuyama, Keiichi; Ishimura, Masataka; Takada, Hidetoshi; Shouval, Dror S; Griffith, Alexandra E; Snapper, Scott B; Yamashita, Yushiro; Yamamoto, Ken

    2016-01-28

    Although deleterious mutations in interleukin-10 and its receptor molecules cause severe infantile-onset inflammatory bowel disease, there are no reports of mutations affecting this signaling pathway in Japanese patients. Here we report a novel exonic mutation in the IL10RA gene that caused unique splicing aberrations in a Japanese patient with infantile-onset of inflammatory bowel disease in association with immune thrombocytopenic purpura and a transient clinical syndrome mimicking juvenile myelomonocytic leukemia. A Japanese boy, who was the first child of non-consanguineous healthy parents, developed bloody diarrhea, perianal fistula, and folliculitis in early infancy and was diagnosed with inflammatory bowel disease. He also developed immune thrombocytopenic purpura and transient features mimicking juvenile myelomonocytic leukemia. The patient failed to respond to various treatments, including elemental diet, salazosulfapyridine, metronidazole, corticosteroid, infliximab, and adalimumab. We identified a novel mutation (c.537G > A, p.T179T) in exon 4 of the IL10RA gene causing unique splicing aberrations and resulting in lack of signaling through the interleukin-10 receptor. At 21 months of age, the patient underwent allogeneic hematopoietic stem cell transplantation and achieved clinical remission. We describe a novel exonic mutation in the IL10RA gene resulting in infantile-onset inflammatory bowel disease. This mutation might also be involved in his early-onset hematologic disorders. Physicians should be familiar with the clinical phenotype of IL-10 signaling defects in order to enable prompt diagnosis at an early age and referral for allogeneic hematopoietic stem cell transplantation.

  19. In vivo mutation of pre-mRNA processing factor 8 (Prpf8) affects transcript splicing, cell survival and myeloid differentiation

    OpenAIRE

    Keightley, Maria-Cristina; Crowhurst, Meredith O.; Layton, Judith E.; Beilharz, Traude; Markmiller, Sebastian; Varma, Sony; Hogan, Benjamin M.; de Jong-Curtain, Tanya A.; Heath, Joan K.; Lieschke, Graham J.

    2013-01-01

    Mutated spliceosome components are recurrently being associated with perturbed tissue development and disease pathogenesis. Cephalophŏnus (cph), is a zebrafish mutant carrying an early premature STOP codon in the spliceosome component Prpf8 (pre-mRNA processing factor 8). Cph initially develops normally, but then develops widespread cell death, especially in neurons, and is embryonic lethal. Cph mutants accumulate aberrantly spliced transcripts retaining both U2- and U12-type introns. Within ...

  20. Neonatal Marfan Syndrome: Report of a Case with an Inherited Splicing Mutation outside the Neonatal Domain.

    Science.gov (United States)

    Le Gloan, Laurianne; Hauet, Quentin; David, Albert; Hanna, Nadine; Arfeuille, Chloé; Arnaud, Pauline; Boileau, Catherine; Romefort, Bénédicte; Benbrik, Nadir; Gournay, Véronique; Joram, Nicolas; Baron, Olivier; Isidor, Bertrand

    2016-02-01

    We report a child and her mother affected by Marfan syndrome. The child presented with a phenotype of neonatal Marfan syndrome, revealed by acute and refractory heart failure, finally leading to death within the first 4 months of life. Her mother had a common clinical presentation. Genetic analysis revealed an inherited FBN1 mutation. This intronic mutation (c.6163+3_6163+6del), undescribed to date, leads to exon 49 skipping, corresponding to in-frame deletion of 42 amino acids (p.Ile2014_Asp2055del). FBN1 next-generation sequencing did not show any argument for mosaicism. Association in the same family of severe neonatal and classical Marfan syndrome illustrates the intrafamilial phenotype variability.

  1. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

    DEFF Research Database (Denmark)

    Hartung, Anne-Mette; Swensen, Jeff; Uriz, Inaki E

    2016-01-01

    identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting...

  2. Aberrant splicing of androgen receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity

    NARCIS (Netherlands)

    Ris-Stalpers, C.; Kuiper, G. G.; Faber, P. W.; Schweikert, H. U.; van Rooij, H. C.; Zegers, N. D.; Hodgins, M. B.; Degenhart, H. J.; Trapman, J.; Brinkmann, A. O.

    1990-01-01

    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symptoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. We report a G----T mutation in the splice donor site of

  3. Novel de novo BRCA2 mutation in a patient with a family history of breast cancer

    DEFF Research Database (Denmark)

    Hansen, Thomas V O; Bisgaard, Marie Luise; Jønson, Lars

    2008-01-01

    BACKGROUND: BRCA2 germ-line mutations predispose to breast and ovarian cancer. Mutations are widespread and unclassified splice variants are frequently encountered. We describe the parental origin and functional characterization of a novel de novo BRCA2 splice site mutation found in a patient...... exhibiting a ductal carcinoma at the age of 40. METHODS: Variations were identified by denaturing high performance liquid chromatography (dHPLC) and sequencing of the BRCA1 and BRCA2 genes. The effect of the mutation on splicing was examined by exon trapping in COS-7 cells and by RT-PCR on RNA isolated from...... leucocytes and carcinoma tissue. RESULTS: A novel BRCA2 variant in the splice donor site of exon 21 (nucleotide 8982+1 G-->A/c.8754+1 G-->A) was identified. Exon trapping showed that the mutation activates a cryptic splice site 46 base pairs 3' of exon 21, resulting in the inclusion of a premature stop codon...

  4. An ENU mutagenesis screen in zebrafish for visual system mutants identifies a novel splice-acceptor site mutation in patched2 that results in Colobomas.

    Science.gov (United States)

    Lee, Jiwoon; Cox, Ben D; Daly, Christina M S; Lee, Chanjae; Nuckels, Richard J; Tittle, Rachel K; Uribe, Rosa A; Gross, Jeffrey M

    2012-12-13

    To identify recessive mutations affecting development and/or maintenance of the zebrafish visual system. A three-generation ENU (N-Nitroso-N-ethylurea)-based forward genetic screen was performed. F3 embryos were screened visually from 1 to 5 days postfertilization (dpf) for ocular abnormalities, and 5 dpf embryos were fixed and processed for cryosectioning, after which eye sections were screened for defects in cellular organization within the retina, lens, and cornea. A combination of PCR and DNA sequencing, in situ hybridization, and pharmacological treatments were used to clone and characterize a coloboma mutant. A total of 126 F2 families were screened, and, from these, 18 recessive mutations were identified that affected eye development. Phenotypes included lens malformations and cataracts, photoreceptor defects, oculocutaneous albinism, microphthalmia, and colobomas. Analysis of one such coloboma mutant, uta(1), identified a splice-acceptor mutation in the patched2 gene that resulted in an in-frame deletion of 19 amino acids that are predicted to contribute to the first extracellular loop of Patched2. ptch2(uta1) mutants possessed elevated Hedgehog (Hh) pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2(uta1) mutant embryos. We have identified 18 recessive mutations affecting development of the zebrafish visual system and we have characterized a novel splice-acceptor site mutation in patched2 that results in enhanced Hh pathway activity and colobomas.

  5. Hepatosplenomegalic lipidosis: what unless Gaucher? Adult cholesteryl ester storage disease (CESD) with anemia, mesenteric lipodystrophy, increased plasma chitotriosidase activity and a homozygous lysosomal acid lipase -1 exon 8 splice junction mutation.

    Science.gov (United States)

    vom Dahl, S; Harzer, K; Rolfs, A; Albrecht, B; Niederau, C; Vogt, C; van Weely, S; Aerts, J; Müller, G; Häussinger, D

    1999-10-01

    A 36-year-old woman was admitted for hepatosplenomegaly and anemia. Bone marrow cytology showed "sea-blue histiocytes", vacuolated macrophages and plasma cells. As primary liver disease, malignancy or hematologic disorders were excluded, and plasma chitotriosidase activity was increased 27-fold over control, the presence of a lysosomal storage disease was suspected. Biochemical analysis of skin fibroblasts revealed normal glucocerebrosidase and sphingomyelinase activity, but lipid analysis showed a more than 15-fold accumulation of cholesterol esters within the cells. The activity of lysosomal acid lipase (LAL) in fibroblast homogenates was decreased to 12% of control subjects. Mutational analysis of the patient's blood showed the homozygous G-->A mutation at position -1 of the exon 8 splice donor site (E8SJM-allele) known for adult cholesteryl ester storage disease (CESD); the polymorphic background was that of the complex haplotype -6Thr, 2Gly, 894 G-->A. Based on clinical, laboratory, cytological and and biochemical findings, CESD can clearly be separated from other more frequent inherited lysosomal storage diseases, e.g. atypical forms of Gaucher disease.

  6. Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.

    Directory of Open Access Journals (Sweden)

    Melissa K Boles

    2009-12-01

    Full Text Available An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn, inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.

  7. Homozygosity for the common GAA gene splice site mutation c.-32-13T>G in Pompe disease is associated with the classical adult phenotypical spectrum.

    Science.gov (United States)

    Musumeci, Olimpia; Thieme, Andrea; Claeys, Kristl G; Wenninger, Stephan; Kley, Rudolf A; Kuhn, Marius; Lukacs, Zoltan; Deschauer, Marcus; Gaeta, Michele; Toscano, Antonio; Gläser, Dieter; Schoser, Benedikt

    2015-09-01

    Homozygosity for the common Caucasian splice site mutation c.-32-13T>G in intron 1 of the GAA gene is rather rare in Pompe patients. We report on the clinical, biochemical, morphological, muscle imaging, and genetic findings of six adult Pompe patients from five unrelated families with the c.-32-13T>G GAA gene mutation in homozygous state. All patients had decreased GAA activity and elevated creatine kinase levels. Five patients, aged between 43 and 61 years (median 53 years), initially presented with myalgia, hyperCKaemia, and/or exercise induced fatigue at an age of onset (12-55 years). All but one had proximal lower limb weakness combined with axial weakness and moderate respiratory insufficiency; the sixth patient presented with hyperCKaemia only. Muscle biopsies showed PAS-positive vacuolar myopathy with lysosomal changes and reduced GAA activity. Muscle MRI of lower limb muscles revealed a moderate adipose substitution of the gluteal muscles, biceps femoris and slight fatty infiltration of all thigh muscles. One MRI of the respiratory muscles revealed a diaphragmatic atrophy with unilateral diaphragm elevation. So, the common Caucasian, so called mild, splice site mutation c.-32-13T>G in intron 1 of the GAA gene in a homozygote status reflects the full adult Pompe disease phenotype severity spectrum. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. In vivo mutation of pre-mRNA processing factor 8 (Prpf8) affects transcript splicing, cell survival and myeloid differentiation

    Science.gov (United States)

    Keightley, Maria-Cristina; Crowhurst, Meredith O.; Layton, Judith E.; Beilharz, Traude; Markmiller, Sebastian; Varma, Sony; Hogan, Benjamin M.; de Jong-Curtain, Tanya A.; Heath, Joan K.; Lieschke, Graham J.

    2013-01-01

    Mutated spliceosome components are recurrently being associated with perturbed tissue development and disease pathogenesis. Cephalophŏnus (cph), is a zebrafish mutant carrying an early premature STOP codon in the spliceosome component Prpf8 (pre-mRNA processing factor 8). Cph initially develops normally, but then develops widespread cell death, especially in neurons, and is embryonic lethal. Cph mutants accumulate aberrantly spliced transcripts retaining both U2- and U12-type introns. Within early haematopoeisis, myeloid differentiation is impaired suggesting Prpf8 is required for haematopoietic development. Cph provides an animal model for zygotic PRPF8 dysfunction diseases and for evaluating therapeutic interventions. PMID:23714367

  9. Mutations in matrix and SP1 repair the packaging specificity of a Human Immunodeficiency Virus Type 1 mutant by reducing the association of Gag with spliced viral RNA

    Directory of Open Access Journals (Sweden)

    Ristic Natalia

    2010-09-01

    Full Text Available Abstract Background The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1 sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants. Results HIV-1 lacking SL1 (NLΔSL1 did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K on day 18 postinfection and C1907T in the SP1 domain (P10L on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-11-07

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

  11. Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11.

    Science.gov (United States)

    Baert, Annelot; Machackova, Eva; Coene, Ilse; Cremin, Carol; Turner, Kristin; Portigal-Todd, Cheryl; Asrat, Marie Jill; Nuk, Jennifer; Mindlin, Allison; Young, Sean; MacMillan, Andree; Van Maerken, Tom; Trbusek, Martin; McKinnon, Wendy; Wood, Marie E; Foulkes, William D; Santamariña, Marta; de la Hoya, Miguel; Foretova, Lenka; Poppe, Bruce; Vral, Anne; Rosseel, Toon; De Leeneer, Kim; Vega, Ana; Claes, Kathleen B M

    2018-04-01

    For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies. © 2017 Wiley Periodicals, Inc.

  12. Characterization of the Wilson disease gene: Genomic organization; alternative splicing; structure/function predictions; and population frequencies of disease-specific mutations

    Energy Technology Data Exchange (ETDEWEB)

    Petrukhin, K.; Chernov, I.; Ross, B.M. [Columbia Univ., New York, NY (United States)] [and others

    1994-09-01

    The Wilson disease (WD) gene has recently been identified as a putative copper-transporting ATPase with high amino acid similarity with the Menkes disease (MNK) gene. We have further characterized the WD gene by extending the 5{prime}-coding and non-coding DNA sequence and elucidating the intron/exon structure and genomic organization. Analysis of RNA transcripts from liver, brain, kidney and placenta reveals extensive alternative splicing which may provide a mechanism to regulate the quantity of functional protein product. Comparative sequence analysis shows that WD and MNK belong to the sub-family of heavy metal-transporting ATPases with several characterizing features which include unique amino acid motifs and distinct N-terminal and C-terminal transmembrane structure. Our data indicate that the 600 amino acid metal binding portion of the WD and MNK proteins was formed by gene duplication events and splicing of the 6 metal binding domain segment to a common ancestral protein. We have raised a WD-specific anti-peptide antibody to the N-terminal region and are beginning to explore the cellular and intracellular location of the WD protein. The metal-binding segment of the WD protein has been expressed in E. coli and metal binding assays are underway to characterize this aspect of the protein`s function. We have identified numerous disease-specific mutations and developed a rapid {open_quotes}reverse dot blot{close_quotes} screening protocol to determine mutation frequencies in different populations. The most common mutation disrupts the characteristic SEHP motif and accounts for more than 40% of WD cases in North American, Russian, and Swedish populations. This mutation has not been observed in our limited Sicilian sample.

  13. The prevalent deep intronic c. 639+919 G>A GLA mutation causes pseudoexon activation and Fabry disease by abolishing the binding of hnRNPA1 and hnRNP A2/B1 to a splicing silencer

    DEFF Research Database (Denmark)

    Palhais, Bruno; Dembic, Maja; Sabaratnam, Rugivan

    2016-01-01

    Fabry disease is an X-linked recessive inborn disorder of the glycosphingolipid metabolism, caused by total or partial deficiency of the lysosomal α-galactosidase A enzyme due to mutations in the GLA gene. The prevalent c.639+919 G>A mutation in GLA leads to pathogenic insertion of a 57bp...... oligonucleotide (SSO) mediated blocking of the pseudoexon 3'ss and 5'ss effectively restores normal GLA splicing. This indicates that SSO based splicing correction may be a therapeutic alternative in the treatment of Fabry disease....

  14. Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

    Energy Technology Data Exchange (ETDEWEB)

    Solera, J. (Unidades de Genetica Molecular, Madrid (Spain)); Magallon, M.; Martin-Villar, J. (Hemofilia Hospital, Madrid (Spain)); Coloma, A. (Departamento deBioquimica de la Facultad de Medicina de la Universidad Autonoma, Madrid (Spain))

    1992-02-01

    DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.

  15. A novel SLC25A20 splicing mutation in patients of different ethnic origin with neonatally lethal carnitine-acylcarnitine translocase (CACT) deficiency.

    Science.gov (United States)

    Korman, Stanley H; Pitt, James J; Boneh, Avihu; Dweikat, Imad; Zater, Mokhtar; Meiner, Vardiella; Gutman, Alisa; Brivet, Michèle

    2006-12-01

    Carnitine-acylcarnitine translocase (CACT) deficiency is a rare disorder of fatty acid oxidation associated with high mortality. Two female newborns of different ethnic origin (the first Anglo-Celtic and the second Palestinian Arab) both died after sudden collapse on day 2 of life. Both had elevated bloodspot long-chain acylcarnitines consistent with either CACT or carnitine palmitoyltransferase II (CPT2) deficiency; the latter was excluded by demonstrating normal CPT2 activity in fibroblasts. Direct sequencing of all SLC25A20 (CACT) gene exons and exon-intron boundaries revealed that Patient 1 was compound heterozygous for a novel c.609-3c>g (IVS6-3c>g) mutation on the paternal allele and a previously described c.326delG mutation on the maternal allele. Patient 2 was homozygous for the same, novel c.609-3c>g mutation. Previously reported SLC25A20 mutations have been almost exclusively confined to a single family or ethnic group. Analysis of fibroblast cDNA by RT-PCR, agarose gel electrophoresis and sequencing of extracted bands showed that both mutations produce aberrant splicing. c.609-3C>G results in exon 7 skipping leading to a frameshift with premature termination seven amino acids downstream. c.326delG was confirmed to produce skipping of exons 3 or 3 plus 4. CACT activity in both patients' fibroblasts was near-zero. For both families, prenatal diagnosis of an unaffected fetus was performed by mutation analysis on CVS tissue in a subsequent pregnancy. Due to the urgency of prenatal diagnosis in the second family, molecular diagnosis was performed prior to demonstration of CACT enzyme deficiency, illustrating that mutation analysis is a rapid and reliable approach to first-line diagnosis of CACT deficiency.

  16. Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

    International Nuclear Information System (INIS)

    Zhang, Wentao; Du, Bojing; Liu, Di; Qi, Xiaoting

    2014-01-01

    Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerance in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd 2+ uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance

  17. Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wentao; Du, Bojing; Liu, Di; Qi, Xiaoting, E-mail: qixiaoting@cnu.edu.cn

    2014-12-12

    Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerance in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd{sup 2+} uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance.

  18. Using a minigene approach to characterize a novel splice site mutation in human F7 gene causing inherited factor VII deficiency in a Chinese pedigree.

    Science.gov (United States)

    Yu, T; Wang, X; Ding, Q; Fu, Q; Dai, J; Lu, Y; Xi, X; Wang, H

    2009-11-01

    Factor VII deficiency which transmitted as an autosomal recessive disorder is a rare haemorrhagic condition. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a Chinese pedigree with FVII deficiency. The proband was diagnosed as inherited coagulation FVII deficiency by reduced plasma levels of FVII activity (4.4%) and antigen (38.5%). All nine exons and their flanking sequence of F7 gene were amplified by polymerase chain reaction (PCR) for the proband and the PCR products were directly sequenced. The compound heterozygous mutations of F7 (NM_000131.3) c.572-1G>A and F7 (NM_000131.3) c.1165T>G; p.Cys389Gly were identified in the proband's F7 gene. To investigate the splicing patterns associated with F7 c.572-1G>A, ectopic transcripts in leucocytes of the proband were analyzed. F7 minigenes, spanning from intron 4 to intron 7 and carrying either an A or a G at position -1 of intron 5, were constructed and transiently transfected into human embryonic kidney (HEK) 293T cells, followed by RT-PCR analysis. The aberrant transcripts from the F7 c.572-1G>A mutant allele were not detected by ectopic transcription study. Sequencing of the RT-PCR products from the mutant transfectant demonstrated the production of an erroneously spliced mRNA with exon 6 skipping, whereas a normal splicing occurred in the wide type transfectant. The aberrant mRNA produced from the F7 c.572-1G>A mutant allele is responsible for the factor VII deficiency in this pedigree.

  19. Two novel exonic point mutations in HEXA identified in a juvenile Tay-Sachs patient: role of alternative splicing and nonsense-mediated mRNA decay.

    Science.gov (United States)

    Levit, A; Nutman, D; Osher, E; Kamhi, E; Navon, R

    2010-06-01

    We have identified three mutations in the beta-hexoseaminidase A (HEXA) gene in a juvenile Tay-Sachs disease (TSD) patient, which exhibited a reduced level of HEXA mRNA. Two mutations are novel, c.814G>A (p.Gly272Arg) and c.1305C>T (p.=), located in exon 8 and in exon 11, respectively. The third mutation, c.1195A>G (p.Asn399Asp) in exon 11, has been previously characterized as a common polymorphism in African-Americans. Hex A activity measured in TSD Glial cells, transfected with HEXA cDNA constructs bearing these mutations, was unaltered from the activity level measured in normal HEXA cDNA. Analysis of RT-PCR products revealed three aberrant transcripts in the patient, one where exon 8 was absent, one where exon 11 was absent and a third lacking both exons 10 and 11. All three novel transcripts contain frameshifts resulting in premature termination codons (PTCs). Transfection of mini-gene constructs carrying the c.814G>A and c.1305C>T mutations proved that the two mutations result in exon skipping. mRNAs that harbor a PTC are detected and degraded by the nonsense-mediated mRNA decay (NMD) pathway to prevent synthesis of abnormal proteins. However, although NMD is functional in the patient's fibroblasts, aberrant transcripts are still present. We suggest that the level of correctly spliced transcripts as well as the efficiency in which NMD degrade the PTC-containing transcripts, apparently plays an important role in the phenotype severity of the unique patient and thus should be considered as a potential target for drug therapy.

  20. A unique LAMB3 splice-site mutation with founder effect from the Balkans causes lethal epidermolysis bullosa in several European countries.

    Science.gov (United States)

    Mayer, B; Silló, P; Mazán, M; Pintér, D; Medvecz, M; Has, C; Castiglia, D; Petit, F; Charlesworth, A; Hatvani, Zs; Pamjav, H; Kárpáti, S

    2016-10-01

    We have encountered repeated cases of recessive lethal generalized severe (Herlitz-type) junctional epidermolysis bullosa (JEB gen sev) in infants born to Hungarian Roma parents residing in a small region of Hungary. To identify the disease-causing mutation and to investigate the genetic background of its unique carrier group. The LAMB3 gene was analysed in peripheral-blood genomic DNA samples, and the pathological consequences of the lethal defect were confirmed by cutaneous LAMB3cDNA sequencing. A median joining haplotype network within the Y chromosome H1a-M82 haplogroup of individuals from the community was constructed, and LAMB3 single-nucleotide polymorphism (SNP) patterns were also determined. An unconventional intronic splice-site mutation (LAMB3, c.1133-22G>A) was identified. Thirty of 64 voluntarily screened Roma from the closed community carried the mutation, but none of the 306 Roma from other regions of the country did. The age of the mutation was estimated to be 548 ± 222 years. Within the last year, more patients with JEB gen sev carrying the same unusual mutation have been identified in three unrelated families, all immigrants from the Balkans. Two were compound heterozygous newborns, in Germany and Italy, and one homozygous newborn died in France. Only the French family recognized their Roma background. LAMB3SNP haplotyping confirmed the link between the apparently unrelated Hungarian, German and Italian male cases, but could not verify the same background in the female newborn from France. The estimated age of the mutation corresponds to the time period when Roma were wandering in the Balkans. © 2016 British Association of Dermatologists.

  1. Familial isolated primary pigmented nodular adrenocortical disease associated with a novel low penetrance PRKAR1A gene splice site mutation

    DEFF Research Database (Denmark)

    Storr, Helen L; Metherell, Louise A; Dias, Renuka

    2010-01-01

    Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating germline protein kinase A regulatory subunit type 1-alpha (PRKAR1A) mutations and loss of heterozygosity at the 17q22-24 locus in approximately 50% patients. PRKAR1A mutations are observed in both isolated PP...... PPNAD (iPPNAD) and Carney complex (CNC). Most mutations result in a functionally null-allele and exhibit high penetrance. We genotyped members of an extended family for a novel PRKAR1A mutation and undertook detailed phenotyping for CNC in the affected individuals....

  2. Single base mutation in the pro. alpha. 2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame pro. alpha. 2(I) chain

    Energy Technology Data Exchange (ETDEWEB)

    Tromp, G.; Prockop, D.J. (Thomas Jefferson Univ., Philadelphia, PA (USA))

    1988-07-01

    Previous observations demonstrated that a lethal variant of osteogenesis imperfecta had two altered alleles for pro{alpha}2(I) chains of type I procollagen. One mutation produced a nonfunctioning allele in that there was synthesis of mRNA but no detectable synthesis of pro{alpha}2(I) chains from the allele. The mutation in the other allele caused synthesis of shortened pro{alpha}2(I) chains that lacked most or all of the 18 amino acids encoded by exon 28. Subclones of the pro{alpha}2(I) gene were prepared from the proband's DNA and the DNA sequence was determined for a 582-base-pair (bp) region that extended from the last 30 bp of intervening sequence 26 to the first 26 bp of intervening sequence 29. Data from six independent subclones demonstrated that all had the same sequence as a previously isolated normal clone for the pro{alpha}2(I) gene except that four subclones had a single base mutation at the 3{prime} end of intervening sequence 27. The mutation was a substitution of guanine for adenine that changed the universal consensus sequence for the 3{prime} splicing site of RNA from -AG- to -GG-. S1 nuclease experiments demonstrated that about half the pro{alpha}2(I) mRNA in the proband's fibroblasts was abnormally spliced and that the major species of abnormal pro{alpha}2(I) mRNA was completely spliced from the last codon of exon 27 to the first codon of exon 29. The mutation is apparently unique among RNA splicing mutations of mammalian systems in producing a shortened polypeptide chain that is in-frame in terms of coding sequences, that is used in the subunit assembly of a protein, and that contributes to a lethal phenotype.

  3. A synonymous mutation in TCOF1 causes Treacher Collins syndrome due to mis-splicing of a constitutive exon.

    Science.gov (United States)

    Macaya, D; Katsanis, S H; Hefferon, T W; Audlin, S; Mendelsohn, N J; Roggenbuck, J; Cutting, G R

    2009-08-01

    Interpretation of the pathogenicity of sequence alterations in disease-associated genes is challenging. This is especially true for novel alterations that lack obvious functional consequences. We report here on a patient with Treacher Collins syndrome (TCS) found to carry a previously reported mutation, c.122C > T, which predicts p.A41V, and a novel synonymous mutation, c.3612A > C. Pedigree analysis showed that the c.122C > T mutation segregated with normal phenotypes in multiple family members while the c.3612A > C was de novo in the patient. Analysis of TCOF1 RNA in lymphocytes showed a transcript missing exon 22. These results show that TCS in the patient is due to haploinsufficiency of TCOF1 caused by the synonymous de novo c.3612A > C mutation. This study highlights the importance of clinical and pedigree evaluation in the interpretation of known and novel sequence alterations. 2009 Wiley-Liss, Inc.

  4. A novel Bruton's tyrosine kinase gene (BTK) invariant splice site mutation in a Malaysian family with X-linked agammaglobulinemia.

    Science.gov (United States)

    Chear, Chai Teng; Gill, Harvindar Kaur; Ramly, Nazatul Haslina; Dhaliwal, Jasbir Singh; Bujang, Noraini; Ripen, Adiratna Mat; Mohamad, Saharuddin Bin

    2013-12-01

    X-linked agammaglobulinemia (XLA) is a rare genetic disorder caused by mutations in the Bruton's tyrosine kinase (BTK) gene. These mutations cause defects in early B cell development. A patient with no circulating B cells and low serum immunoglobulin isotypes was studied as were his mother and sister. Monocyte BTK protein expression was evaluated by flow cytometry. The mutation was determined using PCR and followed by sequencing. Flow cytometry showed the patient lacked BTK protein expression in his monocytes while the mother and sister had 62% and 40% of the monocytes showing BTK protein expressions respectively. The patient had a novel base substitution in the first nucleotide of intron 9 in the BTK gene, and the mutation was IVS9+1Gagammaglobulinemia and may be used for subsequent genetic counseling, carrier detection and prenatal diagnosis.

  5. A novel splice-site mutation in ATP6V0A4 gene in two brothers with ...

    Indian Academy of Sciences (India)

    2014-12-10

    Dec 10, 2014 ... ral hearing loss (SNHL) observed in patients with mutations in the ATP6V1B1 gene (Vargas-Poussou et al. 2006). Indeed,. Stover et al. (2002) demonstrated that young adult patients with ATP6V0A4 mutations could develop a mild SNHL in the long term (Stover et al. 2002). Some dRTA patients without. ∗.

  6. 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.

  7. 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

  8. Allogeneic stem-cell transplantation in patients with NPM1-mutated acute myeloid leukemia: results from a prospective donor versus no-donor analysis of patients after upfront HLA typing within the SAL-AML 2003 trial.

    Science.gov (United States)

    Röllig, Christoph; Bornhäuser, Martin; Kramer, Michael; Thiede, Christian; Ho, Anthony D; Krämer, Alwin; Schäfer-Eckart, Kerstin; Wandt, Hannes; Hänel, Mathias; Einsele, Hermann; Aulitzky, Walter E; Schmitz, Norbert; Berdel, Wolfgang E; Stelljes, Matthias; Müller-Tidow, Carsten; Krug, Utz; Platzbecker, Uwe; Wermke, Martin; Baldus, Claudia D; Krause, Stefan W; Stölzel, Friedrich; von Bonin, Malte; Schaich, Markus; Serve, Hubert; Schetelig, Johannes; Ehninger, Gerhard

    2015-02-10

    The presence of a mutated nucleophosmin-1 gene (NPM1(mut)) in acute myeloid leukemia (AML) is associated with a favorable prognosis. To assess the predictive value with regard to allogeneic stem-cell transplantation (SCT), we compared the clinical course of patients with NPM1(mut) AML eligible for allogeneic SCT in a donor versus no-donor analysis. Of 1,179 patients with AML (age 18 to 60 years) treated in the Study Alliance Leukemia AML 2003 trial, we identified all NPM1(mut) patients with an intermediate-risk karyotype. According to the trial protocol, patients were intended to receive an allogeneic SCT if an HLA-identical sibling donor was available. Patients with no available donor received consolidation or autologous SCT. We compared relapse-free survival (RFS) and overall survival (OS) depending on the availability of a suitable donor. Of 304 eligible patients, 77 patients had a sibling donor and 227 had no available matched family donor. The 3-year RFS rates in the donor and no-donor groups were 71% and 47%, respectively (P = .005); OS rates were 70% and 60%, respectively (P = .114). In patients with normal karyotype and no FLT3 internal tandem duplication (n = 148), the 3-year RFS rates in the donor and no-donor groups were 83% and 53%, respectively (P = .004); and the 3-year OS rates were 81% and 75%, respectively (P = .300). Allogeneic SCT led to a significantly prolonged RFS in patients with NPM1(mut) AML. The absence of a statistically significant difference in OS is most likely a result of the fact that NPM1(mut) patients who experienced relapse responded well to salvage treatment. Allogeneic SCT in first remission has potent antileukemic efficacy and is a valuable treatment option in patients with NPM1(mut) AML with a sibling donor. © 2014 by American Society of Clinical Oncology.

  9. Multiple surface antigen mutations in five blood donors with occult hepatitis B virus infection

    NARCIS (Netherlands)

    Zaaijer, H. L.; Torres, P.; Ontañón, A.; Ponte, L. González; Koppelman, M. H. G. M.; Lelie, P. N.; Hemert, F. J. van; Boot, H. J.

    2008-01-01

    Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA while the HBV surface antigen (HBsAg) remains undetectable. The HBV genomes in five asymptomatic blood donors with occult HBV infection and low viremia ( <10 to 1,000 HBV DNA copies/mL, genotype D) were studied. An

  10. A novel splice site mutation in the dentin sialophosphoprotein gene in a Chinese family with dentinogenesis imperfecta type II

    International Nuclear Information System (INIS)

    Wang Haoyang; Hou Yanning; Cui Yingxia; Huang Yufeng; Shi Yichao; Xia Xinyi; Lu Hongyong; Wang Yunhua; Li Xiaojun

    2009-01-01

    Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM 125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C→A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II

  11. SKIP is a component of the spliceosome linking alternative splicing and the circadian clock in Arabidopsis.

    Science.gov (United States)

    Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C Robertson; Xu, Xiaodong; Ma, Ligeng

    2012-08-01

    Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5' and 3' splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level.

  12. Asymptomatic parental mosaicism for osteogenesis imperfect associated with a new splice site mutation in COL1A2

    DEFF Research Database (Denmark)

    Frederiksen, Anja Lisbeth; Dunø, Morten; Johnsen, Iben Birgit Gade

    2016-01-01

    Recurrent lethal perinatal osteogenesis imperfecta may result from asymptomatic parental mosaicism. A previously unreported mutation in COL1A2 leads to recurrent cases of fetal osteogenesis imperfecta Sillence type IIA, which emphasizes the importance of clinical and genetic evaluation of mosaicism...

  13. Identification of a novel EYA1 splice-site mutation in a Danish branchio-oto-renal syndrome family

    DEFF Research Database (Denmark)

    Henriksen, Ann Marie; Tümer, Zeynep; Tommerup, Niels

    2004-01-01

    Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by variable clinical manifestations including branchial fistulae, preauricular pits, ear malformations, hearing impairment, and renal anomalies. BOR is caused by mutations in the genes EYA1 and SIX1. A Danish BOR fa...

  14. Treacher Collins syndrome may result from insertions, deletions or splicing mutations, which introduce a termination codon into the gene

    NARCIS (Netherlands)

    Gladwin, A. J.; Dixon, J.; Loftus, S. K.; Edwards, S.; Wasmuth, J. J.; Hennekam, R. C.; Dixon, M. J.

    1996-01-01

    Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development the features of which include conductive hearing loss and cleft palate. Recently, the Treacher Collins syndrome gene (TCOF1) has been positionally cloned and a series of five mutations within the coding sequence

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation...

  16. XLA patients with BTK splice-site mutations produce low levels of wild-type BTK transcripts.

    NARCIS (Netherlands)

    Noordzij, J.G.; Bruin-Versteeg, S. de; Hartwig, N.G.; Weemaes, C.M.R.; Gerritsen, E.J.; Bernatowska, E.; Lierde, S. van; Groot, R. de; Dongen, J.J.M. van

    2002-01-01

    X-linked agammaglobulinemia is caused by mutations in the BTK gene, which result in a precursor B-cell differentiation arrest in the bone marrow and the absence of or strongly reduced B lymphocytes in blood. We identified a patient with a mild clinical phenotype, low numbers of B lymphocytes, and a

  17. Asymptomatic parental mosaicism for osteogenesis imperfecta associated with a new splice site mutation in COL1A2

    OpenAIRE

    Frederiksen, Anja Lisbeth; Duno, Morten; Johnsen, Iben B. G.; Nielsen, Morten Frost; Kr?ig?rd, Anne Bruun

    2016-01-01

    Key Clinical Message Recurrent lethal perinatal osteogenesis imperfecta may result from asymptomatic parental mosaicism. A previously unreported mutation in COL1A2 leads to recurrent cases of fetal osteogenesis imperfecta Sillence type IIA, which emphasizes the importance of clinical and genetic evaluation of mosaicism in asymptomatic parents as verified mosaicism highly increases recurrence risk.

  18. Identification of a candidate mutation in the COL1A2 gene of a Chow Chow with osteogenesis imperfecta.

    Science.gov (United States)

    Quist, E M; Doan, R; Pool, R R; Porter, B F; Bannasch, D L; Dindot, S V

    2017-09-19

    Osteogenesis imperfecta (OI) is a genetic disease that occurs in humans and animals. Individuals with OI exhibit signs of extreme bone fragility and osteopenia with frequent fractures and perinatal lethality in severe cases. In this study, we report the clinical diagnosis of OI in a dog and the use targeted next-generation sequencing to identify a candidate autosomal dominant mutation in the COL1A2 gene. A five-month old male Chow Chow was examined with a fractured left humerus and resolving, bilateral femoral fractures. Radiographs revealed generalized osteopenia and bilateral humeral, radial, and femoral fractures. Targeted next-generation sequencing of genes associated with OI in humans (COL1A1, COL1A2, LEPRE1, SERPINH1, and CRTAP) revealed a G>A heterozygous mutation in the splice donor site of exon 18 of the COL1A2 gene (c.936+1G>A). The splice donor mutation was not detected among 91 control dogs representing 21 breeds. A comparative analysis of exon 18 and the exon-intron junction further showed that the mutated splice donor site is conserved among vertebrates. Altogether, these findings reveal a candidate autosomal splice donor site mutation causing OI in an individual Chow Chow. © The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Clinical Expression and New SPINK5 Splicing Defects in Netherton Syndrome : Unmasking a Frequent Founder Synonymous Mutation and Unconventional Intronic Mutations

    NARCIS (Netherlands)

    Lacroix, Matthieu; Lacaze-Buzy, Laetitia; Furio, Laetitia; Tron, Elodie; Valari, Manthoula; Van der Wier, Gerda; Bodemer, Christine; Bygum, Anette; Bursztejn, Anne-Claire; Gaitanis, George; Paradisi, Mauro; Stratigos, Alexander; Weibel, Lisa; Deraison, Celine; Hovnanian, Alain

    Netherton syndrome (NS) is a severe skin disease caused by loss-of-function mutations in SPINK5 (serine protease inhibitor Kazal-type 5) encoding the serine protease inhibitor LEKTI (lympho-epithelial Kazal type-related inhibitor). Here, we disclose new SPINK5 defects in 12 patients, who presented a

  20. The connection between splicing and cancer

    OpenAIRE

    Srebrow, Anabella; Kornblihtt, Alberto Rodolfo

    2017-01-01

    Alternative splicing is a crucial mechanism for generating protein diversity. Different splice variants of a given protein can display different and even antagonistic biological functions. Therefore, appropriate control of their synthesis is required to assure the complex orchestration of cellular processes within multicellular organisms. Mutations in cisacting splicing elements or changes in the activity of regulatory proteins that compromise the accuracy of either constitutive or alternativ...

  1. An RNA-splicing mutation (G{sup +51VS20}) in the Type II collagen gene (COL2A1) in a family with spondyloepiphyseal dysplasia congenita

    Energy Technology Data Exchange (ETDEWEB)

    Tiller, G.E.; Polumbo, P.A. [Vanderbilt Univ. School of Medicine, Nashville, TN (United States); Weis, M.A.; Eyre, D.R. [Univ. of Washington, Seattle, WA (United States); Gruber, H.E.; Rimoin, D.L.; Cohn, D.H. [Cedars-Sinai Medical Center, Los Angeles, CA (United States)]|[Univ. of California School of Medicine, Los Angeles, CA (United States)

    1995-02-01

    Defects in type II collagen have been demonstrated in a phenotypic continuum of chondrodysplasias that includes achondrogenesis II, hypochondrogenesis, spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia, and Stickler syndrome. We have determined that cartilage from a terminated fetus with an inherited form of SEDC contained both normal {alpha}1(II) collagen chains and chains that lacked amino acids 256-273 of the triple-helical domain. PCR amplification of this region of COL2A1, from genomic DNA, yielded products of normal size, while amplification of cDNA yielded a normal sized species and a shorter fragment missing exon 20. Sequence analysis of genomic DNA from the fetus revealed a G{yields}T transversion at position +5 of intron 20; the affected father was also heterozygous for the mutation. Allele-specific PCR and heteroduplex analysis of a VNTR in COL2A1 independently confirmed the unaffected status of a fetus in a subsequent pregnancy. Thermodynamic calculations suggest that the mutation prevents normal splicing of exon 20 by interfering with binding of U{sub 1} small-nuclear RNA to pre-mRNA, thus leading to skipping of exon 20 in transcripts from the mutant allele. Electron micrographs of diseased cartilage showed intracellular inclusion bodies, which were stained by an antibody to {alpha}1(II) procollagen. Our findings support the hypothesis that {alpha}-chain length alterations that preserve the Gly-X-Y repeat motif of the triple helix result in partial intracellular retention of {alpha}1(II) procollagen and produce mild to moderate chondrodysplasia phenotypes. 50 refs., 6 figs., 1 tab.

  2. A splicing mutation in the novel mitochondrial protein DNAJC11 causes motor neuron pathology associated with cristae disorganization, and lymphoid abnormalities in mice.

    Directory of Open Access Journals (Sweden)

    Fotis Ioakeimidis

    Full Text Available Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.

  3. A splicing mutation in the novel mitochondrial protein DNAJC11 causes motor neuron pathology associated with cristae disorganization, and lymphoid abnormalities in mice.

    Science.gov (United States)

    Ioakeimidis, Fotis; Ott, Christine; Kozjak-Pavlovic, Vera; Violitzi, Foteini; Rinotas, Vagelis; Makrinou, Eleni; Eliopoulos, Elias; Fasseas, Costas; Kollias, George; Douni, Eleni

    2014-01-01

    Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.

  4. Severe subacute GM2 gangliosidosis caused by an apparently silent HEXA mutation (V324V) that results in aberrant splicing and reduced HEXA mRNA.

