Combined Use of In Silico and In Vitro Splicing Assays for Interpretation of Genomic Variants of Unknown Significance in Cardiomyopathies and Channelopathies

Abstract

The identification of molecular anomalies in patients with inherited arrhythmias or cardiomyopathies is a <em>multi challenge</em> due to: i) the number of genes involved; ii) the number of polymorphisms and the fact that most mutations are private; and iii) the variable degree of penetrance which complicates family segregation study. Consequently, a number of unclassified variants (UV) are found in patients&rsquo; DNA and some (outside the canonical GT/AG) may affect splicing. Mutational screening on the most prevalent genes involved in arrythmias syndromes or in cardiomyopathies was performed on a cohort made up of 740 unrelated French index probands. To identify splice variants among the identified UVs, a combination of available <em>in silico</em> and <em>in vitro </em>tools were used since transcript is not available. Using this approach, 10 previously identified UVs were reclassified as disease-causing mutations and, more precisely, as haploinsufficiency mutations rather than dominant-negative mutations. Most of them (7 of 10) were observed in MYBPC3. Our study highlighted the importance of the combined use of <em>in silico</em> and <em>in vitro</em> splicing assays to improve the prediction of the functional impact of identified genetic variants. The primary challenge now, with new sequencing technologies, is to distinguish between background polymorphisms and pathogenic mutations. Since splice site mutations can account for almost 50% of disease-causing mutations, recognizing them from among other variations is essential

Sarcomere Mutation-Specific Expression Patterns in Human Hypertrophic Cardiomyopathy

“…4 However, both splice acceptor mutations demonstrated intron inclusion as opposed to exon skipping. 10, 11 For the exon splice site mutation Glu542Gln, we demonstrated not only a splice-defective transcript, but also a full-length missense transcript that was not previously appreciated. 13 Confirmation of the splice products for each mutation also prevented misclassification of mutations in exon splice sites as missense, 8, 14, 15 with important implications for disease pathogenesis.…”

“…Correct predictions of whether and to what degree these mutations alter splicing has critical implications for disease mechanisms elicited by these mutations. Splice predictions by in silico models, in vitro splice reactions, 10 or sequencing of peripheral blood leukocyte mRNA 4, 11 may not accurately predict the consequences of a splice site mutation in the relevant tissue. 12 In this study, we confirmed altered splicing and generation of a PTC from myocardium of all HCM patients harboring MYBPC3 mutations in consensus splice sites.…”

Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies

“…Alamut is a licensed software package available from Interactive Biosoftware (). To determine the robustness of our in silico predictions, we compared the effect on the splicing process of 10 previously reported intron mutations verified by in vitro/in vivo assay [19,20,21,22,23,24] with the outcome of our Alamut analysis, and obtained similar results (Supplementary Table S1). The results obtained with this predictor software for the novel intron variants are summarized in Table 2.…”

Aberrant Splicing Is the Pathogenicity Mechanism of the p.Glu314Lys Variant in CYP11A1 Gene

“…CYP11A1 exon 5 with 313 flanking intronic bases in intron 4 and 241 flanking intronic bases in intron 5 was PCR amplified from the patient genomic DNA (total product size: 714 bp). Wild-type (WT) and mutated polymerase chain reaction (PCR) products were inserted in the Nde I restriction site of the pTB minigene vector as previously reported ( 24 , 25 ). Transfection in HeLa cells, reverse transcription -PCR (RT-PCR) procedures and analysis have been previously described ( 24 , 25 ).…”

“…Wild-type (WT) and mutated polymerase chain reaction (PCR) products were inserted in the Nde I restriction site of the pTB minigene vector as previously reported ( 24 , 25 ). Transfection in HeLa cells, reverse transcription -PCR (RT-PCR) procedures and analysis have been previously described ( 24 , 25 ). Reverse transcription-PCR products were analyzed by agarose gel electrophoresis (2%) and Sanger sequencing.…”