The Experts below are selected from a list of 5613 Experts worldwide ranked by ideXlab platform
Shinya Ishiko - One of the best experts on this subject based on the ideXlab platform.
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pathogenic evaluation of synonymous col4a5 variants in x linked alport syndrome using a Minigene assay
Molecular Genetics & Genomic Medicine, 2020Co-Authors: Tomoko Horinouchi, Shinya Ishiko, China Nagano, Nana Sakakibara, Tomohiko Yamamura, Shogo Minamikawa, Koichi Nakanishi, Yuko Shima, Naoya Morisada, Yuya AotoAbstract:BACKGROUND X-linked Alport syndrome (XLAS) is a progressive, hereditary glomerular nephritis of variable severity caused by pathogenic COL4A5 variants. Currently, genetic testing is widely used for diagnosing XLAS; however, determining the pathogenicity of variants detected by such analyses can be difficult. Intronic variants or synonymous variants may cause inherited diseases by inducing aberrant splicing. Transcript analysis is necessary to confirm the pathogenicity of such variants, but it is sometimes difficult to extract mRNA directly from patient specimens. METHODS In this study, we conducted in vitro splicing analysis using a hybrid Minigene assay and specimens from three XLAS patients with synonymous variants causing aberrant splicing, including previously reported pathogenic mutations in the same codon. The variants were c.876 A>T (p.Gly292=), c.2358 A>G (p.Pro786=), and c.3906 A>G (p.Gln1302=). RESULTS The results from our hybrid Minigene assay were sufficient to predict splicing abnormalities; c.876 A>T cause 17-bp del and 35-bp del, c.2358 A>G cause exon 29 skipping, c.3906 A>G cause exon 42 skipping, which are very likely to cause pathogenicity. Further, patients carrying c.2358 A>G exhibited a mild phenotype that may have been associated with the presence of both normal and abnormally spliced transcripts. CONCLUSION The Minigene system was shown to be a sensitive assay and a useful tool for investigating the pathogenicity of synonymous variants.
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functional analysis of suspected splicing variants in clcn5 gene in dent disease 1
Clinical and Experimental Nephrology, 2020Co-Authors: Tomohiko Inoue, Yuya Aoto, Yugo Shibagaki, Tomoko Horinouchi, Masafumi Matsuo, Daisuke Ichikawa, China Nagano, Nana Sakakibara, Tomohiko Yamamura, Shinya IshikoAbstract:In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into Minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. We conducted functional splicing assays using a hybrid Minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced Minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these Minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our Minigene assay results. We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G). We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.
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Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1
Clinical and Experimental Nephrology, 2020Co-Authors: Tomohiko Inoue, Yuya Aoto, Yugo Shibagaki, Tomoko Horinouchi, Masafumi Matsuo, Daisuke Ichikawa, China Nagano, Nana Sakakibara, Tomohiko Yamamura, Shinya IshikoAbstract:Background In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into Minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. Methods We conducted functional splicing assays using a hybrid Minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5 . We extracted information on these variants from the Human Gene Mutation Database. We reproduced Minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these Minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our Minigene assay results. Results We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G). Conclusion We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.
Franco Pagani - One of the best experts on this subject based on the ideXlab platform.
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binding of dazap1 and hnrnpa1 a2 to an exonic splicing silencer in a natural brca1 exon 18 mutant
Molecular and Cellular Biology, 2008Co-Authors: Elisa Goina, Natasa Skoko, Franco PaganiAbstract:A disease-causing G-to-T transversion at position +6 of BRCA1 exon 18 induces exclusion of the exon from the mRNA and, as has been suggested by in silico analysis, disrupts an ASF/SF2-dependent splicing enhancer. We show here using a pulldown assay with an internal standard that wild-type (WT) and mutant T6 sequences displayed similar ASF/SF2 binding efficiencies, which were significantly lower than that of a typical exonic splicing enhancer derived from the extra domain A exon of fibronectin. Overexpression or small interfering RNA (siRNA)-mediated depletion of ASF/SF2 did not affect the splicing of a WT BRCA1 Minigene but resulted in an increase and decrease of T6 exon 18 inclusion, respectively. Furthermore, extensive mutation analysis using hybrid Minigenes indicated that the T6 mutant creates a sequence with a prevalently inhibitory function. Indeed, RNA-protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a splicing factor-dependent silencer. This sequence specifically binds to the known repressor protein hnRNPA1/A2 and to DAZAP1, the involvement of which in splicing inhibition we have demonstrated. Our results indicate that the binding of the splicing factors hnRNPA1/A2 and DAZAP1 is the primary determinant of T6 BRCA1 exon 18 exclusion.