    Science.gov (United States)

    Wicklow, Brandy A; Ivanovich, Jennifer L; Plews, Margot M; Salo, Timothy J; Noetzel, Michael J; Lueder, Gregg T; Cartegni, Luca; Kaback, Michael M; Sandhoff, Konrad; Steiner, Robert D; Triggs-Raine, Barbara L

    2004-06-01

    We have characterized the molecular basis of beta-hexosaminidase A (HEX A) deficiency in a patient ascertained through an ophthalmologic examination that revealed cherry red spots on his retina. The absence of neurological deficit in this child until 3 3/4 years of age indicated residual HEX A must be present. Three HEXA mutations, 10T > C (S4P) and 972T > A (V324V) on the maternal allele, and 1A > T (M1L) on the paternal allele were identified. The effects of the amino acid substitutions on HEX A expressed in COS-7 cells were analyzed; as expected, no HEX A activity was associated with the M1L mutation but surprisingly, the S4P mutation resulted in 59% of the HEX A activity expressed by the wild type cDNA. The effect of the S4P change was much less than that of another HEXA mutation, G269S, associated with an adult onset form of G(M2) gangliosidosis. This indicated that the S4P change was not the cause of disease and suggested that one of the mutations on the maternal allele, 10T > C or 972T > A, had its effect at the mRNA level. This was confirmed by Northern blot analysis that showed only 7% of the normal level of HEXA mRNA in proband fibroblasts. Analysis of the residual mRNA by RT/PCR and sequencing revealed normal transcripts from both the maternal and paternal allele, as well as a low abundance aberrant transcript from the maternal allele. Sequencing of this aberrant transcript revealed a new exon 8 donor site created by the 972T > A mutation that resulted in a 17 bp deletion and destabilization of the resulting abnormal transcript. The remaining normal mRNA produced from the 972T > A allele must account for the delayed onset of clinical symptoms in this child. Copyright 2004 Wiley-Liss, Inc.

  5. SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

    Science.gov (United States)

    Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

    2015-04-01

    Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Genomic HEXploring allows landscaping of novel potential splicing regulatory elements.

    Science.gov (United States)

    Erkelenz, Steffen; Theiss, Stephan; Otte, Marianne; Widera, Marek; Peter, Jan Otto; Schaal, Heiner

    2014-01-01

    Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based 'HEXplorer score' as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. SKIP Is a Component of the Spliceosome Linking Alternative Splicing and the Circadian Clock in Arabidopsis[W

    Science.gov (United States)

    Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

    2012-01-01

    Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

  8. Deep sequence analysis of non-small cell lung cancer: Integrated analysis of gene expression, alternative splicing, and single nucleotide variations in lung adenocarcinomas with and without oncogenic KRAS mutations

    Directory of Open Access Journals (Sweden)

    Krishna R Kalari

    2012-02-01

    Full Text Available KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC, and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes, alternate splicing (259 genes and SNV-related changes (65 genes in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NFkB, ERK1/2 and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene-gene connections within the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NFkB, ERK1/2 and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPARγ signaling pathways, suggesting that targeted PPARγ antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations.

  9. Deep Sequence Analysis of Non-Small Cell Lung Cancer: Integrated Analysis of Gene Expression, Alternative Splicing, and Single Nucleotide Variations in Lung Adenocarcinomas with and without Oncogenic KRAS Mutations

    International Nuclear Information System (INIS)

    Kalari, Krishna R.; Rossell, David; Necela, Brian M.; Asmann, Yan W.; Nair, Asha

    2012-01-01

    KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NFκB, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene–gene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NFκB, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPARγ signaling pathways, suggesting that targeted PPARγ antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations.

  10. Reversion of the Arabidopsis rpn12a-1 exon-trap mutation by an intragenic suppressor that weakens the chimeric 5’ splice site [v2; ref status: indexed, http://f1000r.es/18y

    Directory of Open Access Journals (Sweden)

    Jasmina Kurepa

    2013-06-01

    Full Text Available Background: In the Arabidopsis 26S proteasome mutant rpn12a-1, an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in the rpn12a-1 mutant, two RPN12a transcripts are produced and they encode the wild-type RPN12a and a chimeric RPN12a-NPTII protein. Both proteins form complexes with other proteasome subunits leading to the formation of wild-type and mutant proteasome versions. The net result of this heterogeneity of proteasome particles is a reduction of total cellular proteasome activity. One of the consequences of reduced proteasomal activity is decreased sensitivity to the major plant hormone cytokinin. Methods: We performed ethyl methanesulfonate mutagenesis of rpn12a-1 and isolated revertants with wild-type cytokinin sensitivity. Results: We describe the isolation and analyses of suppressor of rpn12a-1 (sor1. The sor1 mutation is intragenic and located at the fifth position of the chimeric intron. This mutation weakens the activated 5' splice site associated with the STOP codon and tilts the processing of the RPN12a mRNA back towards polyadenylation. Conclusions: These results validate our earlier interpretation of the unusual nature of the rpn12a-1 mutation. Furthermore, the data show that optimal 26S proteasome activity requires RPN12a accumulation beyond a critical threshold. Finally, this finding reinforces our previous conclusion that proteasome function is critical for the cytokinin-dependent regulation of plant growth.

  11. Alteration of introns in a hyaluronan synthase 1 (HAS1 minigene convert Pre-mRNA [corrected] splicing to the aberrant pattern in multiple myeloma (MM: MM patients harbor similar changes.

    Directory of Open Access Journals (Sweden)

    Jitra Kriangkum

    Full Text Available Aberrant pre-mRNA splice variants of hyaluronan synthase 1 (HAS1 have been identified in malignant cells from cancer patients. Bioinformatic analysis suggests that intronic sequence changes can underlie aberrant splicing. Deletions and mutations were introduced into HAS1 minigene constructs to identify regions that can influence aberrant intronic splicing, comparing the splicing pattern in transfectants with that in multiple myeloma (MM patients. Introduced genetic variations in introns 3 and 4 of HAS1 as shown here can promote aberrant splicing of the type detected in malignant cells from MM patients. HAS1Vd is a novel intronic splice variant first identified here. HAS1Vb, an intronic splice variant previously identified in patients, skips exon 4 and utilizes the same intron 4 alternative 3'splice site as HAS1Vd. For transfected constructs with unaltered introns 3 and 4, HAS1Vd transcripts are readily detectable, frequently to the exclusion of HAS1Vb. In contrast, in MM patients, HAS1Vb is more frequent than HAS1Vd. In the HAS1 minigene, combining deletion in intron 4 with mutations in intron 3 leads to a shift from HAS1Vd expression to HAS1Vb expression. The upregulation of aberrant splicing, exemplified here by the expression of HAS1Vb, is shown here to be influenced by multiple genetic changes in intronic sequences. For HAS1Vb, this includes enhanced exon 4 skipping and increased usage of alternative 3' splice sites. Thus, the combination of introduced mutations in HAS1 intron3 with introduced deletions in HAS1 intron 4 promoted a shift to an aberrant splicing pattern previously shown to be clinically significant. Most MM patients harbor genetic variations in intron 4, and as shown here, nearly half harbor recurrent mutations in HAS1 intron 3. Our work suggests that aberrant intronic HAS1 splicing in MM patients may rely on intronic HAS1 deletions and mutations that are frequent in MM patients but absent from healthy donors.

  12. Alternative splicing in osteoclasts and Paget’s disease of bone

    OpenAIRE

    Klinck, Roscoe; Laberge, Gino; Bisson, Martine; McManus, Stephen; Michou, Laëtitia; Brown, Jacques P; Roux, Sophie

    2014-01-01

    Background Mutations in the SQSTM1/p62 gene have been reported in Paget’s disease of bone (PDB), but they are not sufficient to induce the pagetic osteoclast (OC) phenotype. We hypothesized that specific RNA isoforms of OC-related genes may contribute to the overactivity of pagetic OCs, along with other genetic predisposing factors. Methods Alternative splicing (AS) events were studied using a PCR-based screening strategy in OC cultures from 29 patients with PDB and 26 healthy donors (HD), al...

  13. Mutations in the CEP290 (NPHP6) Gene Are a Frequent Cause of Leber Congenital Amaurosis

    Science.gov (United States)

    den Hollander, Anneke I.; Koenekoop, Robert K.; Yzer, Suzanne; Lopez, Irma; Arends, Maarten L.; Voesenek, Krysta E. J.; Zonneveld, Marijke N.; Strom, Tim M.; Meitinger, Thomas; Brunner, Han G.; Hoyng, Carel B.; van den Born, L. Ingeborgh; Rohrschneider, Klaus; Cremers, Frans P. M.

    2006-01-01

    Leber congenital amaurosis (LCA) is one of the main causes of childhood blindness. To date, mutations in eight genes have been described, which together account for ∼45% of LCA cases. We localized the genetic defect in a consanguineous LCA-affected family from Quebec and identified a splice defect in a gene encoding a centrosomal protein (CEP290). The defect is caused by an intronic mutation (c.2991+1655A→G) that creates a strong splice-donor site and inserts a cryptic exon in the CEP290 messenger RNA. This mutation was detected in 16 (21%) of 76 unrelated patients with LCA, either homozygously or in combination with a second deleterious mutation on the other allele. CEP290 mutations therefore represent one of the most frequent causes of LCA identified so far. PMID:16909394

  14. Frequency of the hemochromatosis HFE mutations C282Y, H63D, and S65C in blood donors in the Faroe Islands

    DEFF Research Database (Denmark)

    Milman, Nils; á Steig, Torkil; Koefoed, Pernille

    2004-01-01

    .3-2.5%). The frequency of the C282Y mutation is high in Faroese blood donors, being close to and not significantly different from the frequencies reported in other Scandinavian countries: Denmark 5.7%, Norway 6.6%, Iceland 5.1%, and Sweden 6.1%. The frequency of the H63D mutation in Faroese subjects is significantly......The aim of the study was to assess the frequencies of the hereditary hemochromatosis HFE mutations C282Y, H63D, and S65C in the population in the Faroe Islands. The series comprised 200 randomly selected blood donors of Faroese heritage. The frequency of the C282Y, H63D, and S65C mutations...... frequency was 8.0% (95% CI 5.3-10.7%). The series contained three (1.5%) H63D homozygous subjects and 60 (30.0%) H63D heterozygous subjects. The H63D allele frequency was 17.5% (95% CI 13.8-21.2%). There were four (2.0%) S65C heterozygous subjects. The S65C allele frequency was 1.0% (95% CI 0...

  15. A novel 'splice site' HCN4 Gene mutation, c.1737+1 G>T, causes familial bradycardia, reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability.

    Science.gov (United States)

    Hategan, Lidia; Csányi, Beáta; Ördög, Balázs; Kákonyi, Kornél; Tringer, Annamária; Kiss, Orsolya; Orosz, Andrea; Sághy, László; Nagy, István; Hegedűs, Zoltán; Rudas, László; Széll, Márta; Varró, András; Forster, Tamás; Sepp, Róbert

    2017-08-15

    The most important molecular determinant of heart rate regulation in sino-atrial pacemaker cells includes hyperpolarization-activated, cyclic nucleotide-gated ion channels, the major isoform of which is encoded by the HCN4 gene. Mutations affecting the HCN4 gene are associated primarily with sick sinus syndrome. A novel c.1737+1 G>T 'splice-site' HCN4 mutation was identified in a large family with familial bradycardia which co-segregated with the disease providing a two-point LOD score of 4.87. Twelve out of the 22 investigated family members [4 males, 8 females average age 36 (SD 6) years] were considered as clinically affected (heart rateheart rates [62 (SD 8) vs. 73 (SD 8) bpm, p=0.0168) were significantly lower in carriers on 24-hour Holter recordings. Under maximum exercise test carriers achieved significantly lower heart rates than non-carrier family members, and percent heart rate reserve and percent corrected heart rate reserve were significantly lower in carriers. Applying rigorous criteria for chronotropic incompetence a higher number of carriers exhibited chronotropic incompetence. Parameters, characterizing short-term variability of heart rate (i.e. rMSSD and pNN50%) were increased in carrier family members, even after normalization for heart rate, in the 24-hour ECG recordings with the same relative increase in 5-minute recordings. The identified novel 'splice site' HCN4 gene mutation, c.1737+1 G>T, causes familial bradycardia and leads to reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability in the mutation carriers. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. A contracted DNA repeat in LHX3 intron 5 is associated with aberrant splicing and pituitary dwarfism in German shepherd dogs.

    Directory of Open Access Journals (Sweden)

    Annemarie M W Y Voorbij

    Full Text Available Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism.

  17. The somatic FAH C.1061C>A change counteracts the frequent FAH c.1062+5G>A mutation and permits U1snRNA-based splicing correction.

    Science.gov (United States)

    Scalet, Daniela; Sacchetto, Claudia; Bernardi, Francesco; Pinotti, Mirko; van de Graaf, Stan F J; Balestra, Dario

    2018-03-01

    In tyrosinaemia type 1(HT1), a mosaic pattern of fumarylacetoacetase (FAH) immunopositive or immunonegative nodules in liver tissue has been reported in many patients. This aspect is generally explained by a spontaneous reversion of the mutation into a normal genotype. In one HT1 patient carrying the frequent FAH c.1062+5G>A mutation, a second somatic change (c.1061C>A) has been reported in the same allele, and found in immunopositive nodules. Here, we demonstrated that the c.1062+5G>A prevents usage of the exon 12 5' splice site (ss), even when forced by an engineered U1snRNA specifically designed on the FAH 5'ss to strengthen its recognition. Noticeably the new somatic c.1061C>A change, in linkage with the c.1062+5G>A mutation, partially rescues the defective 5'ss and is associated to trace level (~5%) of correct transcripts. Interestingly, this combined genetic condition strongly favored the rescue by the engineered U1snRNA, with correct transcripts reaching up to 60%. Altogether, these findings elucidate the molecular basis of HT1 caused by the frequent FAH c.1062+5G>A mutation, and demonstrate the compensatory effect of the c.1061C>A change in promoting exon definition, thus unraveling a rare mechanism leading to FAH immune-reactive mosaicism.

  18. Double-target Antisense U1snRNAs Correct Mis-splicing Due to c.639+861C>T and c.639+919G>A GLA Deep Intronic Mutations

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    Lorenzo Ferri

    2016-01-01

    Full Text Available Fabry disease is a rare X-linked lysosomal storage disorder caused by deficiency of the α-galactosidase A (α-Gal A enzyme, which is encoded by the GLA gene. GLA transcription in humans produces a major mRNA encoding α-Gal A and a minor mRNA of unknown function, which retains a 57-nucleotide-long cryptic exon between exons 4 and 5, bearing a premature termination codon. NM_000169.2:c.639+861C>T and NM_000169.2:c.639+919G>A GLA deep intronic mutations have been described to cause Fabry disease by inducing overexpression of the alternatively spliced mRNA, along with a dramatic decrease in the major one. Here, we built a wild-type GLA minigene and two minigenes that carry mutations c.639+861C>T and c.639+919G>A. Once transfected into cells, the minigenes recapitulate the molecular patterns observed in patients, at the mRNA, protein, and enzymatic level. We constructed a set of specific double-target U1asRNAs to correct c.639+861C>T and c.639+919G>A GLA mutations. Efficacy of U1asRNAs in inducing the skipping of the cryptic exon was evaluated upon their transient co-transfection with the minigenes in COS-1 cells, by real-time polymerase chain reaction (PCR, western blot analysis, and α-Gal A enzyme assay. We identified a set of U1asRNAs that efficiently restored α-Gal A enzyme activity and the correct splicing pathways in reporter minigenes. We also identified a unique U1asRNA correcting both mutations as efficently as the mutation-specific U1asRNAs. Our study proves that an exon skipping-based approach recovering α-Gal A activity in the c.639+861C>T and c.639+919G>A GLA mutations is active.

  19. Hot-spot KIF5A mutations cause familial ALS

    Science.gov (United States)

    Yilmaz, Rüstem; Müller, Kathrin; Grehl, Torsten; Petri, Susanne; Meyer, Thomas; Grosskreutz, Julian; Weydt, Patrick; Ruf, Wolfgang; Neuwirth, Christoph; Weber, Markus; Pinto, Susana; Claeys, Kristl G; Schrank, Berthold; Jordan, Berit; Knehr, Antje; Günther, Kornelia; Hübers, Annemarie; Zeller, Daniel; Kubisch, Christian; Jablonka, Sibylle; Klopstock, Thomas; de Carvalho, Mamede; Sperfeld, Anne; Borck, Guntram; Volk, Alexander E; Dorst, Johannes; Weis, Joachim; Otto, Markus; Schuster, Joachim; Del Tredici, Kelly; Braak, Heiko; Danzer, Karin M; Freischmidt, Axel; Meitinger, Thomas; Strom, Tim M; Ludolph, Albert C; Andersen, Peter M; Weishaupt, Jochen H; Weyen, Ute; Hermann, Andreas; Hagenacker, Tim; Koch, Jan Christoph; Lingor, Paul; Göricke, Bettina; Zierz, Stephan; Baum, Petra; Wolf, Joachim; Winkler, Andrea; Young, Peter; Bogdahn, Ulrich; Prudlo, Johannes; Kassubek., Jan

    2018-01-01

    Abstract Heterozygous missense mutations in the N-terminal motor or coiled-coil domains of the kinesin family member 5A (KIF5A) gene cause monogenic spastic paraplegia (HSP10) and Charcot-Marie-Tooth disease type 2 (CMT2). Moreover, heterozygous de novo frame-shift mutations in the C-terminal domain of KIF5A are associated with neonatal intractable myoclonus, a neurodevelopmental syndrome. These findings, together with the observation that many of the disease genes associated with amyotrophic lateral sclerosis disrupt cytoskeletal function and intracellular transport, led us to hypothesize that mutations in KIF5A are also a cause of amyotrophic lateral sclerosis. Using whole exome sequencing followed by rare variant analysis of 426 patients with familial amyotrophic lateral sclerosis and 6137 control subjects, we detected an enrichment of KIF5A splice-site mutations in amyotrophic lateral sclerosis (2/426 compared to 0/6137 in controls; P = 4.2 × 10−3), both located in a hot-spot in the C-terminus of the protein and predicted to affect splicing exon 27. We additionally show co-segregation with amyotrophic lateral sclerosis of two canonical splice-site mutations in two families. Investigation of lymphoblast cell lines from patients with KIF5A splice-site mutations revealed the loss of mutant RNA expression and suggested haploinsufficiency as the most probable underlying molecular mechanism. Furthermore, mRNA sequencing of a rare non-synonymous missense mutation (predicting p.Arg1007Gly) located in the C-terminus of the protein shortly upstream of the splice donor of exon 27 revealed defective KIF5A pre-mRNA splicing in respective patient-derived cell lines owing to abrogation of the donor site. Finally, the non-synonymous single nucleotide variant rs113247976 (minor allele frequency = 1.00% in controls, n = 6137), also located in the C-terminal region [p.(Pro986Leu) in exon 26], was significantly enriched in familial amyotrophic lateral sclerosis patients (minor

  20. Evaluation of MYBPC3 trans-Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes

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    Maksymilian Prondzynski

    2017-06-01

    Full Text Available Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C. Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5′ or 3′ pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1 the feasibility of trans-splicing, although with low efficiency, and (2 efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.

  1. ALS-causing P56S mutation and splicing variation on the hVAPB MSP domain transform its β-sandwich fold into lipid-interacting helical conformations.

    Science.gov (United States)

    Qin, Haina; Wang, Wei; Song, Jianxing

    2013-02-15

    P56S mutation on VAPB MSP domain causes a familial ALS, characteristic of severe aggregation both in vivo and in vitro. We previously showed that P56S rendered the MSP domain to be predominantly disordered in water. Unexpectedly, here we reveal that P56S-MSP transforms into a highly helical conformation in a membrane environment. This chameleon transformation is shared by a splicing variant VAPB-3 with a truncated MSP domain, which is also highly disordered and buffer insoluble as demonstrated here by NMR. Our discovery provides a mechanism for ALS-causing VAPB mutants/variants to gain novel functions such as to mediate ER structure before significant accumulation of aggregates occurs. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. 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

  3. RNA Splicing: Regulation and Dysregulation in the Heart

    NARCIS (Netherlands)

    van den Hoogenhof, Maarten M. G.; Pinto, Yigal M.; Creemers, Esther E.

    2016-01-01

    RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new

  4. Revealing the Determinants of Widespread Alternative Splicing Perturbation in Cancer

    Directory of Open Access Journals (Sweden)

    Yongsheng Li

    2017-10-01

    Full Text Available It is increasingly appreciated that alternative splicing plays a key role in generating functional specificity and diversity in cancer. However, the mechanisms by which cancer mutations perturb splicing remain unknown. Here, we developed a network-based strategy, DrAS-Net, to investigate more than 2.5 million variants across cancer types and link somatic mutations with cancer-specific splicing events. We identified more than 40,000 driver variant candidates and their 80,000 putative splicing targets deregulated in 33 cancer types and inferred their functional impact. Strikingly, tumors with splicing perturbations show reduced expression of immune system-related genes and increased expression of cell proliferation markers. Tumors harboring different mutations in the same gene often exhibit distinct splicing perturbations. Further stratification of 10,000 patients based on their mutation-splicing relationships identifies subtypes with distinct clinical features, including survival rates. Our work reveals how single-nucleotide changes can alter the repertoires of splicing isoforms, providing insights into oncogenic mechanisms for precision medicine.

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

    DEFF Research Database (Denmark)

    Covaciu, C; Grosso, F; Pisaneschi, E

    2011-01-01

    shoulders. DEB-Pr is caused by either dominant (DDEB-Pr) or recessive mutations in the COL7A1 gene encoding type VII collagen (COLVII). The full spectrum of COL7A1 mutations in DEB-Pr remains elusive and the genotype-phenotype correlation is largely incomplete. Here, we report and functionally characterize...

  6. The BRCA1-Δ11q Alternative Splice Isoform Bypasses Germline Mutations and Promotes Therapeutic Resistance to PARP Inhibition and Cisplatin

    DEFF Research Database (Denmark)

    Wang, Yifan; Bernhardy, Andrea J; Cruz, Cristina

    2016-01-01

    Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and...

  7. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene

    NARCIS (Netherlands)

    Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; van Kuilenburg, André B. P.

    2016-01-01

    Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA

  8. Exome Sequencing Identifies a Novel LMNA Splice-Site Mutation and Multigenic Heterozygosity of Potential Modifiers in a Family with Sick Sinus Syndrome, Dilated Cardiomyopathy, and Sudden Cardiac Death.

    Directory of Open Access Journals (Sweden)

    Michael V Zaragoza

    Full Text Available The goals are to understand the primary genetic mechanisms that cause Sick Sinus Syndrome and to identify potential modifiers that may result in intrafamilial variability within a multigenerational family. The proband is a 63-year-old male with a family history of individuals (>10 with sinus node dysfunction, ventricular arrhythmia, cardiomyopathy, heart failure, and sudden death. We used exome sequencing of a single individual to identify a novel LMNA mutation and demonstrated the importance of Sanger validation and family studies when evaluating candidates. After initial single-gene studies were negative, we conducted exome sequencing for the proband which produced 9 gigabases of sequencing data. Bioinformatics analysis showed 94% of the reads mapped to the reference and identified 128,563 unique variants with 108,795 (85% located in 16,319 genes of 19,056 target genes. We discovered multiple variants in known arrhythmia, cardiomyopathy, or ion channel associated genes that may serve as potential modifiers in disease expression. To identify candidate mutations, we focused on ~2,000 variants located in 237 genes of 283 known arrhythmia, cardiomyopathy, or ion channel associated genes. We filtered the candidates to 41 variants in 33 genes using zygosity, protein impact, database searches, and clinical association. Only 21 of 41 (51% variants were validated by Sanger sequencing. We selected nine confirmed variants with minor allele frequencies G, a novel heterozygous splice-site mutation as the primary mutation with rare or novel variants in HCN4, MYBPC3, PKP4, TMPO, TTN, DMPK and KCNJ10 as potential modifiers and a mechanism consistent with haploinsufficiency.

  9. tRNA splicing

    OpenAIRE

    Abelson, John; Trotta, Christopher R.; Li, Hong

    1998-01-01

    Introns interrupt the continuity of many eukaryal genes, and therefore their removal by splicing is a crucial step in gene expression. Interestingly, even within Eukarya there are at least four splicing mechanisms. mRNA splicing in the nucleus takes place in two phosphotransfer reactions on a complex and dynamic machine, the spliceosome. This reaction is related in mechanism to the two self-splicing mechanisms for Group 1 and Group 2 introns. In fact the Group 2 introns are spliced by an iden...

  10. A novel missense mutation nt737T>G of JK gene with Jk(a-b-) phenotype in Chinese blood donors.

    Science.gov (United States)

    Ma, L; Liu, Y C; Zhu, S W; Hu, W J; Chen, X; Xue, M; Zhen, L; Wu, M H; Liu, Y; Sun, J

    2015-02-01

    The aim of this study was to investigate the molecular mechanism of the JK-null phenotype in the Chinese population. The Jk(a-b-) phenotype is vanishingly rare and the molecular basis differs between ethnic groups. The information regarding the molecular basis of JK-null alleles in the Chinese population is limited. Three unrelated Jk(a-b-) phenotype donors were selected from 52 260 randomly blood samples through the urea lysis test and serological analysis. The JK gene-coding regions were amplified by the polymerase chain reaction and the products were sequenced directly. Sequencing results revealed that one sample of JK(*) B alleles carried the well-known Polynesian Jk(a-b-) mutation IVS5-1g>a. Another null allele, also on the JK(*) B background, presented with two heterozygous missense mutation, including nt222C>A(Asn74Lys) in exon 5 and nt896G>A(Gly299Glu) in exon 9. The third null allele carried two heterozygous missense mutations, nt222C>A and a novel allele nt737T>G(Leu246Arg) in exon 8. The family investigation revealed that the proband was JK(*) A(737T>G)/JK(*) B(222C>A). The Jk(a-b-) phenotype in the Chinese population shows several different molecular mechanisms. A novel missense mutation nt737T>G of JK gene was found as associated with Jk(a-b-) phenotype. © 2015 British Blood Transfusion Society.

  11. Alternative mRNA Splicing in the Pathogenesis of Obesity

    Directory of Open Access Journals (Sweden)

    Chi-Ming Wong

    2018-02-01

    Full Text Available Alternative mRNA splicing is an important mechanism in expansion of proteome diversity by production of multiple protein isoforms. However, emerging evidence indicates that only a limited number of annotated protein isoforms by alternative splicing are detected, and the coding sequence of alternative splice variants usually is only slightly different from that of the canonical sequence. Nevertheless, mis-splicing is associated with a large array of human diseases. Previous reviews mainly focused on hereditary and somatic mutations in cis-acting RNA sequence elements and trans-acting splicing factors. The importance of environmental perturbations contributed to mis-splicing is not assessed. As significant changes in exon skipping and splicing factors expression levels are observed with diet-induced obesity, this review focuses on several well-known alternatively spliced metabolic factors and discusses recent advances in the regulation of the expressions of splice variants under the pathophysiological conditions of obesity. The potential of targeting the alternative mRNA mis-splicing for obesity-associated diseases therapies will also be discussed.

  12. High prevalence of HIV-1 transmitted drug-resistance mutations from proviral DNA massively parallel sequencing data of therapy-naïve chronically infected Brazilian blood donors.

    Directory of Open Access Journals (Sweden)

    Rodrigo Pessôa

    Full Text Available An improved understanding of the prevalence of low-abundance transmitted drug-resistance mutations (TDRM in therapy-naïve HIV-1-infected patients may help determine which patients are the best candidates for therapy. In this study, we aimed to obtain a comprehensive picture of the evolving HIV-1 TDRM across the massive parallel sequences (MPS of the viral entire proviral genome in a well-characterized Brazilian blood donor naïve to antiretroviral drugs.The MPS data from 128 samples used in the analysis were sourced from Brazilian blood donors and were previously classified by less-sensitive (LS or "detuned" enzyme immunoassay as non-recent or longstanding HIV-1 infections. The Stanford HIV Resistance Database (HIVDBv 6.2 and IAS-USA mutation lists were used to interpret the pattern of drug resistance. The minority variants with TDRM were identified using a threshold of ≥ 1.0% and ≤ 20% of the reads sequenced. The rate of TDRM in the MPS data of the proviral genome were compared with the corresponding published consensus sequences of their plasma viruses.No TDRM were detected in the integrase or envelope regions. The overall prevalence of TDRM in the protease (PR and reverse transcriptase (RT regions of the HIV-1 pol gene was 44.5% (57/128, including any mutations to the nucleoside analogue reverse transcriptase inhibitors (NRTI and non-nucleoside analogue reverse transcriptase inhibitors (NNRTI. Of the 57 subjects, 43 (75.4% harbored a minority variant containing at least one clinically relevant TDRM. Among the 43 subjects, 33 (76.7% had detectable minority resistant variants to NRTIs, 6 (13.9% to NNRTIs, and 16 (37.2% to PR inhibitors. The comparison of viral sequences in both sources, plasma and cells, would have detected 48 DNA provirus disclosed TDRM by MPS previously missed by plasma bulk analysis.Our findings revealed a high prevalence of TDRM found in this group, as the use of MPS drastically increased the detection of these

  13. A splicing mutation in the DMD gene detected by next-generation sequencing and confirmed by mRNA and protein analysis.

    Science.gov (United States)

    Roucher Boulez, Florence; Menassa, Rita; Streichenberger, Nathalie; Manel, Véronique; Mallet-Motak, Delphine; Morel, Yves; Michel-Calemard, Laurence

    2015-08-25

    Dystrophinopathies, either the severe Duchenne Muscular Dystrophy (DMD) or the milder Becker Muscular Dystrophy (BMD), are X-linked recessive disorders caused by mutations in the DMD gene. DMD is one of the longest human genes. Large deletions or duplications account for 60-80% of the mutations. Remaining anomalies consist in point mutations or small rearrangements. Routinely, the molecular diagnosis is done by a Multiplex Ligation-dependent Probe Amplification (MLPA) or array Comparative Genome Hybridization (aCGH), followed, if negative, by Sanger sequencing of all exons. In this study, massive parallel sequencing (MPS) or next generation sequencing (NGS) was used to make a rapid and costless molecular diagnosis in a young boy suspected of DMD. A small deletion: NM_004006.2:c.2803+5_2803+8del was identified. The diagnosis was performed in one single manipulation and within a week. The consequence of this intronic mutation is a skipping of exon 21 confirmed by mRNA and protein analysis. NGS appears to be an efficient new strategy in DMD molecular diagnosis. It highlights the major evolution of the diagnostic strategy towards high throughput technologies, where bioinformatics analysis becomes the real challenge for variations detection. This is the first study reporting in vivo impact of this intronic mutation. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. ISVASE: identification of sequence variant associated with splicing event using RNA-seq data.

    Science.gov (United States)

    Aljohi, Hasan Awad; Liu, Wanfei; Lin, Qiang; Yu, Jun; Hu, Songnian

    2017-06-28

    Exon recognition and splicing precisely and efficiently by spliceosome is the key to generate mature mRNAs. About one third or a half of disease-related mutations affect RNA splicing. Software PVAAS has been developed to identify variants associated with aberrant splicing by directly using RNA-seq data. However, it bases on the assumption that annotated splicing site is normal splicing, which is not true in fact. We develop the ISVASE, a tool for specifically identifying sequence variants associated with splicing events (SVASE) by using RNA-seq data. Comparing with PVAAS, our tool has several advantages, such as multi-pass stringent rule-dependent filters and statistical filters, only using split-reads, independent sequence variant identification in each part of splicing (junction), sequence variant detection for both of known and novel splicing event, additional exon-exon junction shift event detection if known splicing events provided, splicing signal evaluation, known DNA mutation and/or RNA editing data supported, higher precision and consistency, and short running time. Using a realistic RNA-seq dataset, we performed a case study to illustrate the functionality and effectiveness of our method. Moreover, the output of SVASEs can be used for downstream analysis such as splicing regulatory element study and sequence variant functional analysis. ISVASE is useful for researchers interested in sequence variants (DNA mutation and/or RNA editing) associated with splicing events. The package is freely available at https://sourceforge.net/projects/isvase/ .

  15. Identification of five novel FBN1 mutations by non-radioactive single-strand conformation analysis

    Energy Technology Data Exchange (ETDEWEB)

    Liu, W.; Qian, C.; Comeau, K.; Francke, U. [Stanford Univ. Medical Center, Stanford, CA (United States)

    1994-09-01

    Marfan syndrome (MFS), one of the most common genetic disorders of connective tissue, is characterized by variable manifestations in skeletal, cardiovascular and ocular systems. Mutations in the fibrillin gene on chromosome 15 (FBN1) have been shown to cause MFS. To examine the relationship between FBN1 gene mutations, fibrillin protein function and MFS phenotypes, we screened for alternations in the fibrillin coding sequence in fibroblast derived cDNA from MFS patients. To date, abnormally migrating bands in more than 20 unrelated MFS patients have been identified by using non-radioactive single-strand conformation analysis and silver staining. Five altered bands have been directly sequenced. Two missense mutations and three splice site mutations have been identified. Both missense mutations substitute another amino acid for a cysteine residue (C1402W and C1672R) in EGF-like motifs of the fibrillin polypeptide chain. The two splice site mutations are at nucleotide positions 6994+1 (G{yields}A), and 7205-2 (A{yields}G) and result in in-frame skipping of exon 56 and 58, respectively. Skipping of exon 56 occurs in 50% of mutant transcripts. Use of a cryptic splice site 51 bp upstream of the normal donor site results in half of the mutant transcripts containing part of exon 56. Both products contain in-frame deletions. Another splice site mutation, identified by exon screening from patient genomic DNA using intron primers, is at nucleotide position 2293+2 (T{yields}A), but the predicted exon skipping has not been detected at the RT-PCR level. This may be due to instability of the mutant transcript. Including the mutations reported here, a total of 8 out of 36 published FBN1 gene mutations involve exon skipping. It may be inferred that FBN1 exon skipping plays an important pathogenic role in MFS.

  16. Clinical significance of intronic variants in BRAF inhibitor resistant melanomas with altered BRAF transcript splicing

    OpenAIRE

    Pupo, Gulietta M.; Boyd, Suzanah C.; Fung, Carina; Carlino, Matteo S.; Menzies, Alexander M.; Pedersen, Bernadette; Johansson, Peter; Hayward, Nicholas K.; Kefford, Richard F.; Scolyer, Richard A.; Long, Georgina V.; Rizos, Helen

    2017-01-01

    Alternate BRAF splicing is the most common mechanism of acquired resistance to BRAF inhibitor treatment in melanoma. Recently, alternate BRAF exon 4?8 splicing was shown to involve an intronic mutation, located 51 nucleotides upstream of BRAF exon 9 within a predicted splicing branch point. This intronic mutation was identified in a single cell line but has not been examined in vivo. Herein we demonstrate that in three melanomas biopsied from patients with acquired resistance to BRAF inhibito...

  17. A recurring dominant negative mutation causes autosomal dominant growth hormone deficiency - a clinical research center study

    Energy Technology Data Exchange (ETDEWEB)

    Cogan, J.D.; Prince, M.; Phillips, J. [Vanderbilt Univ. School of Medicine, Nashville, TN (United States)] [and others

    1995-12-01

    Familial isolated GH deficiency type II (IGHD-II) is an autosomal dominant disorder that has been previously shown in some patients to be caused by heterogeneous GH gene defects that affect GH messenger RNA (mRNA) splicing. We report here our findings of multiple G{r_arrow}A transitions of the first base of the donor splice site of IVS 3 (+1G{r_arrow}A) in IGHD II subjects from three nonrelated kindreds from Sweden, North America, and South Africa. This + 1G{r_arrow}A substitution creates an NlaIII site that was used to demonstrate that all affected individuals in all three families were heterozygous for the mutation. To determine the effect of this mutation of GH mRNA processing, HeLa cells were transfected with expression plasmids containing normal or mutant +1G{r_arrow}A alleles, and complementary DNAs from the resulting GH mRNAs were sequenced. The mutation was found to destroy the GH IVS3 donor splice site, causing skipping of exon 3 and loss of the codons for amino acids 32-71 of the mature GH peptide from the mutant GH mRNA. Our finding of exon 3 skipping in transcripts of the +1G{r_arrow}A mutant allele is identical to our previous report of a different sixth base transition (+6T{r_arrow}C) mutation of the IVS 3 donor splice site that also causes IGHD II. Microsatellite analysis of an affected subjects` DNA from each of the three nonrelated kindreds indicates that the +1G{r_arrow}A mutation arose independently in each family. Finding that neither grandparent has the mutation in the first family suggests that it arose de novo in that family. Our data indicate that (1) +1G{r_arrow}A IVS 3 mutations perturb GH mRNA splicing and cause IGHD II; and (2) these mutations can present as de novo GHD cases. 13 refs., 4 figs., 1 tab.

  18. Novel mutation in the 5' splice site of exon 4 of the TCOF1 gene in the patient with Treacher Collins syndrome.

    Science.gov (United States)

    Marszalek, Bozena; Wisniewski, Slawomir A; Wojcicki, Piotr; Kobus, Kazimierz; Trzeciak, Wieslaw H

    2003-12-01

    Treacher Collins syndrome (TCS) is caused by mutations in the TCOF1 gene. This gene encodes a serine/alanine-rich protein called treacle. The structure of the entire TCOF1 gene was investigated in a patient with TCS. We detected a novel deletion (376delAAGGTGAGTGGGACTGCC) spanning 3 bp of exon 4 and 15 bp of the adjacent intronic sequence. This mutation causes premature termination of translation, resulting in a truncated protein devoid of nucleolar localization signal, and potential phosphorylation sites. Real-time PCR analysis showed different melting temperatures of the amplified fragment containing normal allele and that harboring the 18 bp deletion, thus providing a rapid screening assay for this and other deletions of the TCOF1 gene. Copyright 2003 Wiley-Liss, Inc.

  19. A point mutation in the [2Fe–2S] cluster binding region of the NAF-1 protein (H114C) dramatically hinders the cluster donor properties

    Energy Technology Data Exchange (ETDEWEB)

    Tamir, Sagi; Eisenberg-Domovich, Yael [The Hebrew University of Jerusalem, Edmond J. Safra Campus at Givat Ram, Jerusalem 91904 (Israel); Conlan, Andrea R.; Stofleth, Jason T.; Lipper, Colin H.; Paddock, Mark L. [University of California at San Diego, La Jolla, CA 92093 (United States); Mittler, Ron [University of North Texas, Denton, TX 76203 (United States); Jennings, Patricia A. [University of California at San Diego, La Jolla, CA 92093 (United States); Livnah, Oded, E-mail: oded.livnah@huji.ac.il; Nechushtai, Rachel, E-mail: oded.livnah@huji.ac.il [The Hebrew University of Jerusalem, Edmond J. Safra Campus at Givat Ram, Jerusalem 91904 (Israel)

    2014-06-01

    NAF-1 has been shown to be related with human health and disease, is upregulated in epithelial breast cancer and suppression of its expression significantly suppresses tumor growth. It is shown that replacement of the single His ligand with Cys resulted in dramatic changes to the properties of its 2Fe-2S clusters without any global crystal structural changes. NAF-1 is an important [2Fe–2S] NEET protein associated with human health and disease. A mis-splicing mutation in NAF-1 results in Wolfram Syndrome type 2, a lethal childhood disease. Upregulation of NAF-1 is found in epithelial breast cancer cells, and suppression of NAF-1 expression by knockdown significantly suppresses tumor growth. Key to NAF-1 function is the NEET fold with its [2Fe–2S] cluster. In this work, the high-resolution structure of native NAF-1 was determined to 1.65 Å resolution (R factor = 13.5%) together with that of a mutant in which the single His ligand of its [2Fe–2S] cluster, His114, was replaced by Cys. The NAF-1 H114C mutant structure was determined to 1.58 Å resolution (R factor = 16.0%). All structural differences were localized to the cluster binding site. Compared with native NAF-1, the [2Fe–2S] clusters of the H114C mutant were found to (i) be 25-fold more stable, (ii) have a redox potential that is 300 mV more negative and (iii) have their cluster donation/transfer function abolished. Because no global structural differences were found between the mutant and the native (wild-type) NAF-1 proteins, yet significant functional differences exist between them, the NAF-1 H114C mutant is an excellent tool to decipher the underlying biological importance of the [2Fe–2S] cluster of NAF-1 in vivo.

