Characterisation and interpretation of genetic variants by functional splicing assays in cardiovascular disorders

Abstract

Advancements in sequencing technologies and their application to patients’ care have revolutionised human genetics. A huge number of genetic variants and their association with human diseases have been identified, in particular for cardiovascular disorders (CVDs). CVDs represent a major cause of sudden death, due to genetic alterations of genes encoding for proteins mainly involved in sarcomeric and cytoskeletal function. While genetic variants altering protein function clearly cause disease, not all genetic variants identified in patients are necessarily linked to the development of a cardiac disorders, thus a classification following standard criteria has been introduced at the clinical level by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) which includes classifications of Pathogenic, Likely Pathogenic, and Variants of Unknown Significance (VUS) for variants where is unclear whether they will exert a functional effect on gene expression or function. Many diseases causing variants do not lie within protein coding segments of the genome and cause disease through alteration in RNA transcription and/or processing. For example,38 to 50% of disease causing variants are associated with altered mRNA splicing (Bryen et al., 2022). The evaluation of the potential pathogenicity of variants that may affect mRNA splicing is not easy due to the complexity of splicing mechanisms, providing a significant motivation for conducting functional studies to elucidate the effects of gene variants on splicing. In this thesis, in silico, in vitro and in vivo functional RNA splicing analysis of 24 variants identified in potential candidate genes in patients with CVDs enabled the reclassification of58% Variants of Unknown Significance and 25% of likely pathogenic variants in pathogenic. These findings strongly support the introduction of these assays to determine the pathogenicity of splicing variants in clinical settings. Additionally, these techniques may allow the development of treatments for genetic diseases with defective splicing. For example, the use of small molecule splicing modulators regulating different steps of mRNA splicing. Here, six small molecules were tested on a series of mini gene constructs, to determine whether they can rescue effects of pathogenic variants on mRNA splicing of cardiac genes. Of the compounds tested, 6-Benzyladenine and Branaplam were the most efficient in restoring splicing. Altogether, these results demonstrated that small molecule compounds could represent a valid therapeutic approach by correcting aberrant splicing.

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Identifiers

ORCID for Diana Baralle: orcid.org/0000-0003-3217-4833

ORCID for John Holloway: orcid.org/0000-0001-9998-0464

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