Dawn S Chandler - One of the best experts on this subject based on the ideXlab platform.
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a humanized smn gene containing the smn2 nucleotide alteration in exon 7 mimics smn2 splicing and the sma disease phenotype
Human Molecular Genetics, 2010Co-Authors: Jordan T Gladman, Thomas W Bebee, Chris Edwards, Dawn S Chandler, Mark M Rich, Zarife Sahenk, Xueyong WangAbstract:Proximal spinal muscular atrophy (SMA) is a neurodegenerative disease caused by low levels of the survival motor neuron (SMN) protein. In humans, SMN1 and SMN2 encode the SMN protein. In SMA patients, the SMN1 gene is lost and the remaining SMN2 gene only partially compensates. Mediated by a C>T nucleotide transition in SMN2, the inefficient recognition of exon 7 by the splicing machinery results in low levels of SMN. Because the SMN2 gene is capable of expressing SMN protein, correction of SMN2 splicing is an attractive therapeutic option. Although current mouse models of SMA characterized by Smn knock-out alleles in combination with SMN2 transgenes adequately model the disease phenotype, their complex genetics and short lifespan have hindered the development and testing of therapies aimed at SMN2 splicing correction. Here we show that the mouse and human Minigenes are regulated similarly by conserved elements within in exon 7 and its downstream intron. Importantly, the C>T mutation is sufficient to induce exon 7 skipping in the mouse Minigene as in the human SMN2. When the mouse Smn gene was humanized to carry the C>T mutation, keeping it under the control of the endogenous promoter, and in the natural genomic context, the resulting mice exhibit exon 7 skipping and mild adult onset SMA characterized by muscle weakness, decreased activity and an alteration of the muscle fibers size. This Smn C>T mouse represents a new model for an adult onset form of SMA (type III/IV) also know as the Kugelberg–Welander disease.
Tomohiko Inoue - One of the best experts on this subject based on the ideXlab platform.
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functional analysis of suspected splicing variants in clcn5 gene in dent disease 1
Clinical and Experimental Nephrology, 2020Co-Authors: Tomohiko Inoue, Yuya Aoto, Yugo Shibagaki, Tomoko Horinouchi, Masafumi Matsuo, Daisuke Ichikawa, China Nagano, Nana Sakakibara, Tomohiko Yamamura, Shinya IshikoAbstract:In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into Minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. We conducted functional splicing assays using a hybrid Minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced Minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these Minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our Minigene assay results. We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G). We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.
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Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1
Clinical and Experimental Nephrology, 2020Co-Authors: Tomohiko Inoue, Yuya Aoto, Yugo Shibagaki, Tomoko Horinouchi, Masafumi Matsuo, Daisuke Ichikawa, China Nagano, Nana Sakakibara, Tomohiko Yamamura, Shinya IshikoAbstract:Background In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into Minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. Methods We conducted functional splicing assays using a hybrid Minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5 . We extracted information on these variants from the Human Gene Mutation Database. We reproduced Minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these Minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our Minigene assay results. Results We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G). Conclusion We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.
Felix Claverie-martin - One of the best experts on this subject based on the ideXlab platform.