  20. A point mutation in the [2Fe–2S] cluster binding region of the NAF-1 protein (H114C) dramatically hinders the cluster donor properties

    International Nuclear Information System (INIS)

    Tamir, Sagi; Eisenberg-Domovich, Yael; Conlan, Andrea R.; Stofleth, Jason T.; Lipper, Colin H.; Paddock, Mark L.; Mittler, Ron; Jennings, Patricia A.; Livnah, Oded; Nechushtai, Rachel

    2014-01-01

    NAF-1 has been shown to be related with human health and disease, is upregulated in epithelial breast cancer and suppression of its expression significantly suppresses tumor growth. It is shown that replacement of the single His ligand with Cys resulted in dramatic changes to the properties of its 2Fe-2S clusters without any global crystal structural changes. NAF-1 is an important [2Fe–2S] NEET protein associated with human health and disease. A mis-splicing mutation in NAF-1 results in Wolfram Syndrome type 2, a lethal childhood disease. Upregulation of NAF-1 is found in epithelial breast cancer cells, and suppression of NAF-1 expression by knockdown significantly suppresses tumor growth. Key to NAF-1 function is the NEET fold with its [2Fe–2S] cluster. In this work, the high-resolution structure of native NAF-1 was determined to 1.65 Å resolution (R factor = 13.5%) together with that of a mutant in which the single His ligand of its [2Fe–2S] cluster, His114, was replaced by Cys. The NAF-1 H114C mutant structure was determined to 1.58 Å resolution (R factor = 16.0%). All structural differences were localized to the cluster binding site. Compared with native NAF-1, the [2Fe–2S] clusters of the H114C mutant were found to (i) be 25-fold more stable, (ii) have a redox potential that is 300 mV more negative and (iii) have their cluster donation/transfer function abolished. Because no global structural differences were found between the mutant and the native (wild-type) NAF-1 proteins, yet significant functional differences exist between them, the NAF-1 H114C mutant is an excellent tool to decipher the underlying biological importance of the [2Fe–2S] cluster of NAF-1 in vivo

  1. spliceR

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Porse, Bo Torben; Sandelin, Albin

    2014-01-01

    RNA-seq data is currently underutilized, in part because it is difficult to predict the functional impact of alternate transcription events. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternative splicing...

  2. A novel NHS mutation causes Nance-Horan Syndrome in a Chinese family.

    Science.gov (United States)

    Tian, Qi; Li, Yunping; Kousar, Rizwana; Guo, Hui; Peng, Fenglan; Zheng, Yu; Yang, Xiaohua; Long, Zhigao; Tian, Runyi; Xia, Kun; Lin, Haiying; Pan, Qian

    2017-01-07

    Nance-Horan Syndrome (NHS) (OMIM: 302350) is a rare X-linked developmental disorder characterized by bilateral congenital cataracts, with occasional dental anomalies, characteristic dysmorphic features, brachymetacarpia and mental retardation. Carrier females exhibit similar manifestations that are less severe than in affected males. Here, we report a four-generation Chinese family with multiple affected individuals presenting Nance-Horan Syndrome. Whole-exome sequencing combined with RT-PCR and Sanger sequencing was used to search for a genetic cause underlying the disease phenotype. Whole-exome sequencing identified in all affected individuals of the family a novel donor splicing site mutation (NM_198270: c.1045 + 2T > A) in intron 4 of the gene NHS, which maps to chromosome Xp22.13. The identified mutation results in an RNA processing defect causing a 416-nucleotide addition to exon 4 of the mRNA transcript, likely producing a truncated NHS protein. The donor splicing site mutation NM_198270: c.1045 + 2T > A of the NHS gene is the causative mutation in this Nance-Horan Syndrome family. This research broadens the spectrum of NHS gene mutations, contributing to our understanding of the molecular genetics of NHS.

  3. Aberrant and alternative splicing in skeletal system disease.

    Science.gov (United States)

    Fan, Xin; Tang, Liling

    2013-10-01

    The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. © 2013 Elsevier B.V. All rights reserved.

  4. A mutation at IVS1 + 5 of the von Hippel-Lindau gene resulting in intron retention in transcripts is not pathogenic in a patient with a tongue cancer?: case report

    Directory of Open Access Journals (Sweden)

    Asakawa Takeshi

    2012-03-01

    Full Text Available Abstract Background Von Hippel-Lindau disease (VHL is a dominantly inherited familial cancer syndrome predisposing the patient to a variety of malignant and benign neoplasms, most frequently hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic tumors. VHL is caused by mutations of the VHL tumor suppressor gene on the short arm of chromosome 3, and clinical manifestations develop if both alleles are inactivated according to the two-hit hypothesis. VHL mutations are more frequent in the coding region and occur occasionally in the splicing region of the gene. Previously, we reported that the loss of heterozygosity (LOH of the VHL gene is common in squamous cell carcinoma tissues of the tongue. Case Presentation We describe a case of squamous cell carcinoma in the tongue caused by a point mutation in the splicing region of the VHL gene and discuss its association with VHL disease. Sequence analysis of DNA extracted from the tumor and peripheral blood of the patient with squamous cell carcinoma revealed a heterozygous germline mutation (c. 340 + 5 G > C in the splice donor sequence in intron 1 of the VHL gene. RT-PCR analysis of the exon1/intron1 junction in RNA from tumor tissue detected an unspliced transcript. Analysis of LOH using a marker with a heterozygous mutation of nucleotides (G or C revealed a deletion of the mutant C allele in the carcinoma tissues. Conclusions The fifth nucleotide G of the splice donor site of the VHL gene is important for the efficiency of splicing at that site. The development of tongue cancer in this patient was not associated with VHL disease because the mutation occurred in only a single allele of the VHL gene and that allele was deleted in tumor cells.

  5. Multiset splicing systems.

    Science.gov (United States)

    Dassow, Jürgen; Vaszil, György

    2004-01-01

    We consider splicing systems reflecting two important aspects of the behaviour of DNA molecules in nature or in laboratory experiments which so far have not been studied in the literature. We examine the effect of splicing rules applied to finite multisets of words using sequential and different types of parallel derivation strategies and compare the sets of words or sets of multisets which can be obtained.

  6. Keratin 1 gene mutation detected in epidermal nevus with epidermolytic hyperkeratosis.

    Science.gov (United States)

    Tsubota, Akiko; Akiyama, Masashi; Sakai, Kaori; Goto, Maki; Nomura, Yukiko; Ando, Satomi; Abe, Masataka; Sawamura, Daisuke; Shimizu, Hiroshi

    2007-06-01

    Since 1994, four cases of epidermal nevus with epidermolytic hyperkeratosis (EH) caused by keratin 10 gene mutations have been reported, although no keratin 1 (K1) gene mutation has yet been reported. We detected a K1 gene (KRT1) mutation in epidermal nevus with EH in a 10-year-old Japanese male. The patient showed well-demarcated verrucous, hyperkeratotic plaques mainly on the trunk, covering 15% of the entire body surface. No hyperkeratosis was seen on the palms or soles. He had no family history of skin disorders. His lesional skin showed typical granular degeneration and, ultrastructurally, clumped keratin filaments were observed in the upper epidermis. Direct sequence analysis of genomic DNA extracted from lesional skin revealed a heterozygous 5' donor splice site mutation c.591+2T>A in KRT1. This mutation was not detected in genomic DNA samples from the patient's peripheral blood leukocytes or those of other family members. The identical splice mutation was previously reported in a family with palmoplantar keratoderma and mild ichthyosis, and was demonstrated to result in a 22 amino-acid deletion p.Val175_Lys196del in the H1 and 1A domains of K1. To our knowledge, the present patient is the first reported case of epidermal nevus associated with EH caused by a K1 gene mutation in a mosaic pattern.

  7. First report of a novel LMNA mutation in a Chinese family with limb ...

    Indian Academy of Sciences (India)

    2014-12-12

    shown in figure 3), including 36 mis- sense mutations, 13 truncated mutations and four splicing mutations. Mutation p.W520R reported in this study is a novel finding. The correlation between the phenotypes and localiza-.

  8. Analysis and recognition of 5 ' UTR intron splice sites in human pre-mRNA

    DEFF Research Database (Denmark)

    Eden, E.; Brunak, Søren

    2004-01-01

    Prediction of splice sites in non-coding regions of genes is one of the most challenging aspects of gene structure recognition. We perform a rigorous analysis of such splice sites embedded in human 5' untranslated regions (UTRs), and investigate correlations between this class of splice sites...... and other features found in the adjacent exons and introns. By restricting the training of neural network algorithms to 'pure' UTRs (not extending partially into protein coding regions), we for the first time investigate the predictive power of the splicing signal proper, in contrast to conventional splice...... in the synaptic weights of the neural networks trained to identify UTR donor sites. Conventional splice site prediction methods perform poorly in UTRs because the reading frame pattern is absent. The NetUTR method presented here performs 2-.3-fold better compared with NetGene2 and GenScan in 5' UTRs. We also...

  9. A method of predicting changes in human gene splicing induced by genetic variants in context of cis-acting elements

    Directory of Open Access Journals (Sweden)

    Hicks Chindo

    2010-01-01

    Full Text Available Abstract Background Polymorphic variants and mutations disrupting canonical splicing isoforms are among the leading causes of human hereditary disorders. While there is a substantial evidence of aberrant splicing causing Mendelian diseases, the implication of such events in multi-genic disorders is yet to be well understood. We have developed a new tool (SpliceScan II for predicting the effects of genetic variants on splicing and cis-regulatory elements. The novel Bayesian non-canonical 5'GC splice site (SS sensor used in our tool allows inference on non-canonical exons. Results Our tool performed favorably when compared with the existing methods in the context of genes linked to the Autism Spectrum Disorder (ASD. SpliceScan II was able to predict more aberrant splicing isoforms triggered by the mutations, as documented in DBASS5 and DBASS3 aberrant splicing databases, than other existing methods. Detrimental effects behind some of the polymorphic variations previously associated with Alzheimer's and breast cancer could be explained by changes in predicted splicing patterns. Conclusions We have developed SpliceScan II, an effective and sensitive tool for predicting the detrimental effects of genomic variants on splicing leading to Mendelian and complex hereditary disorders. The method could potentially be used to screen resequenced patient DNA to identify de novo mutations and polymorphic variants that could contribute to a genetic disorder.

  10. Identification of three novel NHS mutations in families with Nance-Horan syndrome.

    Science.gov (United States)

    Huang, Kristen M; Wu, Junhua; Brooks, Simon P; Hardcastle, Alison J; Lewis, Richard Alan; Stambolian, Dwight

    2007-03-27

    Nance-Horan Syndrome (NHS) is an infrequent and often overlooked X-linked disorder characterized by dense congenital cataracts, microphthalmia, and dental abnormalities. The syndrome is caused by mutations in the NHS gene, whose function is not known. The purpose of this study was to identify the frequency and distribution of NHS gene mutations and compare genotype with Nance-Horan phenotype in five North American NHS families. Genomic DNA was isolated from white blood cells from NHS patients and family members. The NHS gene coding region and its splice site donor and acceptor regions were amplified from genomic DNA by PCR, and the amplicons were sequenced directly. We identified three unique NHS coding region mutations in these NHS families. This report extends the number of unique identified NHS mutations to 14.

  11. Rapid screening of yeast mutants with reporters identifies new splicing phenotypes.

    Science.gov (United States)

    Dreumont, Natacha; Séraphin, Bertrand

    2013-06-01

    Nuclear precursor mRNA splicing requires the stepwise assembly of a large complex, the spliceosome. Recent large-scale analyses, including purification of splicing complexes, high-throughput genetic screens and interactomic studies, have linked numerous factors to this dynamic process, including a well-defined core conserved from yeast to human. Intriguingly, despite extensive studies, no splicing defects were reported for some of the corresponding yeast mutants. To resolve this paradox, we screened a collection of viable yeast strains carrying mutations in splicing-related factors with a set of reporters including artificial constructs carrying competing splice sites. Previous analyses have indeed demonstrated that this strategy identifies yeast factors able to regulate alternative splicing and whose properties are conserved in human cells. The method, sensitive to subtle defects, revealed new splicing phenotypes for most analyzed factors such as the Urn1 protein. Interestingly, a mutant of PRP8 specifically lacking an N-terminal proline-rich region stimulated the splicing of a reporter containing competing branchpoint/3' splice site regions. Thus, using appropriate reporters, yeast can be used to quickly delineate the effect of various factors on splicing and identify those with the propensity to regulate alternative splicing events. © 2013 FEBS.

  12. A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo

    DEFF Research Database (Denmark)

    Damgaard, Christian Kroun; Kahns, Søren; Lykke-Andersen, Søren

    2008-01-01

    Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or β-globin mRNAs, harb...... a promoter-proximal 5′ splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing preinitiation complex assembly.......Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or β-globin mRNAs......, harboring wild-type or various 5′ splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5′ splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced...

  13. Minor class splicing shapes the zebrafish transcriptome during development

    DEFF Research Database (Denmark)

    Markmiller, Sebastian; Cloonan, Nicole; Lardelli, Rea M

    2014-01-01

    Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities...... known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we...... as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays...

  14. The neurogenetics of alternative splicing.

    Science.gov (United States)

    Vuong, Celine K; Black, Douglas L; Zheng, Sika

    2016-05-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain.

  15. Alternative Splicing in Lung Cancer

    OpenAIRE

    Pio, Ruben; Montuenga, Luis M.

    2009-01-01

    Abstract: Alterations in alternative splicing affect essential biologic processes and are the basis for a number of pathologic conditions, including cancer. In this review we will summarize the evidence supporting the relevance of alternative splicing in lung cancer. An example that illustrates this relevance is the altered balance between Bcl-xL and Bcl-xS, two splice variants of the apoptosis regulator Bcl-x. Splice modifications in cancer-related genes can be associated ...

  16. Where splicing joins chromatin

    Czech Academy of Sciences Publication Activity Database

    Hnilicová, Jarmila; Staněk, David

    2011-01-01

    Roč. 2, č. 3 (2011), s. 182-188 ISSN 1949-1034 R&D Projects: GA ČR GAP305/10/0424; GA AV ČR KAN200520801 Institutional research plan: CEZ:AV0Z50520514 Keywords : chromatin * exon * alternative splicing * transcription * snRNP Subject RIV: EB - Genetics ; Molecular Biology

  17. Expressiveness of basic Splice

    NARCIS (Netherlands)

    J.C. van de Pol (Jaco)

    2000-01-01

    textabstractWe study a simple software architecture, in which application processes are coordinated by writing into and reading from a global set. This architecture underlies Splice, which is developed and used at the company Hollandse Signaalapparaten. Our approach is distinguished by viewing the

  18. Trans-Splicing Improvement by the Combined Application of Antisense Strategies

    Directory of Open Access Journals (Sweden)

    Ulrich Koller

    2015-01-01

    Full Text Available Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs, presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB. We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG, lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

  19. Genomic characterisation of the Jk(a−b−) phenotype in Thai blood donors

    Science.gov (United States)

    Sriwanitchrak, Pramote; Sriwanitchrak, Kanchana; Tubrod, Jintana; Kupatawintu, Pawinee; Kaset, Chollanot; Nathalang, Oytip

    2012-01-01

    Background The Kidd (JK) blood group antigens are encoded by the JK gene. The rare Jk(a−b−) phenotype can be caused by homozygosity for a silent JK allele. Currently, JKnull alleles have been identified among different populations; however, information on its presence among Thais is not available. Materials and methods Screening for the Jk(a−b−) phenotype by the urea lysis test was performed in 25,340 blood samples from Thai blood donors. The Jk(a−b−) phenotypes were confirmed by an indirect antiglobulin test (IAT). Additionally, polymerase chain reaction amplification and sequence analysis of the JK gene were performed using previously described methods. Results Five samples were confirmed as having a Jk(a−b−) phenotype by a urea lysis test and IAT; four of these samples were investigated. Two samples of JK*02 alleles were homozygous for a g>a mutation at the 3′ acceptor splice site of intron 5 of the JK gene, as in previous studies in Asians and Polynesians. Moreover, one sample of JK*02 alleles was homozygous for an 896G>A mutation at exon 9 (Gly299Glu), as in a previous study in Polynesians. Interestingly, missense dual mutations of JK*01 alleles from a female blood donor were identified. The first mutation was 956C>T (Thr319Met) in exon 10, as in a recent study in African-Americans. The second mutation was 130G>A (Glu44Lys) at exon 4, as in previous studies among Caucasians. Conclusion There are various different molecular bases of the Jk(a−b−) phenotype. This is the first report of JKnull alleles among Thais. The information presented in this study could be beneficial in planning genotyping strategies for blood donors and patients. PMID:22153692

  20. Genomic characterisation of the Jk(a-b-) phenotype in Thai blood donors.

    Science.gov (United States)

    Sriwanitchrak, Pramote; Sriwanitchrak, Kanchana; Tubrod, Jintana; Kupatawintu, Pawinee; Kaset, Chollanot; Nathalang, Oytip

    2012-04-01

    The Kidd (JK) blood group antigens are encoded by the JK gene. The rare Jk(a-b-) phenotype can be caused by homozygosity for a silent JK allele. Currently, JK(null) alleles have been identified among different populations; however, information on its presence among Thais is not available. Screening for the Jk(a-b-) phenotype by the urea lysis test was performed in 25,340 blood samples from Thai blood donors. The Jk(a-b-) phenotypes were confirmed by an indirect antiglobulin test (IAT). Additionally, polymerase chain reaction amplification and sequence analysis of the JK gene were performed using previously described methods. Five samples were confirmed as having a Jk(a-b-) phenotype by a urea lysis test and IAT; four of these samples were investigated. Two samples of JK*02 alleles were homozygous for a g>a mutation at the 3' acceptor splice site of intron 5 of the JK gene, as in previous studies in Asians and Polynesians. Moreover, one sample of JK*02 alleles was homozygous for an 896G>A mutation at exon 9 (Gly299Glu), as in a previous study in Polynesians. Interestingly, missense dual mutations of JK*01 alleles from a female blood donor were identified. The first mutation was 956C>T (Thr319Met) in exon 10, as in a recent study in African-Americans. The second mutation was 130G>A (Glu44Lys) at exon 4, as in previous studies among Caucasians. There are various different molecular bases of the Jk(a-b-) phenotype. This is the first report of JK(null) alleles among Thais. The information presented in this study could be beneficial in planning genotyping strategies for blood donors and patients.

  1. An intronic ABCA3 mutation that is responsible for respiratory disease.

    Science.gov (United States)

    Agrawal, Amit; Hamvas, Aaron; Cole, F Sessions; Wambach, Jennifer A; Wegner, Daniel; Coghill, Carl; Harrison, Keith; Nogee, Lawrence M

    2012-06-01

    Member A3 of the ATP-binding cassette family of transporters (ABCA3) is essential for surfactant metabolism. Nonsense, missense, frameshift, and splice-site mutations in the ABCA3 gene (ABCA3) have been reported as causes of neonatal respiratory failure (NRF) and interstitial lung disease. We tested the hypothesis that mutations in noncoding regions of ABCA3 may cause lung disease. ABCA3-specific cDNA was generated and sequenced from frozen lung tissue from a child with fatal lung disease with only one identified ABCA3 mutation. ABCA3 was sequenced from genomic DNA prepared from blood samples obtained from the proband, parents, and other children with NRF. ABCA3 cDNA from the proband contained sequences derived from intron 25 that would be predicted to alter the structure and function of the ABCA3 protein. Genomic DNA sequencing revealed a heterozygous C>T transition in intron 25 trans to the known mutation, creating a new donor splice site. Seven additional infants with an ABCA3-deficient phenotype and inconclusive genetic findings had this same variant, which was not found in 2,132 control chromosomes. These findings support that this variant is a disease-causing mutation that may account for additional cases of ABCA3 deficiency with negative genetic studies.

  2. Alternative REST Splicing Underappreciated

    OpenAIRE

    Chen, Guo-Lin; Miller, Gregory

    2017-01-01

    As a major orchestrator of the cellular epigenome, the repressor element-1 silencing transcription factor (REST) can either repress or activate thousands of genes depending on cellular context, suggesting a highly context-dependent REST function tuned by environmental cues. While REST shows cell-type non-selective active transcription, an N-terminal REST4 isoform caused by alternative splicing - inclusion of an extra exon (N3c) which introduces a pre-mature stop codon - has been implicated in...

  3. The neurogenetics of alternative splicing

    OpenAIRE

    Vuong, Celine K.; Black, Douglas L.; Zheng, Sika

    2016-01-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that r...

  4. Work organization for splice consolidation

    CERN Document Server

    Bertinelli, F

    2011-01-01

    The Splices Task Force has worked in 2010 to prepare the necessary interventions for 7 TeV operation. The design solution for consolidating the main interconnection splices is well advanced. The required activities to implement it are described, highlighting working assumptions, missing resources and schedule considerations. Progress has also been made in assessing other splices, 6 kA praying hands and corrector circuits: results and ongoing work are presented, highlighting priorities for the remaining work.

  5. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef; Hehl, Reinhard

    2010-09-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain.

  6. Widening the mutation spectrum of EVC and EVC2: ectopic expression of Weyer variants in NIH 3T3 fibroblasts disrupts Hedgehog signaling.

    Science.gov (United States)

    Valencia, Maria; Lapunzina, Pablo; Lim, Derek; Zannolli, Raffaella; Bartholdi, Deborah; Wollnik, Bernd; Al-Ajlouni, Othman; Eid, Suhair S; Cox, Helen; Buoni, Sabrina; Hayek, Joseph; Martinez-Frias, Maria L; Antonio, Perez-Aytes; Temtamy, Samia; Aglan, Mona; Goodship, Judith A; Ruiz-Perez, Victor L

    2009-12-01

    Autosomal recessive Ellis-van Creveld syndrome and autosomal dominant Weyer acrodental dysostosis are allelic conditions caused by mutations in EVC or EVC2. We performed a mutation screening study in 36 EvC cases and 3 cases of Weyer acrodental dysostosis, and identified pathogenic changes either in EVC or in EVC2 in all cases. We detected 40 independent EVC/EVC2 mutations of which 29 were novel changes in Ellis-van Creveld cases and 2 were novel mutations identified in Weyer pedigrees. Of interest one EvC patient had a T>G nucleotide substitution in intron 7 of EVC (c.940-150T>G), which creates a new donor splice site and results in the inclusion of a new exon. The T>G substitution is at nucleotide +5 of the novel 5' splice site. The three Weyer mutations occurred in the final exon of EVC2 (exon 22), suggesting that specific residues encoded by this exon are a key part of the protein. Using murine versions of EVC2 exon 22 mutations we demonstrate that the expression of a Weyer variant, but not the expression of a truncated protein that mimics an Ellis-van Creveld syndrome mutation, impairs Hedgehog signal transduction in NIH 3T3 cells in keeping with its dominant effect.

  7. Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

    Directory of Open Access Journals (Sweden)

    Delphine Trochet

    2016-01-01

    Full Text Available Dynamin 2 (DNM2 is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.

  8. TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

    Directory of Open Access Journals (Sweden)

    Pavan Kumar P

    2014-03-01

    Full Text Available TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

  9. TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

    Science.gov (United States)

    Kumar P, Pavan; Franklin, Sarah; Emechebe, Uchenna; Hu, Hao; Moore, Barry; Lehman, Chris; Yandell, Mark; Moon, Anne M

    2014-03-01

    TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

  10. The proper splicing of RNAi factors is critical for pericentric heterochromatin assembly in fission yeast.

    Directory of Open Access Journals (Sweden)

    Scott P Kallgren

    Full Text Available Heterochromatin preferentially assembles at repetitive DNA elements, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNAi machinery is required for heterochromatin assembly in a diverse range of organisms. In fission yeast, RNA splicing factors are also required for pericentric heterochromatin assembly, and a prevailing model is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, by screening the fission yeast deletion library, we discovered four novel splicing factors that are required for pericentric heterochromatin assembly. Sequencing total cellular RNAs from the strongest of these mutants, cwf14Δ, showed intron retention in mRNAs of several RNAi factors. Moreover, introducing cDNA versions of RNAi factors significantly restored pericentric heterochromatin in splicing mutants. We also found that mutations of splicing factors resulted in defective telomeric heterochromatin assembly and mis-splicing the mRNA of shelterin component Tpz1, and that replacement of tpz1+ with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus, proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres.

  11. Minor class splicing shapes the zebrafish transcriptome during development.

    Science.gov (United States)

    Markmiller, Sebastian; Cloonan, Nicole; Lardelli, Rea M; Doggett, Karen; Keightley, Maria-Cristina; Boglev, Yeliz; Trotter, Andrew J; Ng, Annie Y; Wilkins, Simon J; Verkade, Heather; Ober, Elke A; Field, Holly A; Grimmond, Sean M; Lieschke, Graham J; Stainier, Didier Y R; Heath, Joan K

    2014-02-25

    Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.

  12. Alternative Splicing of NOX4 in the Failing Human Heart

    Directory of Open Access Journals (Sweden)

    Zoltán V. Varga

    2017-11-01

    Full Text Available Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5′-3′ end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure.

  13. Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

    DEFF Research Database (Denmark)

    Bestas, Burcu; Moreno, Pedro M D; Blomberg, K Emelie M

    2014-01-01

    X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense...

  14. Analysis of a splice array experiment elucidates roles of chromatin elongation factor Spt4-5 in splicing.

    Directory of Open Access Journals (Sweden)

    Yuanyuan Xiao

    2005-09-01

    Full Text Available Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4-5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4-5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4-Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function.

  15. A Novel Mutation in a Critical Region for the Methyl Donor Binding in DNMT3B Causes Immunodeficiency, Centromeric Instability, and Facial Anomalies Syndrome (ICF).

    Science.gov (United States)

    Rechavi, Erez; Lev, Atar; Eyal, Eran; Barel, Ortal; Kol, Nitzan; Barhom, Sarit Farage; Pode-Shakked, Ben; Anikster, Yair; Somech, Raz; Simon, Amos J

    2016-11-01

    Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is an extremely rare autosomal recessive disease. The immune phenotype is characterized by hypogammaglobulinemia in the presence of B cells. T cell lymphopenia also develops in some patients. We sought to further investigate the immune defect in an ICF patient with a novel missense mutation in DNMT3B and a severe phenotype. Patient lymphocytes were examined for subset counts, immunoglobulin levels, T and B cell de novo production (via excision circles) and receptor repertoire diversity. Mutated DNMT3B protein structure was modeled to assess the effect of a mutation located outside of the catalytic region on protein function. A novel homozygous missense mutation, Ala585Thr, was found in DNMT3B. The patient had decreased B cell counts with hypogammaglobulinemia, and normal T cell counts. CD4 + T cells decreased over time, leading to an inversion of the CD4 + to CD8 + ratio. Excision circle copy numbers were normal, signifying normal de novo lymphocyte production, but the ratio between naïve and total B cells was low, indicating decreased in vivo B cell replication. T and B cell receptor repertoires displayed normal diversity. Computerized modeling of the mutated Ala585 residue suggested reduced thermostability, possibly affecting the enzyme kinetics. Our results highlight the existence of a T cell defect that develops over time in ICF patient, in addition to the known B cell dysfunction. With intravenous immunoglobulin (IVIG) treatment ameliorating the B cell defect, the extent of CD4 + lymphopenia may determine the severity of ICF immunodeficiency.

  16. Donor Tag Game

    Science.gov (United States)

    ... Donor Community > Games > Donor Tag Game Donor Tag Game This feature requires version 6 or later of ... of Needles LGBTQ+ Donors Blood Donor Community SleevesUp Games Facebook Avatars and Badges Banners eCards Make a ...

  17. Evaluation of a high throughput method for the detection of mutations associated with thrombosis and hereditary hemochromatosis in Brazilian blood donors.

    Directory of Open Access Journals (Sweden)

    Vivian Dionisio Tavares Niewiadonski

    Full Text Available The aim of this study was to evaluate the OpenArray platform for genetic testing of blood donors and to assess the genotype frequencies of nucleotide-polymorphisms (SNPs associated with venous thrombosis (G1691A and G20210A, hyperhomocysteinemia (C677T, A1298C, and hereditary hemochromatosis (C282Y, H63D and S65C in blood donors from Sao Paulo, Brazil.We examined 400 blood donor samples collected from October to November 2011. The SNPs were detected using OpenArray technology. The blood samples were also examined using a real-time PCR-FRET system to compare the results and determine the accuracy of the OpenArray method.We observed 100% agreement in all assays tested, except HFE C282Y, which showed 99.75% agreement. The HFE C282Y assay was further confirmed through direct sequencing, and the results showed that OpenArray analysis was accurate. The calculated frequencies of each SNP were FV G1691A 98.8% (G/G, 1.2% (G/A; FII G2021A 99.5% (G/G, 0.5% (G/A; MTHFR C677T 45.5% (C/C, 44.8% (C/T, 9.8% (T/T; MTHFR A1298C 60.3% (A/A, 33.6% (A/C, 6.1% (C/C; HFE C282Y 96%(G/G, 4%(G/A, HFE H63D 78.1%(C/C, 20.3% (C/G, 1.6% (G/G; and HFE S65C 98.1% (A/A, 1.9% (A/T.Taken together, these results describe the frequencies of SNPs associated with diseases and are important to enhance our current knowledge of the genetic profiles of Brazilian blood donors, although a larger study is needed for a more accurate determination of the frequency of the alleles. Furthermore, the OpenArray platform showed a high concordance rate with standard FRET RT-PCR.

  18. Evaluation of a High Throughput Method for the Detection of Mutations Associated with Thrombosis and Hereditary Hemochromatosis in Brazilian Blood Donors

    Science.gov (United States)

    Dionisio Tavares Niewiadonski, Vivian; dos Santos Bianchi, Juliana Vieira; de Almeida-Neto, Cesar; Gaburo, Nelson; Sabino, Ester Cerdeira

    2015-01-01

    Background The aim of this study was to evaluate the OpenArray platform for genetic testing of blood donors and to assess the genotype frequencies of nucleotide-polymorphisms (SNPs) associated with venous thrombosis (G1691A and G20210A), hyperhomocysteinemia (C677T, A1298C), and hereditary hemochromatosis (C282Y, H63D and S65C) in blood donors from Sao Paulo, Brazil. Methods We examined 400 blood donor samples collected from October to November 2011. The SNPs were detected using OpenArray technology. The blood samples were also examined using a real-time PCR–FRET system to compare the results and determine the accuracy of the OpenArray method. Results We observed 100% agreement in all assays tested, except HFE C282Y, which showed 99.75% agreement. The HFE C282Y assay was further confirmed through direct sequencing, and the results showed that OpenArray analysis was accurate. The calculated frequencies of each SNP were FV G1691A 98.8% (G/G), 1.2% (G/A); FII G2021A 99.5% (G/G), 0.5% (G/A); MTHFR C677T 45.5% (C/C), 44.8% (C/T), 9.8% (T/T); MTHFR A1298C 60.3% (A/A), 33.6% (A/C), 6.1% (C/C); HFE C282Y 96%(G/G), 4%(G/A), HFE H63D 78.1%(C/C), 20.3% (C/G), 1.6% (G/G); and HFE S65C 98.1% (A/A), 1.9% (A/T). Conclusion Taken together, these results describe the frequencies of SNPs associated with diseases and are important to enhance our current knowledge of the genetic profiles of Brazilian blood donors, although a larger study is needed for a more accurate determination of the frequency of the alleles. Furthermore, the OpenArray platform showed a high concordance rate with standard FRET RT-PCR. PMID:25955572

  19. The infection staging and profile of genotypic distribution and drug resistance mutation among the human immunodeficiency virus-1 infected blood donors from five Chinese blood centers, 2012-2014.

    Directory of Open Access Journals (Sweden)

    Peibin Zeng

    Full Text Available The increasing complexity and diversity of the human immunodeficiency virus-1 (HIV-1 infections challenge the disease control and anti-retrovirus treatment in China. The infection stages and molecular characteristics of HIV-1 from infected Chinese blood donors were examined to shed light on the HIV genotype distribution and the status of drug resistance mutations (DRMs in the changing HIV epidemic in China. Western blot (WB confirmed HIV-1 positive plasma samples were collected from blood donors at five Chinese blood centers from April 16, 2012, through June 30, 2014. The HIV infection stages were determined using the Lag-avidity assay. HIV Pol regions including whole protease and partial reverse transcriptase (RT were amplified and sequenced to establish the profile of genotype distribution and drug resistance mutations (DRMs. Viral loads were determined using the ROCHE COBAS system. Of the 259 HIV-1 positive samples tested by the Lag-avidity assay, 23.6% (61/259 were identified as recent infections. A total of 205 amplified sequences displayed the following genotype distributions: circulating recombinant form (CRF 07_BC (61.5%, CRF08_BC (8.3%, CRF01_AE (20%, B (6.3%, and 01B (3.9%. There was no significant difference in genotype distribution between recent and long-term infections. 31 DRMs were identified from 27 samples including four protease inhibitors (PIs accessory DRMs, two PIs major DRMs (M46I, two nucleoside RT inhibitors DRMs (K219R and K70Q, and 23 nonnucleoside RT inhibitors DRMs. 27 samples had DRMs, yielding a drug resistance prevalence of 13.2% (27/205. Our findings provide important information for developing strategies for comprehensive HIV control and improving anti-retroviral treatment in China.

  20. Intronic Alus influence alternative splicing.

    Directory of Open Access Journals (Sweden)

    Galit Lev-Maor

    2008-09-01

    Full Text Available Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

  1. Misregulation of Alternative Splicing in a Mouse Model of Rett Syndrome.

    Directory of Open Access Journals (Sweden)

    Ronghui Li

    2016-06-01

    Full Text Available Mutations in the human MECP2 gene cause Rett syndrome (RTT, a severe neurodevelopmental disorder that predominantly affects girls. Despite decades of work, the molecular function of MeCP2 is not fully understood. Here we report a systematic identification of MeCP2-interacting proteins in the mouse brain. In addition to transcription regulators, we found that MeCP2 physically interacts with several modulators of RNA splicing, including LEDGF and DHX9. These interactions are disrupted by RTT causing mutations, suggesting that they may play a role in RTT pathogenesis. Consistent with the idea, deep RNA sequencing revealed misregulation of hundreds of splicing events in the cortex of Mecp2 knockout mice. To reveal the functional consequence of altered RNA splicing due to the loss of MeCP2, we focused on the regulation of the splicing of the flip/flop exon of Gria2 and other AMPAR genes. We found a significant splicing shift in the flip/flop exon toward the flop inclusion, leading to a faster decay in the AMPAR gated current and altered synaptic transmission. In summary, our study identified direct physical interaction between MeCP2 and splicing factors, a novel MeCP2 target gene, and established functional connection between a specific RNA splicing change and synaptic phenotypes in RTT mice. These results not only help our understanding of the molecular function of MeCP2, but also reveal potential drug targets for future therapies.

  2. Spliceman2: a computational web server that predicts defects in pre-mRNA splicing.

    Science.gov (United States)

    Cygan, Kamil Jan; Sanford, Clayton Hendrick; Fairbrother, William Guy

    2017-09-15

    Most pre-mRNA transcripts in eukaryotic cells must undergo splicing to remove introns and join exons, and splicing elements present a large mutational target for disease-causing mutations. Splicing elements are strongly position dependent with respect to the transcript annotations. In 2012, we presented Spliceman, an online tool that used positional dependence to predict how likely distant mutations around annotated splice sites were to disrupt splicing. Here, we present an improved version of the previous tool that will be more useful for predicting the likelihood of splicing mutations. We have added industry-standard input options (i.e. Spliceman now accepts variant call format files), which allow much larger inputs than previously available. The tool also can visualize the locations-within exons and introns-of sequence variants to be analyzed and the predicted effects on splicing of the pre-mRNA transcript. In addition, Spliceman2 integrates with RNAcompete motif libraries to provide a prediction of which trans -acting factors binding sites are disrupted/created and links out to the UCSC genome browser. In summary, the new features in Spliceman2 will allow scientists and physicians to better understand the effects of single nucleotide variations on splicing. Freely available on the web at http://fairbrother.biomed.brown.edu/spliceman2 . Website implemented in PHP framework-Laravel 5, PostgreSQL, Apache, and Perl, with all major browsers supported. william_fairbrother@brown.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  3. cis-Acting and trans-acting modulation of equine infectious anemia virus alternative RNA splicing

    International Nuclear Information System (INIS)

    Liao, Huey-Jane; Baker, Carl C.; Princler, Gerald L.; Derse, David

    2004-01-01

    Equine infectious anemia virus (EIAV), a lentivirus distantly related to HIV-1, encodes regulatory proteins, EIAV Tat (ETat) and Rev (ERev), from a four-exon mRNA. Exon 3 of the tat/rev mRNA contains a 30-nucleotide purine-rich element (PRE) which binds both ERev and SF2/ASF, a member of the SR family of RNA splicing factors. To better understand the role of this element in the regulation of EIAV pre-mRNA splicing, we quantified the effects of mutation or deletion of the PRE on exon 3 splicing in vitro and on alternative splicing in vivo. We also determined the branch point elements upstream of exons 3 and 4. In vitro splicing of exon 3 to exon 4 was not affected by mutation of the PRE, and addition of purified SR proteins enhanced splicing independently of the PRE. In vitro splicing of exon 2 to exon 3 was dependent on the PRE; under conditions of excess SR proteins, either the PRE or the 5' splice site of exon 3 was sufficient to activate splicing. We applied isoform-specific primers in real-time RT-PCR reactions to quantitatively analyze alternative splicing in cells transfected with rev-minus EIAV provirus constructs. In the context of provirus with wild-type exon 3, greater than 80% of the viral mRNAs were multiply spliced, and of these, less than 1% excluded exon 3. Deletion of the PRE resulted in a decrease in the relative amount of multiply spliced mRNA to about 40% of the total and approximately 39% of the viral mRNA excluded exon 3. Ectopic expression of ERev caused a decrease in the relative amount of multiply spliced mRNA to approximately 50% of the total and increased mRNAs that excluded exon 3 to about 4%. Over-expression of SF2/ASF in cells transfected with wild-type provirus constructs inhibited splicing but did not significantly alter exon 3 skipping

  4. Prenatal diagnosis and molecular genetic analysis of short rib-polydactyly syndrome type III (Verma-Naumoff in a second-trimester fetus with a homozygous splice site mutation in intron 4 in the NEK1 gene

    Directory of Open Access Journals (Sweden)

    Chih-Ping Chen

    2012-06-01

    Conclusion: Polydactyly, micromelia, metaphyseal spurs, widened humeral metaphyses, and shortened ribs can be prominent prenatal ultrasound findings of SRPS III. The present case provides evidence for a correlation of a mutation in the NEK1 gene with SRPS III.

  5. Pre-mRNA splicing is a determinant of histone H3K36 methylation.

    Science.gov (United States)

    Kim, Soojin; Kim, Hyunmin; Fong, Nova; Erickson, Benjamin; Bentley, David L

    2011-08-16

    A chromatin code appears to mark introns and exons with distinct patterns of nucleosome enrichment and histone methylation. We investigated whether a causal relationship exists between splicing and chromatin modification by asking whether splice-site mutations affect the methylation of histone H3K36. Deletions of the 3' splice site in intron 2 or in both introns 1 and 2 of an integrated β-globin reporter gene caused a shift in relative distribution of H3K36 trimethylation away from 5' ends and toward 3' ends. The effects of splice-site mutations correlated with enhanced retention of a U5 snRNP subunit on transcription complexes downstream of the gene. In contrast, a poly(A) site mutation did not affect H3K36 methylation. Similarly, global inhibition of splicing by spliceostatin A caused a rapid repositioning of H3K36me3 away from 5' ends in favor of 3' ends. These results suggest that the cotranscriptional splicing apparatus influences establishment of normal patterns of histone modification.

  6. Mutations in the lysosomal [beta]-galactosidase gene that cause the adult form of GMI gangliosidosis

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, S.; Rafi, M.A.; Wenger, D.A. (Thomas Jefferson Univ., Philadelphia, PA (United States))

    1994-06-01

    Three adult patients with acid-galactosidase deficiency/GM1 gangliosidosis who were from two unrelated families of Scandinavian descent were found to share a common point mutation in the coding region of the corresponding gene. The patients share common clinical features, including early dysarthria, mild ataxia, and bone abnormalities. When cDNA from the two patients in family 1 was PCR amplified and sequenced, most (39/41) of the clones showed a C-to-T transition (C[yields]T) at nucleotide 245 (counting from the initiation codon). This mutation changes the codon for the Thr(ACG) to Met(ATG). Mutant and normal sequences were also found in that position in genomic DNA, indicating the presence of another mutant allele. Genomic DNA from the patient in family 2 revealed the same point mutation in one allele. It was determined that in each family only the father carried the C[yields]T mutation. Expression studies showed that this mutation produced 3%-4% of [beta]-galactosidase activity, confirming its deleterious effects. The cDNA clones from the patients in family 1 that did not contain the C[yields]T revealed a 20-bp insertion of intronic sequence between nucleotides 75 and 76, the location of the first intron. Further analysis showed the insertion of a T near the 5[prime] splice donor site which led to the use of a cryptic splice site. It appears that the C[yields]T mutation results in enough functional enzyme to produce a mild adult form of the disease, even in the presence of a second mutation that likely produces nonfunctional enzyme. 31 refs., 7 figs., 1 tab.

  7. Handbook of knotting and splicing

    CERN Document Server

    Hasluck, Paul N

    2005-01-01

    Clearly written and amply illustrated with 208 figures, this classic guide ranges from simple and useful knots to complex varieties. Additional topics include rope splicing, working cordage, hammock making, more.

