The CYSMA web server: An example of integrative tool for in silico analysis of missense variants identified in Mendelian disorders

Abstract

Exome sequencing used for molecular diagnosis of Mendelian disorders considerably increases the number of missense variants of unclear significance, whose pathogenicity can be assessed by a variety of prediction tools. As the performance of algorithms may vary according to the datasets, complementary specific resources are needed to improve variant interpretation. As a model, we were interested in the cystic fibrosis transmembrane conductance regulator gene (CFTR) causing cystic fibrosis, in which at least 40% of missense variants are reported. Cystic fibrosis missense analysis (CYSMA) is a new web server designed for online estimation of the pathological relevance of CFTR missense variants. CYSMA generates a set of computationally derived data, ranging from evolutionary conservation to functional observations from three-dimensional structures, provides all available allelic frequencies, clinical observations, and references for functional studies. Compared to software classically used in analysis pipelines on a dataset of 141 well-characterized missense variants, CYSMA was the most efficient tool to discriminate benign missense variants, with a specificity of 85%, and very good sensitivity of 89%. These results suggest that such integrative tools could be adapted to numbers of genes involved in Mendelian disorders to improve the interpretation of missense variants identified in the context of diagnosis. K E Y W O R D S3D models, 3D structures, bioinformatics, cystic fibrosis transmembrane conductance regulator (CFTR), rare missense variants, sequence alignment

Molecular Diagnosis and Genetic Counseling of Cystic Fibrosis and Related Disorders: New Challenges

“…There are some limitations with using in silico tools, notably because of challenges in prioritizing one tool over the other and the lack of reliable standard interpretation guidelines [ 19 ]. Cystic fibrosis missense analysis (CYSMA) is a recently developed website dedicated to CFTR missense variants based on integrated in-house bioinformatics tools, which proved efficient to predict the impact of CFTR variants [ 46 ].…”

Next-Generation Sequencing for Screening Analysis of Cystic Fibrosis: Spectrum and Novel Variants in a South–Central Italian Cohort

“…Previously unreported variants were defined as “novel”, and the impact of each missense sequence mutation was predicted using the CYSMA biological tool [ 22 ]. This tool computes the impact of the sequence variation in terms of Ortholog conservation, shared domain conservation, secondary structure analysis and 3D analysis forecasting [ 23 ]. Analogous observations have also been computed to assess the impact of the sequence variation on the protein structure.…”

The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy

“…We did not observe any impact on protein synthesis and activity of the mutant proteins carrying I175V and E403D (missense variants). Even if I175 and E403 are two highly conserved amino acids among orthologs, the absence of impact was suspected by the similarity of physicochemical properties of the WT and mutant amino acids in the Venn Diagram and the absence of deleterious effect predicted by the three-dimensional model [21] . As expected, the two shorter proteins resulting from the aberrant splicing, CFTR-del57bpEx5 and CFTR-delEx9, were not correctly folded (no C band in Western Blot (WB)) (Fig 2A).…”

“…S3). Regarding the 3D structural model [21], the amino acid Glu92 is involved in the pore construction and participates in a salt bridge ( Fig 3C ), that may result in a gating defect if a very low proportion of mature protein could be addressed to the plasma membrane in vivo . The variant E92K can therefore concomitantly be considered as a class II variant, due to the splicing defect and the amino acid substitution, and possibly class III of the functional classification.…”