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Exonic CLDN16 mutations associated with familial hypomagnesemia with hypercalciuria and nephrocalcinosis can induce deleterious mRNA alterations
BMC Medical Genetics, 2019Co-Authors: Ana Perdomo-ramirez, Elena Ramos-trujillo, Marian De Armas-ortiz, Lorena Suarez-artiles, Felix Claverie-martinAbstract:Background Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis type 1 is an autosomal recessive disease characterized by excessive renal magnesium and calcium excretion, bilateral nephrocalcinosis, and progressive chronic renal failure. This rare disease is caused by mutations in CLDN16 that encodes claudin-16, a tight-junction protein involved in paracellular reabsorption of magnesium and calcium in the renal tubule. Most of these variants are located in exons and have been classified as missense mutations. The functional consequences of some of these claudin-16 mutant proteins have been analysed after heterologous expression showing indeed a significant loss of function compared to the wild-type claudin-16. We hypothesize that a number of CLDN16 exonic mutations can be responsible for the disease phenotype by disrupting the pre-mRNA splicing process. Methods We selected 12 previously described presumed CLDN16 missense mutations and analysed their potential effect on pre-mRNA splicing using a Minigene assay. Results Our results indicate that five of these mutations induce significant splicing alterations. Mutations c.453G > T and c.446G > T seem to inactivate exonic splicing enhancers and promote the use of an internal cryptic acceptor splice site resulting in inclusion of a truncated exon 3 in the mature mRNA. Mutation c.571G > A affects an exonic splicing enhancer resulting in partial skipping of exon 3. Mutations c.593G > C and c.593G > A disturb the acceptor splice site of intron 3 and cause complete exon 4 skipping. Conclusions To our knowledge, this is the first report of CLDN16 exonic mutations producing alterations in splicing. We suggest that in the absence of patients RNA samples, splicing functional assays with Minigenes could be valuable for evaluating the effect of exonic CLDN16 mutations on pre-mRNA splicing.
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Exonic CLDN16 mutations associated with familial hypomagnesemia with hypercalciuria and nephrocalcinosis can induce deleterious mRNA alterations.
BMC medical genetics, 2019Co-Authors: Ana Perdomo-ramirez, Elena Ramos-trujillo, Marian De Armas-ortiz, Lorena Suarez-artiles, Felix Claverie-martinAbstract:Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis type 1 is an autosomal recessive disease characterized by excessive renal magnesium and calcium excretion, bilateral nephrocalcinosis, and progressive chronic renal failure. This rare disease is caused by mutations in CLDN16 that encodes claudin-16, a tight-junction protein involved in paracellular reabsorption of magnesium and calcium in the renal tubule. Most of these variants are located in exons and have been classified as missense mutations. The functional consequences of some of these claudin-16 mutant proteins have been analysed after heterologous expression showing indeed a significant loss of function compared to the wild-type claudin-16. We hypothesize that a number of CLDN16 exonic mutations can be responsible for the disease phenotype by disrupting the pre-mRNA splicing process. We selected 12 previously described presumed CLDN16 missense mutations and analysed their potential effect on pre-mRNA splicing using a Minigene assay. Our results indicate that five of these mutations induce significant splicing alterations. Mutations c.453G > T and c.446G > T seem to inactivate exonic splicing enhancers and promote the use of an internal cryptic acceptor splice site resulting in inclusion of a truncated exon 3 in the mature mRNA. Mutation c.571G > A affects an exonic splicing enhancer resulting in partial skipping of exon 3. Mutations c.593G > C and c.593G > A disturb the acceptor splice site of intron 3 and cause complete exon 4 skipping. To our knowledge, this is the first report of CLDN16 exonic mutations producing alterations in splicing. We suggest that in the absence of patients RNA samples, splicing functional assays with Minigenes could be valuable for evaluating the effect of exonic CLDN16 mutations on pre-mRNA splicing.
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Additional file 1: of Exonic CLDN16 mutations associated with familial hypomagnesemia with hypercalciuria and nephrocalcinosis can induce deleterious mRNA alterations
2019Co-Authors: Ana Perdomo-ramirez, Elena Ramos-trujillo, Marian De Armas-ortiz, Lorena Suarez-artiles, Felix Claverie-martinAbstract:Table S1. Primers used in the construction of the Minigene and the site directed mutagenesis. (PDF 102 kb