  8. Fibrillin mutations in the Marfan syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Price, C.E.; Wang, M.; Wang, J.; Godfrey, M. [Univ. of Nebraska Medical Center, Omaha, NE (United States)

    1994-09-01

    The Marfan syndrome (MFS) is an autosomal dominant heritable disorder of connective tissue manifested by variable and pleiotropic defect in the skeletal, ocular, and cardiovascular systems. We have recently begun to use intron-specific primers that have become available through the International Marfan Syndrome Consortium to screen for fibrillin mutations in MFS patients. Using the genomic PCR-based approach in addition to RT-PCR methodologies, we have identified several novel mutations. A single base insertion was identified in all affected individuals of one family. The insertion of an {open_quote}A{close_quote} at position 1891 in exon 15 causes a premature stop codon and thus a truncated polypeptide. The truncated protein of 617 amino acids has an expected molecular weight of 63 kD. Metabolic labeling and immunoprecipitation studies are in progress. A C{r_arrow}T transition at position 1634 in exon 12 causing a 5th position Cys to Phe substitution in an EGF-like motif was observed in another MFS patient. Finally, we have identified a G{r_arrow}A transition at the +1 position of the donor splice site that causes the deletion of fibrillin exon 32 in a patient with the neonatal form of MFS. Exon 32 is a precursor EGF-like calcium binding motif that is located in a single stretch of 12 similar domains. We had previously identified the skipping of this exon due to an A{r_arrow}T transversion at the -2 position of the consensus acceptor splice site in another patient with neonatal MFS. The reason that the skipping of exon 32 causes a neonatal lethal MFS phenotype is presently unclear. These studies will help elucidate the role of diverse regions of fibrillin.

  9. Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome

    Directory of Open Access Journals (Sweden)

    Semina Elena V

    2006-07-01

    Full Text Available Abstract Background Axenfeld-Rieger syndrome (ARS is associated with mutations in the PITX2 gene that encodes a homeobox transcription factor. Several intronic PITX2 mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested. Methods We present two new families with recurrent PITX2 intronic mutations and use PITX2c minigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. Three PITX2 mutations have been analyzed: a G>T mutation within the AG 3' splice site (ss junction associated with exon 4 (IVS4-1G>T, a G>C mutation at position +5 of the 5' (ss of exon 4 (IVS4+5G>C, and a previously reported A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G. Results Mutation IVS4+5G>C showed 71% retention of the intron between exons 4 and 5, and poorly expressed protein. Wild-type protein levels were proportionally expressed from correctly spliced mRNA. The G>T mutation within the exon 4 AG 3'ss junction shifted splicing exclusively to a new AG and resulted in a severely truncated, poorly expressed protein. Finally, the A>G substitution at position -11 of the 3'ss of exon 5 shifted splicing exclusively to a newly created upstream AG and resulted in generation of a protein with a truncated homeodomain. Conclusion This is the first direct evidence to support aberrant RNA splicing as the mechanism underlying the disorder in some patients and suggests that the magnitude of the splicing defect may contribute to the variability of ARS phenotypes, in support of a gene dosage model of Axenfeld-Rieger syndrome.

  10. A novel mutation of the GATA site in the erythroid cell-specific regulatory element of the ABO gene in a blood donor with the Am B phenotype.

    Science.gov (United States)

    Oda, A; Isa, K; Ogasawara, K; Kameyama, K; Okuda, K; Hirashima, M; Ishii, H; Kimura, K; Matsukura, H; Hirayama, F; Kawa, K

    2015-05-01

    The Am and Bm phenotypes are characterized by weak expression of the A or B antigens, respectively, by red blood cells with a normal expression by the saliva of secretors. Deletion of the regulatory element in the first intron of the ABO gene and disruption of the GATA motif in the element were found to be responsible. In this study, we identified a novel mutation within the GATA motif (G>C substitution at position c.28 + 5830) in the regulatory element of the A allele that might diminish transcription activity causing the generation of the Am B phenotype. © 2014 International Society of Blood Transfusion.

  11. A genome-wide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets.

    Science.gov (United States)

    Adamia, Sophia; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bar-Natan, Michal; Pevzner, Samuel; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Burke, John; Galinsky, Ilene; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Stone, Richard; Griffin, James D

    2014-03-01

    Despite new treatments, acute myeloid leukemia (AML) remains an incurable disease. More effective drug design requires an expanded view of the molecular complexity that underlies AML. Alternative splicing of RNA is used by normal cells to generate protein diversity. Growing evidence indicates that aberrant splicing of genes plays a key role in cancer. We investigated genome-wide splicing abnormalities in AML and based on these abnormalities, we aimed to identify novel potential biomarkers and therapeutic targets. We used genome-wide alternative splicing screening to investigate alternative splicing abnormalities in two independent AML patient cohorts [Dana-Farber Cancer Institute (DFCI) (Boston, MA) and University Hospital de Nantes (UHN) (Nantes, France)] and normal donors. Selected splicing events were confirmed through cloning and sequencing analysis, and than validated in 193 patients with AML. Our results show that approximately 29% of expressed genes genome-wide were differentially and recurrently spliced in patients with AML compared with normal donors bone marrow CD34(+) cells. Results were reproducible in two independent AML cohorts. In both cohorts, annotation analyses indicated similar proportions of differentially spliced genes encoding several oncogenes, tumor suppressor proteins, splicing factors, and heterogeneous-nuclear-ribonucleoproteins, proteins involved in apoptosis, cell proliferation, and spliceosome assembly. Our findings are consistent with reports for other malignances and indicate that AML-specific aberrations in splicing mechanisms are a hallmark of AML pathogenesis. Overall, our results suggest that aberrant splicing is a common characteristic for AML. Our findings also suggest that splice variant transcripts that are the result of splicing aberrations create novel disease markers and provide potential targets for small molecules or antibody therapeutics for this disease. ©2013 AACR

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-09-01

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

  13. Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model.

    Science.gov (United States)

    Bestas, Burcu; Moreno, Pedro M D; Blomberg, K Emelie M; Mohammad, Dara K; Saleh, Amer F; Sutlu, Tolga; Nordin, Joel Z; Guterstam, Peter; Gustafsson, Manuela O; Kharazi, Shabnam; Piątosa, Barbara; Roberts, Thomas C; Behlke, Mark A; Wood, Matthew J A; Gait, Michael J; Lundin, Karin E; El Andaloussi, Samir; Månsson, Robert; Berglöf, Anna; Wengel, Jesper; Smith, C I Edvard

    2014-09-01

    X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

  14. Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches

    Science.gov (United States)

    Lorain, Stéphanie; Peccate, Cécile; Le Hir, Maëva; Griffith, Graziella; Philippi, Susanne; Précigout, Guillaume; Mamchaoui, Kamel; Jollet, Arnaud; Voit, Thomas; Garcia, Luis

    2013-01-01

    RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans-splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre–trans-splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans-splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv, illustrating that a given PTM can be suitable for a variety of mutations. PMID:23861443

  15. The emergence of alternative 3' and 5' splice site exons from constitutive exons.

    Directory of Open Access Journals (Sweden)

    Eli Koren

    2007-05-01

    Full Text Available Alternative 3' and 5' splice site (ss events constitute a significant part of all alternative splicing events. These events were also found to be related to several aberrant splicing diseases. However, only few of the characteristics that distinguish these events from alternative cassette exons are known currently. In this study, we compared the characteristics of constitutive exons, alternative cassette exons, and alternative 3'ss and 5'ss exons. The results revealed that alternative 3'ss and 5'ss exons are an intermediate state between constitutive and alternative cassette exons, where the constitutive side resembles constitutive exons, and the alternative side resembles alternative cassette exons. The results also show that alternative 3'ss and 5'ss exons exhibit low levels of symmetry (frame-preserving, similar to constitutive exons, whereas the sequence between the two alternative splice sites shows high symmetry levels, similar to alternative cassette exons. In addition, flanking intronic conservation analysis revealed that exons whose alternative splice sites are at least nine nucleotides apart show a high conservation level, indicating intronic participation in the regulation of their splicing, whereas exons whose alternative splice sites are fewer than nine nucleotides apart show a low conservation level. Further examination of these exons, spanning seven vertebrate species, suggests an evolutionary model in which the alternative state is a derivative of an ancestral constitutive exon, where a mutation inside the exon or along the flanking intron resulted in the creation of a new splice site that competes with the original one, leading to alternative splice site selection. This model was validated experimentally on four exons, showing that they indeed originated from constitutive exons that acquired a new competing splice site during evolution.

  16. Splice Expression Variation Analysis (SEVA) for Inter-tumor Heterogeneity of Gene Isoform Usage in Cancer.

    Science.gov (United States)

    Afsari, Bahman; Guo, Theresa; Considine, Michael; Florea, Liliana; Kagohara, Luciane T; Stein-O'Brien, Genevieve L; Kelley, Dylan; Flam, Emily; Zambo, Kristina D; Ha, Patrick K; Geman, Donald; Ochs, Michael F; Califano, Joseph A; Gaykalova, Daria A; Favorov, Alexander V; Fertig, Elana J

    2018-01-12

    Current bioinformatics methods to detect changes in gene isoform usage in distinct phenotypes compare the relative expected isoform usage in phenotypes. These statistics model differences in isoform usage in normal tissues, which have stable regulation of gene splicing. Pathological conditions, such as cancer, can have broken regulation of splicing that increases the heterogeneity of the expression of splice variants. Inferring events with such differential heterogeneity in gene isoform usage requires new statistical approaches. We introduce Splice Expression Variability Analysis (SEVA) to model increased heterogeneity of splice variant usage between conditions (e.g., tumor and normal samples). SEVA uses a rank-based multivariate statistic that compares the variability of junction expression profiles within one condition to the variability within another. Simulated data show that SEVA is unique in modeling heterogeneity of gene isoform usage, and benchmark SEVA's performance against EBSeq, DiffSplice, and rMATS that model differential isoform usage instead of heterogeneity. We confirm the accuracy of SEVA in identifying known splice variants in head and neck cancer and perform cross-study validation of novel splice variants. A novel comparison of splice variant heterogeneity between subtypes of head and neck cancer demonstrated unanticipated similarity between the heterogeneity of gene isoform usage in HPV-positive and HPV-negative subtypes and anticipated increased heterogeneity among HPV-negative samples with mutations in genes that regulate the splice variant machinery. These results show that SEVA accurately models differential heterogeneity of gene isoform usage from RNA-seq data. SEVA is implemented in the R/Bioconductor package GSReg. bahman@jhu.edu, ejfertig@jhmi.edu. Supplementary data are available at Bioinformatics online. © The Author (2018). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  17. Spliceosome mutations exhibit specific associations with epigenetic modifiers and proto-oncogenes mutated in myelodysplastic syndrome.

    Science.gov (United States)

    Mian, Syed A; Smith, Alexander E; Kulasekararaj, Austin G; Kizilors, Aytug; Mohamedali, Azim M; Lea, Nicholas C; Mitsopoulos, Konstantinos; Ford, Kevin; Nasser, Erick; Seidl, Thomas; Mufti, Ghulam J

    2013-07-01

    The recent identification of acquired mutations in key components of the spliceosome machinery strongly implicates abnormalities of mRNA splicing in the pathogenesis of myelodysplastic syndromes. However, questions remain as to how these aberrations functionally combine with the growing list of mutations in genes involved in epigenetic modification and cell signaling/transcription regulation identified in these diseases. In this study, amplicon sequencing was used to perform a mutation screen in 154 myelodysplastic syndrome patients using a 22-gene panel, including commonly mutated spliceosome components (SF3B1, SRSF2, U2AF1, ZRSR2), and a further 18 genes known to be mutated in myeloid cancers. Sequencing of the 22-gene panel revealed that 76% (n=117) of the patients had mutations in at least one of the genes, with 38% (n=59) having splicing gene mutations and 49% (n=75) patients harboring more than one gene mutation. Interestingly, single and specific epigenetic modifier mutations tended to coexist with SF3B1 and SRSF2 mutations (P<0.03). Furthermore, mutations in SF3B1 and SRSF2 were mutually exclusive to TP53 mutations both at diagnosis and at the time of disease transformation. Moreover, mutations in FLT3, NRAS, RUNX1, CCBL and C-KIT were more likely to co-occur with splicing factor mutations generally (P<0.02), and SRSF2 mutants in particular (P<0.003) and were significantly associated with disease transformation (P<0.02). SF3B1 and TP53 mutations had varying impacts on overall survival with hazard ratios of 0.2 (P<0.03, 95% CI, 0.1-0.8) and 2.1 (P<0.04, 95% CI, 1.1-4.4), respectively. Moreover, patients with splicing factor mutations alone had a better overall survival than those with epigenetic modifier mutations, or cell signaling/transcription regulator mutations with and without coexisting mutations of splicing factor genes, with worsening prognosis (P<0.001). These findings suggest that splicing factor mutations are maintained throughout disease

  18. FUBP1: a new protagonist in splicing regulation of the DMD gene.

    Science.gov (United States)

    Miro, Julie; Laaref, Abdelhamid Mahdi; Rofidal, Valérie; Lagrafeuille, Rosyne; Hem, Sonia; Thorel, Delphine; Méchin, Déborah; Mamchaoui, Kamel; Mouly, Vincent; Claustres, Mireille; Tuffery-Giraud, Sylvie

    2015-02-27

    We investigated the molecular mechanisms for in-frame skipping of DMD exon 39 caused by the nonsense c.5480T>A mutation in a patient with Becker muscular dystrophy. RNase-assisted pull down assay coupled with mass spectrometry revealed that the mutant RNA probe specifically recruits hnRNPA1, hnRNPA2/B1 and DAZAP1. Functional studies in a human myoblast cell line transfected with DMD minigenes confirmed the splicing inhibitory activity of hnRNPA1 and hnRNPA2/B1, and showed that DAZAP1, also known to activate splicing, acts negatively in the context of the mutated exon 39. Furthermore, we uncovered that recognition of endogenous DMD exon 39 in muscle cells is promoted by FUSE binding protein 1 (FUBP1), a multifunctional DNA- and RNA-binding protein whose role in splicing is largely unknown. By serial deletion and mutagenesis studies in minigenes, we delineated a functional intronic splicing enhancer (ISE) in intron 38. FUBP1 recruitment to the RNA sequence containing the ISE was established by RNA pull down and RNA EMSA, and further confirmed by RNA-ChIP on endogenous DMD pre-mRNA. This study provides new insights about the splicing regulation of DMD exon 39, highlighting the emerging role of FUBP1 in splicing and describing the first ISE for constitutive exon inclusion in the mature DMD transcript. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia

    Science.gov (United States)

    Axelrod, Felicia B.; Liebes, Leonard; Gold-von Simson, Gabrielle; Mendoza, Sandra; Mull, James; Leyne, Maire; Norcliffe-Kaufmann, Lucy; Kaufmann, Horacio; Slaugenhaupt, Susan A.

    2011-01-01

    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex associated protein/ elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase wild-type IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine if oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/day for 28 days. An increase in wild-type IKBKAP mRNA expression in leukocytes was noted after eight days in six of eight individuals; after 28 days the mean increase as compared to baseline was significant (p=0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients, but also that effect appears to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine if kinetin will prove therapeutic in FD patients. PMID:21775922

  20. Attitudes of sperm, egg and embryo donors and recipients towards genetic information and screening of donors.

    Science.gov (United States)

    Amor, David J; Kerr, Annabelle; Somanathan, Nandini; McEwen, Alison; Tome, Marianne; Hodgson, Jan; Lewis, Sharon

    2018-02-09

    Gamete and embryo donors undergo genetic screening procedures in order to maximise the health of donor-conceived offspring. In the era of genomic medicine, expanded genetic screening may be offered to donors for the purpose of avoiding transmission of harmful genetic mutations. The objective of this study was to explore the attitudes of donors and recipients toward the expanded genetic screening of donors. Qualitative interview study with thematic analysis, undertaken in a tertiary fertility centre. Semi-structured in-depth qualitative interviews were conducted with eleven recipients and nine donors from three different cohorts (sperm, egg and embryo donors/recipients). Donors and recipients acknowledged the importance of genetic information and were comfortable with the existing level of genetic screening of donors. Recipients recognised some potential benefits of expanded genetic screening of donors; however both recipients and donors were apprehensive about extended genomic technologies, with concerns about how this information would be used and the ethics of genetic selectivity. Participants in donor programs support some level of genetic screening of donors, but are wary of expanding genetic screening beyond current levels.

  1. 0-6652 : spliced Texas girder bridges.

    Science.gov (United States)

    2015-02-01

    Spliced girder technology continues to attract : attention due to its versatility over traditional : prestressed concrete highway bridge construction. : By joining multiple precast concrete girders using : post-tensioning, spliced girder technology :...

  2. NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML.

    Science.gov (United States)

    Adamia, Sophia; Bar-Natan, Michal; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bach, Christian; Pevzner, Samuel; Calimeri, Teresa; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Galinsky, Ilene; Mathews, Steven; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Tenen, Daniel G; Stone, Richard M; Griffin, James D

    2014-05-01

    Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34(+) bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics.

  3. Recruitment of feces donors among blood donors

    DEFF Research Database (Denmark)

    Dahl Jørgensen, Simon Mark; Erikstrup, Christian; Dinh, Khoa Manh

    2018-01-01

    As the use of fecal microbiota transplantation (FMT) has gained momentum, an increasing need for continuous access to healthy feces donors has developed. Blood donors constitute a healthy subset of the general population and may serve as an appropriate group for recruitment. In this study, we...... investigated the suitability of blood donors as feces donors. In a prospective cohort study, we recruited blood donors onsite at a public Danish blood bank. Following their consent, the blood donors underwent a stepwise screening process: First, blood donors completed an electronic pre-screening questionnaire...... to rule out predisposing risk factors. Second, eligible blood donors had blood and fecal samples examined. Of 155 blood donors asked to participate, 137 (88%) completed the electronic pre-screening questionnaire, 16 declined, and 2 were excluded. Of the 137 donors who completed the questionnaire, 79 (58...

  4. A genetic cluster of patients with variant xeroderma pigmentosum with two different founder mutations.

    Science.gov (United States)

    Munford, V; Castro, L P; Souto, R; Lerner, L K; Vilar, J B; Quayle, C; Asif, H; Schuch, A P; de Souza, T A; Ienne, S; Alves, F I A; Moura, L M S; Galante, P A F; Camargo, A A; Liboredo, R; Pena, S D J; Sarasin, A; Chaibub, S C; Menck, C F M

    2017-05-01

    Xeroderma pigmentosum (XP) is a rare human syndrome associated with hypersensitivity to sunlight and a high frequency of skin tumours at an early age. We identified a community in the state of Goias (central Brazil), a sunny and tropical region, with a high incidence of XP (17 patients among approximately 1000 inhabitants). To identify gene mutations in the affected community and map the distribution of the affected alleles, correlating the mutations with clinical phenotypes. Functional analyses of DNA repair capacity and cell-cycle responses after ultraviolet exposure were investigated in cells from local patients with XP, allowing the identification of the mutated gene, which was then sequenced to locate the mutations. A specific assay was designed for mapping the distribution of these mutations in the community. Skin primary fibroblasts showed normal DNA damage removal but abnormal DNA synthesis after ultraviolet irradiation and deficient expression of the Polη protein, which is encoded by POLH. We detected two different POLH mutations: one at the splice donor site of intron 6 (c.764 +1 G>A), and the other in exon 8 (c.907 C>T, p.Arg303X). The mutation at intron 6 is novel, whereas the mutation at exon 8 has been previously described in Europe. Thus, these mutations were likely brought to the community long ago, suggesting two founder effects for this rare disease. This work describes a genetic cluster involving POLH, and, particularly unexpected, with two independent founder mutations, including one that likely originated in Europe. © 2016 British Association of Dermatologists.

  5. Splice-correction strategies for treatment of X-linked agammaglobulinemia.

    Science.gov (United States)

    Bestas, Burcu; Turunen, Janne J; Blomberg, K Emelie M; Wang, Qing; Månsson, Robert; El Andaloussi, Samir; Berglöf, Anna; Smith, C I Edvard

    2015-03-01

    X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option.

  6. Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.

    Directory of Open Access Journals (Sweden)

    Helen M Wise

    Full Text Available Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42 with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

  7. GC content around splice sites affects splicing through pre-mRNA secondary structures

    Directory of Open Access Journals (Sweden)

    Chen Liang

    2011-01-01

    Full Text Available Abstract Background Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens, mice (Mus musculus, fruit flies (Drosophila melanogaster, and nematodes (Caenorhabditis elegans to further investigate this phenomenon. Results We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures. Conclusion All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

  8. Titin Diversity—Alternative Splicing Gone Wild

    Directory of Open Access Journals (Sweden)

    Wei Guo

    2010-01-01

    Full Text Available Titin is an extremely large protein found in highest concentrations in heart and skeletal muscle. The single mammalian gene is expressed in multiple isoforms as a result of alternative splicing. Although titin isoform expression is controlled developmentally and in a tissue specific manner, the vast number of potential splicing pathways far exceeds those described in any other alternatively spliced gene. Over 1 million human splice pathways for a single individual can be potentially derived from the PEVK region alone. A new splicing pattern for the human cardiac N2BA isoform type has been found in which the PEVK region includes only the N2B type exons. The alterations in splicing and titin isoform expression in human heart disease provide impetus for future detailed study of the splicing mechanisms for this giant protein.

  9. Recessive mutations of TMC1 associated with moderate to severe hearing loss.

    Science.gov (United States)

    Imtiaz, Ayesha; Maqsood, Azra; Rehman, Atteeq U; Morell, Robert J; Holt, Jeffrey R; Friedman, Thomas B; Naz, Sadaf

    2016-04-01

    TMC1 encodes a protein required for the normal function of mechanically activated channels that enable sensory transduction in auditory and vestibular hair cells. TMC1 protein is localized at the tips of the hair cell stereocilia, the site of conventional mechanotransduction. In many populations, loss-of-function recessive mutations of TMC1 are associated with profound deafness across all frequencies tested. In six families reported here, variable moderate-to-severe or moderate-to-profound hearing loss co-segregated with STR (short tandem repeats) markers at the TMC1 locus DFNB7/11. Massively parallel and Sanger sequencing of genomic DNA revealed each family co-segregating hearing loss with a homozygous TMC1 mutation: two reported mutations (p.R34X and p.R389Q) and three novel mutations (p.S596R, p.N199I, and c.1404 + 1G > T). TMC1 cDNA sequence from affected subjects homozygous for the donor splice site transversion c.1404 + 1G > T revealed skipping of exon 16, deleting 60 amino acids from the TMC1 protein. Since the mutations in our study cause less than profound hearing loss, we speculate that there is hypo-functional TMC1 mechanotransduction channel activity and that other even less damaging variants of TMC1 may be associated with more common mild-to-severe sensorineural hearing loss.

  10. Mutations in Dnaaf1 and Lrrc48 Cause Hydrocephalus, Laterality Defects, and Sinusitis in Mice

    Directory of Open Access Journals (Sweden)

    Seungshin Ha

    2016-08-01

    Full Text Available We have previously described a forward genetic screen in mice for abnormalities of brain development. Characterization of two hydrocephalus mutants by whole-exome sequencing after whole-genome SNP mapping revealed novel recessive mutations in Dnaaf1 and Lrrc48. Mouse mutants of these two genes have not been previously reported. The Dnaaf1 mutant carries a mutation at the splice donor site of exon 4, which results in abnormal transcripts. The Lrrc48 mutation is a missense mutation at a highly conserved leucine residue, which is also associated with a decrease in Lrrc48 transcription. Both Dnaaf1 and Lrrc48 belong to a leucine-rich repeat-containing protein family and are components of the ciliary axoneme. Their Chlamydomonas orthologs are known to be required for normal ciliary beat frequency or flagellar waveform, respectively. Some Dnaaf1 or Lrrc48 homozygote mutants displayed laterality defects, suggesting a motile cilia defect in the embryonic node. Mucus accumulation and neutrophil infiltration in the maxillary sinuses suggested sinusitis. Dnaaf1 mutants showed postnatal lethality, and none survived to weaning age. Lrrc48 mutants survive to adulthood, but had male infertility. ARL13B immunostaining showed the presence of motile cilia in the mutants, and the distal distribution of DNAH9 in the axoneme of upper airway motile cilia appeared normal. The phenotypic abnormalities suggest that mutations in Dnaaf1 and Lrrc48 cause defects in motile cilia function.

  11. Novel homozygous AIRE mutation in a German patient with severe APECED.

    Science.gov (United States)

    von Schnurbein, J; Lahr, G; Posovszky, C; Debatin, K M; Wabitsch, M

    2008-10-01

    Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disorder typically presenting with chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal failure variably accompanied by other symptoms. APECED is caused by a mutation in the autoimmune regulator gene (AIRE). Today over 60 different mutations are known world-wide, most of them localized in exons 2, 8, and 10. We report here a German girl with rheumatoid factor positive arthritis, chronic mucocutaneous candidiasis, autoimmune hepatitis, chronic diarrhea, vitiligo, hypothyroidism, hypoparathyroidism, and adrenal failure who is homozygous for a novel mutation at the end of exon 3 of the AIRE gene (c.462G>A), within the conserved splice donor sequence. This mutation probably introduces a frameshift after amino acid 154 (p.Pro154fs) by skipping exon 4. In addition, we analyzed five other family members out of three generations for the AIRE gene mutation and for polymorphisms in the cytotoxic T lymphocyte antigen 4 (CTLA4) gene region and lymphoid protein tyrosine phosphatase (PTPN22) gene, which are associated with the occurrence of sporadic autoimmune Addison's disease, type 1 diabetes mellitus, and generalized vitiligo.

  12. Interplay between exonic splicing enhancers, mRNA processing, and mRNA surveillance in the dystrophic Mdx mouse.

    Directory of Open Access Journals (Sweden)

    Massimo Buvoli

    2007-05-01

    Full Text Available Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5' and 3' splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD, lacks dystrophin by virtue of a premature termination codon (PTC in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described.Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3' splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS, but induces exclusively nonsense-mediated mRNA decay (NMD. Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24.Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exon-exon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites.

  13. Novel mutations in the transmembrane natriuretic peptide receptor ...

    Indian Academy of Sciences (India)

    It is an autosomal recessive disorder due to mutation in NPR2 gene which impairs skeletal growth. To screen the mutations in the gene NPR2, all of its coding exons and splice junction sites were PCR amplified from genomic DNA of affected individuals of four families and sequenced. Four homozygous mutations in four ...

  14. Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo.

    Science.gov (United States)

    Waldsich, C; Semrad, K; Schroeder, R

    1998-12-01

    The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.

  15. Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides

    Science.gov (United States)

    Liu, Jun; Bhadra, Malini; Sinnakannu, Joanna Rajeswary; Yue, Wan Lin; Tan, Cheryl Weiqi; Rigo, Frank; Ong, S.Tiong; Roca, Xavier

    2017-01-01

    Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis-acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM-polymorphism-associated TKI-resistant CML and other cancers. PMID:29100409

  16. Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides.

    Science.gov (United States)

    Liu, Jun; Bhadra, Malini; Sinnakannu, Joanna Rajeswary; Yue, Wan Lin; Tan, Cheryl Weiqi; Rigo, Frank; Ong, S Tiong; Roca, Xavier

    2017-09-29

    Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis -acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM -polymorphism-associated TKI-resistant CML and other cancers.

  17. Changes in Alternative Splicing as Pharmacodynamic Markers for Sudemycin D6

    Directory of Open Access Journals (Sweden)

    Morgan Thurman

    2017-09-01

    Full Text Available Objective: The aim of the study was to define pharmacodynamic markers for sudemycin D6, an experimental cancer drug that changes alternative splicing in human blood. Methods: Blood samples from 12 donors were incubated with sudemycin D6 for up to 24 hours, and at several time points total RNA from lymphocytes was prepared and the pre-messenger RNA (mRNA splicing patterns were analyzed with reverse transcription-polymerase chain reaction. Results: Similar to immortalized cells, blood lymphocytes change alternative splicing due to sudemycin D6 treatment. However, lymphocytes in blood respond slower than immortalized cultured cells. Conclusions: Exon skipping in the DUSP11 and SRRM1 pre-mRNAs are pharmacodynamic markers for sudemycin D6 treatment and show effects beginning at 9 hours after treatment.

  18. Histone H3 lysine 36 methylation affects temperature-induced alternative splicing and flowering in plants.

    Science.gov (United States)

    Pajoro, A; Severing, E; Angenent, G C; Immink, R G H

    2017-06-01

    Global warming severely affects flowering time and reproductive success of plants. Alternative splicing of pre-messenger RNA (mRNA) is an important mechanism underlying ambient temperature-controlled responses in plants, yet its regulation is poorly understood. An increase in temperature promotes changes in plant morphology as well as the transition from the vegetative to the reproductive phase in Arabidopsis thaliana via changes in splicing of key regulatory genes. Here we investigate whether a particular histone modification affects ambient temperature-induced alternative splicing and flowering time. We use a genome-wide approach and perform RNA-sequencing (RNA-seq) analyses and histone H3 lysine 36 tri-methylation (H3K36me3) chromatin immunoprecipitation sequencing (ChIP-seq) in plants exposed to different ambient temperatures. Analysis and comparison of these datasets reveal that temperature-induced differentially spliced genes are enriched in H3K36me3. Moreover, we find that reduction of H3K36me3 deposition causes alteration in temperature-induced alternative splicing. We also show that plants with mutations in H3K36me3 writers, eraser, or readers have altered high ambient temperature-induced flowering. Our results show a key role for the histone mark H3K36me3 in splicing regulation and plant plasticity to fluctuating ambient temperature. Our findings open new perspectives for the breeding of crops that can better cope with environmental changes due to climate change.

  19. Gene Therapy via Trans-Splicing for LMNA-Related Congenital Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Feriel Azibani

    2018-03-01

    Full Text Available We assessed the potential of Lmna-mRNA repair by spliceosome-mediated RNA trans-splicing as a therapeutic approach for LMNA-related congenital muscular dystrophy. This gene therapy strategy leads to reduction of mutated transcript expression for the benefit of corresponding wild-type (WT transcripts. We developed 5′-RNA pre-trans-splicing molecules containing the first five exons of Lmna and targeting intron 5 of Lmna pre-mRNA. Among nine pre-trans-splicing molecules, differing in the targeted sequence in intron 5 and tested in C2C12 myoblasts, three induced trans-splicing events on endogenous Lmna mRNA and confirmed at protein level. Further analyses performed in primary myotubes derived from an LMNA-related congenital muscular dystrophy (L-CMD mouse model led to a partial rescue of the mutant phenotype. Finally, we tested this approach in vivo using adeno-associated virus (AAV delivery in newborn mice and showed that trans-splicing events occurred in WT mice 50 days after AAV delivery, although at a low rate. Altogether, while these results provide the first evidence for reprogramming LMNA mRNA in vitro, strategies to improve the rate of trans-splicing events still need to be developed for efficient application of this therapeutic approach in vivo.

  20. In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin

    Directory of Open Access Journals (Sweden)

    Delphine Laustriat

    2015-01-01

    Full Text Available Major physiological changes are governed by alternative splicing of RNA, and its misregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication and demonstrate the effects of the biguanide metformin, on alternative splicing. The mechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1, a multisystemic disease considered to be a spliceopathy. We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. The drug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing.

  1. RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing.

    Science.gov (United States)

    Maatz, Henrike; Jens, Marvin; Liss, Martin; Schafer, Sebastian; Heinig, Matthias; Kirchner, Marieluise; Adami, Eleonora; Rintisch, Carola; Dauksaite, Vita; Radke, Michael H; Selbach, Matthias; Barton, Paul J R; Cook, Stuart A; Rajewsky, Nikolaus; Gotthardt, Michael; Landthaler, Markus; Hubner, Norbert

    2014-08-01

    Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3' and 5' splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure.

  2. Integrative analysis revealed the molecular mechanism underlying RBM10-mediated splicing regulation.

    Science.gov (United States)

    Wang, Yongbo; Gogol-Döring, Andreas; Hu, Hao; Fröhler, Sebastian; Ma, Yunxia; Jens, Marvin; Maaskola, Jonas; Murakawa, Yasuhiro; Quedenau, Claudia; Landthaler, Markus; Kalscheuer, Vera; Wieczorek, Dagmar; Wang, Yang; Hu, Yuhui; Chen, Wei

    2013-09-01

    RBM10 encodes an RNA binding protein. Mutations in RBM10 are known to cause multiple congenital anomaly syndrome in male humans, the TARP syndrome. However, the molecular function of RBM10 is unknown. Here we used PAR-CLIP to identify thousands of binding sites of RBM10 and observed significant RBM10-RNA interactions in the vicinity of splice sites. Computational analyses of binding sites as well as loss-of-function and gain-of-function experiments provided evidence for the function of RBM10 in regulating exon skipping and suggested an underlying mechanistic model, which could be subsequently validated by minigene experiments. Furthermore, we demonstrated the splicing defects in a patient carrying an RBM10 mutation, which could be explained by disrupted function of RBM10 in splicing regulation. Overall, our study established RBM10 as an important regulator of alternative splicing, presented a mechanistic model for RBM10-mediated splicing regulation and provided a molecular link to understanding a human congenital disorder. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

  3. Cerebrotendinous xanthomatosis (CTX): an association of pulverulent cataracts and pseudo-dominant developmental delay in a family with a splice site mutation in CYP27A1--a case report.

    Science.gov (United States)

    Bourkiza, Rabia; Joyce, Sarah; Patel, Himanshu; Chan, Michelle; Meyer, Esther; Maher, Eamonn R; Reddy, M Ashwin

    2010-06-01

    A 15-year-old boy with developmental delay presented to the pediatric ophthalmology clinic with bilateral pulverulent cataracts. The family was examined for developmental delay, cataracts and systemic problems. The parents were consanguineous and originally from Bangladesh. All the children were born in the UK. The mother and 5 children had developmental delay. Three children had global developmental delay, diarrhea and pulverulent cataracts. Two children had microcephaly, developmental delay, constipation and no cataracts. The mother did not have microcephaly, cataracts or gastrointestinal problems. Linkage analysis via autozygosity testing was performed for detection of loci and candidate genes. The patients with cataracts were segregated with homozygous mutations in the CYP27A1 (G to A substitution at position +1 of intron 6). The complex nature of this family's findings suggested that it had an unusual autosomal dominant condition with variable expression. Autozygosity testing demonstrated that three members had Cerebrotendinous xanthomatosis (CTX), which is inherited in an autosomal recessive manner. The aetiology of the developmental delay in other family members remains unknown. Cerebrotendinous xanthomatosis is a rare autosomal recessive condition that can result in neurological deficits and early death if left untreated. In view of the reversible nature of the condition with appropriate treatment, there needs to be a high level of suspicion of CTX for any child with cataracts and developmental delay even if the pattern of inheritance is not straightforward at initial assessment.

  4. Alternative Splicing in Plant Immunity

    Directory of Open Access Journals (Sweden)

    Shengming Yang

    2014-06-01

    Full Text Available Alternative splicing (AS occurs widely in plants and can provide the main source of transcriptome and proteome diversity in an organism. AS functions in a range of physiological processes, including plant disease resistance, but its biological roles and functional mechanisms remain poorly understood. Many plant disease resistance (R genes undergo AS, and several R genes require alternatively spliced transcripts to produce R proteins that can specifically recognize pathogen invasion. In the finely-tuned process of R protein activation, the truncated isoforms generated by AS may participate in plant disease resistance either by suppressing the negative regulation of initiation of immunity, or by directly engaging in effector-triggered signaling. Although emerging research has shown the functional significance of AS in plant biotic stress responses, many aspects of this topic remain to be understood. Several interesting issues surrounding the AS of R genes, especially regarding its functional roles and regulation, will require innovative techniques and additional research to unravel.

  5. VEGF Spliced Variants: Possible Role of Anti-Angiogenesis Therapy

    Directory of Open Access Journals (Sweden)

    Caroline Hilmi

    2012-01-01

    Full Text Available Angiogenesis has been targeted in retinopathies, psoriasis, and a variety of cancers (colon, breast, lung, and kidney. Among these tumour types, clear cell renal cell carcinomas (RCCs are the most vascularized tumours due to mutations of the von Hippel Lindau gene resulting in HIF-1 alpha stabilisation and overexpression of Vascular Endothelial Growth Factor (VEGF. Surgical nephrectomy remains the most efficient curative treatment for patients with noninvasive disease, while VEGF targeting has resulted in varying degrees of success for treating metastatic disease. VEGF pre-mRNA undergoes alternative splicing generating pro-angiogenic isoforms. However, the recent identification of novel splice variants of VEGF with anti-angiogenic properties has provided some insight for the lack of current treatment efficacy. Here we discuss an explanation for the relapse to anti-angiogenesis treatment as being due to either an initial or acquired resistance to the therapy. We also discuss targeting angiogenesis via SR (serine/arginine-rich proteins implicated in VEGF splicing.

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

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    Lan Lin

    2008-10-01

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

  7. IPEX due to an exon 7 skipping FOXP3 mutation with autoimmune diabetes mellitus cured by selective TReg cell engraftment.

    Science.gov (United States)

    Magg, Thomas; Wiebking, Volker; Conca, Raffaele; Krebs, Stefan; Arens, Stefan; Schmid, Irene; Klein, Christoph; Albert, Michael H; Hauck, Fabian

    2018-03-19

    Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare inherited disorder leading to severe organ-specific autoimmunity. IPEX is caused by hemizygous mutations in FOXP3, which codes for a master transcription factor of regulatory T (T Reg ) cell development and function. We describe a four-year-old boy with typical but slightly delayed-onset of IPEX with autoimmune diabetes mellitus, enteropathy, hepatitis and skin disease. We found the unreported FOXP3 splice site mutation c.816+2T>A that leads to the loss of leucine-zipper coding exon 7. RNA-Seq revealed that FOXP3Δ7 leads to differential expression of FOXP3 regulated genes. After myeloablative conditioning the patient underwent allogeneic HSCT from a matched unrelated donor. HSCT led to the resolution of all IPEX symptoms including insulin requirement despite persisting autoantibody levels. After initial full donor engraftment nearly complete autologous reconstitution was documented, but donor-derived T Reg cells persisted with a lineage-specific chimerism of >70% and the patient remained in clinical remission. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Mutation testing in Treacher Collins Syndrome.

    Science.gov (United States)

    Ellis, P E; Dawson, M; Dixon, M J

    2002-12-01

    To report on a study where 97 subjects were screened for mutations in the Treacher Collins syndrome (TCS) gene TCOF1. Ninety-seven subjects with a clinical diagnosis of TCS were screened for potential mutations in TCOF1, by means of single strand conformation polymorphism (SSCP) analysis. In those subjects where potential mutations were detected, sequence analysis was performed to determine the site and type of mutation present. Thirty-six TCS-specific mutations are reported including 27 deletions, six point mutations, two splice junction mutations, and one insertion/deletion. This brings the total number of mutations reported to date to 105. The importance of detection of these mutations is mainly in postnatal diagnosis and genetic counselling. Knowledge of the family specific mutation may also be used in prenatal diagnosis to confirm whether the foetus is affected or not, and give the parents the choice of whether to continue with the pregnancy.

  9. Capacity of columns with splice imperfections

    International Nuclear Information System (INIS)

    Popov, E.P.; Stephen, R.M.

    1977-01-01

    To study the behavior of spliced columns subjected to tensile forces simulating situations which may develop in an earthquake, all of the spliced specimens were tested to failure in tension after first having been subjected to large compressive loads. The results of these tests indicate that the lack of perfect contact at compression splices of columns may not be important, provided that the gaps are shimmed and welding is used to maintain the sections in alignment

  10. Bi-allelic Mutations in PKD1L1 Are Associated with Laterality Defects in Humans.

    Science.gov (United States)

    Vetrini, Francesco; D'Alessandro, Lisa C A; Akdemir, Zeynep C; Braxton, Alicia; Azamian, Mahshid S; Eldomery, Mohammad K; Miller, Kathryn; Kois, Chelsea; Sack, Virginia; Shur, Natasha; Rijhsinghani, Asha; Chandarana, Jignesh; Ding, Yan; Holtzman, Judy; Jhangiani, Shalini N; Muzny, Donna M; Gibbs, Richard A; Eng, Christine M; Hanchard, Neil A; Harel, Tamar; Rosenfeld, Jill A; Belmont, John W; Lupski, James R; Yang, Yaping

    2016-10-06

    Disruption of the establishment of left-right (L-R) asymmetry leads to situs anomalies ranging from situs inversus totalis (SIT) to situs ambiguus (heterotaxy). The genetic causes of laterality defects in humans are highly heterogeneous. Via whole-exome sequencing (WES), we identified homozygous mutations in PKD1L1 from three affected individuals in two unrelated families. PKD1L1 encodes a polycystin-1-like protein and its loss of function is known to cause laterality defects in mouse and medaka fish models. Family 1 had one fetus and one deceased child with heterotaxy and complex congenital heart malformations. WES identified a homozygous splicing mutation, c.6473+2_6473+3delTG, which disrupts the invariant splice donor site in intron 42, in both affected individuals. In the second family, a homozygous c.5072G>C (p.Cys1691Ser) missense mutation was detected in an individual with SIT and congenital heart disease. The p.Cys1691Ser substitution affects a highly conserved cysteine residue and is predicted by molecular modeling to disrupt a disulfide bridge essential for the proper folding of the G protein-coupled receptor proteolytic site (GPS) motif. Damaging effects associated with substitutions of this conserved cysteine residue in the GPS motif have also been reported in other genes, namely GPR56, BAI3, and PKD1 in human and lat-1 in C. elegans, further supporting the likely pathogenicity of p.Cys1691Ser in PKD1L1. The identification of bi-allelic PKD1L1 mutations recapitulates previous findings regarding phenotypic consequences of loss of function of the orthologous genes in mice and medaka fish and further expands our understanding of genetic contributions to laterality defects in humans. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  11. Nyretransplantation med levende donor

    DEFF Research Database (Denmark)

    Kamper, A L; Løkkegaard, H; Rasmussen, F

    2000-01-01

    In recent years transplantation from living donors has accounted for 25-30% of all kidney transplants in Denmark corresponding to 40-45 per year. Most of these living donors are parents or siblings, although internationally an increasing number are unrelated donors. Donor nephrectomy is associated...

