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Anna Chiara Nascimbeni Marina Fanin Elisabetta Tasca Corrado Angelini 《Human mutation》2010,31(9):E1658-E1669
Variants of unknown significance in the CAPN3 gene constitute a significant challenge for genetic counselling. Despite the frequency of intronic nucleotide changes in this gene (15‐25% of all mutations), so far their pathogenicity has only been inferred by in‐silico analysis, and occasionally, proven by RNA analysis. In this study, 5 different intronic variants (one novel) that bioinformatic tools predicted would affect RNA splicing, underwent comprehensive studies which were designed to prove they are disease‐causing. Muscle mRNA from 15 calpainopathy patients was analyzed by RT‐PCR and splicing‐specific‐PCR tests. We established the previously unrecognized pathogenicity of these mutations, which caused aberrant splicing, most frequently by the activation of cryptic splicing sites or, occasionally, by exon skipping. The absence or severe reduction of protein demonstrated their deleterious effect at translational level. We concluded that bioinformatic tools are valuable to suggest the potential effects of intronic variants; however, the experimental demonstration of the pathogenicity is not always easy to do even when using RNA analysis (low abundance, degradation mechanisms), and it might not be successful unless splicing‐specific‐PCR tests are used. A comprehensive approach is therefore recommended to identify and describe unclassified variants in order to offer essential data for basic and clinical geneticists. © 2010 Wiley‐Liss, Inc. 相似文献
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The molecular genetic cause of over 3,000 monogenic disorders is currently unknown. This review discusses how novel genomic techniques like Next‐Generation DNA Sequencing (NGS) and genotyping arrays open new avenues in the elucidation of genetic defects causing monogenic disorders. They will not only speed up disease gene identification but will enable us to systematically tackle previously intractable monogenic disorders. These are mainly disorders not amenable to classic linkage analysis, for example, due to insufficient family size. Most monogenic diseases are caused by exonic mutations or splice‐site mutations changing the amino acid sequence of the affected gene. These mutations can be identified by sequencing of all exons in the human genome (exome sequencing) rendering whole genome sequencing unnecessary in most cases. Genotyping arrays containing 105–2×106 single nucleotide polymorphisms (SNPs) and nonpolymorphic markers allow highly accurate mapping of genomic deletions and duplications not detectable by exome sequencing, which are the second most common cause of monogenic disorders. However, several hundred rare, previously unknown sequence variants affecting the amino acid sequence of the encoded protein are found in the exome of every human individual. Therefore, the main challenge will be the differentiation between the many rare benign variants detected by novel genomic techniques and disease causing mutations. Hum Mutat 32:144–151, 2011. © 2011 Wiley‐Liss, Inc. 相似文献
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Mutations in SNRPB,Encoding Components of the Core Splicing Machinery,Cause Cerebro‐Costo‐Mandibular Syndrome
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Séverine Bacrot Mathilde Doyard Céline Huber Olivier Alibeu Niklas Feldhahn Daphné Lehalle Didier Lacombe Sandrine Marlin Patrick Nitschke Florence Petit Marie‐Paule Vazquez Arnold Munnich Valérie Cormier‐Daire 《Human mutation》2015,36(2):187-190
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Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha‐glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre‐messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.‐32‐13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment. 相似文献
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Leveraging splice‐affecting variant predictors and a minigene validation system to identify Mendelian disease‐causing variants among exon‐captured variants of uncertain significance
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Zachry T. Soens Justin Branch Shijing Wu Zhisheng Yuan Yumei Li Hui Li Keqing Wang Mingchu Xu Lavan Rajan Fabiana L. Motta Renata T. Simões Irma Lopez‐Solache Radwan Ajlan David G. Birch Peiquan Zhao Fernanda B. Porto Juliana Sallum Robert K. Koenekoop Ruifang Sui Rui Chen 《Human mutation》2017,38(11):1521-1533
