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

Abstract

Genomic variants which disrupt splicing are a major cause of rare genetic disease. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. 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 show that splicing branchpoints are highly constrained by purifying selection, and harbour 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. We use phenotype matching and RNA studies to confirm a new diagnosis for six individuals to date. In summary, we demonstrate the clinical value of examining non-canonical splicing variants in participants with unsolved rare diseases.

Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies

“…For each variant, four predicted probabilities were obtained: probabilities of introducing a splice Acceptor Gain (AG), Acceptor Loss (AL), Donor Gain (DG), or Donor Loss (DL) accompanied by a predicted position of such change relative to each variant. We computed an aggregate SpliceAI score to incorporate contributions from each of the 4 predicted categories using a previously described formula 20 : We considered a high likelihood of disrupting splicing to be greater than 0.80, and a low likelihood less than 0.25.…”

Quantifying negative selection in human 3’ UTRs uncovers constrained targets of RNA-binding proteins

“…While this type of approach has been applied to identify genetic variation under strong selection in non-coding regions such as 5’ UTRs (Whiffin et al 2020) and introns at splice sites (Blakes et al 2022; Lord et al 2019), it has not yet been applied to 3’ UTRs, suggesting that signals of negative selection may be challenging to uncover in these regions. Instead, the few instances where negative selection in 3’ UTRs has been inferred have been limited in scope, did not adjust for mutability, and were secondary to other efforts (Zhang et al 2020; Park et al 2021; Kainov et al 2016).…”

Transcriptome analysis provides critical answers to the “variants of uncertain significance” conundrum

“…Without additional functional data, the large number of variants existing further down‐ or upstream would lead to low overall precision. However, evidence is mounting in support of the fact that deep‐intronic, noncanonical splicing variants can drive disease pathology (Blakes et al, 2022 ; Koster et al, 2021 ).…”