  12. Alternative Splicing in Neurogenesis and Brain Development

    Directory of Open Access Journals (Sweden)

    Chun-Hao Su

    2018-02-01

    Full Text Available Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

  13. The mutation spectrum of the JK-null phenotype in the Chinese population.

    Science.gov (United States)

    Guo, Zhonghui; Wang, Chen; Yan, Kangfeng; Xie, Jingwen; Shen, Wei; Li, Qin; Zhang, Jiamin; Ye, Luyi; Zhu, Ziyan

    2013-03-01

    This study aimed to analyze the mutation spectrum of the JK-null phenotype in the Chinese population. The JK gene encoding the Kidd blood group antigen protein and JK*A/JK*B polymorphism caused by a G-to-A mutation at nt838 are well described. However, the molecular basis of the JK-null phenotype in Chinese populations remains unclear. Sixteen unrelated JK-null phenotype donors detected by red blood cell urea lysis resistance assay of 201,194 Chinese blood donors were confirmed in serologic agglutination tests. JK-null alleles were analyzed by MnlI polymerase chain reaction-restriction fragment length polymorphism and sequencing of all JK gene coding regions. In addition to the well-known Polynesian JK-null allele JK*B(IVS5-1g>a) and two alleles discovered in Taiwan, JK*B(896G>A) and JK*B(222C>A), seven JK-null allele types were detected in this study including four novel JK-null alleles: a nonsense mutation, JK*B(512G>A); two types of missense point mutations, JK*B(536C>G) and JK*B(437T>C); and a splice mutation, JK*A(IVS8+5g>a), resulting in skipping of Exon 8. This study demonstrates the frequency and heterogeneity of the JK-null phenotype in Chinese populations. Based on our findings, the mechanisms underlying the Chinese Jk(a-b-) phenotype are quite different from other ethnic groups. The two most common types of JK-null alleles were JK*B(IVS5-1g>a) and JK*B(896G>A) in Chinese persons. Four novel JK-null alleles were noted to be associated with the Jk(a-b-) phenotype. © 2012 American Association of Blood Banks.

  14. Mutations in a new gene in Ellis-van Creveld syndrome and Weyers acrodental dysostosis.

    Science.gov (United States)

    Ruiz-Perez, V L; Ide, S E; Strom, T M; Lorenz, B; Wilson, D; Woods, K; King, L; Francomano, C; Freisinger, P; Spranger, S; Marino, B; Dallapiccola, B; Wright, M; Meitinger, T; Polymeropoulos, M H; Goodship, J

    2000-03-01

    Ellis-van Creveld syndrome (EvC, MIM 225500) is an autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly and dysplastic nails and teeth. Congenital cardiac defects, most commonly a defect of primary atrial septation producing a common atrium, occur in 60% of affected individuals. The disease was mapped to chromosome 4p16 in nine Amish subpedigrees and single pedigrees from Mexico, Ecuador and Brazil. Weyers acrodental dysostosis (MIM 193530), an autosomal dominant disorder with a similar but milder phenotype, has been mapped in a single pedigree to an area including the EvC critical region. We have identified a new gene (EVC), encoding a 992-amino-acid protein, that is mutated in individuals with EvC. We identified a splice-donor change in an Amish pedigree and six truncating mutations and a single amino acid deletion in seven pedigrees. The heterozygous carriers of these mutations did not manifest features of EvC. We found two heterozygous missense mutations associated with a phenotype, one in a man with Weyers acrodental dysostosis and another in a father and his daughter, who both have the heart defect characteristic of EvC and polydactyly, but not short stature. We suggest that EvC and Weyers acrodental dysostosis are allelic conditions.

  15. A novel mutation in CLCN1 associated with feline myotonia congenita.

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    Barbara Gandolfi

    Full Text Available Myotonia congenita (MC is a skeletal muscle channelopathy characterized by inability of the muscle to relax following voluntary contraction. Worldwide population prevalence in humans is 1:100,000. Studies in mice, dogs, humans and goats confirmed myotonia associated with functional defects in chloride channels and mutations in a skeletal muscle chloride channel (CLCN1. CLCN1 encodes for the most abundant chloride channel in the skeletal muscle cell membrane. Five random bred cats from Winnipeg, Canada with MC were examined. All cats had a protruding tongue, limited range of jaw motion and drooling with prominent neck and proximal limb musculature. All cats had blepharospasm upon palpebral reflex testing and a short-strided gait. Electromyograms demonstrated myotonic discharges at a mean frequency of 300 Hz resembling the sound of a 'swarm of bees'. Muscle histopathology showed hypertrophy of all fiber types. Direct sequencing of CLCN1 revealed a mutation disrupting a donor splice site downstream of exon 16 in only the affected cats. In vitro translation of the mutated protein predicted a premature truncation and partial lack of the highly conserved CBS1 (cystathionine β-synthase domain critical for ion transport activity and one dimerization domain pivotal in channel formation. Genetic screening of the Winnipeg random bred population of the cats' origin identified carriers of the mutation. A genetic test for population screening is now available and carrier cats from the feral population can be identified.

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

    Science.gov (United States)

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

    2014-08-01

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

  17. Optimization of Peptide Nucleic Acid Antisense Oligonucleotides for Local and Systemic Dystrophin Splice Correction in the mdx Mouse

    Science.gov (United States)

    Yin, HaiFang; Betts, Corinne; Saleh, Amer F; Ivanova, Gabriela D; Lee, Hyunil; Seow, Yiqi; Kim, Dalsoo; Gait, Michael J; Wood, Matthew JA

    2010-01-01

    Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration. Major challenges in developing this splice correction therapy are to optimize AO chemistry and to develop more effective systemic AO delivery. Peptide nucleic acid (PNA) AOs are an alternative AO chemistry with favorable in vivo biochemical properties and splice correcting abilities. Here, we show long-term splice correction of the DMD gene in mdx mice following intramuscular PNA delivery and effective splice correction in aged mdx mice. Further, we report detailed optimization of systemic PNA delivery dose regimens and PNA AO lengths to yield splice correction, with 25-mer PNA AOs providing the greatest splice correcting efficacy, restoring dystrophin protein in multiple peripheral muscle groups. PNA AOs therefore provide an attractive candidate AO chemistry for DMD exon skipping therapy. PMID:20068555

  18. A novel splice variant in the N-propeptide of COL5A1 causes an EDS phenotype with severe kyphoscoliosis and eye involvement.

    Directory of Open Access Journals (Sweden)

    Sofie Symoens

    Full Text Available BACKGROUND: The Ehlers-Danlos Syndrome (EDS is a heritable connective tissue disorder characterized by hyperextensible skin, joint hypermobility and soft tissue fragility. The classic subtype of EDS is caused by mutations in one of the type V collagen genes (COL5A1 and COL5A2. Most mutations affect the type V collagen helical domain and lead to a diminished or structurally abnormal type V collagen protein. Remarkably, only two mutations were reported to affect the extended, highly conserved N-propeptide domain, which plays an important role in the regulation of the heterotypic collagen fibril diameter. We identified a novel COL5A1 N-propeptide mutation, resulting in an unusual but severe classic EDS phenotype and a remarkable splicing outcome. METHODOLOGY/PRINCIPAL FINDINGS: We identified a novel COL5A1 N-propeptide acceptor-splice site mutation (IVS6-2A>G, NM_000093.3_c.925-2A>G in a patient with cutaneous features of EDS, severe progressive scoliosis and eye involvement. Two mutant transcripts were identified, one with an exon 7 skip and one in which exon 7 and the upstream exon 6 are deleted. Both transcripts are expressed and secreted into the extracellular matrix, where they can participate in and perturb collagen fibrillogenesis, as illustrated by the presence of dermal collagen cauliflowers. Determination of the order of intron removal and computational analysis showed that simultaneous skipping of exons 6 and 7 is due to the combined effect of delayed splicing of intron 7, altered pre-mRNA secondary structure, low splice site strength and possibly disturbed binding of splicing factors. CONCLUSIONS/SIGNIFICANCE: We report a novel COL5A1 N-propeptide acceptor-splice site mutation in intron 6, which not only affects splicing of the adjacent exon 7, but also causes a splicing error of the upstream exon 6. Our findings add further insights into the COL5A1 splicing order and show for the first time that a single COL5A1 acceptor-splice site

  19. Protein truncation test: analysis of two novel point mutations at the carboxy-terminus of the human dystrophin gene associated with mental retardation.

    Science.gov (United States)

    Tuffery, S; Lenk, U; Roberts, R G; Coubes, C; Demaille, J; Claustres, M

    1995-01-01

    Approximately one-third of the mutations responsible for Duchenne muscular dytrophy (DMD) do not involve gross rearrangements of the dystrophin gene. Methods for intensive mutation screening have recently been applied to this immense gene, which resulted in the identification of a number of point mutations in DMD patients, mostly translation-terminating mutations. A number of data raised the possibility that the C-terminal region of dystrophin might be involved in some cases of mental retardation associated with DMD. Using single-strand conformation analysis of products amplified by polymerase chain reaction (PCR-SSCA) to screen the terminal domains of the dystrophin gene (exons 60-79) of 20 unrelated patients with DMD or BMD, we detected two novel point mutations in two mentally retarded DMD patients: a 1-bp deletion in exon 70 (10334delC) and a 5' splice donor site alteration in intron 69 (10294 + 1G-->T). Both mutations should result in a premature translation termination of dystrophin. The possible effects on the reading frame were analyzed by the study of reverse transcripts amplified from peripheral blood lymphocytes mRNA and by the protein truncation test.

  20. Multiple splice defects in ABCA1 cause low HDL-C in a family with Hypoalphalipoproteinemia and premature coronary disease

    Directory of Open Access Journals (Sweden)

    Miller Michael

    2009-01-01

    Full Text Available Abstract Background Mutations at splice junctions causing exon skipping are uncommon compared to exonic mutations, and two intronic mutations causing an aberrant phenotype have rarely been reported. Despite the high number of functional ABCA1 mutations reported to date, splice variants have been reported infrequently. We screened DNA from a 41 year-old male with low HDL-C (12 mg/dL [0.31 mmol/L] and a family history of premature coronary heart disease (CHD using polymerase chain reaction single-strand conformation polymorphism (SSCP analysis. Methods Family members with low levels of HDL-C (n = 6 were screened by SSCP for mutations in ABCA1. Samples with altered SSCP patterns were sequenced directly using either an ABI 3700 or ABI3730Xl DNA Analyzer. To screen for splicing defects, cDNA was isolated from the proband's RNA and was sequenced as above. A series of minigenes were constructed to determine the contribution of normal and defective alleles. Results Two novel splice variants in ABCA1 were identified. The first mutation was a single base pair change (T->C in IVS 7, 6 bps downstream from the exon7/intron7 junction. Amplification of cDNA and allelic subcloning identified skipping of Exon 7 that results in the elimination of 59 amino acids from the first extracellular loop of the ABCA1 protein. The second mutation was a single base pair change (G->C at IVS 31 -1, at the intron/exon junction of exon 32. This mutation causes skipping of exon 32, resulting in 8 novel amino acids followed by a stop codon and a predicted protein size of 1496 AA, compared to normal (2261 AA. Bioinformatic studies predicted an impact on splicing as confirmed by in vitro assays of constitutive splicing. Conclusion In addition to carnitine-acylcarnitine translocase (CACT deficiency and Hermansky-Pudlak syndrome type 3, this represents only the third reported case in which 2 different splice mutations has resulted in an aberrant clinical phenotype.

  1. Alcoholism and Alternative Splicing of Candidate Genes

    Directory of Open Access Journals (Sweden)

    Toshikazu Sasabe

    2010-03-01

    Full Text Available Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism.

  2. A DSPP Mutation Causing Dentinogenesis Imperfecta and Characterization of the Mutational Effect

    Directory of Open Access Journals (Sweden)

    Sook-Kyung Lee

    2013-01-01

    Full Text Available Mutations in the DSPP gene have been identified in nonsyndromic hereditary dentin defects, but the genotype-phenotype correlations are not fully understood. Recently, it has been demonstrated that the mutations of DSPP affecting the IPV leader sequence result in mutant DSPP retention in rough endoplasmic reticulum (ER. In this study, we identified a Korean family with dentinogenesis imperfecta type III. To identify the disease causing mutation in this family, we performed mutational analysis based on candidate gene sequencing. Exons and exon-intron boundaries of DSPP gene were sequenced, and the effects of the identified mutation on the pre-mRNA splicing and protein secretion were investigated. Candidate gene sequencing revealed a mutation (c.50C > T, p.P17L in exon 2 of the DSPP gene. The splicing assay showed that the mutation did not influence pre-mRNA splicing. However, the mutation interfered with protein secretion and resulted in the mutant protein remaining largely in the ER. These results suggest that the mutation affects ER-to-Golgi apparatus export and results in the reduction of secreted DSPP and ER overload. This may induce cell stress and damage processing and/or transport of dentin matrix proteins or other critical proteins.

  3. Spliced

    DEFF Research Database (Denmark)

    Addison, Courtney Page

    2017-01-01

    Human gene therapy (HGT) aims to cure disease by inserting or editing the DNA of patients with genetic conditions. Since foundational genetic techniques came into use in the 1970s, the field has developed to the point that now three therapies have market approval, and over 1800 clinical trials have...... been initiated. In this article I present a brief history of HGT, showing how the ethical and practical viability of the field was achieved by key scientific and regulatory actors. These parties carefully articulated gene therapy’s scope, limiting it to therapeutic interventions on somatic cells......, and cultivated alliances and divisions that bolstered the field’s legitimacy. At times these measures faltered, and then practitioners and sometimes patients would invoke an ethical imperative, posing gene therapy as the best solution to life and death problems. I suggest that we consider how boundary...

  4. Depolarization-mediated regulation of alternative splicing

    Directory of Open Access Journals (Sweden)

    Alok eSharma

    2011-12-01

    Full Text Available Alternative splicing in eukaryotes plays an important role in regulating gene expression by selectively including alternative exons. A wealth of information has been accumulated that explains how alternative exons are selected in a developmental stage- or tissue-specific fashion. However, our knowledge of how cells respond to environmental changes to alter alternative splicing is very limited. For example, although a number of alternative exons have been shown to be regulated by calcium level alterations, the underlying mechanisms are not well understood. As calcium signaling in neurons plays a crucial role in essential neuronal functions such as learning and memory formation, it is important to understand how this process is regulated at every level in gene expression. The significance of the dynamic control of alternative splicing in response to changes of calcium levels has been largely unappreciated. In this communication, we will summarize the recent advances in calcium signaling-mediated alternative splicing that have provided some insights into the important regulatory mechanisms. In addition to describing the cis-acting RNA elements on the pre-mRNA molecules that respond to changes of intracellular calcium levels, we will summarize how splicing regulators change and affect alternative splicing in this process. We will also discuss a novel mode of calcium-mediated splicing regulation at the level of chromatin structure and transcription.

  5. A novel splicing mutation in the V2 vasopressin receptor

    DEFF Research Database (Denmark)

    Kamperis, Konstantinos; Siggaard, C; Herlin, Troels

    2000-01-01

    In order to elucidate the molecular basis and the clinical characteristics of X-linked recessive nephrogenic diabetes insipidus (CNDI) in a kindred of Danish descent, we performed direct sequencing of the arginine vasopressin receptor 2 (AVPR2) gene in five members of the family, as well as clini......In order to elucidate the molecular basis and the clinical characteristics of X-linked recessive nephrogenic diabetes insipidus (CNDI) in a kindred of Danish descent, we performed direct sequencing of the arginine vasopressin receptor 2 (AVPR2) gene in five members of the family, as well...

  6. Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids

    DEFF Research Database (Denmark)

    Lonkar, Pallavi; Kim, Ki-Hyun; Kuan, Jean Y

    2009-01-01

    Beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures...... hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta......-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair...

  7. 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 ...

  8. RAGE splicing variants in mammals.

    Science.gov (United States)

    Sterenczak, Katharina Anna; Nolte, Ingo; Murua Escobar, Hugo

    2013-01-01

    The receptor for advanced glycation end products (RAGE) is a multiligand receptor of environmental stressors which plays key roles in pathophysiological processes, including immune/inflammatory disorders, Alzheimer's disease, diabetic arteriosclerosis, tumorigenesis, and metastasis. Besides the full-length RAGE protein in humans nearly 20 natural occurring RAGE splicing variants were described on mRNA and protein level. These naturally occurring isoforms are characterized by either N-terminally or C-terminally truncations and are discussed as possible regulators of the full-length RAGE receptor either by competitive ligand binding or by displacing the full-length protein in the membrane. Accordingly, expression deregulations of the naturally occurring isoforms were supposed to have significant effect on RAGE-mediated disorders. Thereby the soluble C-truncated RAGE isoforms present in plasma and tissues are the mostly focused isoforms in research and clinics. Deregulations of the circulating levels of soluble RAGE forms were reported in several RAGE-associated pathological disorders including for example atherosclerosis, diabetes, renal failure, Alzheimer's disease, and several cancer types. Regarding other mammalian species, the canine RAGE gene showed high similarities to the corresponding human structures indicating RAGE to be evolutionary highly conserved between both species. Similar to humans the canine RAGE showed a complex and extensive splicing activity leading to a manifold pattern of RAGE isoforms. Due to the similarities seen in several canine and human diseases-including cancer-comparative structural and functional analyses allow the development of RAGE and ligand-specific therapeutic approaches beneficial for human and veterinary medicine.

  9. Splicing pattern - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us ASTRA Splicing pattern Data detail Data name Splicing pattern DOI 10.18908/lsdba.nbdc00371-0...04 Description of data contents The patterns of alternative splicing/transcriptional initiation Data file Fi...le name: astra_splicing_pattern.zip File URL: ftp://ftp.biosciencedbc.jp/archive/astra/LATEST/astra_splicing_patte...ogodb/view/astra_splicing_pattern#en Data acquisition method For the five organisms (H. sapiens, M. musculus...apping data into bit arrays, detection of splicing patterns and distribution to t

  10. Divergent mitochondrial and endoplasmic reticulum association of DMPK splice isoforms depends on unique sequence arrangements in tail anchors.

    NARCIS (Netherlands)

    Herpen, R.E.M.A. van; Oude Ophuis, R.J.A.; Wijers-Rouw, M.J.P.; Bennink, M.B.; Loo, F.A.J. van de; Fransen, J.; Wieringa, B.; Wansink, D.G.

    2005-01-01

    Myotonic dystrophy protein kinase (DMPK) is a Ser/Thr-type protein kinase with unknown function, originally identified as the product of the gene that is mutated by triplet repeat expansion in patients with myotonic dystrophy type 1 (DM1). Alternative splicing of DMPK transcripts results in multiple

  11. Regular languages, regular grammars and automata in splicing systems

    Science.gov (United States)

    Mohamad Jan, Nurhidaya; Fong, Wan Heng; Sarmin, Nor Haniza

    2013-04-01

    Splicing system is known as a mathematical model that initiates the connection between the study of DNA molecules and formal language theory. In splicing systems, languages called splicing languages refer to the set of double-stranded DNA molecules that may arise from an initial set of DNA molecules in the presence of restriction enzymes and ligase. In this paper, some splicing languages resulted from their respective splicing systems are shown. Since all splicing languages are regular, languages which result from the splicing systems can be further investigated using grammars and automata in the field of formal language theory. The splicing language can be written in the form of regular languages generated by grammar. Besides that, splicing systems can be accepted by automata. In this research, two restriction enzymes are used in splicing systems namely BfuCI and NcoI.

  12. Causes of iron overload in blood donors - a clinical study

    DEFF Research Database (Denmark)

    Laursen, A H; Bjerrum, O W; Friis-Hansen, L

    2018-01-01

    of hyperferritinaemia in the blood donor population and explore the value of extensive HH mutational analyses. MATERIALS AND METHODS: Forty-nine consecutive donors (f = 6, m = 43) were included prospectively from the Capital Regional Blood Center. Inclusion criteria were a single ferritin value >1000 μg/l or repeated......BACKGROUND AND OBJECTIVES: Despite the obligate iron loss from blood donation, some donors present with hyperferritinaemia that can result from a wide range of acute and chronic conditions including hereditary haemochromatosis (HH). The objective of our study was to investigate the causes...... four donors had apparent alternative causes of hyperferritinaemia. CONCLUSION: HH-related mutations were the most frequent cause of hyperferritinaemia in a Danish blood donor population, and it appears that several different HH-genotypes can contribute to hyperferritinaemia. HH screening in blood...

  13. Computational Recognition of RNA Splice Sites by Exact Algorithms for the Quadratic Traveling Salesman Problem

    Directory of Open Access Journals (Sweden)

    Anja Fischer

    2015-06-01

    Full Text Available One fundamental problem of bioinformatics is the computational recognition of DNA and RNA binding sites. Given a set of short DNA or RNA sequences of equal length such as transcription factor binding sites or RNA splice sites, the task is to learn a pattern from this set that allows the recognition of similar sites in another set of DNA or RNA sequences. Permuted Markov (PM models and permuted variable length Markov (PVLM models are two powerful models for this task, but the problem of finding an optimal PM model or PVLM model is NP-hard. While the problem of finding an optimal PM model or PVLM model of order one is equivalent to the traveling salesman problem (TSP, the problem of finding an optimal PM model or PVLM model of order two is equivalent to the quadratic TSP (QTSP. Several exact algorithms exist for solving the QTSP, but it is unclear if these algorithms are capable of solving QTSP instances resulting from RNA splice sites of at least 150 base pairs in a reasonable time frame. Here, we investigate the performance of three exact algorithms for solving the QTSP for ten datasets of splice acceptor sites and splice donor sites of five different species and find that one of these algorithms is capable of solving QTSP instances of up to 200 base pairs with a running time of less than two days.

  14. Splicing defects in ABCD1 gene leading to both exon skipping and partial intron retention in X-linked adrenoleukodystrophy Tunisian patient.

    Science.gov (United States)

    Kallabi, Fakhri; Hadj Salem, Ikhlass; Ben Chehida, Amel; Ben Salah, Ghada; Ben Turkia, Hadhami; Tebib, Neji; Keskes, Leila; Kamoun, Hassen

    2015-08-01

    X-linked adrenoleukodystrophy (X-ALD) affects the nervous system white matter and adrenal cortex secondary to mutations in the ABCD1 gene that encodes a peroxisomal membrane protein: the adrenoleukodystrophy protein. The disease is characterized by high concentrations of very long-chain fatty acids in plasma, adrenal, testicular and nervous tissues. Various types of mutations have been identified in the ABCD1 gene: point mutations, insertions, and deletions. To date, more than 40 point mutations have been reported at the splice junctions of the ABCD1 gene; only few functional studies have been performed to explore these types of mutations. In this study, we have identified de novo splice site mutation c.1780+2T>G in ABCD1 gene in an X-ALD Tunisian patient. Sequencing analysis of cDNA showed a minor transcript lacking exon 7 and a major transcript with a partial intron 7 retention due to activation of a new intronic cryptic splice site. Both outcomes lead to frameshifts with premature stop codon generation in exon 8 and intron 7 respectively. To the best of our knowledge, the current study demonstrates that a single splicing mutation affects the ABCD1 transcripts and the ALDP protein function. Copyright © 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

  15. Structural basis for regulation of GPR56/ADGRG1 by its alternatively spliced extracellular domains

    OpenAIRE

    Salzman, Gabriel S.; Ackerman, Sarah D.; Ding, Chen; Koide, Akiko; Leon, Katherine; Luo, Rong; Stoveken, Hannah M.; Fernandez, Celia G.; Tall, Gregory G.; Piao, Xianhua; Monk, Kelly R.; Koide, Shohei; Araç, Demet

    2016-01-01

    Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. H...

  16. Protein splicing and its evolution in eukaryotes

    Directory of Open Access Journals (Sweden)

    Starokadomskyy P. L.

    2010-02-01

    Full Text Available Inteins, or protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins being spliced together. This process was called protein splicing. Originally inteins were found in prokaryotic or unicellular eukaryotic organisms. But the general principles of post-translation protein rearrangement are evolving yielding different post-translation modification of proteins in multicellular organisms. For clarity, these non-intein mediated events call either protein rearrangements or protein editing. The most intriguing example of protein editing is proteasome-mediated splicing of antigens in vertebrates that may play important role in antigen presentation. Other examples of protein rearrangements are maturation of Hg-proteins (critical receptors in embryogenesis as well as maturation of several metabolic enzymes. Despite a lack of experimental data we try to analyze some intriguing examples of protein splicing evolution.

  17. HOLLYWOOD: a comparative relational database of alternative splicing.

    Science.gov (United States)

    Holste, Dirk; Huo, George; Tung, Vivian; Burge, Christopher B

    2006-01-01

    RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at http://hollywood.mit.edu.

  18. Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Li, Long; Pintel, David J., E-mail: pinteld@missouri.edu

    2012-04-25

    Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.

  19. Characterization of cryptic splicing in germline PTEN intronic variants in Cowden syndrome.

    Science.gov (United States)

    Chen, Hannah Jinlian; Romigh, Todd; Sesock, Kaitlin; Eng, Charis

    2017-10-01

    Germline mutations in the tumor-suppressor gene PTEN predispose to subsets of Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome, and autism. Evidence-based classification of PTEN variants as either deleterious or benign is urgently needed for accurate molecular diagnosis and gene-informed genetic counseling. We studied 34 different germline PTEN intronic variants from 61 CS patients, characterized their PTEN mRNA processing, and analyzed PTEN expression and downstream readouts of P-AKT and P-ERK1/2. While we found that many mutations near splice junctions result in exon skipping, we also identified the presence of cryptic splicing that resulted in premature termination or a shift in isoform usage. PTEN protein expression is significantly lower in the group with splicing changes while P-AKT, but not P-ERK1/2, is significantly increased. Our observations of these PTEN intronic variants should contribute to the determination of pathogenicity of PTEN intronic variants and aid in genetic counseling. © 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.

  20. Nyretransplantation med levende donor

    DEFF Research Database (Denmark)

    Kamper, A L; Løkkegaard, H; Rasmussen, F

    2000-01-01

    In recent years transplantation from living donors has accounted for 25-30% of all kidney transplants in Denmark corresponding to 40-45 per year. Most of these living donors are parents or siblings, although internationally an increasing number are unrelated donors. Donor nephrectomy is associate...... in cadaver transplantation. The ethical and psychological aspects related to transplantation from a living donor are complex and need to be carefully evaluated when this treatment is offered to the patients.......In recent years transplantation from living donors has accounted for 25-30% of all kidney transplants in Denmark corresponding to 40-45 per year. Most of these living donors are parents or siblings, although internationally an increasing number are unrelated donors. Donor nephrectomy is associated...... with only few complications. The long-term outcome for kidney donors is good without increase in mortality or risk for development of hypertension and renal failure; proteinuria may be seen. Living kidney transplantation is the optimal treatment of end-stage renal disease with better graft survival than...

  1. Murine muscular dystrophy caused by a mutation in the laminin alpha 2 (Lama2) gene

    DEFF Research Database (Denmark)

    Xu, H; Wu, X R; Wewer, U M

    1994-01-01

    The classic murine muscular dystrophy strain, dy, was first described almost 40 years ago. We have identified the molecular basis of an allele of dy, called dy2J, by detecting a mutation in the laminin alpha 2 chain gene--the first identified mutation in laminin-2. The G to A mutation in a splice...

  2. Mechanism of alternative splicing and its regulation.

    Science.gov (United States)

    Wang, Yan; Liu, Jing; Huang, B O; Xu, Yan-Mei; Li, Jing; Huang, Lin-Feng; Lin, Jin; Zhang, Jing; Min, Qing-Hua; Yang, Wei-Ming; Wang, Xiao-Zhong

    2015-03-01

    Alternative splicing of precursor mRNA is an essential mechanism to increase the complexity of gene expression, and it plays an important role in cellular differentiation and organism development. Regulation of alternative splicing is a complicated process in which numerous interacting components are at work, including cis-acting elements and trans-acting factors, and is further guided by the functional coupling between transcription and splicing. Additional molecular features, such as chromatin structure, RNA structure and alternative transcription initiation or alternative transcription termination, collaborate with these basic components to generate the protein diversity due to alternative splicing. All these factors contributing to this one fundamental biological process add up to a mechanism that is critical to the proper functioning of cells. Any corruption of the process may lead to disruption of normal cellular function and the eventuality of disease. Cancer is one of those diseases, where alternative splicing may be the basis for the identification of novel diagnostic and prognostic biomarkers, as well as new strategies for therapy. Thus, an in-depth understanding of alternative splicing regulation has the potential not only to elucidate fundamental biological principles, but to provide solutions for various diseases.

  3. Thermopriming Triggers Splicing Memory in Arabidopsis

    KAUST Repository

    Ling, Yu

    2018-02-20

    Abiotic and biotic stresses limit crop productivity. Exposure to a non-lethal stress, referred to as priming, can allow plants to survive subsequent and otherwise lethal conditions; the priming effect persists even after a prolonged stress-free period. However, the molecular mechanisms underlying priming are not fully understood. Here, we investigated the molecular basis of heat shock memory and the role of priming in Arabidopsisthaliana. Comprehensive analysis of transcriptome-wide changes in gene expression and alternative splicing in primed and non-primed plants revealed that alternative splicing functions as a novel component of heat shock memory. We show that priming of plants with a non-lethal heat stress results in de-repression of splicing after a second exposure to heat stress. By contrast, non-primed plants showed significant repression of splicing. These observations link ‘splicing memory’ to the ability of plants to survive subsequent and otherwise lethal heat stress. This newly discovered priming-induced splicing memory may represent a general feature of heat stress responses in plants and other organisms as many of the key components of heat shock responses are conserved among eukaryotes. Furthermore, this finding could facilitate the development of novel approaches to improve plant survival under extreme heat stress.

  4. Novel Alternative Splice Variants of Mouse Cdk5rap2.

    Directory of Open Access Journals (Sweden)

    Nadine Kraemer

    Full Text Available Autosomal recessive primary microcephaly (MCPH is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

  5. An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA.

    Science.gov (United States)

    Sirand-Pugnet, P; Durosay, P; Brody, E; Marie, J

    1995-09-11

    Computer analysis of human intron sequences have revealed a 50 nucleotide (nt) GC-rich region downstream of the 5' splice site; the trinucleotide GGG occurs almost four times as frequently as it would in a random sequence. The 5' part of a beta-tropomyosin intron exhibits six repetitions of the motif (A/U)GGG. In order to test whether these motifs play a role in the splicing process we have mutated some or all of them. Mutated RNAs show a lower in vitro splicing efficiency when compared with the wild-type, especially when all six motifs are mutated (> 70% inhibition). Assembly of the spliceosome complex B and, to a lesser extent, of the pre-spliceosome complex A also appears to be strongly affected by this mutation. A 55 kDa protein within HeLa cell nuclear extract is efficiently cross-linked to the G-rich region. This protein is present in the splicing complexes and its cross-linking to the pre-mRNA requires the presence of one or several snRNP. Altogether our results suggest that the G-rich sequences present in the 5' part of introns may act as an enhancer of the splicing reaction at the level of spliceosome assembly.

  6. Temporal and tissue specific regulation of RP-associated splicing factor genes PRPF3, PRPF31 and PRPC8--implications in the pathogenesis of RP.

    Directory of Open Access Journals (Sweden)

    Huibi Cao

    2011-01-01

    Full Text Available Genetic mutations in several ubiquitously expressed RNA splicing genes such as PRPF3, PRP31 and PRPC8, have been found to cause retina-specific diseases in humans. To understand this intriguing phenomenon, most studies have been focused on testing two major hypotheses. One hypothesis assumes that these mutations interrupt retina-specific interactions that are important for RNA splicing, implying that there are specific components in the retina interacting with these splicing factors. The second hypothesis suggests that these mutations have only a mild effect on the protein function and thus affect only the metabolically highly active cells such as retinal photoreceptors.We examined the second hypothesis using the PRPF3 gene as an example. We analyzed the spatial and temporal expression of the PRPF3 gene in mice and found that it is highly expressed in retinal cells relative to other tissues and its expression is developmentally regulated. In addition, we also found that PRP31 and PRPC8 as well as snRNAs are highly expressed in retinal cells.Our data suggest that the retina requires a relatively high level of RNA splicing activity for optimal tissue-specific physiological function. Because the RP18 mutation has neither a debilitating nor acute effect on protein function, we suggest that retinal degeneration is the accumulative effect of decades of suboptimal RNA splicing due to the mildly impaired protein.

  7. Laparoscopic donor nephrectomy

    Directory of Open Access Journals (Sweden)

    Gupta Nitin

    2005-01-01

    Full Text Available Of the various options for patients with end stage renal disease, kidney transplantation is the treatment of choice for a suitable patient. The kidney for transplantation is retrieved from either a cadaver or a live donor. Living donor nephrectomy has been developed as a method to address the shortfall in cadaveric kidneys available for transplantation. Laparoscopic living donor nephrectomy (LLDN, by reducing postoperative pain, shortening convalescence, and improving the cosmetic outcome of the donor nephrectomy, has shown the potential to increase the number of living kidney donations further by removing some of the disincentives inherent to donation itself. The technique of LLDN has undergone evolution at different transplant centers and many modifications have been done to improve donor safety and recipient outcome. Virtually all donors eligible for an open surgical procedure may also undergo the laparoscopic operation. Various earlier contraindications to LDN, such as right donor kidney, multiple vessels, anomalous vasculature and obesity have been overcome with increasing experience. Laparoscopic live donor nephrectomy can be done transperitoneally or retroperitoneally on either side. The approach is most commonly transperitoneal, which allows adequate working space and easy dissection. A review of literature and our experience with regards to standard approach and the modifications is presented including a cost saving model for the developing countries. An assessment has been made, of the impact of LDN on the outcome of donor and the recipient.

  8. Mismatched single stranded antisense oligonucleotides can induce efficient dystrophin splice switching

    Directory of Open Access Journals (Sweden)

    Kole Ryszard

    2011-10-01

    Full Text Available Abstract Background Antisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds. Methods Here we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25. Results It was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer. Conclusion This raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.

  9. BRCA1 Exon 11, a CERES (Composite Regulatory Element of Splicing Element Involved in Splice Regulation

    Directory of Open Access Journals (Sweden)

    Claudia Tammaro

    2014-07-01

    Full Text Available Unclassified variants (UV of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a “silent” change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES.

  10. Vitamin D and alternative splicing of RNA.

    Science.gov (United States)

    Zhou, Rui; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Xu, Jianzhong; Adams, John S; Hewison, Martin

    2015-04-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

    Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

  12. Alternative splicing of the cardiac sodium channel creates multiple variants of mutant T1620K channels.

    Directory of Open Access Journals (Sweden)

    Stefan Walzik

    2011-04-01

    Full Text Available Alternative splicing creates several Na(v1.5 transcripts in the mammalian myocardium and in various other tissues including brain, dorsal root ganglia, breast cancer cells as well as neuronal stem cell lines. In total nine Na(v1.5 splice variants have been discovered. Four of them, namely Na(v1.5a, Na(v1.5c, Na(v1.5d, and Na(v1.5e, generate functional channels in heterologous expression systems. The significance of alternatively spliced transcripts for cardiac excitation, in particular their role in SCN5A channelopathies, is less well understood. In the present study, we systematically investigated electrophysiological properties of mutant T1620K channels in the background of all known functional Na(v1.5 splice variants in HEK293 cells. This mutation has been previously associated with two distinct cardiac excitation disorders: with long QT syndrome type 3 (LQT3 and isolated cardiac conduction disease (CCD. When investigating the effect of the T1620K mutation, we noticed similar channel defects in the background of hNa(v1.5, hNa(v1.5a, and hNa(v1.5c. In contrast, the hNa(v1.5d background produced differential effects: In the mutant channel, some gain-of-function features did not emerge, whereas loss-of-function became more pronounced. In case of hNa(v1.5e, the neonatal variant of hNa(v1.5, both the splice variant itself as well as the corresponding mutant channel showed electrophysiological properties that were distinct from the wild-type and mutant reference channels, hNa(v1.5 and T1620K, respectively. In conclusion, our data show that alternative splicing is a mechanism capable of generating a variety of functionally distinct wild-type and mutant hNa(v1.5 channels. Thus, the cellular splicing machinery is a potential player affecting genotype-phenotype correlations in SCN5A channelopathies.

  13. Exome sequencing identifies mutations in the gene TTC7A in French-Canadian cases with hereditary multiple intestinal atresia.

    Science.gov (United States)

    Samuels, Mark E; Majewski, Jacek; Alirezaie, Najmeh; Fernandez, Isabel; Casals, Ferran; Patey, Natalie; Decaluwe, Hélène; Gosselin, Isabelle; Haddad, Elie; Hodgkinson, Alan; Idaghdour, Youssef; Marchand, Valerie; Michaud, Jacques L; Rodrigue, Marc-André; Desjardins, Sylvie; Dubois, Stéphane; Le Deist, Francoise; Awadalla, Philip; Raymond, Vincent; Maranda, Bruno

    2013-05-01

    Congenital multiple intestinal atresia (MIA) is a severe, fatal neonatal disorder, involving the occurrence of obstructions in the small and large intestines ultimately leading to organ failure. Surgical interventions are palliative but do not provide long-term survival. Severe immunodeficiency may be associated with the phenotype. A genetic basis for MIA is likely. We had previously ascertained a cohort of patients of French-Canadian origin, most of whom were deceased as infants or in utero. The goal of the study was to identify the molecular basis for the disease in the patients of this cohort. We performed whole exome sequencing on samples from five patients of four families. Validation of mutations and familial segregation was performed using standard Sanger sequencing in these and three additional families with deceased cases. Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing. Five patients from four different families were each homozygous for a four base intronic deletion in the gene TTC7A, immediately adjacent to a consensus GT splice donor site. The deletion was demonstrated to have deleterious effects on splicing causing the skipping of the attendant upstream coding exon, thereby leading to a predicted severe protein truncation. Parents were heterozygous carriers of the deletion in these families and in two additional families segregating affected cases. In a seventh family, an affected case was compound heterozygous for the same 4bp deletion and a second missense mutation p.L823P, also predicted as pathogenic. No other sequenced genes possessed deleterious variants explanatory for all patients in the cohort. Neither mutation was seen in a large set of control chromosomes. Based on our genetic results, TTC7A is the likely causal gene for MIA.

  14. Homologous SV40 RNA trans-splicing: Special case or prime example of viral RNA trans-splicing?

    Directory of Open Access Journals (Sweden)

    Sushmita Poddar

    2014-06-01

    Full Text Available To date the Simian Virus 40 (SV40 is the only proven example of a virus that recruits the mechanism of RNA trans-splicing to diversify its sequences and gene products. Thereby, two identical viral transcripts are efficiently joined by homologous trans-splicing triggering the formation of a highly transforming 100 kDa super T antigen. Sequences of other viruses including HIV-1 and the human adenovirus type 5 were reported to be involved in heterologous trans-splicing towards cellular or viral sequences but the meaning of these events remains unclear. We computationally and experimentally investigated molecular features associated with viral RNA trans-splicing and identified a common pattern: Viral RNA trans-splicing occurs between strong cryptic or regular viral splice sites and strong regular or cryptic splice sites of the trans-splice partner sequences. The majority of these splice sites are supported by exonic splice enhancers. Splice sites that could compete with the trans-splicing sites for cis-splice reactions are weaker or inexistent. Finally, all but one of the trans-splice reactions seem to be facilitated by one or more complementary binding domains of 11 to 16 nucleotides in length which, however occur with a statistical probability close to one for the given length of the involved sequences. The chimeric RNAs generated via heterologous viral RNA trans-splicing either did not lead to fusion proteins or led to proteins of unknown function. Our data suggest that distinct viral RNAs are highly susceptible to trans-splicing and that heterologous viral trans-splicing, unlike homologous SV40 trans-splicing, represents a chance event.

  15. Identification and Functional Characterization of Two Intronic NIPBL Mutations in Two Patients with Cornelia de Lange Syndrome

    Directory of Open Access Journals (Sweden)

    María E. Teresa-Rodrigo

    2016-01-01

    Full Text Available Cornelia de Lange syndrome (CdLS is a rare genetically heterogeneous disorder with a high phenotypic variability including mental retardation, developmental delay, and limb malformations. The genetic causes in about 30% of patients with CdLS are still unknown. We report on the functional characterization of two intronic NIPBL mutations in two patients with CdLS that do not affect a conserved splice-donor or acceptor site. Interestingly, mRNA analyses showed aberrantly spliced transcripts missing exon 28 or 37, suggesting the loss of the branch site by the c.5329-15A>G transition and a disruption of the polypyrimidine by the c.6344del(-13_(-8 deletion. While the loss of exon 28 retains the reading frame of the NIBPL transcript resulting in a shortened protein, the loss of exon 37 shifts the reading frame with the consequence of a premature stop of translation. Subsequent quantitative PCR analysis demonstrated a 30% decrease of the total NIPBL mRNA levels associated with the frameshift transcript. Consistent with our results, this patient shows a more severe phenotype compared to the patient with the aberrant transcript that retains its reading frame. Thus, intronic variants identified by sequencing analysis in CdLS diagnostics should carefully be examined before excluding them as nonrelevant to disease.

