Spectrum of splicing variants in disease genes and the ability of RNA analysis to reduce uncertainty in clinical interpretation

Abstract

The complexities of gene expression pose challenges for the clinical interpretation of splicing variants. To better understand splicing variants and their contribution to hereditary disease, we evaluated their prevalence, clinical classifications, and associations with diseases, inheritance, and functional characteristics in a 689,321-person clinical cohort and two large public datasets. In the clinical cohort, splicing variants represented 13% of all variants classified as pathogenic (P), likely pathogenic (LP), or variants of uncertain significance (VUSs). Most splicing variants were outside essential splice sites and were classified as VUSs. Among all individuals tested, 5.4% had a splicing VUS. If RNA analysis were to contribute supporting evidence to variant interpretation, we estimated that splicing VUSs would be reclassified in 1.7% of individuals in our cohort. This would result in a clinically significant result (i.e., P/LP) in 0.1% of individuals overall because most reclassifications would change VUSs to likely benign. In ClinVar, splicing VUSs were 4.8% of reported variants and could benefit from RNA analysis. In the Genome Aggregation Database (gnomAD), splicing variants comprised 9.4% of variants in protein-coding genes; most were rare, precluding unambiguous classification as benign. Splicing variants were depleted in genes associated with dominant inheritance and haploinsufficiency, although some genes had rare variants at essential splice sites or had common splicing variants that were most likely compatible with normal gene function. Overall, we describe the contribution of splicing variants to hereditary disease, the potential utility of RNA analysis for reclassifying splicing VUSs, and how natural variation may confound clinical interpretation of splicing variants.

Keywords: RNA analysis; functional studies; gene panel; genetic testing; in silico prediction; next-generation sequencing; splice site; splicing; variant classification; variants of uncertain significance.

Figure 1

Clinically classified splicing variants in a large clinical cohort Number of splicing variants at exonic or intronic positions indicated among 689,321 individuals tested for a variety of inherited diseases. All exonic splicing variants are grouped together; the intronic splicing variants are grouped by distance from the intron-exon junction in base pairs (bp). Note that intronic variants more than 10 bp from the intron-exon junction may not be detected because of reportable range of the sequencing assay; therefore, splice variants ±10 bp intronic are most likely underrepresented. P/LP, pathogenic/likely pathogenic; VUS, variant(s) of uncertain significance. Colors within each bar indicate the number classified as P/LP (blue) or VUSs (green).

Figure 2

Distribution of variant types and their clinical classifications in a clinical cohort of 689,321 individuals tested for genetic disease (A–C) Number and proportion of variants by type and clinical classification among (A) all observed variants, (B) unique variants, and (C) patients. Splicing variants are shown both as a group and split into ESS and non-ESS variants. VUS + RNA potential indicates splicing VUSs that have the potential to be reclassified with the addition of evidence from RNA analysis; these are included in the splicing VUSs total. ESS, essential splice site.

Figure 3

Frequencies of splice variants in the healthy human genome Splicing variants were identified in gnomAD (v.2.0.2) via the Ensembl Variant Effect Predictor (v.85). (A) Bar graph indicating the absolute number of variants identified in gnomAD within coding regions and ±8 bp of intronic sequence. Splicing variants include variants at the ESS (at intronic positions ±1–2) and at non-ESS locations (at intronic positions ±3–8 bp and exonic positions ±1–3 bp). Other includes in-frame indels and alterations to stop and start codons. (B) Allele frequencies for splicing variants as determined by “popmax” and grouped as common (>1%), rare (0.1%–1%), and very rare (<0.1%). (C) Outlier boxplot showing the distribution of splicing and truncating variants among hereditary disease genes by inheritance patterns. (D) Outlier boxplot showing the distribution of splicing and truncating variants in exons of all gnomAD genes with high pLI scores (pLI > 0.9) and low pLI scores (pLI ≤ 0.9). AD, autosomal dominant; AR, autosomal recessive; ESS, essential splice site; gnomAD, Genome Aggregation Database; pLI, probability of loss-of-function intolerant; XL, X-linked.