A systematic analysis of splicing variants identifies new diagnoses in the 100,000 Genomes Project

Abstract

Background Genomic variants which disrupt splicing are a major cause of rare genetic diseases. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. Improving the clinical interpretation of non-canonical splicing variants offers a major opportunity to uplift diagnostic yields from whole genome sequencing data. Methods Here, we examine the landscape of splicing variants in whole-genome sequencing data from 38,688 individuals in the 100,000 Genomes Project and assess the contribution of non-canonical splicing variants to rare genetic diseases. We use a variant-level constraint metric (the mutability-adjusted proportion of singletons) to identify constrained functional variant classes near exon–intron junctions and at putative splicing branchpoints. To identify new diagnoses for individuals with unsolved rare diseases in the 100,000 Genomes Project, we identified individuals with de novo single-nucleotide variants near exon–intron boundaries and at putative splicing branchpoints in known disease genes. We identified candidate diagnostic variants through manual phenotype matching and confirmed new molecular diagnoses through clinical variant interpretation and functional RNA studies. Results We show that near-splice positions and splicing branchpoints are highly constrained by purifying selection and harbour potentially damaging non-coding variants which are amenable to systematic analysis in sequencing data. From 258 de novo splicing variants in known rare disease genes, we identify 35 new likely diagnoses in probands with an unsolved rare disease. To date, we have confirmed a new diagnosis for six individuals, including four in whom RNA studies were performed. Conclusions Overall, we demonstrate the clinical value of examining non-canonical splicing variants in individuals with unsolved rare diseases.

Case report: A novel STXBP1 splice variant and the landscape of splicing-involved STXBP1-related disorders

“…It seems that smaller deletion (one case of c.795-2A>T, 12bp deletion, and moderate delay with seizure onset at 2.5 months) causes phenotypes less severe than larger deletion (two cases of c.795-2A>G, 108bp deletion, severe delay with seizure onset at 0.5 or 0.35 month). Different changes at the same splice site (c.795-2A>T/G) could cause different molecular consequences enough for significant clinical differences, supporting a previous notion that the functional effect of splicing variants is on a continuum rather than binary ( 10 ). Similarly, c.1030-1G > A is predicted to result in skipping of the whole 81-bp Exon13, compared to a deletion of only 27bp caused by c.1030-1G>T. More drastic differences likely exist between c.1462-2A>G (12bp in-frame deletion) and c.1462-2A>T (86bp frameshift deletion), but unfortunately, no clinical data are available to assess the genotype–phenotype relationship.…”

“…Our identified variant, STXBP1 NM_001032221.4:c.578+2T>C, is located at a canonical splice site and leads to abnormal splicing. In general, canonical splice variants, within 2bp of exon–intron junction, are widely annotated as “loss of function” (LoF) variants and are known to be strong diagnostic candidates in LoF disorders ( 10 ). For example, +2T>C variants have been frequently reported to cause human genetic disease and are routinely scored as pathogenic splicing mutations.…”

“…To obtain a comprehensive understanding of the genotype–phenotype relationship for STXBP1 splice variants, we compiled a list of 54 canonical and 203 non-canonical splice variants ( Supplementary Table 2 ) from the Clinvar database. We evaluated those variants through spliceAI, a deep neural network that accurately predicts splice sites based on pre-mRNA sequence, which proves to be a highly accurate and informative prediction tool for potential splicing changes ( 10 , 12 , 13 ). Most canonical splice variants (44 out of 54) cause frameshift insertion or deletion and thus disruptive changes in STXBP1 expression.…”

Improving Hereditary Hemorrhagic Telangiectasia Molecular Diagnosis: A Referral Center Experience

“…Splicing is a key process in eukaryotic cells and its disruption is one of the main molecular causes of rare genetic diseases [ 33 , 34 ]. Disturbances in the splicing could lead to the introduction of a premature stop codon or variations in the protein structure due to in-frame changes.…”

The Genetics of Intellectual Disability

“…The most promising interpretation strategies have so far been hypothesis-driven approaches rather than searching for statistical enrichment in non-coding sequence. For SNVs, these included the identification of non-coding variants affecting the so-called gene body, where rare (de novo) variants affected, for instance, sequences of the UTRs disrupting the wild-type translation start site [96,97] or (de novo) variants in introns, where they introduced cryptic splice sites leading to abnormal splicing and the inclusion of poison exons [98][99][100]. For non-coding structural variants, the molecular mechanism leading to disease has mainly been the dysregulation of spatial 3D-organization [101] and gene regulation, for instance, through the disruption of topological-associated domain structures [102][103][104].…”