  16. Identification and Functional Characterization of Two Intronic NIPBL Mutations in Two Patients with Cornelia de Lange Syndrome.

    Science.gov (United States)

    Teresa-Rodrigo, María E; Eckhold, Juliane; Puisac, Beatriz; Pozojevic, Jelena; Parenti, Ilaria; Baquero-Montoya, Carolina; Gil-Rodríguez, María C; Braunholz, Diana; Dalski, Andreas; Hernández-Marcos, María; Ayerza, Ariadna; Bernal, María L; Ramos, Feliciano J; Wieczorek, Dagmar; Gillessen-Kaesbach, Gabriele; Pié, Juan; Kaiser, Frank J

    2016-01-01

    Cornelia de Lange syndrome (CdLS) is a rare genetically heterogeneous disorder with a high phenotypic variability including mental retardation, developmental delay, and limb malformations. The genetic causes in about 30% of patients with CdLS are still unknown. We report on the functional characterization of two intronic NIPBL mutations in two patients with CdLS that do not affect a conserved splice-donor or acceptor site. Interestingly, mRNA analyses showed aberrantly spliced transcripts missing exon 28 or 37, suggesting the loss of the branch site by the c.5329-15A>G transition and a disruption of the polypyrimidine by the c.6344del(-13)_(-8) deletion. While the loss of exon 28 retains the reading frame of the NIBPL transcript resulting in a shortened protein, the loss of exon 37 shifts the reading frame with the consequence of a premature stop of translation. Subsequent quantitative PCR analysis demonstrated a 30% decrease of the total NIPBL mRNA levels associated with the frameshift transcript. Consistent with our results, this patient shows a more severe phenotype compared to the patient with the aberrant transcript that retains its reading frame. Thus, intronic variants identified by sequencing analysis in CdLS diagnostics should carefully be examined before excluding them as nonrelevant to disease.

  17. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Yi, E-mail: yihooyi@gmail.com; Ericsson, Ida, E-mail: ida.ericsson@ntnu.no; Doseth, Berit, E-mail: berit.doseth@ntnu.no; Liabakk, Nina B., E-mail: nina.beate.liabakk@ntnu.no; Krokan, Hans E., E-mail: hans.krokan@ntnu.no; Kavli, Bodil, E-mail: bodil.kavli@ntnu.no

    2014-03-10

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation.

  18. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    International Nuclear Information System (INIS)

    Hu, Yi; Ericsson, Ida; Doseth, Berit; Liabakk, Nina B.; Krokan, Hans E.; Kavli, Bodil

    2014-01-01

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation

  19. Defective splicing, disease and therapy: searching for master checkpoints in exon definition.

    Science.gov (United States)

    Buratti, Emanuele; Baralle, Marco; Baralle, Francisco E

    2006-01-01

    The number of aberrant splicing processes causing human disease is growing exponentially and many recent studies have uncovered some aspects of the unexpectedly complex network of interactions involved in these dysfunctions. As a consequence, our knowledge of the various cis- and trans-acting factors playing a role on both normal and aberrant splicing pathways has been enhanced greatly. However, the resulting information explosion has also uncovered the fact that many splicing systems are not easy to model. In fact we are still unable, with certainty, to predict the outcome of a given genomic variation. Nonetheless, in the midst of all this complexity some hard won lessons have been learned and in this survey we will focus on the importance of the wide sequence context when trying to understand why apparently similar mutations can give rise to different effects. The examples discussed in this summary will highlight the fine 'balance of power' that is often present between all the various regulatory elements that define exon boundaries. In the final part, we shall then discuss possible therapeutic targets and strategies to rescue genetic defects of complex splicing systems.

  20. Human aldehyde dehydrogenase genes: alternatively spliced transcriptional variants and their suggested nomenclature.

    Science.gov (United States)

    Black, William J; Stagos, Dimitrios; Marchitti, Satori A; Nebert, Daniel W; Tipton, Keith F; Bairoch, Amos; Vasiliou, Vasilis

    2009-11-01

    The human aldehyde dehydrogenase (ALDH) gene superfamily consists of 19 genes encoding enzymes critical for NAD(P)-dependent oxidation of endogenous and exogenous aldehydes, including drugs and environmental toxicants. Mutations in ALDH genes are the molecular basis of several disease states (e.g. Sjögren-Larsson syndrome, pyridoxine-dependent seizures, and type II hyperprolinemia) and may contribute to the etiology of complex diseases such as cancer and Alzheimer's disease. The aim of this nomenclature update was to identify splice transcriptional variants principally for the human ALDH genes. Data-mining methods were used to retrieve all human ALDH sequences. Alternatively spliced transcriptional variants were determined based on (i) criteria for sequence integrity and genomic alignment; (ii) evidence of multiple independent cDNA sequences corresponding to a variant sequence; and (iii) if available, empirical evidence of variants from the literature. Alternatively spliced transcriptional variants and their encoded proteins exist for most of the human ALDH genes; however, their function and significance remain to be established. When compared with the human genome, rat and mouse include an additional gene, Aldh1a7, in the ALDH1A subfamily. To avoid confusion when identifying splice variants in various genomes, nomenclature guidelines for the naming of such alternative transcriptional variants and proteins are recommended herein. In addition, a web database (www.aldh.org) has been developed to provide up-to-date information and nomenclature guidelines for the ALDH superfamily.

  1. Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

    Science.gov (United States)

    Hüttner, Clemens; Murauer, Eva M.; Hainzl, Stefan; Kocher, Thomas; Neumayer, Anna; Reichelt, Julia; Bauer, Johann W.; Koller, Ulrich

    2016-01-01

    RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3′ and 5′ RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders. PMID:27669223

  2. Systems of donor transfer

    NARCIS (Netherlands)

    F.T. de Charro (Frank); J.E.M. Akveld (Hans); E. Hessing (Ellen)

    1993-01-01

    textabstractThe development of medical knowledge has resulted in a demand in society for donor organs, but the recruitment of donor organs for transplantation is difficult. This paper aims to provide some general insights into the complex interaction processes involved. A laissez-faire policy, in

  3. Dealing with Donor Anger.

    Science.gov (United States)

    McNamee, Mike

    1995-01-01

    Techniques that reduce donors' resistance to college fund-raising requests, either direct mail or telephone solicitations, are offered. These include: respecting the prospects' concerns about privacy; offering nonintrusive giving options; honesty and clarity of communication; reinforcing donor sense of control; connecting with prospects'…

  4. Genomic organization of Tropomodulins 2 and 4 and unusual intergenic and intraexonic splicing of YL-1 and Tropomodulin 4

    Directory of Open Access Journals (Sweden)

    Zoghbi Huda Y

    2001-10-01

    Full Text Available Abstract Background The tropomodulins (TMODs are a family of proteins that cap the pointed ends of actin filaments. Four TMODs have been identified in humans, with orthologs in mice. Mutations in actin or actin-binding proteins have been found to cause several human diseases, ranging from hypertrophic cardiomyopathy to immunodefiencies such as Wiskott-Aldrich syndrome. We had previously mapped Tropomodulin 2 (TMOD2 to the genomic region containing the gene for amyotrophic lateral sclerosis 5 (ALS5. We determined the genomic structure of Tmod2 in order to better analyze patient DNA for mutations; we also determined the genomic structure of Tropomodulin 4 (TMOD4. Results In this study, we determined the genomic structure of TMOD2 and TMOD4 and found the organization of both genes to be similar. Sequence analysis of TMOD2 revealed no mutations or polymorphisms in ALS5 patients or controls. Interestingly, we discovered that another gene, YL-1, intergenically splices into TMOD4. YL-1 encodes six exons, the last of which is 291 bp from a 5' untranslated exon of TMOD4. We used 5' RACE and RT-PCR from TMOD4 to identify several intergenic RACE products. YL-1 was also found to undergo unconventional splicing using non-canonical splice sites within exons (intraexonic splicing to produce several alternative transcripts. Conclusions The genomic structure of TMOD2 and TMOD4 have been delineated. This should facilitate future mutational analysis of these genes. In addition, intergenic splicing at TMOD4/YL-1 was discovered, demonstrating yet another level of complexity of gene organization and regulation.

  5. Description of a PCR-based technique for DNA splicing and mutagenesis by producing 5' overhangs with run through stop DNA synthesis utilizing Ara-C

    Directory of Open Access Journals (Sweden)

    Silverman Mel

    2005-09-01

    Full Text Available Abstract Background Splicing of DNA molecules is an important task in molecular biology that facilitates cloning, mutagenesis and creation of chimeric genes. Mutagenesis and DNA splicing techniques exist, some requiring restriction enzymes, and others utilize staggered reannealing approaches. Results A method for DNA splicing and mutagenesis without restriction enzymes is described. The method is based on mild template-dependent polymerization arrest with two molecules of cytosine arabinose (Ara-C incorporated into PCR primers. Two rounds of PCR are employed: the first PCR produces 5' overhangs that are utilized for DNA splicing. The second PCR is based on polymerization running through the Ara-C molecules to produce the desired final product. To illustrate application of the run through stop mutagenesis and DNA splicing technique, we have carried out splicing of two segments of the human cofilin 1 gene and introduced a mutational deletion into the product. Conclusion We have demonstrated the utility of a new PCR-based method for carrying out DNA splicing and mutagenesis by incorporating Ara-C into the PCR primers.

  6. Widespread evolutionary conservation of alternatively spliced exons in caenorhabditis

    DEFF Research Database (Denmark)

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

    2007-01-01

    Alternative splicing (AS) contributes to increased transcriptome and proteome diversity in various eukaryotic lineages. Previous studies showed low levels of conservation of alternatively spliced (cassette) exons within mammals and within dipterans. We report a strikingly different pattern in Cae...

  7. The candidate splicing factor Sfswap regulates growth and patterning of inner ear sensory organs.

    Directory of Open Access Journals (Sweden)

    Yalda Moayedi

    2014-01-01

    Full Text Available The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1. We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.

  8. Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11-48K and U11/U12-65K genes.

    Science.gov (United States)

    Niemelä, Elina H; Verbeeren, Jens; Singha, Prosanta; Nurmi, Visa; Frilander, Mikko J

    2015-01-01

    Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11-48K and U11/U12-65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5' splice sites (5'ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25-50 nt downstream from the target 3' splice site (3'ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11-35K protein for 3'ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5'ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5'ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.

  9. Identification of Common Genetic Variation That Modulates Alternative Splicing

    OpenAIRE

    Hull, Jeremy; Campino, Susana; Rowlands, Kate; Chan, Man-Suen; Copley, Richard R; Taylor, Martin S; Rockett, Kirk; Elvidge, Gareth; Keating, Brendan; Knight, Julian; Kwiatkowski, Dominic

    2007-01-01

    Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by nat...

  10. Universal Alternative Splicing of Noncoding Exons

    DEFF Research Database (Denmark)

    Deveson, Ira W; Brunck, Marion E; Blackburn, James

    2018-01-01

    The human transcriptome is so large, diverse, and dynamic that, even after a decade of investigation by RNA sequencing (RNA-seq), we have yet to resolve its true dimensions. RNA-seq suffers from an expression-dependent bias that impedes characterization of low-abundance transcripts. We performed......, indicative of regulation by a deeply conserved splicing code. We propose that noncoding exons are functionally modular, with alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution....

  11. SPA: a probabilistic algorithm for spliced alignment.

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because they use ad hoc scoring models that cannot correctly trade off the likelihoods of various sequencing errors against the probabilities of different gene structures. Here we develop a Bayesian probabilistic approach to cDNA-to-genome alignment. Gene structures are assigned prior probabilities based on the lengths of their introns and exons, and based on the sequences at their splice boundaries. A likelihood model for sequencing errors takes into account the rates at which misincorporation, as well as insertions and deletions of different lengths, occurs during sequencing. The parameters of both the prior and likelihood model can be automatically estimated from a set of cDNAs, thus enabling our method to adapt itself to different organisms and experimental procedures. We implemented our method in a fast cDNA-to-genome alignment program, SPA, and applied it to the FANTOM3 dataset of over 100,000 full-length mouse cDNAs and a dataset of over 20,000 full-length human cDNAs. Comparison with the results of four other mapping programs shows that SPA produces alignments of significantly higher quality. In particular, the quality of the SPA alignments near splice boundaries and SPA's mapping of the 5' and 3' ends of the cDNAs are highly improved, allowing for more accurate identification of transcript starts and ends, and accurate identification of subtle splice variations. Finally, our splice boundary analysis on the human dataset suggests the existence of a novel non

  12. Marginal kidney donor

    Directory of Open Access Journals (Sweden)

    Ganesh Gopalakrishnan

    2007-01-01

    Full Text Available Renal transplantation is the treatment of choice for a medically eligible patient with end stage renal disease. The number of renal transplants has increased rapidly over the last two decades. However, the demand for organs has increased even more. This disparity between the availability of organs and waitlisted patients for transplants has forced many transplant centers across the world to use marginal kidneys and donors. We performed a Medline search to establish the current status of marginal kidney donors in the world. Transplant programs using marginal deceased renal grafts is well established. The focus is now on efforts to improve their results. Utilization of non-heart-beating donors is still in a plateau phase and comprises a minor percentage of deceased donations. The main concern is primary non-function of the renal graft apart from legal and ethical issues. Transplants with living donors outnumbered cadaveric transplants at many centers in the last decade. There has been an increased use of marginal living kidney donors with some acceptable medical risks. Our primary concern is the safety of the living donor. There is not enough scientific data available to quantify the risks involved for such donation. The definition of marginal living donor is still not clear and there are no uniform recommendations. The decision must be tailored to each donor who in turn should be actively involved at all levels of the decision-making process. In the current circumstances, our responsibility is very crucial in making decisions for either accepting or rejecting a marginal living donor.

  13. Influence of donor age on induced pluripotent stem cells.

    Science.gov (United States)

    Lo Sardo, Valentina; Ferguson, William; Erikson, Galina A; Topol, Eric J; Baldwin, Kristin K; Torkamani, Ali

    2017-01-01

    Induced pluripotent stem cells (iPSCs) are being pursued as a source of cells for autologous therapies, many of which will be aimed at aged patients. To explore the impact of age on iPSC quality, we produced iPSCs from blood cells of 16 donors aged 21-100. We find that iPSCs from older donors retain an epigenetic signature of age, which can be reduced through passaging. Clonal expansion via reprogramming also enables the discovery of somatic mutations present in individual donor cells, which are missed by bulk sequencing methods. We show that exomic mutations in iPSCs increase linearly with age, and all iPSC lines analyzed carry at least one gene-disrupting mutation, several of which have been associated with cancer or dysfunction. Unexpectedly, elderly donors (>90 yrs) harbor fewer mutations than predicted, likely due to a contracted blood progenitor pool. These studies establish that donor age is associated with an increased risk of abnormalities in iPSCs and will inform clinical development of reprogramming technology.

  14. Abnormalities in alternative splicing of angiogenesis-related genes and their role in HIV-related cancers

    Directory of Open Access Journals (Sweden)

    Mthembu NN

    2017-03-01

    Full Text Available Nonkululeko N Mthembu,1 Zukile Mbita,2 Rodney Hull,1 Zodwa Dlamini1 1Research, Innovation and Engagements, Mangosuthu University of Technology, Durban, 2Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, Sovenga, South Africa Abstract: Alternative splicing of mRNA leads to an increase in proteome biodiversity by allowing the generation of multiple mRNAs, coding for multiple protein isoforms of various structural and functional properties from a single primary pre-mRNA transcript. The protein isoforms produced are tightly regulated in normal development but are mostly deregulated in various cancers. In HIV-infected individuals with AIDS, there is an increase in aberrant alternative splicing, resulting in an increase in HIV/AIDS-related cancers, such as Kaposi’s sarcoma, non-Hodgkin’s lymphoma, and cervical cancer. This aberrant splicing leads to abnormal production of protein and is caused by mutations in cis-acting elements or trans-acting factors in angiogenesis-related genes. Restoring the normal regulation of alternative splicing of angiogenic genes would alter the expression of protein isoforms and may confer normal cell physiology in patients with these cancers. This review highlights the abnormalities in alternative splicing of angiogenesis-related genes and their implication in HIV/AIDS-related cancers. This allows us to gain an insight into the pathogenesis of HIV/AIDS-related cancer and in turn elucidate the therapeutic potential of alternatively spliced genes in HIV/AIDS-related malignancies. Keywords: vascular endothelial growth factor, oncogenic viruses, hypoxia induced factor 1, Kaposi’s sarcoma, non-Hodgkin’s lymphoma, therapies targeting alternative splicing

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

    Science.gov (United States)

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

    2012-03-31

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

  16. Epigenetic Modification of the Repair Donor Regulates Targeted Gene Correction

    Directory of Open Access Journals (Sweden)

    Olivier Humbert

    2012-01-01

    Full Text Available Optimizing design of vectors is critical to effective gene therapy. In targeted gene correction (TGC, cleavage of chromosomal DNA near a mutation stimulates homology-directed repair of a target gene using a donor provided in trans. We have systematically addressed epigenetic parameters of donor design, using a flow-based assay to quantify correction frequencies and expression levels of a green fluorescent protein (GFP reporter gene in a human cell line. We show that active transcription of the donor increased correction frequency by threefold, establishing that a proximal promoter enhances donor use. Conversely, CpG methylation of the donor diminished correction frequency and reduced expression of the repaired gene. However, bisulfite sequencing of the target revealed no transfer of methylation marks during repair with a methylated donor. Treatment with histone deacetylase (HDAC inhibitors can partially compensate for epigenetic inactivation, suggesting a role for class I and II HDACs in regulation of donor use. These results establish that epigenetic status of a trans-donor determines both the efficiency and outcome of gene correction, and identify and clarify parameters that should guide donor design for targeted gene therapy.

  17. Mutation analysis of Swedish haemophilia B families - high frequency of unique mutations.

    Science.gov (United States)

    Mårtensson, A; Letelier, A; Halldén, C; Ljung, R

    2016-05-01

    Haemophilia B is caused by a heterogeneous spectrum of mutations. Mutation characterization is important in genetic counselling, prenatal diagnosis and to predict risk of inhibitor development. To study the mutation spectrum, frequency of unique recurrent mutations, genotype-phenotype association and inhibitor development in a population-based study of the complete Swedish haemophilia B population. The study included, facilitated by centralized DNA diagnostics, the complete registered Swedish haemophilia B population (113 families: 47 severe, 22 moderate and 44 mild), each represented by a single patient. Mutation characterization was performed by conventional sequencing of all exons and haplotyping by genotyping of single nucleotide variants and microsatellites. A mutation was found in every family: eight had large deletions, three had small deletions (mutations were found and were predicted to be deleterious. Sixteen mutations (one total gene deletion, 14 substitutions and one acceptor splice site) were present in more than one family. Of the single nucleotide mutations (37/102), 36% arose at CpG sites. Haplotyping of families with identical mutations and present analyses showed that the frequency of unique mutations was at least 65%. Inhibitors developed in 9/47 (19%) patients with severe haemophilia B. The spectrum of haemophilia B mutations reveals at least 65% of the families carry a unique mutation, but with more inhibitor patients than reported internationally, probably as a result of many 'null' mutations. © 2015 John Wiley & Sons Ltd.

  18. Two TP53 germline mutations in a classical Li-Fraumeni syndrome family.

    Science.gov (United States)

    van Hest, Liselotte P; Ruijs, Mariëlle W G; Wagner, Anja; van der Meer, Conny A; Verhoef, Senno; van't Veer, Laura J; Meijers-Heijboer, Hanne

    2007-01-01

    Li-Fraumeni syndrome (LFS) is an autosomal dominantly inherited cancer predisposition syndrome characterized by a combination of tumors including sarcoma, breast cancer, brain tumors, adrenocortical carcinoma and leukemia. Germline mutations in the tumor suppressor gene TP53 are associated with LFS. We present a family with LFS in which initially a novel germline TP53 intron 5 splice site mutation was found. A second germline TP53 mutation, the exon 7 Asn235Ser (704A-->G) mutation, was detected in this family through pre-symptomatic DNA testing. This latter mutation has been reported repeatedly in the literature as a pathogenic mutation involved in LFS. We provide evidence for pathogenicity of the novel intron 5 splice site mutation, whereas this evidence is lacking for the exon 7 Asn235Ser (704A-->G) mutation. Our findings emphasize the importance of performing additional tests in case of germline sequence variants with uncertain functional effects.

  19. An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

    Science.gov (United States)

    Philippe, Lucas; Pandarakalam, George C; Fasimoye, Rotimi; Harrison, Neale; Connolly, Bernadette; Pettitt, Jonathan; Müller, Berndt

    2017-08-21

    Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. 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...

  1. Increased frequency of FBN1 truncating and splicing variants in Marfan syndrome patients with aortic events.

    Science.gov (United States)

    Baudhuin, Linnea M; Kotzer, Katrina E; Lagerstedt, Susan A

    2015-03-01

    Marfan syndrome is a systemic disorder that typically involves FBN1 mutations and cardiovascular manifestations. We investigated FBN1 genotype-phenotype correlations with aortic events (aortic dissection and prophylactic aortic surgery) in patients with Marfan syndrome. Genotype and phenotype information from probands (n = 179) with an FBN1 pathogenic or likely pathogenic variant were assessed. A higher frequency of truncating or splicing FBN1 variants was observed in Ghent criteria-positive patients with an aortic event (n = 34) as compared with all other probands (n = 145) without a reported aortic event (79 vs. 39%; P Marfan syndrome patients with FBN1 truncating and splicing variants.Genet Med 17 3, 177-187.

  2. The hnRNP 2H9 gene, which is involved in the splicing reaction, is a multiply spliced gene

    DEFF Research Database (Denmark)

    Honoré, B

    2000-01-01

    The hnRNP 2H9 gene products are involved in the splicing process and participate in early heat shock-induced splicing arrest. By combining low/high stringency hybridisation, database search, Northern and Western blotting it is shown that the gene is alternatively spliced into at least six transcr...

  3. Auxiliary splice factor U2AF26 and transcription factor Gfi1 cooperate directly in regulating CD45 alternative splicing.

    NARCIS (Netherlands)

    Heyd, F.; Dam, G.B. ten; Moroy, T.

    2006-01-01

    By alternative splicing, different isoforms of the transmembrane tyrosine phosphatase CD45 are generated that either enhance or limit T cell receptor signaling. We report here that CD45 alternative splicing is regulated by cooperative action of the splice factor U2AF26 and the transcription factor

  4. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida

    2014-01-01

    of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...

  5. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    Energy Technology Data Exchange (ETDEWEB)

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.

    2013-02-01

    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  6. Survey of gene splicing algorithms based on reads.

    Science.gov (United States)

    Si, Xiuhua; Wang, Qian; Zhang, Lei; Wu, Ruo; Ma, Jiquan

    2017-11-02

    Gene splicing is the process of assembling a large number of unordered short sequence fragments to the original genome sequence as accurately as possible. Several popular splicing algorithms based on reads are reviewed in this article, including reference genome algorithms and de novo splicing algorithms (Greedy-extension, Overlap-Layout-Consensus graph, De Bruijn graph). We also discuss a new splicing method based on the MapReduce strategy and Hadoop. By comparing these algorithms, some conclusions are drawn and some suggestions on gene splicing research are made.

  7. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    by facilitating or hindering the interaction with factors and small nuclear ribonucleoproteins (snRNPs) that regulate splicing. Moreover, the secondary structure could play a fundamental role in the splicing of yeast species, which lack many of the regulatory splicing factors present in metazoans. This chapter......In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either...

  8. Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival.

    Directory of Open Access Journals (Sweden)

    Dennis Liang Fei

    2016-10-01

    Full Text Available We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3' splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3' splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele.

  9. Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing

    DEFF Research Database (Denmark)

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

    2007-01-01

    , and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional...... classes, cellular locations, intron/exon structures and evolutionary origins. RESULTS: For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants) show high levels of alternative splicing. Genes with products...

  10. Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

    Directory of Open Access Journals (Sweden)

    Gomez-Roman Javier

    2010-06-01

    Full Text Available Abstract Background Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. Results The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. Conclusions This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.

  11. HPV-18 E2circumflexE4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Chye Ling [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Gunaratne, Jayantha [Mass Spectrometry and Systems Biology Laboratory, Institute of Molecular and Cell Biology, A-STAR, Biopolis, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore); Lai, Deborah [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Carthagena, Laetitia [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Wang, Qian [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Xue, Yue Zhen; Quek, Ling Shih [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Doorbar, John [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Bachelerie, Francoise [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Thierry, Francoise, E-mail: francoise.thierry@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Bellanger, Sophie, E-mail: sophie.bellanger@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)

    2012-07-20

    The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1circumflexE4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2circumflexE4 transcripts resulting from 2 splice donor sites in the 5 Prime part of E2, while the splice acceptor site is the one used for E1circumflexE4. Both E2circumflexE4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2circumflexE4-S and E2circumflexE4-L. Whereas we could not differentiate E2circumflexE4-S from E1circumflexE4 in vivo, E2circumflexE4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2circumflexE4 products.

  12. Alternative splicing of T cell receptor (TCR) alpha chain transcripts containing V alpha 1 or V alpha 14 elements.

    Science.gov (United States)

    Mahotka, C; Hansen-Hagge, T E; Bartram, C R

    1995-10-01

    Human acute lymphoblastic leukemia cell lines represent valuable tools to investigate distinct steps of the complex regulatory pathways underlying T cell receptor recombination and expression. A case in point are V delta 2D delta 3 and subsequent V delta 2D delta 3J alpha rearrangements observed in human leukemic pre-B cells as well as in normal lymphopoiesis. The functional expression of these unusual (VD) delta (JC) alpha hybrids is almost exclusively prevented by alternative splicing events. In this report we show that alternative splicing at cryptic splice donor sites within V elements is not a unique feature of hybrid TCR delta/alpha transcripts. Among seven V alpha families analyzed by RT-PCR, alternatively spliced products were observed in TCR alpha recombinations containing V alpha 1 or V alpha 14 elements. In contrast to normal peripheral blood cells and thymocytes, the leukemia cell line JM expressing functional V alpha 1J alpha 3C alpha transcripts lacked evidence of aberrant TCR alpha RNA species.

  13. An engineered U1 small nuclear RNA rescues splicing-defective coagulation F7 gene expression in mice

    Science.gov (United States)

    Balestra, D; Faella, A; Margaritis, P; Cavallari, N; Pagani, F; Bernardi, F; Arruda, V R; Pinotti, M

    2014-01-01

    Background The ability of the spliceosomal small nuclear RNA U1 (U1snRNA) to rescue pre-mRNA splicing impaired by mutations makes it an attractive therapeutic molecule. Coagulation factor deficiencies due to splicing mutations are relatively frequent and could therefore benefit from this strategy. However, the effects of U1snRNAs in vivo remain unknown. Objectives To assess the rescue of the F7 c.859+5G>A splicing mutation (FVII+5A), causing severe human factor VII (hFVII) deficiency, by the modified U1snRNA+5a (U1+5a) in a murine model. Methods Mice expressing the human F7 c.859+5G>A mutant were generated following liver-directed expression by plasmid or recombinant adeno-associated viral (AAV) vector administration. The rescue of the splice-site defective pre-mRNA by U1+5a was monitored in liver and plasma through hFVII-specific assays. Results Injection of plasmids encoding the U1+5a rescued plasma hFVII levels, which increased from undetectable to ∼8.5% of those obtained with the wild-type hFVII plasmid control. To assess long-term effects, mice were injected with low and high doses of two AAV vectors encoding the FVII+5A splice site mutant as template to be corrected by U1+5a. This strategy resulted in hFVII plasma levels of 3.9 ± 0.8 or 23.3 ± 5.1 ng mL−1 in a dose-dependent manner, corresponding in patients to circulating FVII levels of ∼1–4.5% of normal. Moreover, in both experimental models, we also detected correctly spliced hFVII transcripts and hFVII-positive cells in liver cells. Conclusions Here we provide the first in vivo proof-of-principle of the rescue of the expression of a splicing-defective F7 mutant by U1snRNAs, thus highlighting their therapeutic potential in coagulation disorders. PMID:24738135

  14. An engineered U1 small nuclear RNA rescues splicing defective coagulation F7 gene expression in mice.

    Science.gov (United States)

    Balestra, D; Faella, A; Margaritis, P; Cavallari, N; Pagani, F; Bernardi, F; Arruda, V R; Pinotti, M

    2014-02-01

    The ability of the spliceosomal small nuclear RNA U1 (U1snRNA) to rescue pre-mRNA splicing impaired by mutations makes it an attractive therapeutic molecule. Coagulation factor deficiencies due to splicing mutations are relatively frequent and could therefore benefit from this strategy. However, the effects of U1snRNAs in vivo remain unknown. To assess the rescue of the F7 c.859+5G>A splicing mutation (FVII+5A), causing severe human factor VII (hFVII) deficiency, by the modified U1snRNA+5a (U1+5a) in a murine model. Mice expressing the human F7 c.859+5G>A mutant were generated following liver-directed expression by plasmid or recombinant adeno-associated viral (AAV) vector administration. The rescue of the splice-site defective pre-mRNA by U1+5a was monitored in liver and plasma through hFVII-specific assays. Injection of plasmids encoding the U1+5a rescued plasma hFVII levels, which increased from undetectable to ~8.5% of those obtained with the wild-type hFVII plasmid control. To assess long-term effects, mice were injected with low and high doses of two AAV vectors encoding the FVII+5A splice site mutant as template to be corrected by U1+5a. This strategy resulted in hFVII plasma levels of 3.9 ± 0.8 or 23.3 ± 5.1 ng mL⁻¹ in a dose-dependent manner, corresponding in patients to circulating FVII levels of ~1-4.5% of normal. Moreover, in both experimental models, we also detected correctly spliced hFVII transcripts and hFVII-positive cells in liver cells. Here we provide the first in vivo proof of-principle of the rescue of the expression of a splicing-defective F7 mutant by U1snRNAs, thus highlighting their therapeutic potential in coagulation disorders.

  15. Independent donor ethical assessment: aiming to standardize donor advocacy.

    Science.gov (United States)

    Choudhury, Devasmita; Jotterand, Fabrice; Casenave, Gerald; Smith-Morris, Carolyn

    2014-06-01

    Living organ donation has become more common across the world. To ensure an informed consent process, given the complex issues involved with organ donation, independent donor advocacy is required. The choice of how donor advocacy is administered is left up to each transplant center. This article presents the experience and process of donor advocacy at University of Texas Southwestern Medical Center administered by a multidisciplinary team consisting of physicians, surgeons, psychologists, medical ethicists and anthropologists, lawyers, a chaplain, a living kidney donor, and a kidney transplant recipient. To ensure that advocacy remains fair and consistent for all donors being considered, the donor advocacy team at University of Texas Southwestern Medical Center developed the Independent Donor Ethical Assessment, a tool that may be useful to others in rendering donor advocacy. In addition, the tool may be modified as circumstances arise to improve donor advocacy and maintain uniformity in decision making.

  16. A mutation in the RIEG1 gene associated with Peters' anomaly

    OpenAIRE

    Doward, W; Perveen, R; Lloyd, I; Ridgway, A; Wilson, L; Black, G

    1999-01-01

    Mutations within the RIEG1 homeobox gene on chromosome 4q25 have previously been reported in association with Rieger syndrome. We report a 3' splice site mutation within the 3rd intron of the RIEG1 gene which is associated with unilateral Peters' anomaly. The mutation is a single base substition of A to T at the invariant -2 site of the 3' splice site. Peters' anomaly, which is characterised by ocular anterior segment dysgenesis and central corneal opacification, is distinct from Rieger anoma...

  17. Novel mutation in ATP-binding domain of ABCD1 gene in ...

    Indian Academy of Sciences (India)

    Introduction. In adrenoleucodystorphy, more than one thousand mutations in ABCD1 gene have been reported from all over the world, of which 50% are unique (Moser and Kemp 1999). Recently, we had reported a splice-site mutation in ABCD1 gene. Here, we report the first case of adolescent cerebral adrenoleu-.

  18. Evolution of Nova-dependent splicing regulation in the brain.

    Directory of Open Access Journals (Sweden)

    Nejc Jelen

    2007-10-01

    Full Text Available A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs.

  19. Mutation Analysis in Classical Phenylketonuria Patients Followed by Detecting Haplotypes Linked to Some PAH Mutations.

    Science.gov (United States)

    Dehghanian, Fatemeh; Silawi, Mohammad; Tabei, Seyed M B

    2017-02-01

    Deficiency of phenylalanine hydroxylase (PAH) enzyme and elevation of phenylalanine in body fluids cause phenylketonuria (PKU). The gold standard for confirming PKU and PAH deficiency is detecting causal mutations by direct sequencing of the coding exons and splicing involved sequences of the PAH gene. Furthermore, haplotype analysis could be considered as an auxiliary approach for detecting PKU causative mutations before direct sequencing of the PAH gene by making comparisons between prior detected mutation linked-haplotypes and new PKU case haplotypes with undetermined mutations. In this study, 13 unrelated classical PKU patients took part in the study detecting causative mutations. Mutations were identified by polymerase chain reaction (PCR) and direct sequencing in all patients. After that, haplotype analysis was performed by studying VNTR and PAHSTR markers (linked genetic markers of the PAH gene) through application of PCR and capillary electrophoresis (CE). Mutation analysis was performed successfully and the detected mutations were as follows: c.782G>A, c.754C>T, c.842C>G, c.113-115delTCT, c.688G>A, and c.696A>G. Additionally, PAHSTR/VNTR haplotypes were detected to discover haplotypes linked to each mutation. Mutation detection is the best approach for confirming PAH enzyme deficiency in PKU patients. Due to the relatively large size of the PAH gene and high cost of the direct sequencing in developing countries, haplotype analysis could be used before DNA sequencing and mutation detection for a faster and cheaper way via identifying probable mutated exons.

  20. Intronic alternative splicing regulators identified by comparative genomics in nematodes.

    Directory of Open Access Journals (Sweden)

    Jennifer L Kabat

    2006-07-01

    Full Text Available Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high-scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (TGCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis

  1. Eight nucleotide substitutions inhibit splicing to HPV-16 3'-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Xiaoze Li

    Full Text Available The most commonly used 3'-splice site on the human papillomavirus type 16 (HPV-16 genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16.

  2. Identification of Splicing Factors Involved in DMD Exon Skipping Events Using an In Vitro RNA Binding Assay.

    Science.gov (United States)

    Miro, Julie; Bourgeois, Cyril F; Claustres, Mireille; Koenig, Michel; Tuffery-Giraud, Sylvie

    2018-01-01

    Mutation-induced exon skipping in the DMD gene can modulate the severity of the phenotype in patients with Duchenne or Becker Muscular Dystrophy. These alternative splicing events are most likely the result of changes in recruitment of splicing factors at cis-acting elements in the mutated DMD pre-mRNA. The identification of proteins involved can be achieved by an affinity purification procedure. Here, we provide a detailed protocol for the in vitro RNA binding assay that we routinely apply to explore molecular mechanisms underlying splicing defects in the DMD gene. In vitro transcribed RNA probes containing either the wild type or mutated sequence are oxidized and bound to adipic acid dihydrazide-agarose beads. Incubation with a nuclear extract allows the binding of nuclear proteins to the RNA probes. The unbound proteins are washed off and then the specifically RNA-bound proteins are released from the beads by an RNase treatment. After separation by SDS-PAGE, proteins that display differential binding affinities for the wild type and mutant RNA probes are identified by mass spectrometry.

  3. Splicing variants of porcine synphilin-1

    DEFF Research Database (Denmark)

    Larsen, Knud Erik; Madsen, Lone Bruhn; Farajzadeh, Leila

    2015-01-01

    %) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel...... splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation....... with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA...

  4. Resolving deconvolution ambiguity in gene alternative splicing

    Directory of Open Access Journals (Sweden)

    Hubbell Earl

    2009-08-01

    Full Text Available Abstract Background For many gene structures it is impossible to resolve intensity data uniquely to establish abundances of splice variants. This was empirically noted by Wang et al. in which it was called a "degeneracy problem". The ambiguity results from an ill-posed problem where additional information is needed in order to obtain an unique answer in splice variant deconvolution. Results In this paper, we analyze the situations under which the problem occurs and perform a rigorous mathematical study which gives necessary and sufficient conditions on how many and what type of constraints are needed to resolve all ambiguity. This analysis is generally applicable to matrix models of splice variants. We explore the proposal that probe sequence information may provide sufficient additional constraints to resolve real-world instances. However, probe behavior cannot be predicted with sufficient accuracy by any existing probe sequence model, and so we present a Bayesian framework for estimating variant abundances by incorporating the prediction uncertainty from the micro-model of probe responsiveness into the macro-model of probe intensities. Conclusion The matrix analysis of constraints provides a tool for detecting real-world instances in which additional constraints may be necessary to resolve splice variants. While purely mathematical constraints can be stated without error, real-world constraints may themselves be poorly resolved. Our Bayesian framework provides a generic solution to the problem of uniquely estimating transcript abundances given additional constraints that themselves may be uncertain, such as regression fit to probe sequence models. We demonstrate the efficacy of it by extensive simulations as well as various biological data.

  5. DNA computing based on splicing: universality results.

    Science.gov (United States)

    Csuhaj-Varjú, E; Freund, R; Kari, L; Păun, G

    1996-01-01

    The paper extends some of the most recently obtained results on the computational universality of specific variants of H systems (e.g. with regular sets of rules) and proves that we can construct universal computers based on various types of H systems with a finite set of splicing rules as well as a finite set of axioms, i.e. we show the theoretical possibility to design programmable universal DNA computers based on the splicing operation. For H systems working in the multiset style (where the numbers of copies of all available strings are counted) we elaborate how a Turing machine computing a partial recursive function can be simulated by an equivalent H system computing the same function; in that way, from a universal Turning machine we obtain a universal H system. Considering H systems as language generating devices we have to add various simple control mechanisms (checking the presence/absence of certain symbols in the spliced strings) to systems with a finite set of splicing rules as well as with a finite set of axioms in order to obtain the full computational power, i.e. to get a characterization of the family of recursively enumerable languages. We also introduce test tube systems, where several H systems work in parallel in their tubes and from time to time the contents of each tube are redistributed to all tubes according to certain separation conditions. By the construction of universal test tube systems we show that also such systems could serve as the theoretical basis for the development of biological (DNA) computers.

  6. Stochastic principles governing alternative splicing of RNA.

    OpenAIRE

    Jianfei Hu; Eli Boritz; William Wylie; Daniel C Douek

    2017-01-01

    Author summary Alternative RNA splicing within eukaryotic cells enables each gene to generate multiple different mature transcripts which further encode proteins with distinct or even opposing functions. The relative frequencies of the transcript isoforms generated by a particular gene are essential to the maintenance of normal cellular physiology; however, the underlying mechanisms and principles that govern these frequencies are unknown. We analyzed the frequency distribution of all transcr...

  7. Polymorphism in the Alternative Donor Site of the Cryptic Exon of LHCGR: Functional Consequences and Associations with Testosterone Level.

    Science.gov (United States)

    Liu, Wei; Han, Bing; Zhu, Wenjiao; Cheng, Tong; Fan, Mengxia; Wu, Jiajun; Yang, Ying; Zhu, Hui; Si, Jiqiang; Lyu, Qifeng; Chai, Weiran; Zhao, Shuangxia; Song, Huaidong; Kuang, Yanping; Qiao, Jie

    2017-04-03

    Selective splicing is a feature of luteinizing hormone receptor (LHCGR). A cryptic exon (LHCGR-exon 6A) was found to be derived from alternative splicing in intron 6 of the LHCGR gene, which including two transcripts LHCGR-exon 6A-long and LHCGR-exon 6A-short. We addressed the functional consequences of SNP rs68073206, located at the +5 position of an alternative 5' splice donor site, and observed its association with male infertility in the subjects with azoospermia, oligoasthenozoospermia and normozoospermia. The translation product of splicing variant LHCGR-exon 6A was expressed in the cytoplasm and exhibited no affinity with [ 125 I]-hCG. No dominant negative effect was observed in cells co-expressed with LHCGR-exon 6A and wild-type LHCGR. The long transcript (LHCGR-exon 6A-long) was significantly elevated in the granulosa cells with G/G genotypes, which could be reproduced in vitro by mini-gene construct transfection. Genotyping analysis showed no association between rs68073206 and male infertility. However, this polymorphism was significantly associated with testosterone levels in normozoospermic subjects (n = 210). In conclusion, SNP rs68073206 in the splicing site of the cryptic exon 6A of the LHCGR gene affect the splicing pattern in the gene, which may play a role in the modulation of the LHCGR sensitivity in the gonads.