The genetic heterogeneity of Mendelian disorders results in a significant proportion of patients that are unable to be assigned a confident molecular diagnosis after conventional exon sequencing and variant interpretation. Here, we evaluated how many patients with an inherited retinal disease (IRD) have variants of uncertain significance (VUS) that are disrupting splicing in a known IRD gene by means other than affecting the canonical dinucleotide splice site. Three in silico splice‐affecting variant predictors were leveraged to annotate and prioritize variants for splicing functional validation. An in vitro minigene system was used to assay each variant's effect on splicing. Starting with 745 IRD patients lacking a confident molecular diagnosis, we validated 23 VUS as splicing variants that likely explain disease in 26 patients. Using our results, we optimized in silico score cutoffs to guide future variant interpretation. Variants that alter base pairs other than the canonical GT‐AG dinucleotide are often not considered for their potential effect on RNA splicing but in silico tools and a minigene system can be utilized for the prioritization and validation of such splice‐disrupting variants. These variants can be overlooked causes of human disease but can be identified using conventional exon sequencing with proper interpretation guidelines. 相似文献
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Kevin T. Booth Hela Azaiez Kimia Kahrizi Donghong Wang Yuzhou Zhang Kathy Frees Carla Nishimura Hossein Najmabadi Richard J Smith 《Human mutation》2018,39(3):433-440
Dysregulation of splicing is a common factor underlying many inherited diseases including deafness. For one deafness‐associated gene, DFNA5, perturbation of exon 8 splicing results in a constitutively active truncated protein. To date, only intronic mutations have been reported to cause exon 8 skipping in patients with DFNA5‐related deafness. In five families with postlingual progressive autosomal dominant non‐syndromic hearing loss, we employed two next‐generation sequencing platforms—OtoSCOPE and whole exome sequencing—followed by variant filtering and prioritization based on both minor allele frequency and functional consequence using a customized bioinformatics pipeline to identify three novel and two recurrent mutations in DFNA5 that segregated with hearing loss in these families. The three novel mutations are all missense variants within exon 8 that are predicted computationally to decrease splicing efficiency or abolish it completely. We confirmed their functional impact in vitro using mini‐genes carrying each mutant DFNA5 exon 8. In so doing, we present the first exonic mutations in DFNA5 to cause deafness, expand the mutational spectrum of DFNA5‐related hearing loss, and highlight the importance of assessing the effect of coding variants on splicing. 相似文献
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T.B. Gagliardi F.E. Paula M.A. Iwamoto J.L. Proença‐Modena A.E. Santos A.A. Camara M.C. Cervi O.A.L. Cintra E. Arruda 《Journal of medical virology》2013,85(10):1852-1859
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Marshall JD Hinman EG Collin GB Beck S Cerqueira R Maffei P Milan G Zhang W Wilson DI Hearn T Tavares P Vettor R Veronese C Martin M So WV Nishina PM Naggert JK 《Human mutation》2007,28(11):1114-1123
Alstr?m syndrome is a monogenic recessive disorder featuring an array of clinical manifestations, with systemic fibrosis and multiple organ involvement, including retinal degeneration, hearing loss, childhood obesity, diabetes mellitus, dilated cardiomyopathy (DCM), urological dysfunction, and pulmonary, hepatic, and renal failure. We evaluated a large cohort of patients with Alstr?m syndrome for mutations in the ALMS1 gene. In total, 79 disease-causing variants were identified, of which 55 are novel mutations. The variants are primarily clustered in exons 8, 10, and 16, although we also identified novel mutations in exons 12 and 18. Most alleles were identified only once (45/79), but several were found recurrently. Founder effects are likely in families of English and Turkish descent. We also identified 66 SNPs and assessed the functional significance of these variants based on the conserved identity of the protein and the severity of the resulting amino acid substitution. A genotype-phenotype association study examining 18 phenotypic parameters in a subset of 58 patients found suggestive associations between disease-causing variants in exon 16 and the onset of retinal degeneration before the age of 1 year (P = 0.02), the occurrence of urological dysfunction (P = 0.02), of DCM (P = 0.03), and of diabetes (P = 0.03). A significant association was found between alterations in exon 8 and absent, mild, or delayed renal disease (P = 0.0007). This data may have implications for the understanding of the molecular mechanisms of ALMS1 and provides the basis for further investigation of how alternative splicing of ALMS1 contributes to the severity of the disease. 相似文献
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Extension of the Pompe mutation database by linking disease‐associated variants to clinical severity
Monica Y. Nio Stijn L.M. in 't Groen Atze J. Bergsma Nadine A.M.E. van der Beek Marian Kroos Marianne Hoogeveen‐Westerveld Ans T. van der Ploeg W.W.M. Pim Pijnappel 《Human mutation》2019,40(11):1954-1967
