Prevalence, parameters, and pathogenic mechanisms for splice-altering acceptor variants that disrupt the AG exclusion zone

• Published in: HGG advances Vol. 3; no. 4; p. 100125
• Main Authors: Bryen, Samantha J., Yuen, Michaela, Joshi, Himanshu, Dawes, Ruebena, Zhang, Katharine, Lu, Jessica K., Jones, Kristi J., Liang, Christina, Wong, Wui-Kwan, Peduto, Anthony J., Waddell, Leigh B., Evesson, Frances J., Cooper, Sandra T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.10.2022; Elsevier
• Subjects: AG exclusion zone; extended acceptor splice-site variants; minigene; pre-mRNA splicing; pseudo-exon; RNA diagnostics; extended acceptor splice-site variants; pseudo-exon; RNA diagnostics; AG exclusion zone; minigene; pre-mRNA splicing
• ISSN: 2666-2477; 2666-2477
• DOI: 10.1016/j.xhgg.2022.100125

Predicting the pathogenicity of acceptor splice-site variants outside the essential AG is challenging, due to high sequence diversity of the extended splice-site region. Critical analysis of 24,445 intronic extended acceptor splice-site variants reported in ClinVar and the Leiden Open Variation Database (LOVD) demonstrates 41.9% of pathogenic variants create an AG dinucleotide between the predicted branchpoint and acceptor (AG-creating variants in the AG exclusion zone), 28.4% result in loss of a pyrimidine at the −3 position, and 15.1% result in loss of one or more pyrimidines in the polypyrimidine tract. Pathogenicity of AG-creating variants was highly influenced by their position. We define a high-risk zone for pathogenicity: > 6 nucleotides downstream of the predicted branchpoint and >5 nucleotides upstream from the acceptor, where 93.1% of pathogenic AG-creating variants arise and where naturally occurring AG dinucleotides are concordantly depleted (5.8% of natural AGs). SpliceAI effectively predicts pathogenicity of AG-creating variants, achieving 95% sensitivity and 69% specificity. We highlight clinical examples showing contrasting mechanisms for mis-splicing arising from AG variants: (1) cryptic acceptor created; (2) splicing silencer created: an introduced AG silences the acceptor, resulting in exon skipping, intron retention, and/or use of an alternative existing cryptic acceptor; and (3) splicing silencer disrupted: loss of a deep intronic AG activates inclusion of a pseudo-exon. In conclusion, we establish AG-creating variants as a common class of pathogenic extended acceptor variant and outline factors conferring critical risk for mis-splicing for AG-creating variants in the AG exclusion zone, between the branchpoint and acceptor.