  8. Rbfox1 downregulation and altered calpain 3 splicing by FRG1 in a mouse model of Facioscapulohumeral muscular dystrophy (FSHD.

    Directory of Open Access Journals (Sweden)

    Mariaelena Pistoni

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1 in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6- is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.

  9. Alternative splicing regulated by butyrate in bovine epithelial cells.

    Directory of Open Access Journals (Sweden)

    Sitao Wu

    Full Text Available As a signaling molecule and an inhibitor of histone deacetylases (HDACs, butyrate exerts its impact on a broad range of biological processes, such as apoptosis and cell proliferation, in addition to its critical role in energy metabolism in ruminants. This study examined the effect of butyrate on alternative splicing in bovine epithelial cells using RNA-seq technology. Junction reads account for 11.28 and 12.32% of total mapped reads between the butyrate-treated (BT and control (CT groups. 201,326 potential splicing junctions detected were supported by ≥ 3 junction reads. Approximately 94% of these junctions conformed to the consensus sequence (GT/AG while ~3% were GC/AG junctions. No AT/AC junctions were observed. A total of 2,834 exon skipping events, supported by a minimum of 3 junction reads, were detected. At least 7 genes, their mRNA expression significantly affected by butyrate, also had exon skipping events differentially regulated by butyrate. Furthermore, COL5A3, which was induced 310-fold by butyrate (FDR <0.001 at the gene level, had a significantly higher number of junction reads mapped to Exon#8 (Donor and Exon#11 (Acceptor in BT. This event had the potential to result in the formation of a COL5A3 mRNA isoform with 2 of the 69 exons missing. In addition, 216 differentially expressed transcript isoforms regulated by butyrate were detected. For example, Isoform 1 of ORC1 was strongly repressed by butyrate while Isoform 2 remained unchanged. Butyrate physically binds to and inhibits all zinc-dependent HDACs except HDAC6 and HDAC10. Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control. Moreover, thirteen gene fusion events differentially affected by butyrate were identified. Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC

  10. Repair of Mybpc3 mRNA by 5′-trans-splicing in a Mouse Model of Hypertrophic Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Giulia Mearini

    2013-01-01

    Full Text Available RNA trans-splicing has been explored as a therapeutic option for a variety of genetic diseases, but not for cardiac genetic disease. Hypertrophic cardiomyopathy (HCM is an autosomal-dominant disease, characterized by left ventricular hypertrophy (LVH and diastolic dysfunction. MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C is frequently mutated. We evaluated the 5′-trans-splicing strategy in a mouse model of HCM carrying a Mybpc3 mutation. 5′-trans-splicing was induced between two independently transcribed molecules, the mutant endogenous Mypbc3 pre-mRNA and an engineered pre-trans-splicing molecule (PTM carrying a FLAG-tagged wild-type (WT Mybpc3 cDNA sequence. PTMs were packaged into adeno-associated virus (AAV for transduction of cultured cardiac myocytes and the heart in vivo. Full-length repaired Mybpc3 mRNA represented up to 66% of total Mybpc3 transcripts in cardiac myocytes and 0.14% in the heart. Repaired cMyBP-C protein was detected by immunoprecipitation in cells and in vivo and exhibited correct incorporation into the sarcomere in cardiac myocytes. This study provides (i the first evidence of successful 5′-trans-splicing in vivo and (ii proof-of-concept of mRNA repair in the most prevalent cardiac genetic disease. Since current therapeutic options for HCM only alleviate symptoms, these findings open new horizons for causal therapy of the severe forms of the disease.

  11. Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

    DEFF Research Database (Denmark)

    Bruun, Gitte H; Doktor, Thomas K; Borch-Jensen, Jonas

    2016-01-01

    for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing...... regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP......-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site...

  12. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15.

    Science.gov (United States)

    Han, Ting; Goralski, Maria; Gaskill, Nicholas; Capota, Emanuela; Kim, Jiwoong; Ting, Tabitha C; Xie, Yang; Williams, Noelle S; Nijhawan, Deepak

    2017-04-28

    Indisulam is an aryl sulfonamide drug with selective anticancer activity. Its mechanism of action and the basis for its selectivity have so far been unknown. Here we show that indisulam promotes the recruitment of RBM39 (RNA binding motif protein 39) to the CUL4-DCAF15 E3 ubiquitin ligase, leading to RBM39 polyubiquitination and proteasomal degradation. Mutations in RBM39 that prevent its recruitment to CUL4-DCAF15 increase RBM39 stability and confer resistance to indisulam's cytotoxicity. RBM39 associates with precursor messenger RNA (pre-mRNA) splicing factors, and inactivation of RBM39 by indisulam causes aberrant pre-mRNA splicing. Many cancer cell lines derived from hematopoietic and lymphoid lineages are sensitive to indisulam, and their sensitivity correlates with DCAF15 expression levels. Two other clinically tested sulfonamides, tasisulam and chloroquinoxaline sulfonamide, share the same mechanism of action as indisulam. We propose that DCAF15 expression may be a useful biomarker to guide clinical trials of this class of drugs, which we refer to as SPLAMs (splicing inhibitor sulfonamides). Copyright © 2017, American Association for the Advancement of Science.

  13. BLOOD DONORS CAMPAIGN

    CERN Document Server

    2002-01-01

    Wednesday 13 November 2002 in restaurant nr 2, from 8.30 to 16.30 hrs will be held a blood donors campaign, organized by the Etablissement de Transfusion de Haute-Savoie If you already have a card giving your blood group, please bring this with you.

  14. BLOOD DONORS CAMPAIGN

    CERN Multimedia

    Medical Service

    2002-01-01

    Tuesday 19 March 2002 in restaurant nr 2, from 9.00 to 16.30 hrs A blood donors campaign, organized by the Centre de Transfusion sanguine of Geneva If you already have a card giving your blood group, please bring this with you.

  15. BLOOD DONORS CAMPAIGN

    CERN Document Server

    2001-01-01

    A blood donors campaign, organized by the Centre de Transfusion Sanguine of Geneva will be held at CERN on Tuesday 13 March 2001 in restaurant nr 2, from 9.00 to 16.30 hrs If you already have a card giving your blood group, please bring this with you.

  16. BLOOD DONORS CAMPAIGN

    CERN Document Server

    2001-01-01

    A blood donors campaign, organized by the Centre de Transfusion d'Annemasse will be held at CERN on Tuesday 14 November 2001 in restaurant nr 2, from 9.00 to 16.30 hrs If you already have a card giving your blood group, please bring this with you.

  17. BLOOD DONORS CAMPAIGN

    CERN Document Server

    2000-01-01

    A blood donors campaign, organized by the Établissement de Transfusion de Rhône-Alpes will be held at CERN on Tuesday 14 November 2000 in restaurant nr 2, from 8.30 to 16.30 hrs If you already have a card giving your blood group, please bring this with you.

  18. Donor transplant programme

    International Nuclear Information System (INIS)

    Abu Bakar Sulaiman

    1999-01-01

    The transplantation of organs and tissues from one human to another human has become an essential and well established form of therapy for many types of organ and tissue failure. In Malaysia, kidney, cornea and bone marrow transplantation are well established. Recently, liver, bone and heart transplanation have been performed. Unfortunately, because of the lack of cadaveric organ donation, only a limited number of solid organ transplantation have been performed. The cadaveric organ donor rate in Malaysia is low at less than one per million population. The first tissue transplanted in Malaysia was the cornea which was performed in the early 1970s. At that time and even now the majority of corneas came from Sri Lanka. The first kidney transplant was performed in 1975 from a live related donor. The majority of the 629 kidney transplants done at Hospital Kuala Lumpur to date have been from live related donors. Only 35 were from cadaver donors. Similarly, the liver transplantation programme which started in 1995 are from live related donors. A more concerted effort has been made recently to increase the awareness of the public and the health professionals on organ and tissue donation. This national effort to promote organ and tissue donation seems to have gathered momentum in 1997 with the first heart transplant successfully performed at the National Heart Institute. The rate of cadaveric donors has also increased from a previous average of I to 2 per year to 6 per year in the last one year. These developments are most encouraging and may signal the coming of age of our transplantati on programme. The Ministry of Health in conjunction with various institutions, organizations and professional groups, have taken a number of proactive measures to facilitate the development of the cadaveric organ donation programme. Efforts to increase public awareness and to overcome the negative cultural attitude towards organ donation have been intensified. Equally important are efforts

  19. Splicing modulation therapy in the treatment of genetic diseases

    Directory of Open Access Journals (Sweden)

    Arechavala-Gomeza V

    2014-12-01

    Full Text Available Virginia Arechavala-Gomeza,1 Bernard Khoo,2 Annemieke Aartsma-Rus3 1Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain; 2Endocrinology, Division of Medicine, University College London, London, UK; 3Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands All authors contributed equally to this manuscript Abstract: Antisense-mediated splicing modulation is a tool that can be exploited in several ways to provide a potential therapy for rare genetic diseases. This approach is currently being tested in clinical trials for Duchenne muscular dystrophy and spinal muscular atrophy. The present review outlines the versatility of the approach to correct cryptic splicing, modulate alternative splicing, restore the open reading frame, and induce protein knockdown, providing examples of each. Finally, we outline a possible path forward toward the clinical application of this approach for a wide variety of inherited rare diseases. Keywords: splicing, therapy, antisense oligonucleotides, cryptic splicing, alternative splicing

  20. The emerging role of alternative splicing in senescence and aging.

    Science.gov (United States)

    Deschênes, Mathieu; Chabot, Benoit

    2017-10-01

    Deregulation of precursor mRNA splicing is associated with many illnesses and has been linked to age-related chronic diseases. Here we review recent progress documenting how defects in the machinery that performs intron removal and controls splice site selection contribute to cellular senescence and organismal aging. We discuss the functional association linking p53, IGF-1, SIRT1, and ING-1 splice variants with senescence and aging, and review a selection of splicing defects occurring in accelerated aging (progeria), vascular aging, and Alzheimer's disease. Overall, it is becoming increasingly clear that changes in the activity of splicing factors and in the production of key splice variants can impact cellular senescence and the aging phenotype. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  1. Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA.

    Directory of Open Access Journals (Sweden)

    Stephen H Munroe

    Full Text Available The α-thyroid hormone receptor gene (TRα codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3' end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30 located downstream of the alternative 3'splice site of TRα2 mRNA and antisense to the 3'UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing.

  2. Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA

    Science.gov (United States)

    Munroe, Stephen H.; Morales, Christopher H.; Duyck, Tessa H.; Waters, Paul D.

    2015-01-01

    The α-thyroid hormone receptor gene (TRα) codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3’ end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30) located downstream of the alternative 3’splice site of TRα2 mRNA and antisense to the 3’UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing. PMID:26368571

  3. Study of USH1 splicing variants through minigenes and transcript analysis from nasal epithelial cells.

    Directory of Open Access Journals (Sweden)

    María José Aparisi

    Full Text Available Usher syndrome type I (USH1 is an autosomal recessive disorder characterized by congenital profound deafness, vestibular areflexia and prepubertal retinitis pigmentosa. The first purpose of this study was to determine the pathologic nature of eighteen USH1 putative splicing variants found in our series and their effect in the splicing process by minigene assays. These variants were selected according to bioinformatic analysis. The second aim was to analyze the USH1 transcripts, obtained from nasal epithelial cells samples of our patients, in order to corroborate the observed effect of mutations by minigenes in patient's tissues. The last objective was to evaluate the nasal ciliary beat frequency in patients with USH1 and compare it with control subjects. In silico analysis were performed using four bioinformatic programs: NNSplice, Human Splicing Finder, NetGene2 and Spliceview. Afterward, minigenes based on the pSPL3 vector were used to investigate the implication of selected changes in the mRNA processing. To observe the effect of mutations in the patient's tissues, RNA was extracted from nasal epithelial cells and RT-PCR analyses were performed. Four MYO7A (c.470G>A, c.1342_1343delAG, c.5856G>A and c.3652G>A, three CDH23 (c.2289+1G>A, c.6049G>A and c.8722+1delG and one PCDH15 (c.3717+2dupTT variants were observed to affect the splicing process by minigene assays and/or transcripts analysis obtained from nasal cells. Based on our results, minigenes are a good approach to determine the implication of identified variants in the mRNA processing, and the analysis of RNA obtained from nasal epithelial cells is an alternative method to discriminate neutral Usher variants from those with a pathogenic effect on the splicing process. In addition, we could observe that the nasal ciliated epithelium of USH1 patients shows a lower ciliary beat frequency than control subjects.

  4. Splicing Express: a software suite for alternative splicing analysis using next-generation sequencing data

    Directory of Open Access Journals (Sweden)

    Jose E. Kroll

    2015-11-01

    Full Text Available Motivation. Alternative splicing events (ASEs are prevalent in the transcriptome of eukaryotic species and are known to influence many biological phenomena. The identification and quantification of these events are crucial for a better understanding of biological processes. Next-generation DNA sequencing technologies have allowed deep characterization of transcriptomes and made it possible to address these issues. ASEs analysis, however, represents a challenging task especially when many different samples need to be compared. Some popular tools for the analysis of ASEs are known to report thousands of events without annotations and/or graphical representations. A new tool for the identification and visualization of ASEs is here described, which can be used by biologists without a solid bioinformatics background.Results. A software suite named Splicing Express was created to perform ASEs analysis from transcriptome sequencing data derived from next-generation DNA sequencing platforms. Its major goal is to serve the needs of biomedical researchers who do not have bioinformatics skills. Splicing Express performs automatic annotation of transcriptome data (GTF files using gene coordinates available from the UCSC genome browser and allows the analysis of data from all available species. The identification of ASEs is done by a known algorithm previously implemented in another tool named Splooce. As a final result, Splicing Express creates a set of HTML files composed of graphics and tables designed to describe the expression profile of ASEs among all analyzed samples. By using RNA-Seq data from the Illumina Human Body Map and the Rat Body Map, we show that Splicing Express is able to perform all tasks in a straightforward way, identifying well-known specific events.Availability and Implementation.Splicing Express is written in Perl and is suitable to run only in UNIX-like systems. More details can be found at: http://www.bioinformatics-brazil.org/splicingexpress.

  5. Identification of common genetic variation that modulates alternative splicing.

    Directory of Open Access Journals (Sweden)

    Jeremy Hull

    2007-06-01

    Full Text Available Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs. In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

  6. Alternative Splicing of FOXP3-Virtue and Vice.

    Science.gov (United States)

    Mailer, Reiner K W

    2018-01-01

    FOXP3 is the lineage-defining transcription factor of CD4+ CD25+ regulatory T cells. While many aspects of its regulation, interaction, and function are conserved among species, alternatively spliced FOXP3 isoforms are expressed only in human cells. This review summarizes current knowledge about alternative splicing of FOXP3 and the specific functions of FOXP3 isoforms in health and disease. Future perspectives in research and the therapeutic potential of manipulating alternative splicing of FOXP3 are discussed.

  7. Cardiac CRFR1 Expression Is Elevated in Human Heart Failure and Modulated by Genetic Variation and Alternative Splicing

    Science.gov (United States)

    Lewis, Kathy A.; Perrin, Marilyn H.; Sweet, Wendy E.; Moravec, Christine S.; Tang, W. H. Wilson; Huising, Mark O.; Troughton, Richard W.; Cameron, Vicky A.

    2016-01-01

    Corticotropin-releasing factor (CRF) and the CRF-related peptides, urocortin (Ucn)-1, Ucn2, and Ucn3 signal through receptors CRFR1 and CRFR2 to restore homeostasis in response to stress. The Ucns exert potent cardioprotective effects and may have clinical utility in heart failure. To explore the activity of this system in the heart, we measured the levels of myocardial gene expression of the CRF/Ucn family of ligands/receptors and investigated genetic variation and alternative splicing of CRFR1 in 110 heart failure patients and 108 heart donors. Using quantitative real-time PCR, we detected CRFR1, CRFR2, CRF, Ucn1, Ucn2, and Ucn3 in all samples. CRFR2α was the most abundant receptor and Ucn3 the most abundant ligand, both in patients and donors. Compared with donors, cardiac expression of CRFR1, CRF, and Ucn3 was higher (P heart failure and may contribute to the activation of the CRF/Ucn system in these patients. A common variant within the CRFR1 gene and a novel CRFR1 splice variant may modulate CRFR1 expression and signaling. PMID:27754786

  8. Cardiac CRFR1 Expression Is Elevated in Human Heart Failure and Modulated by Genetic Variation and Alternative Splicing.

    Science.gov (United States)

    Pilbrow, Anna P; Lewis, Kathy A; Perrin, Marilyn H; Sweet, Wendy E; Moravec, Christine S; Tang, W H Wilson; Huising, Mark O; Troughton, Richard W; Cameron, Vicky A

    2016-12-01

    Corticotropin-releasing factor (CRF) and the CRF-related peptides, urocortin (Ucn)-1, Ucn2, and Ucn3 signal through receptors CRFR1 and CRFR2 to restore homeostasis in response to stress. The Ucns exert potent cardioprotective effects and may have clinical utility in heart failure. To explore the activity of this system in the heart, we measured the levels of myocardial gene expression of the CRF/Ucn family of ligands/receptors and investigated genetic variation and alternative splicing of CRFR1 in 110 heart failure patients and 108 heart donors. Using quantitative real-time PCR, we detected CRFR1, CRFR2, CRF, Ucn1, Ucn2, and Ucn3 in all samples. CRFR2α was the most abundant receptor and Ucn3 the most abundant ligand, both in patients and donors. Compared with donors, cardiac expression of CRFR1, CRF, and Ucn3 was higher (P heart failure and may contribute to the activation of the CRF/Ucn system in these patients. A common variant within the CRFR1 gene and a novel CRFR1 splice variant may modulate CRFR1 expression and signaling.

  9. PPS, a large multidomain protein, functions with sex-lethal to regulate alternative splicing in Drosophila.

    Science.gov (United States)

    Johnson, Matthew L; Nagengast, Alexis A; Salz, Helen K

    2010-03-05

    Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL-mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance.

  10. Genetic analysis of complement C1s deficiency associated with systemic lupus erythematosus highlights alternative splicing of normal C1s gene

    DEFF Research Database (Denmark)

    Armano, MT; Ferriani, VP; Florido, MP

    2008-01-01

    ' fibroblasts when analyzed by confocal microscopy. We show that all four siblings are homozygous for a mutation at position 938 in exon 6 of the C1s cDNA that creates a premature stop codon. Our investigations led us to reveal the presence of previously uncharacterized splice variants of C1s mRNA transcripts...... in normal human cells. These variants are derived from the skipping of exon 3 and from the use of an alternative 3' splice site within intron 1 which increases the size of exon 2 by 87 nucleotides....

  11. Conservation and Sex-Specific Splicing of the transformer Gene in the Calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata

    Science.gov (United States)

    Li, Fang; Vensko, Steven P.; Belikoff, Esther J.; Scott, Maxwell J.

    2013-01-01

    Transformer (TRA) promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly and Drosophila melanogaster. tra transcripts are sex-specifically spliced such that only the female form encodes full length functional protein. The presence of six predicted TRA/TRA2 binding sites in the sex-specific female intron of the L. cuprina gene suggested that tra splicing is auto-regulated as in medfly and housefly. With the aim of identifying conserved motifs that may play a role in tra sex-specific splicing, here we have isolated and characterized the tra gene from three additional blowfly species, L. sericata, Cochliomyia hominivorax and C. macellaria. The blowfly adult male and female transcripts differ in the choice of splice donor site in the first intron, with males using a site downstream of the site used in females. The tra genes all contain a single TRA/TRA2 site in the male exon and a cluster of four to five sites in the male intron. However, overall the sex-specific intron sequences are poorly conserved in closely related blowflies. The most conserved regions are around the exon/intron junctions, the 3′ end of the intron and near the cluster of TRA/TRA2 sites. We propose a model for sex specific regulation of tra splicing that incorporates the conserved features identified in this study. In L. sericata embryos, the male tra transcript was first detected at around the time of cellular blastoderm formation. RNAi experiments showed that tra is required for female development in L. sericata and C. macellaria. The isolation of the tra gene from the New World screwworm fly C. hominivorax, a major livestock pest, will facilitate the development of a “male-only” strain for genetic control programs. PMID:23409170

  12. Conservation and sex-specific splicing of the transformer gene in the calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata.

    Directory of Open Access Journals (Sweden)

    Fang Li

    Full Text Available Transformer (TRA promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly and Drosophila melanogaster. tra transcripts are sex-specifically spliced such that only the female form encodes full length functional protein. The presence of six predicted TRA/TRA2 binding sites in the sex-specific female intron of the L. cuprina gene suggested that tra splicing is auto-regulated as in medfly and housefly. With the aim of identifying conserved motifs that may play a role in tra sex-specific splicing, here we have isolated and characterized the tra gene from three additional blowfly species, L. sericata, Cochliomyia hominivorax and C. macellaria. The blowfly adult male and female transcripts differ in the choice of splice donor site in the first intron, with males using a site downstream of the site used in females. The tra genes all contain a single TRA/TRA2 site in the male exon and a cluster of four to five sites in the male intron. However, overall the sex-specific intron sequences are poorly conserved in closely related blowflies. The most conserved regions are around the exon/intron junctions, the 3' end of the intron and near the cluster of TRA/TRA2 sites. We propose a model for sex specific regulation of tra splicing that incorporates the conserved features identified in this study. In L. sericata embryos, the male tra transcript was first detected at around the time of cellular blastoderm formation. RNAi experiments showed that tra is required for female development in L. sericata and C. macellaria. The isolation of the tra gene from the New World screwworm fly C. hominivorax, a major livestock pest, will facilitate the development of a "male-only" strain for genetic control programs.

  13. 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.

  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. The strength of intron donor splice sites in human genes displays a bell-shaped pattern

    DEFF Research Database (Denmark)

    Wang, Kai; Wernersson, Rasmus; Brunak, Søren

    2011-01-01

    introns. Interestingly, when analysing the intron containing gene pool from mouse consisting of >15 000 genes, we found the convex pattern to be conserved despite >75 million years of evolutionary divergence between the two organisms. We also analysed an interesting, novel class of chimeric genes which...

  16. HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages

    Directory of Open Access Journals (Sweden)

    Purcell Damian FJ

    2008-02-01

    Full Text Available Abstract Background Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1 differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2 control of HIV-1 alternative splicing, which is essential for optimal viral replication. Results Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins and inhibitory factors (members of the hnRNP family. While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. Conclusion While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative

  17. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

    Directory of Open Access Journals (Sweden)

    Katja Meyer

    2015-07-01

    Full Text Available Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

  18. Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.

    Science.gov (United States)

    Juan, Wen Chun; Roca, Xavier; Ong, S Tiong

    2014-01-01

    Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3' end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.

  19. Transcription rate strongly affects splicing fidelity and cotranscriptionality in budding yeast

    OpenAIRE

    Aslanzadeh, Vahid; Huang, Yuanhua; Sanguinetti, Guido; Beggs, Jean D.

    2018-01-01

    The functional consequences of alternative splicing on altering the transcription rate have been the subject of intensive study in mammalian cells but less is known about effects of splicing on changing the transcription rate in yeast. We present several lines of evidence showing that slow RNA polymerase II elongation increases both cotranscriptional splicing and splicing efficiency and that faster elongation reduces cotranscriptional splicing and splicing efficiency in budding yeast, suggest...

  20. Dynamic Distribution and Interaction of the Arabidopsis SRSF1 Subfamily Splicing Factors.

    Science.gov (United States)

    Stankovic, Nancy; Schloesser, Marie; Joris, Marine; Sauvage, Eric; Hanikenne, Marc; Motte, Patrick

    2016-02-01

    Ser/Arg-rich (SR) proteins are essential nucleus-localized splicing factors. Our prior studies showed that Arabidopsis (Arabidopsis thaliana) RSZ22, a homolog of the human SRSF7 SR factor, exits the nucleus through two pathways, either dependent or independent on the XPO1 receptor. Here, we examined the expression profiles and shuttling dynamics of the Arabidopsis SRSF1 subfamily (SR30, SR34, SR34a, and SR34b) under control of their endogenous promoter in Arabidopsis and in transient expression assay. Due to its rapid nucleocytoplasmic shuttling and high expression level in transient assay, we analyzed the multiple determinants that regulate the localization and shuttling dynamics of SR34. By site-directed mutagenesis of SR34 RNA-binding sequences and Arg/Ser-rich (RS) domain, we further show that functional RRM1 or RRM2 are dispensable for the exclusive protein nuclear localization and speckle-like distribution. However, mutations of both RRMs induced aggregation of the protein whereas mutation in the RS domain decreased the stability of the protein and suppressed its nuclear accumulation. Furthermore, the RNA-binding motif mutants are defective for their export through the XPO1 (CRM1/Exportin-1) receptor pathway, but retain nucleocytoplasmic mobility. We performed a yeast two hybrid screen with SR34 as bait and discovered SR45 as a new interactor. SR45 is an unusual SR splicing factor bearing two RS domains. These interactions were confirmed in planta by FLIM-FRET and BiFC and the roles of SR34 domains in protein-protein interactions were further studied. Altogether, our report extends our understanding of shuttling dynamics of Arabidopsis SR splicing factors. © 2016 American Society of Plant Biologists. All Rights Reserved.

  1. Dynamic Distribution and Interaction of the Arabidopsis SRSF1 Subfamily Splicing Factors1

    Science.gov (United States)

    Stankovic, Nancy; Schloesser, Marie; Joris, Marine; Sauvage, Eric; Hanikenne, Marc; Motte, Patrick

    2016-01-01

    Ser/Arg-rich (SR) proteins are essential nucleus-localized splicing factors. Our prior studies showed that Arabidopsis (Arabidopsis thaliana) RSZ22, a homolog of the human SRSF7 SR factor, exits the nucleus through two pathways, either dependent or independent on the XPO1 receptor. Here, we examined the expression profiles and shuttling dynamics of the Arabidopsis SRSF1 subfamily (SR30, SR34, SR34a, and SR34b) under control of their endogenous promoter in Arabidopsis and in transient expression assay. Due to its rapid nucleocytoplasmic shuttling and high expression level in transient assay, we analyzed the multiple determinants that regulate the localization and shuttling dynamics of SR34. By site-directed mutagenesis of SR34 RNA-binding sequences and Arg/Ser-rich (RS) domain, we further show that functional RRM1 or RRM2 are dispensable for the exclusive protein nuclear localization and speckle-like distribution. However, mutations of both RRMs induced aggregation of the protein whereas mutation in the RS domain decreased the stability of the protein and suppressed its nuclear accumulation. Furthermore, the RNA-binding motif mutants are defective for their export through the XPO1 (CRM1/Exportin-1) receptor pathway, but retain nucleocytoplasmic mobility. We performed a yeast two hybrid screen with SR34 as bait and discovered SR45 as a new interactor. SR45 is an unusual SR splicing factor bearing two RS domains. These interactions were confirmed in planta by FLIM-FRET and BiFC and the roles of SR34 domains in protein-protein interactions were further studied. Altogether, our report extends our understanding of shuttling dynamics of Arabidopsis SR splicing factors. PMID:26697894

  2. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we...... compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs...

  3. Diagnosis and treatment of sideroblastic anemias: from defective heme synthesis to abnormal RNA splicing.

    Science.gov (United States)

    Cazzola, Mario; Malcovati, Luca

    2015-01-01

    The sideroblastic anemias are a heterogeneous group of inherited and acquired disorders characterized by the presence of ring sideroblasts in the bone marrow. X-linked sideroblastic anemia (XLSA) is caused by germline mutations in ALAS2. Hemizygous males have a hypochromic microcytic anemia, which is generally mild to moderate and is caused by defective heme synthesis and ineffective erythropoiesis. XLSA is a typical iron-loading anemia; although most patients are responsive to pyridoxine, treatment of iron overload is also important in the management of these patients. Autosomal recessive sideroblastic anemia attributable to mutations in SLC25A38, a member of the mitochondrial carrier family, is a severe disease: patients present in infancy with microcytic anemia, which soon becomes transfusion dependent. Conservative therapy includes regular red cell transfusion and iron chelation, whereas allogenic stem cell transplantation represents the only curative treatment. Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome characterized mainly by anemia attributable to ineffective erythropoiesis. The clinical course of RARS is generally indolent, but there is a tendency to worsening of anemia over time, so that most patients become transfusion dependent in the long run. More than 90% of these patients carry somatic mutations in SF3B1, a gene encoding a core component of the RNA splicing machinery. These mutations cause misrecognition of 3' splice sites in downstream genes, resulting in truncated gene products and/or decreased expression attributable to nonsense-mediated RNA decay; this explains the multifactorial pathogenesis of RARS. Variants of RARS include refractory cytopenia with multilineage dysplasia and ring sideroblasts, and RARS associated with marked thrombocytosis; these variants involve additional genetic lesions. Inhibitors of molecules of the transforming growth factor-β superfamily have been shown recently to target ineffective

  4. ASpedia: a comprehensive encyclopedia of human alternative splicing.

    Science.gov (United States)

    Hyung, Daejin; Kim, Jihyun; Cho, Soo Young; Park, Charny

    2018-01-04

    Alternative splicing confers the human genome complexity by increasing the diversity of expressed mRNAs. Hundreds or thousands of splicing regions have been identified through differential alternative splicing analysis of high-throughput datasets. However, it is hard to explain the functional impact of each splicing event. Protein domain formation and nonsense-mediated decay are considered the main functional features of splicing. However, other functional features such as miRNA target sites, phosphorylation sites and single-nucleotide variations are directly affected by alternative splicing and affect downstream function. Hence, we established ASpedia: a comprehensive database for human alternative splicing annotation, which encompasses a range of functions, from genomic annotation to isoform-specific function (ASpedia, http://combio.snu.ac.kr/aspedia). The database provides three features: (i) genomic annotation extracted from DNA, RNA and proteins; (ii) transcription and regulation elements analyzed from next-generation sequencing datasets; and (iii) isoform-specific functions collected from known and published datasets. The ASpedia web application includes three components: an annotation database, a retrieval system and a browser specialized in the identification of human alternative splicing events. The retrieval system supports multiple AS event searches resulting from high-throughput analysis and the AS browser comprises genome tracks. Thus, ASpedia facilitates the systemic annotation of the functional impacts of multiple AS events. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Quantitative regulation of alternative splicing in evolution and development

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob L; Roy, Scott W

    2009-01-01

    Alternative splicing (AS) is a widespread mechanism with an important role in increasing transcriptome and proteome diversity by generating multiple different products from the same gene. Evolutionary studies of AS have focused primarily on the conservation of alternatively spliced sequences or o...

  6. Molecular heterogeneity of the Jk(null) phenotype: expression analysis of the Jk(S291P) mutation found in Finns.

    Science.gov (United States)

    Sidoux-Walter, F; Lucien, N; Nissinen, R; Sistonen, P; Henry, S; Moulds, J; Cartron, J P; Bailly, P

    2000-08-15

    Polymerase chain reaction genotyping of 32 unrelated Jk(null) individuals originating predominantly from Polynesia and Finland indicated that all were homozygous for the JK*B polymorphism and that 17 of 32, including the 14 Polynesians, carried a 3'-acceptor splice site mutation of intron 5 that resulted in the skipping of exon 6 (called mutation Jk delta 6). The remaining 15 Jk(null) donors from Finland were homozygous for a new T871C transition resulting in a S291P amino acid substitution at a consensus N-glycosylation site of the Jk polypeptide. Transcription-translation assays revealed that the Jk(S291P) mutant was translated into a glycosylated component as efficiently as the wild-type Jk polypeptide (wt Jk)] in the presence of microsomes, thus indicating that the S291P mutation has no effect on the N-glycosylation pattern of the Jk protein. Expression studies in Xenopus oocytes revealed that the Jk(S291P) polypeptide functions as a urea transporter, but the transport activity and the membrane expression level of the mutant protein was reduced to a similar extent. A substantial fraction of the mutant protein was retained intracellularly suggesting that the transit to the plasma membrane was reduced, presumably because of the S-->P mutation. After transfection in erythroleukemia K562 cells the wild-type, but not the mutant, protein was efficiently expressed at the cell surface. Because the Jk(S291P) mutant polypeptide was not present in human red cells from Jk(null) individuals, expression data in the erythroid context clearly indicates that the S-->P mutation is the molecular basis of the Finnish Jk(null) phenotype. (Blood. 2000;96:1566-1573)

  7. Clinical and biological implications of driver mutations in myelodysplastic syndromes.

    Science.gov (United States)

    Papaemmanuil, Elli; Gerstung, Moritz; Malcovati, Luca; Tauro, Sudhir; Gundem, Gunes; Van Loo, Peter; Yoon, Chris J; Ellis, Peter; Wedge, David C; Pellagatti, Andrea; Shlien, Adam; Groves, Michael John; Forbes, Simon A; Raine, Keiran; Hinton, Jon; Mudie, Laura J; McLaren, Stuart; Hardy, Claire; Latimer, Calli; Della Porta, Matteo G; O'Meara, Sarah; Ambaglio, Ilaria; Galli, Anna; Butler, Adam P; Walldin, Gunilla; Teague, Jon W; Quek, Lynn; Sternberg, Alex; Gambacorti-Passerini, Carlo; Cross, Nicholas C P; Green, Anthony R; Boultwood, Jacqueline; Vyas, Paresh; Hellstrom-Lindberg, Eva; Bowen, David; Cazzola, Mario; Stratton, Michael R; Campbell, Peter J

    2013-11-21

    Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic hematological malignancies characterized by dysplasia, ineffective hematopoiesis and a variable risk of progression to acute myeloid leukemia. Sequencing of MDS genomes has identified mutations in genes implicated in RNA splicing, DNA modification, chromatin regulation, and cell signaling. We sequenced 111 genes across 738 patients with MDS or closely related neoplasms (including chronic myelomonocytic leukemia and MDS-myeloproliferative neoplasms) to explore the role of acquired mutations in MDS biology and clinical phenotype. Seventy-eight percent of patients had 1 or more oncogenic mutations. We identify complex patterns of pairwise association between genes, indicative of epistatic interactions involving components of the spliceosome machinery and epigenetic modifiers. Coupled with inferences on subclonal mutations, these data suggest a hypothesis of genetic "predestination," in which early driver mutations, typically affecting genes involved in RNA splicing, dictate future trajectories of disease evolution with distinct clinical phenotypes. Driver mutations had equivalent prognostic significance, whether clonal or subclonal, and leukemia-free survival deteriorated steadily as numbers of driver mutations increased. Thus, analysis of oncogenic mutations in large, well-characterized cohorts of patients illustrates the interconnections between the cancer genome and disease biology, with considerable potential for clinical application.

  8. A study of alternative splicing in the pig

    DEFF Research Database (Denmark)

    Hillig, Ann-Britt Nygaard; Cirera Salicio, Susanna; Gilchrist, Michael J.

    2010-01-01

    BACKGROUND: Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible...... alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence. RESULTS: The pig EST data generated by the Sino...... transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific. CONCLUSIONS: In accordance with human...

  9. Detecting Image Splicing Using Merged Features in Chroma Space

    Directory of Open Access Journals (Sweden)

    Bo Xu

    2014-01-01

    Full Text Available Image splicing is an image editing method to copy a part of an image and paste it onto another image, and it is commonly followed by postprocessing such as local/global blurring, compression, and resizing. To detect this kind of forgery, the image rich models, a feature set successfully used in the steganalysis is evaluated on the splicing image dataset at first, and the dominant submodel is selected as the first kind of feature. The selected feature and the DCT Markov features are used together to detect splicing forgery in the chroma channel, which is convinced effective in splicing detection. The experimental results indicate that the proposed method can detect splicing forgeries with lower error rate compared to the previous literature.

  10. Some relations between two stages DNA splicing languages

    Science.gov (United States)

    Mudaber, Mohammad Hassan; Yusof, Yuhani; Mohamad, Mohd Sham

    2014-06-01

    A new symbolization of Yusof-Goode (Y-G) rule, which is associated with Y-G splicing system, was introduced by Yusof in 2012 under the framework of formal language theory. The purpose of this investigation is to present the biological process of DNA splicing in a translucent way. In this study, two stages splicing languages are introduced based on Y-G approach and some relations between stage one and stage two splicing languages are presented, given as theorems. Additionally, the existing relations between two stages splicing languages based on crossings and contexts of restriction enzymes factors with respect to two initial strings (having two cutting sites) and two rules are presented as subset.

  11. SpliceDetector: a software for detection of alternative splicing events in human and model organisms directly from transcript IDs.

    Science.gov (United States)

    Baharlou Houreh, Mandana; Ghorbani Kalkhajeh, Payam; Niazi, Ali; Ebrahimi, Faezeh; Ebrahimie, Esmaeil

    2018-03-22

    In eukaryotes, different combinations of exons lead to multiple transcripts with various functions in protein level, in a process called alternative splicing (AS). Unfolding the complexity of functional genomics through genome-wide profiling of AS and determining the altered ultimate products provide new insights for better understanding of many biological processes, disease progress as well as drug development programs to target harmful splicing variants. The current available tools of alternative splicing work with raw data and include heavy computation. In particular, there is a shortcoming in tools to discover AS events directly from transcripts. Here, we developed a Windows-based user-friendly tool for identifying AS events from transcripts without the need to any advanced computer skill or database download. Meanwhile, due to online working mode, our application employs the updated SpliceGraphs without the need to any resource updating. First, SpliceGraph forms based on the frequency of active splice sites in pre-mRNA. Then, the presented approach compares query transcript exons to SpliceGraph exons. The tool provides the possibility of statistical analysis of AS events as well as AS visualization compared to SpliceGraph. The developed application works for transcript sets in human and model organisms.

  12. Reversible optic neuropathy with OPA1 exon 5b mutation

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  13. Donor attention to reading materials.

    Science.gov (United States)

    O'Brien, S F; Osmond, L; Choquet, K; Yi, Q-L; Goldman, M

    2015-11-01

    Mandatory predonation reading materials inform donors about risk factors for transmissible disease, possible complications of donation and changes to the donation process. We aimed to assess the attention to predonation reading materials and factors which may affect attention. A national survey in 2008 of 18,108 blood donors asked about self-assessed attention to reading the materials. In face-to-face interviews, 441 donors completed additional questions about reading the materials and a literacy test. Qualitative interviews of 27 donors assessed their approach to reading. In the national survey, most of the first-time donors said they read all or most of the materials (90.9% first-time vs. 57.6% repeat donors, P reading them carefully (P read materials carefully, skimmed or did not read, most knew that donors are informed of positive transmissible disease test results (97.1%, 95.5, 98.0 P > 0.05), but fewer recalled seeing the definition of sex (77.2%, 56.9, 24.2 P read materials carefully, skimmed or did not read were compared (P > 0.05). Qualitative interviews showed that donors are reluctant to read any more than necessary and decide based on perceived importance or relevance. Attention to predonation reading materials tends to be better among first-time donors. The effectiveness is limited by low motivation to read, especially for repeat donors, as well as poor literacy. © 2015 International Society of Blood Transfusion.

  14. New mutations in the NHS gene in Nance-Horan Syndrome families from the Netherlands.

    Science.gov (United States)

    Florijn, Ralph J; Loves, Willem; Maillette de Buy Wenniger-Prick, Liesbeth J J M; Mannens, Marcel M A M; Tijmes, Nel; Brooks, Simon P; Hardcastle, Alison J; Bergen, Arthur A B

    2006-09-01

    Mutations in the NHS gene cause Nance-Horan Syndrome (NHS), a rare X-chromosomal recessive disorder with variable features, including congenital cataract, microphthalmia, a peculiar form of the ear and dental anomalies. We investigated the NHS gene in four additional families with NHS from the Netherlands, by dHPLC and direct sequencing. We identified an unique mutation in each family. Three out of these four mutations were not reported before. We report here the first splice site sequence alteration mutation and three protein truncating mutations. Our results suggest that X-linked cataract and NHS are allelic disorders.

  15. Detection of T lymphocytes with a second-site mutation in skin lesions of atypical X-linked severe combined immunodeficiency mimicking Omenn syndrome.

    Science.gov (United States)

    Wada, Taizo; Yasui, Masahiro; Toma, Tomoko; Nakayama, Yuko; Nishida, Mika; Shimizu, Masaki; Okajima, Michiko; Kasahara, Yoshihito; Koizumi, Shoichi; Inoue, Masami; Kawa, Keisei; Yachie, Akihiro

    2008-09-01

    X-linked severe combined immunodeficiency (XSCID) is caused by mutations of the common gamma chain (gammac) and usually characterized by the absence of T and natural killer (NK) cells. Here, we report an atypical case of XSCID presenting with autologous T and NK cells and Omenn syndrome-like manifestations. The patient carried a splice-site mutation (IVS1+5G>A) that caused most of the mRNA to be incorrectly spliced but produced normally spliced transcript in lesser amount, leading to residual gammac expression and development of T and NK cells. The skin biopsy specimen showed massive infiltration of revertant T cells. Those T cells were found to have a second-site mutation and result in complete restoration of correct splicing. These findings suggest that the clinical spectrum of XSCID is quite broad and includes atypical cases mimicking Omenn syndrome, and highlight the importance of revertant mosaicism as a possible cause for variable phenotypic expression.