Pompe disease is an autosomal recessive lysosomal storage disorder caused by disease‐associated variants in the acid alpha‐glucosidase (GAA) gene. The current Pompe mutation database provides a severity rating of GAA variants based on in silico predictions and expression studies. Here, we extended the database with clinical information of reported phenotypes. We added additional in silico predictions for effects on splicing and protein function and for cross reactive immunologic material (CRIM) status, minor allele frequencies, and molecular analyses. We analyzed 867 patients and 562 GAA variants. Based on their combination with a GAA null allele (i.e., complete deficiency of GAA enzyme activity), 49% of the 422 disease‐associated variants could be linked to classic infantile, childhood, or adult phenotypes. Predictions and immunoblot analyses identified 131 CRIM negative and 216 CRIM positive variants. While disease‐associated missense variants were found throughout the GAA protein, they were enriched up to seven‐fold in the catalytic site. Fifteen percent of disease‐associated missense variants were predicted to affect splicing. This should be confirmed using splicing assays. Inclusion of clinical severity rating in the Pompe mutation database provides an invaluable tool for diagnosis, prognosis of disease progression, treatment regimens, and the future development of personalized medicine for Pompe disease. 相似文献
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Capillary Electrophoresis Analysis of Conventional Splicing Assays: IARC Analytical and Clinical Classification of 31 BRCA2 Genetic Variants
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Gorka Ruiz de Garibay Alberto Acedo Zaida García‐Casado Sara Gutiérrez‐Enríquez Alicia Tosar Atocha Romero Pilar Garre Gemma Llort Mads Thomassen Orland Díez Pedro Pérez‐Segura Eduardo Díaz‐Rubio Eladio A. Velasco Trinidad Caldés Miguel de la Hoya 《Human mutation》2014,35(1):53-57
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A noncoding variant in GANAB explains isolated polycystic liver disease (PCLD) in a large family
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Whitney Besse Jungmin Choi Dina Ahram Shrikant Mane Simone Sanna‐Cherchi Vicente Torres Stefan Somlo 《Human mutation》2018,39(3):378-382
Expanded mutation detection and novel gene discovery for isolated polycystic liver disease (PCLD) are necessary as 50% of cases do not have identified mutations in the seven published disease genes. We investigated a family with five affected siblings for which no loss‐of‐function variants were identified by whole exome sequencing analysis. SNP genotyping and linkage analysis narrowed the candidate regions to ~8% of the genome, which included two published PCLD genes in close proximity to each other, GANAB and LRP5. Based on these findings, we re‐evaluated the exome sequencing data and identified a novel intronic nine base pair deletion in the vicinity of the GANAB exon 24 splice donor that had initially been discarded by the sequence analysis pipelines. We used a minigene assay to show that this deletion leads to skipping of exon 24 in cell lines and primary human cholangiocytes. These findings prompt genomic evaluation beyond the coding region to enhance mutation detection in PCLD and to avoid premature implication of other genes in linkage disequilibrium. 相似文献
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Anatoly Tiulpakov Natalia Zubkova Nina Makretskaya Tatiana S. Krasnova Anna I. Melnikova Alena S. Fedyaeva Evgeny Vasilyev Vasily M. Petrov Petr M. Rubtsov 《Human mutation》2020,41(1):129-132
The next‐generation sequencing (NGS) has become a routine method for diagnostics of inherited disorders. However, assessment of the discovered variants may be challenging, especially when they are not predicted to change the protein sequence. Here we performed a functional analysis of 20 novel or rare intronic and synonymous glucokinase (GCK) gene variants identified by targeted NGS in 1,130 patients with maturity‐onset diabetes of the young. Human Splicing Finder, ver 3.1 and a precomputed index of splicing variants (SPIDEX) were used for in silico prediction. In vitro effects of GCK gene variants on splicing were tested using a minigene expression approach. In vitro effect on splicing was shown for 9 of 20 variants, including two synonymous substitutions. In silico and in vitro results matched in about 50% of cases. The results demonstrate that novel or rare apparently benign GCK gene variants should be regarded as potential splicing mutations. 相似文献
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Daniela Di Giacomo Pascaline Gaildrat Anna Abuli Julie Abdat Thierry Frébourg Mario Tosi Alexandra Martins 《Human mutation》2013,34(11):1547-1557
Exonic variants can alter pre‐mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5′ splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers’ scores recently established by Ke et al. ( 2011 ). Comparisons of hexamer‐based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high‐throughput sequencing. 相似文献