  16. Hydrocephalus, agenesis of the corpus callosum, and cleft lip/palate represent frequent associations in fetuses with Peters' plus syndrome and B3GALTL mutations. Fetal PPS phenotypes, expanded by Dandy Walker cyst and encephalocele.

    Science.gov (United States)

    Schoner, Katharina; Kohlhase, Juergen; Müller, Annette M; Schramm, Thomas; Plassmann, Margit; Schmitz, Ralf; Neesen, Juergen; Wieacker, Peter; Rehder, Helga

    2013-01-01

    Fetal pathology aims to recognize syndromal patterns of anomalies for goal-directed mutation analyses, genetic counseling, and early prenatal diagnosis in consecutive pregnancies. Here, we report on five fetuses with Peters' plus syndrome (PPS) from two distinct families aborted after prenatal ultrasound diagnosis of hydrocephaly. We performed fetal autopsies and molecular analyses. Among 44 fetuses with prenatally diagnosed hydrocephaly, four fetuses of 16 to 21 gestational weeks presented with additional cleft lip/palate and/or agenesis of the corpus callosum. Other features were growth retardation, hypertelorism, anomalies of the eyes, in part consistent with Peters' anterior chamber anomalies, mild brachymelia, brachydactyly, and also internal anomalies. Suspected PPS was confirmed by detection of B3GALTL mutation in these four fetuses and in one additional sib fetus, revealing homozygosity for the common c.660 + 1G > A donor splice site mutation in intron 8. Autosomal-recessive PPS has not yet been diagnosed prenatally. We want to alert ultrasonographers to the diagnosis of this disorder in growth-retarded fetuses with (recurrent) hydrocephaly, agenesis of the corpus callosum, and cleft lip/palate and stress the more severe fetal manifestation, describing a first such case with additional Dandy-Walker cyst and occult meningoencephalocele. © 2012 John Wiley & Sons, Ltd.

  17. Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

    Directory of Open Access Journals (Sweden)

    Douglas A Schober

    Full Text Available Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S. Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

  18. Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

    Science.gov (United States)

    Schober, Douglas A; Croy, Carrie H; Ruble, Cara L; Tao, Ran; Felder, Christian C

    2017-01-01

    Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

  19. The mutational spectrum in Treacher Collins syndrome reveals a predominance of mutations that create a premature-termination codon

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, S.J.; Gladwin, A.J.; Dixon, M.J. [Univ. of Manchester (United Kingdom)

    1997-03-01

    Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. The TCS locus has been mapped to human chromosome 5q31.3-32 and the mutated gene identified. In the current investigation, 25 previously undescribed mutations, which are spread throughout the gene, are presented. This brings the total reported to date to 35, which represents a detection rate of 60%. Of the mutations that have been reported to date, all but one result in the introduction of a premature-termination codon into the predicted protein, treacle. Moreover, the mutations are largely family specific, although a common 5-bp deletion in exon 24 (seven different families) and a recurrent splicing mutation in intron 3 (two different families) have been identified. This mutational spectrum supports the hypothesis that TCS results from haploin-sufficiency. 49 refs., 4 figs., 3 tabs.

  20. Splicing Express: a software suite for alternative splicing analysis using next-generation sequencing data

    OpenAIRE

    Kroll, Jose E.; Kim, Jihoon; Ohno-Machado, Lucila; de Souza, Sandro J.

    2015-01-01

    Motivation. Alternative splicing events (ASEs) are prevalent in the transcriptome of eukaryotic species and are known to influence many biological phenomena. The identification and quantification of these events are crucial for a better understanding of biological processes. Next-generation DNA sequencing technologies have allowed deep characterization of transcriptomes and made it possible to address these issues. ASEs analysis, however, represents a challenging task especially when many dif...

  1. Global Splicing Pattern Reversion during Somatic Cell Reprogramming

    Directory of Open Access Journals (Sweden)

    Sho Ohta

    2013-10-01

    Full Text Available Alternative splicing generates multiple transcripts from a single gene, and cell-type-specific splicing profiles are important for the properties and functions of the cells. Recently, somatic cells have been shown to undergo dedifferentiation after the forced expression of transcription factors. However, it remains unclear whether somatic cell splicing is reorganized during reprogramming. Here, by combining deep sequencing with high-throughput absolute qRT-PCR, we show that somatic splicing profiles revert to pluripotent ones during reprogramming. Remarkably, the splicing pattern in pluripotent stem cells resembles that in testes, and the regulatory regions have specific characteristics in length and sequence. Furthermore, our siRNA screen has identified RNA-binding proteins that regulate splicing events in iPSCs. We have then demonstrated that two of the RNA-binding proteins, U2af1 and Srsf3, play a role in somatic cell reprogramming. Our results indicate that the drastic alteration in splicing represents part of the molecular network involved in the reprogramming process.

  2. Herboxidiene triggers splicing repression and abiotic stress responses in plants

    KAUST Repository

    Alshareef, Sahar

    2017-03-27

    Background Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and stresses. Small-molecule inhibitors that perturb splicing provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. Results Here, we show that herboxidiene (GEX1A) inhibits both constitutive and alternative splicing. Moreover, GEX1A activates genome-wide transcriptional patterns involved in abiotic stress responses in plants. GEX1A treatment -activated ABA-inducible promoters, and led to stomatal closure. Interestingly, GEX1A and pladienolide B (PB) elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. Conclusions Our study establishes GEX1A as a potent splicing inhibitor in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

  3. Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals

    NARCIS (Netherlands)

    Borensztajn, Keren; Sobrier, Marie-Laure; Duquesnoy, Philippe; Fischer, Anne-Marie; Tapon-Bretaudière, Jacqueline; Amselem, Serge

    2006-01-01

    Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5' splice sites are accurately identified remain controversial and incompletely resolved. The human F7

  4. A Novel c.554+5C>T Mutation in the DUOXA2 Gene Combined with p.R885Q Mutation in the DUOX2 Gene Causing Congenital Hypothyroidism.

    Science.gov (United States)

    Zheng, Xiao; Ma, Shao Gang; Qiu, Ya Li; Guo, Man Li; Shao, Xiao Juan

    2016-06-05

    The coexistence of mutations in the dual oxidase maturation factor 2 (DUOXA2) and dual oxidase 2 (DUOX2) genes is rarely identified in congenital hypothyroidism (CH). This study reports a boy with CH due to a novel splice-site mutation in the DUOXA2 gene and a missense mutation in the DUOX2 gene. A four-year-old boy was diagnosed with CH at neonatal screening and was enrolled in this study. The DUOXA2, DUOX2, thyroid peroxidase (TPO), and thyrotropin receptor (TSHR) genes were considered for genetic defects screening. Genomic DNA was extracted from peripheral blood leukocytes, and Sanger sequencing was used to screen the mutations in the exon fragments. Family members of the patient and the controls were also enrolled and evaluated. The boy harbored compound heterozygous mutations including a novel splice-site mutation c.554+5C>T in the maternal DUOXA2 allele and c.2654G>A (p.R885Q) in the paternal DUOX2 allele. The germline mutations from his parents were consistent with an autosomal recessive inheritance pattern. No mutations in the TPO and TSHR genes were detected. A novel splice-site mutation c.554+5C>T in the DUOXA2 gene and a mutation p.R885Q in the DUOX2 gene were identified in a 4-year-old patient with goitrous CH.

  5. Preventing Mitochondrial Diseases: Embryo-Sparing Donor-Independent Options.

    Science.gov (United States)

    Adashi, Eli Y; Cohen, I Glenn

    2018-05-01

    Mutant mitochondrial DNA gives rise to a broad range of incurable inborn maladies. Prevention may now be possible by replacing the mutation-carrying mitochondria of zygotes or oocytes at risk with donated unaffected counterparts. However, mitochondrial replacement therapy is being held back by theological, ethical, and safety concerns over the loss of human zygotes and the involvement of a donor. These concerns make it plain that the identification, validation, and regulatory adjudication of novel embryo-sparing donor-independent technologies remains a pressing imperative. This Opinion highlights three emerging embryo-sparing donor-independent options that stand to markedly allay theological, ethical, and safety concerns raised by mitochondrial replacement therapy. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Splicing landscape of the eight collaborative cross founder strains.

    Science.gov (United States)

    Zheng, Christina L; Wilmot, Beth; Walter, Nicole Ar; Oberbeck, Denesa; Kawane, Sunita; Searles, Robert P; McWeeney, Shannon K; Hitzemann, Robert

    2015-02-05

    The Collaborative Cross (CC) is a large panel of genetically diverse recombinant inbred mouse strains specifically designed to provide a systems genetics resource for the study of complex traits. In part, the utility of the CC stems from the extensive genome-wide annotations of founder strain sequence and structural variation. Still missing, however, are transcriptome-specific annotations of the CC founder strains that could further enhance the utility of this resource. We provide a comprehensive survey of the splicing landscape of the 8 CC founder strains by leveraging the high level of alternative splicing within the brain. Using deep transcriptome sequencing, we found that a majority of the splicing landscape is conserved among the 8 strains, with ~65% of junctions being shared by at least 2 strains. We, however, found a large number of potential strain-specific splicing events as well, with an average of ~3000 and ~500 with ≥3 and ≥10 sequence read coverage, respectively, within each strain. To better understand strain-specific splicing within the CC founder strains, we defined criteria for and identified high-confidence strain-specific splicing events. These splicing events were defined as exon-exon junctions 1) found within only one strain, 2) with a read coverage ≥10, and 3) defined by a canonical splice site. With these criteria, a total of 1509 high-confidence strain-specific splicing events were identified, with the majority found within two of the wild-derived strains, CAST and PWK. Strikingly, the overwhelming majority, 94%, of these strain-specific splicing events are not yet annotated. Strain-specific splicing was also located within genomic regions recently reported to be over- and under-represented within CC populations. Phenotypic characterization of CC populations is increasing; thus these results will not only aid in further elucidating the transcriptomic architecture of the individual CC founder strains, but they will also help in guiding

  7. International collaborative donor project.

    Science.gov (United States)

    Ríos Zambudio, Antonio

    2018-02-01

    The International Donor Collaborative Project (PCID) research group was created in 1996 in Spain with the aim of promoting research in the field of organ donation and transplantation, led by Spanish surgeons. During this period they have developed the questionnaires of the PCID, both the attitude towards cadaver and live donation, which are the most used questionnaires in publications in indexed journals. They have been the driving group of stratified studies representative of the populations under study, and of the performance of multivariate statistical analyzes in the field of psycho-social research in organ donation and transplantation. The main contributions of the group focus on the analysis of health center professionals and emerging migrant groups. In recent years, studies have been extended to the United States, Latin America (mainly Mexico) and Europe. Copyright © 2017 AEC. Publicado por Elsevier España, S.L.U. All rights reserved.

  8. Alternative splicing variations in mouse CAPS2: differential expression and functional properties of splicing variants

    Directory of Open Access Journals (Sweden)

    Furuichi Teiichi

    2007-04-01

    Full Text Available Abstract Background Ca2+-dependent activator protein 2 (CAPS2/CADPS2 is a secretory vesicle-associated protein involved in the release of neurotrophin. We recently reported that an aberrant, alternatively spliced CAPS2 mRNA that lacks exon 3 (CAPS2Δexon3 is detected in some patients with autism. Splicing variations in mouse CAPS2 and their expression and functions remain unclear. Results In this study, we defined 31 exons in the mouse CAPS2 gene and identified six alternative splicing variants, CAPS2a-f. CAPS2a is an isoform lacking exons 22 and 25, which encode part of the Munc13-1-homologous domain (MHD. CAPS2b lacks exon 25. CAPS2c lacks exons 11 and 22. CAPS2d, 2e, and 2f have C-terminal deletions from exon 14, exon 12, and exon 5, respectively. On the other hand, a mouse counterpart of CAPS2Δexon3 was not detected in the mouse tissues tested. CAPS2b was expressed exclusively in the brain, and the other isoforms were highly expressed in the brain, but also in some non-neural tissues. In the brain, all isoforms showed predominant expression patterns in the cerebellum. In the developing cerebellum, CAPS2b showed an up-regulated expression pattern, whereas the other isoforms exhibited transiently peaked expression patterns. CAPS2 proteins were mostly recovered in soluble fractions, but some were present in membrane fractions, except for CAPS2c and 2f, both of which lack the PH domain, suggesting that the PH domain is important for membrane association. In contrast to CAPS2a and 2b, CAPS2c showed slightly decreased BDNF-releasing activity, which is likely due to the C-terminal truncation of the PH domain in CAPS2c. Conclusion This study indicates that, in mouse, there are six splicing variants of CAPS2 (CAPS2a-f, and that these are subdivided into two groups: a long form containing the C-terminal MHD and a short form lacking the C-terminal MHD. These results demonstrate that the splicing variations correlate with their expression patterns and

  9. Donor selection criteria and procurement

    International Nuclear Information System (INIS)

    Agcaoili, N.R.

    1999-01-01

    Donor selection is one of the most important aspects of tissue banking practice. Without a good donor selection criteria, the results of any effort of trying to preserve tissues will have disastrous outcome for the recipient of these tissues. While with a very good and strict donor selection the Tissue Bank can guarantee safe and effective tissue allografts. There are significant aspects in the history and physical examination of the donor that must be emphasized. A donor exclusion criteria has also been formulated together with a list of all the needed laboratory examinations to eliminate possible diseases that may be transferred from the donor. The methods of procurement of tissue allografts from living and cadaver donors will be described. The limitations and advantages of each will be taken.There are also special restrictions that are important in the practice of removing the tissues from the donors. All the necessary equipment should be ready and the potential risk on the personnel should be known to all doing Tissue Banking

  10. [DNA probes for the alternative splicing region of the 6th exon of the human CSF-1 gene. Polymerase chain reaction and subcloning].

    Science.gov (United States)

    Lanina, T P; Aleksandrova, N M; Surin, V L; Iasenskaia, E V; Grineva, N I

    1993-12-01

    The DNA probes--pA6-CSF-1 and pA2-CSF-1 for the alternative splicing region of the 6 exon human CSF-1 gene were prepared using PCR and subsequent subcloning in pUC19 plasmid at the XmaI/BamHI sites. Due to the insert sequencing and blotting of human leukocytes DNA, the DNA probes obtained can be useful for screening of mutations in the human CSF-1 gene.

  11. Heart transplantation from older donors

    Directory of Open Access Journals (Sweden)

    V. N. Poptsov

    2017-01-01

    Full Text Available In the current situation of the shortage of suitable donor organs, heart transplantation from older donors is one of the ways to increase the performance of more heart transplants, particularly, in patients with urgent need of transplantation. While planning a heart transplantation from older donor one should consider increased risk of early cardiac allograft dysfunction, preexisting coronary artery disease, accelerated transplant vasculopathy which may adversely affect early and long-term survival of recipients. Subject to careful selection of donor–recipient pairs, effective prevention and treatment of early cardiac allograft dysfunction, pre-existing atherosclerosis and transplant vasculopathy the early and long-term survival of heart transplant recipients from older donors is comparable to heart transplantation from young donors.

  12. Dermal fibroblasts in Hutchinson-Gilford progeria syndrome with the lamin A G608G mutation have dysmorphic nuclei and are hypersensitive to heat stress

    Directory of Open Access Journals (Sweden)

    Worman Howard J

    2005-06-01

    Full Text Available Abstract Background Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670 is a rare sporadic disorder with an incidence of approximately 1 per 8 million live births. The phenotypic appearance consists of short stature, sculptured nose, alopecia, prominent scalp veins, small face, loss of subcutaneous fat, faint mid-facial cyanosis, and dystrophic nails. HGPS is caused by mutations in LMNA, the gene that encodes nuclear lamins A and C. The most common mutation in subjects with HGPS is a de novo single-base pair substitution, G608G (GGC>GGT, within exon 11 of LMNA. This creates an abnormal splice donor site, leading to expression of a truncated protein. Results We studied a new case of a 5 year-old girl with HGPS and found a heterozygous point mutation, G608G, in LMNA. Complementary DNA sequencing of RNA showed that this mutation resulted in the deletion of 50 amino acids in the carboxyl-terminal tail domain of prelamin A. We characterized a primary dermal fibroblast cell line derived from the subject's skin. These cells expressed the mutant protein and exhibited a normal growth rate at early passage in primary culture but showed alterations in nuclear morphology. Expression levels and overall distributions of nuclear lamins and emerin, an integral protein of the inner nuclear membrane, were not dramatically altered. Ultrastructural analysis of the nuclear envelope using electron microscopy showed that chromatin is in close association to the nuclear lamina, even in areas with abnormal nuclear envelope morphology. The fibroblasts were hypersensitive to heat shock, and demonstrated a delayed response to heat stress. Conclusion Dermal fibroblasts from a subject with HGPS expressing a mutant truncated lamin A have dysmorphic nuclei, hypersensitivity to heat shock, and delayed response to heat stress. This suggests that the mutant protein, even when expressed at low levels, causes defective cell stability, which may be responsible for phenotypic

  13. Haplotype and quantitative transcript analyses of Portuguese breast/ovarian cancer families with the BRCA1 R71G founder mutation of Galician origin.

    Science.gov (United States)

    Santos, Catarina; Peixoto, Ana; Rocha, Patrícia; Vega, Ana; Soares, Maria José; Cerveira, Nuno; Bizarro, Susana; Pinheiro, Manuela; Pereira, Deolinda; Rodrigues, Helena; Castro, Fernando; Henrique, Rui; Teixeira, Manuel R

    2009-01-01

    We investigated the functional effect of the missense variant c.211A>G (R71G) localized at position -2 of exon 5 donor splice site in the BRCA1 gene and evaluated whether Portuguese and Galician families with this mutation share a common ancestry. Three unrelated Portuguese breast/ovarian cancer families carrying this variant were studied through qualitative and quantitative transcript analyses. We also evaluated the presence of loss of heterozigosity and the histopathologic characteristics of the carcinomas in those families. Informative families (two from Portugal and one from Galicia) were genotyped for polymorphic microsatellite markers flanking BRCA1 to reconstruct haplotypes. Qualitative RNA analysis revealed the presence of two alternative transcripts both in carriers of the BRCA1 R71G variant and in controls. Semi-quantitative fragment analysis and real-time RT-PCR showed a significant increase of the transcript with an out of frame deletion of the last 22nt of exon 5 (BRCA1-Delta22ntex5) and a decrease of the full-length transcript (BRCA1-ex5FL) in patients carrying the R71G mutation as compared to controls, whereas no significant differences were found for the transcript with in frame skipping of exon 5 (BRCA1-Deltaex5). One haplotype was found to segregate in the two informative Portuguese families and in the Galician family. We demonstrate that disruption of alternative transcript ratios is the mechanism causing hereditary breast/ovarian cancer associated with the BRCA1 R71G mutation. Furthermore, our findings indicate a common ancestry of the Portuguese and Galician families sharing this mutation.

  14. Androgen Receptor Splice Variants and Resistance to Taxane Chemotherapy

    Science.gov (United States)

    2016-10-01

    report. Inventions, patent applications, and/or licenses Nothing to report. Others Nothing to report. 7. Participants & Other... Brand LJ et al: Androgen receptor splice variants mediate enzalutamide resistance in castration-resistant prostate cancer cell lines. Cancer Res

  15. An unusual splice defect in the mitofusin 2 gene (MFN2 is associated with degenerative axonopathy in Tyrolean Grey cattle.

    Directory of Open Access Journals (Sweden)

    Cord Drögemüller

    Full Text Available Tyrolean Grey cattle represent a local breed with a population size of ∼5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2, is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.

  16. [c.2381-3T>C mutation of DMD gene: a rare SNP without significant pathogenicity].

    Science.gov (United States)

    Yan, Yuanlong; Yang, Yuan

    2015-02-01

    To clarify the nature of a DMD splice acceptor mutation c.2381-3T>C. Genomic DNA was extracted from 5 members of a family affected with DMD. For an obligatory carrier, after excluding gross deletion and duplication of the DMD gene with multiplex ligation-dependent probe amplification (MLPA) method, all coding and splice site sequences of the DMD gene were analyzed with Next Generation Sequencing followed by confirmation with targeted Sanger sequencing. Mutations of the carrier were detected in other 4 members. For the splice site mutation, mini-gene was constructed and expressed in vitro to detect the number of transcript and cDNA sequence. A known nonsense mutation (c.8038C>T, p.Arg2680Ter) was identified in the carrier, her sister and the mother. The rest 4 members, except for the mother from the first generation, have all carried the c.2381-3T>C mutation. The latter has been described as a splice site mutation to cause DMD. One of 135 male adults without DMD was also detected to have carried the c.2381-3T>C mutation. No additional transcript was produced by the mini-genes containing c.2381-3T>C mutation. The c.8038C>T(p.Arg2680Ter)mutation of DMD gene probably underlies the disease in this family. The presence of the c.2381-3T>C mutation in a asymptomatic male and a non-DMD male control, together with the normal in vitro expression of the mini-gene carrying the c.2381-3T>C, strongly suggested that the c.2381-3T>C mutation collected in the Human Gene Mutation Database is a rare SNP without significant pathogenicity.

  17. Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains.

    Science.gov (United States)

    Salzman, Gabriel S; Ackerman, Sarah D; Ding, Chen; Koide, Akiko; Leon, Katherine; Luo, Rong; Stoveken, Hannah M; Fernandez, Celia G; Tall, Gregory G; Piao, Xianhua; Monk, Kelly R; Koide, Shohei; Araç, Demet

    2016-09-21

    Adhesion G protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we term Pentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development in vivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Alternative Splicing of CHEK2 and Codeletion with NF2 Promote Chromosomal Instability in Meningioma

    Directory of Open Access Journals (Sweden)

    Hong Wei Yang

    2012-01-01

    Full Text Available Mutations of the NF2 gene on chromosome 22q are thought to initiate tumorigenesis in nearly 50% of meningiomas, and 22q deletion is the earliest and most frequent large-scale chromosomal abnormality observed in these tumors. In aggressive meningiomas, 22q deletions are generally accompanied by the presence of large-scale segmental abnormalities involving other chromosomes, but the reasons for this association are unknown. We find that large-scale chromosomal alterations accumulate during meningioma progression primarily in tumors harboring 22q deletions, suggesting 22q-associated chromosomal instability. Here we show frequent codeletion of the DNA repair and tumor suppressor gene, CHEK2, in combination with NF2 on chromosome 22q in a majority of aggressive meningiomas. In addition, tumor-specific splicing of CHEK2 in meningioma leads to decreased functional Chk2 protein expression. We show that enforced Chk2 knockdown in meningioma cells decreases DNA repair. Furthermore, Chk2 depletion increases centrosome amplification, thereby promoting chromosomal instability. Taken together, these data indicate that alternative splicing and frequent codeletion of CHEK2 and NF2 contribute to the genomic instability and associated development of aggressive biologic behavior in meningiomas.

  19. Ire1 Has Distinct Catalytic Mechanisms for XBP1/HAC1 Splicing and RIDD

    Directory of Open Access Journals (Sweden)

    Arvin B. Tam

    2014-11-01

    Full Text Available An evolutionarily conserved unfolded protein response (UPR component, IRE1, cleaves XBP1/HAC1 introns in order to generate spliced mRNAs that are translated into potent transcription factors. IRE1 also cleaves endoplasmic-reticulum-associated RNAs leading to their decay, an activity termed regulated IRE1-dependent decay (RIDD; however, the mechanism by which IRE1 differentiates intron cleavage from RIDD is not well understood. Using in vitro experiments, we found that IRE1 has two different modes of action: XBP1/HAC1 is cleaved by IRE1 subunits acting cooperatively within IRE1 oligomers, whereas a single subunit of IRE1 performs RIDD without cooperativity. Furthermore, these distinct activities can be separated by complementation of catalytically inactive IRE1 RNase and mutations at oligomerization interfaces. Using an IRE1 RNase inhibitor, STF-083010, selective inhibition of XBP1 splicing indicates that XBP1 promotes cell survival, whereas RIDD leads to cell death, revealing modulation of IRE1 activities as a drug-development strategy.

  20. Width of gene expression profile drives alternative splicing.

    Directory of Open Access Journals (Sweden)

    Daniel Wegmann

    Full Text Available Alternative splicing generates an enormous amount of functional and proteomic diversity in metazoan organisms. This process is probably central to the macromolecular and cellular complexity of higher eukaryotes. While most studies have focused on the molecular mechanism triggering and controlling alternative splicing, as well as on its incidence in different species, its maintenance and evolution within populations has been little investigated. Here, we propose to address these questions by comparing the structural characteristics as well as the functional and transcriptional profiles of genes with monomorphic or polymorphic splicing, referred to as MS and PS genes, respectively. We find that MS and PS genes differ particularly in the number of tissues and cell types where they are expressed.We find a striking deficit of PS genes on the sex chromosomes, particularly on the Y chromosome where it is shown not to be due to the observed lower breadth of expression of genes on that chromosome. The development of a simple model of evolution of cis-regulated alternative splicing leads to predictions in agreement with these observations. It further predicts the conditions for the emergence and the maintenance of cis-regulated alternative splicing, which are both favored by the tissue specific expression of splicing variants. We finally propose that the width of the gene expression profile is an essential factor for the acquisition of new transcript isoforms that could later be maintained by a new form of balancing selection.

  1. Cell-Type-Specific Splicing of Piezo2 Regulates Mechanotransduction

    Directory of Open Access Journals (Sweden)

    Marcin Szczot

    2017-12-01

    Full Text Available Summary: Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which transduction is tuned, permitting the detection of a variety of mechanosensory stimuli. : Szczot et al. find that the mechanoreceptor Piezo2 is extensively alternatively spliced, generating multiple distinct isoforms. Their findings indicate that these splice products have specific tissue and cell type expression patterns and exhibit differences in receptor properties. Keywords: Piezo, touch, sensation, ion-channel, splicing

  2. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Zodwa Dlamini

    2015-11-01

    Full Text Available Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets.

  3. Voluntary whole-blood donors, and compensated platelet donors and plasma donors: motivation to donate, altruism and aggression.

    Science.gov (United States)

    Trimmel, Michael; Lattacher, Helene; Janda, Monika

    2005-10-01

    To establish if voluntary whole-blood donors and compensated platelet donors and plasma donors may differ in their motivation to donate, altruism, aggression and autoaggression. Whole-blood (n=51), platelet (n=52) and plasma donors (n=48) completed a battery of validated questionnaires while waiting to donate. Bivariate and multivariate analyses of variance and t-tests were performed to detect differences between groups as noted. Altruism (mean=40.2) was slightly higher in whole-blood donors than in platelet (mean=38.3) and plasma donors (mean=39.1) (p=0.07). Blood donors (mean=2.8) scored lower in the spontaneous aggression measure than platelet (mean=4.1) and plasma donors (mean=4.4) (p=0.01). Plasma donors (mean=4.9) had higher auto-aggression than whole-blood donors and platelet donors (mean for both groups=3.4) (p=0.01). Differences between the three groups were mediated by sociodemographic variables (MANCOVA). Whole-blood donors donated to help others, platelet and plasma donors mostly to receive the compensation. However, those platelet and plasma donors, who would continue to donate without compensation were similar in altruism and aggression to whole-blood donors. While most platelet donors and plasma donors were motivated by the compensation, those who stated that they would continue to donate without compensation had altruism and aggression scores similar to voluntary whole-blood donors.

  4. A statistical framework for genome-wide discovery of biomarker splice variations with GeneChip Human Exon 1.0 ST Arrays.

    Science.gov (United States)

    Yoshida, Ryo; Numata, Kazuyuki; Imoto, Seiya; Nagasaki, Masao; Doi, Atsushi; Ueno, Kazuko; Miyano, Satoru

    2006-01-01

    Alternative splicing is an important regulatory mechanism that generates multiple mRNA transcripts which are transcribed into functionally diverse proteins. According to the current studies, aberrant transcripts due to splicing mutations are known to cause for 15% of genetic diseases. Therefore understanding regulatory mechanism of alternative splicing is essential for identifying potential biomarkers for several types of human diseases. Most recently, advent of GeneChip Human Exon 1.0 ST Array enables us to measure genome-wide expression profiles of over one million exons. With this new microarray platform, analysis of functional gene expressions could be extended to detect not only differentially expressed genes, but also a set of specific-splicing events that are differentially observed between one or more experimental conditions, e.g. tumor or normal control cells. In this study, we address the statistical problems to identify differentially observed splicing variations from exon expression profiles. The proposed method is organized according to the following process: (1) Data preprocessing for removing systematic biases from the probe intensities. (2) Whole transcript analysis with the analysis of variance (ANOVA) to identify a set of loci that cause the alternative splicing-related to a certain disease. We test the proposed statistical approach on exon expression profiles of colorectal carcinoma. The applicability is verified and discussed in relation to the existing biological knowledge. This paper intends to highlight the potential role of statistical analysis of all exon microarray data. Our work is an important first step toward development of more advanced statistical technology. Supplementary information and materials are available from http://bonsai.ims.u-tokyo.ac.jp/~yoshidar/IBSB2006_ExonArray.htm.

  5. De novo ABCD1 gene mutation in an Indian patient with adrenoleukodystrophy.

    Science.gov (United States)

    Kumar, Neeraj; Shukla, Pallavi; Taneja, Krishna K; Kalra, Veena; Bansal, Surendra K

    2008-10-01

    A large number of ABCD1 gene mutations have been reported all over the world, but not previously in India. We report on the first known patient with childhood cerebral adrenoleukodystrophy and a de novo 3' splice-site mutation in this gene. Magnetic resonance imaging of the brain revealed large, confluent, hyperintense areas in the bilateral cerebral white matter, predominantly parieto-occipital, with extensions into posterior regions that led to breakdown of the blood-brain barrier. An increased level of very long chain fatty acids was also consistent with the biochemical defect for adrenoleukodystrophy. Sequencing of the ABCD1 gene of this patient identified a 3' splice-site mutation in the intervening sequence 4 (-2a > g). We did not find any mutation in the gene of the proband's mother, which confirms its de novo occurrence.

  6. CRISPR/Cas9-mediated target validation of the Splicing Inhibitor Pladienolide B

    KAUST Repository

    Aouida, Mustapha

    2016-02-24

    CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation.

  7. Novel female-specific trans-spliced and alternative splice forms of dsx in the silkworm Bombyx mori.

    Science.gov (United States)

    Duan, Jianping; Xu, Hanfu; Wang, Feng; Ma, Sanyuan; Zha, Xingfu; Guo, Huizhen; Zhao, Ping; Xia, Qingyou

    2013-02-15

    The Bombyx mori doublesex gene (Bmdsx) plays an important role in somatic sexual development. Its pre-mRNA splices in a sex-specific manner to generate two female-specific and one male-specific splice forms. The present study investigated six novel dsx variants generated by trans-splicing between female dsx transcripts and two additional novel genes, dsr1 and dsr2. Expression analysis indicated that Bmdsx-dsr1 represented splicing noise, whereas dsr2, which trans-spliced with dsx to generate five variants, regulated the expression of the female-specific B. mori dsx transcript Bmdsx(F)s. We unexpectedly found a novel exon 2n insertion during Bmdsx transcription, which did not influence the validity of the novel protein, BmDSX(F3). Ectopic expression of BmDSX(F3) repressed the pheromone-binding protein gene and the testis-specific gene A2 in males, and activated of the storage protein 1 gene. Our findings suggest that trans-splicing is a novel regulatory function of Bmdsx, which participates in female sexual development by regulating the expression of three BmDSX(F) proteins. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. [Preliminary analysis of mutations in X-linked adrenoleukodystrophy gene(ABCD1) in Chinese patients].

    Science.gov (United States)

    Xiong, Hui; Pan, Hong; Zhang, Yue-hua; Wu, Xi-ru

    2003-10-01

    To detect the mutations in exon 6 of ABCD1 gene encoding adrenoleukodystrophy protein(ALDP) in Chinese X-linked adrenoleukodystrophy (ALD MIM 300100) patients. Genomic DNA from 14 unrelated patients and two patients' parents with X-linked ALD was extracted using standard procedures from the peripheral blood leukocytes. Polymerase chain reaction (PCR) and DNA direct sequencing were employed to analyze exon 6 of ABCD1 gene. Three mutations in exon 6 were identified in 3 of 14 patients. One mutation was deleted 1 base pair at splice acceptor-site (1489-6 del C). It was not clear what the effect of this mutation is on the ALD protein, maybe induce splicing error. One missense mutation: T1559A(L520Q). These two patients' mothers were heterozygous. The third patient had a mutation: G1548A (L516 L), which is a known polymorphism. It was not a disease causing mutation, so there should be another mutation in this patient. For the first time, mutations in ABCD1 are identified in Chinese ALD patients in the mainland of China. No major gene deletion or rearrangement is detected in exon 6. Despite many mutations having been identified in patients with these clinical phenotypes, the genotype-phenotype correlations have not been clarified, suggesting that other genetic or environmental factors may also be involved in determining phenotypic expression in ALD. Two carriers are also confirmed.

  9. First report of a novel LMNA mutation in a Chinese family with limb ...

    Indian Academy of Sciences (India)

    2014-12-12

    Dec 12, 2014 ... LMNA gene encodes the ubiquitous protein lamin A/C, which is a component of the fibrous meshwork of interme- diate filaments located at the inner surface of the nuclear envelope (Worman 2012). Lamin A/C are splicing vari- ants encoded by the LMNA gene and share the first. 566 amino acids. Mutations ...

  10. Uncovering the role of p53 splice variants in human malignancy: a clinical perspective

    Directory of Open Access Journals (Sweden)

    Surget S

    2013-12-01

    Full Text Available Sylvanie Surget,1,2 Marie P Khoury,1,2 Jean-Christophe Bourdon1,21Dundee Cancer Centre, 2Jacqui Wood Cancer Centre, Ninewells Hospital, University of Dundee, Dundee, UKAbstract: Thirty-five years of research on p53 gave rise to more than 68,000 articles and reviews, but did not allow the uncovering of all the mysteries that this major tumor suppressor holds. How p53 handles the different signals to decide the appropriate cell fate in response to a stress and its implication in tumorigenesis and cancer progression remains unclear. Nevertheless, the uncovering of p53 isoforms has opened new perspectives in the cancer research field. Indeed, the human TP53 gene encodes not only one but at least twelve p53 protein isoforms, which are produced in normal tissues through alternative initiation of translation, usage of alternative promoters, and alternative splicing. In recent years, it became obvious that the different p53 isoforms play an important role in regulating cell fate in response to different stresses in normal cells by differentially regulating gene expression. In cancer cells, abnormal expression of p53 isoforms contributes actively to cancer formation and progression, regardless of TP53 mutation status. They can also be associated with response to treatment, depending on the cell context. The determination of p53 isoform expression and p53 mutation status helps to define different subtypes within a particular cancer type, which would have different responses to treatment. Thus, the understanding of the regulation of p53 isoform expression and their biological activities in relation to the cellular context would constitute an important step toward the improvement of the diagnostic, prognostic, and predictive values of p53 in cancer treatment. This review aims to summarize the involvement of p53 isoforms in cancer and to highlight novel potential therapeutic targets.Keywords: p53, isoforms, p63, p73, alternative splicing, cancer

  11. Donor milk: current perspectives

    Directory of Open Access Journals (Sweden)

    Giuliani F

    2014-07-01

    Full Text Available Francesca Giuliani,1 Ilaria Rovelli,1 Chiara Peila,1 Stefania Alfonsina Liguori,2 Enrico Bertino,1 Alessandra Coscia1 1SCDU Neonatologia, Dipartimento di Scienze Pediatriche e dell'Adolescenza, Università degli Studi di Torino, Torino, Italy; 2SC Neonatologia, Ospedale Maria Vittoria, Torino, Italy Abstract: Mother's own milk is widely recognized as the optimal feeding for term infants, but increasing evidence exists of its benefits also for sick and preterm infants in neonatal intensive care units. However, the nutritional needs for appropriate growth and neurodevelopmental outcomes of such a particular population of infants should be attentively evaluated, considering also the indication to an appropriate fortification of human milk. The target is to achieve growth potential for preterm newborns while ensuring good metabolic outcomes and normal neurological development. When mother's milk is unavailable or in short supply, donor human milk (DHM represents the second best choice and, although somewhat modified by the Holder pasteurization process, it preserves many benefits when compared to formula, as documented by more and more reports, randomized controlled trials, and meta-analyses published in the past few years. Evidence exists of the protection exerted by DHM from necrotizing enterocolitis, while further studies are required to look at possible beneficial effects regarding infections, bronchopulmonary dysplasia, long-term cardiovascular risk factors, feeding tolerance, neurological outcome, and allergy. Finally, the concern that the use of DHM might decrease preterm infant breastfeeding is being raised. Conversely, publications exist showing that the use of DHM in the neonatal unit increases breastfeeding rates at discharge for infants of very low birth weight. Keywords: human milk, preterm infant feeding, milk bank, breast milk, mother's own milk, pasteurized human milk, fortification

  12. Iron deficiency among blood donors

    DEFF Research Database (Denmark)

    Rigas, A S; Pedersen, O B; Magnussen, K

    2017-01-01

    and menopausal status are the strongest predictors of iron deficiency. Only little information on the health effects of iron deficiency in blood donors exits. Possibly, after a standard full blood donation, a temporarily reduced physical performance for women is observed. However, iron deficiency among blood...... donors is not reflected in a reduced self-perceived mental and physical health. In general, the high proportion of iron-deficient donors can be alleviated either by extending the inter-donation intervals or by guided iron supplementation. The experience from Copenhagen, the Capital Region of Denmark......, is that routine ferritin measurements and iron supplementation are feasible and effective ways of reducing the proportion of donors with low haemoglobin levels....

  13. Research Award: Donor Partnerships Division

    International Development Research Centre (IDRC) Digital Library (Canada)

    Corey Piccioni

    2013-08-07

    . The successful candidate must be willing to work as a member of the DPD team in its ongoing acvies, including providing research analysis and coordinang the producon and disseminaon of informaon related to donor.

  14. The SPF27 homologue Num1 connects splicing and kinesin 1-dependent cytoplasmic trafficking in Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Nikola Kellner

    2014-01-01

    Full Text Available The conserved NineTeen protein complex (NTC is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking.

  15. Method of predicting Splice Sites based on signal interactions

    Directory of Open Access Journals (Sweden)

    Deogun Jitender S

    2006-04-01

    Full Text Available Abstract Background Predicting and proper ranking of canonical splice sites (SSs is a challenging problem in bioinformatics and machine learning communities. Any progress in SSs recognition will lead to better understanding of splicing mechanism. We introduce several new approaches of combining a priori knowledge for improved SS detection. First, we design our new Bayesian SS sensor based on oligonucleotide counting. To further enhance prediction quality, we applied our new de novo motif detection tool MHMMotif to intronic ends and exons. We combine elements found with sensor information using Naive Bayesian Network, as implemented in our new tool SpliceScan. Results According to our tests, the Bayesian sensor outperforms the contemporary Maximum Entropy sensor for 5' SS detection. We report a number of putative Exonic (ESE and Intronic (ISE Splicing Enhancers found by MHMMotif tool. T-test statistics on mouse/rat intronic alignments indicates, that detected elements are on average more conserved as compared to other oligos, which supports our assumption of their functional importance. The tool has been shown to outperform the SpliceView, GeneSplicer, NNSplice, Genio and NetUTR tools for the test set of human genes. SpliceScan outperforms all contemporary ab initio gene structural prediction tools on the set of 5' UTR gene fragments. Conclusion Designed methods have many attractive properties, compared to existing approaches. Bayesian sensor, MHMMotif program and SpliceScan tools are freely available on our web site. Reviewers This article was reviewed by Manyuan Long, Arcady Mushegian and Mikhail Gelfand.

  16. Refractory macrocytic anemias in patients with clonal hematopoietic disorders and isolated mutations of the spliceosome gene ZRSR2.

    Science.gov (United States)

    Fleischman, Roger A; Stockton, Shannon S; Cogle, Christopher R

    2017-10-01

    Although mutations in RNA splicing genes occur frequently in patients with clonal cytopenias of unknown significance (CCUS) and myelodysplastic syndromes (MDS), very often additional common myeloid gene driver mutations are present at diagnosis. Thus, the clinical significance of isolated mutations in the most commonly mutated RNA splicing genes remains unknown. Here we report five unusual patients with an isolated mutation causing a loss of function of ZRSR2, a protein required for recognition of a functional 3' splice site. Two of the patients had a diagnosis of CCUS and three patients had an MDS disorder characterized by low risk features and absence of complex cytogenetic abnor-malities. Notably, all five cases were characterized predominantly by macrocytic anemia. In addition, one CCUS patient followed for more than 15 years with a transfusion dependent macrocytic anemia was found to have an inactivating ZRSR2 mutation with an allele frequency of >60%. We conclude that the common clinical features of patients with an isolated mutation of ZRSR2 are a macrocytic anemia without leukopenia, thrombocytopenia or an increase in marrow blast percentage. At least in some cases, the presence of an isolated ZRSR2 mutation can accompany a dominant hematopoietic clone with a low risk for transformation to frank dysplasia or acute leukemia. Copyright © 2017. Published by Elsevier Ltd.

  17. Convergent origins and rapid evolution of spliced leader trans-splicing in metazoa: insights from the ctenophora and hydrozoa.

    Scie