Utilizing ExAC to Assess the Hidden Contribution of Variants of Unknown Significance to Sanfilippo Type B Incidence

Abstract

Given the large and expanding quantity of publicly available sequencing data, it should be possible to extract incidence information for monogenic diseases from allele frequencies, provided one knows which mutations are causal. We tested this idea on a rare, monogenic, lysosomal storage disorder, Sanfilippo Type B (Mucopolysaccharidosis type IIIB).Sanfilippo Type B is caused by mutations in the gene encoding α-N-acetylglucosaminidase (NAGLU). There were 189 NAGLU missense variants found in the ExAC dataset that comprises roughly 60, 000 individual exomes. Only 24 of the 189 missense variants were known to be pathogenic; the remaining 165 variants were of unknown significance (VUS), and their potential contribution to disease is unknown.To address this problem, we measured enzymatic activities of 164 NAGLU missense VUS in the ExAC dataset and developed a statistical framework for estimating disease incidence with associated confidence intervals. We found that 25% of VUS decreased the activity of NAGLU to levels consistent with Sanfilippo Type B pathogenic alleles. We found that a substantial fraction of Sanfilippo Type B incidence (67%) could be accounted for by novel mutations not previously identified in patients, illustrating the utility of combining functional activity data for VUS with population-wide allele frequency data in estimating disease incidence.

Assessment of predicted enzymatic activity of α‐ N ‐acetylglucosaminidase variants of unknown significance for CAGI 2016

“…We used 0.15 f wt activity as a threshold with which we distinguished pathogenic from benign variables. This level of f wt activity is consistent with what we observed from previously identified pathogenic mutations as described in Clark et al (). We also calculated AUC and F ‐max values for thresholds ranging from 0.05 to 0.95 in increments of 0.05 (Table S2, Table S3).…”

“…Our current in vitro enzyme activity assay is limited to testing missense coding variants, and as shown by Clark et al (), there is a very good agreement with observed activity and pathogenicity of known and well‐annotated disease variants. However, the in vitro assay may not always correlate with enzyme activities tested directly from patient samples.…”

“…When calculating ( AUC ) we utilized 0.15 f wt activity thresholds at which variants were designated as either neutral or disease‐causing for our primary analysis. This threshold was based on the upper limit of f wt activity measured in previously observed pathogenic mutations (Clark et al, ). We also calculated AUC and F ‐max for thresholds ranging from 0.05 to 0.95 in increments of 0.05 (Table S2, Table S3).…”

“…For the CAGI challenge, we attempted to select missense variants that were both observed in the population and which were of unknown disease significance (Clark et al, ). To do this, we relied on the v0.3 release of the Exome Aggregation Consortium's (ExAC) collection of exome sequencing data comprising 60,706 individuals as a source for observed missense mutations (Lek et al, ).…”

“…Variants were selected for testing based on being present in the ExAC version v0.3 and not being present in Human Gene Mutation Database (HGMD; Lek et al, ). The enzymatic activity of these missense mutations in NAGLU had been previously measured in transfected cell lysates (Clark, Yu, Aoyagi‐Scharber, & LeBowitz, ). Of the 163 VUS tested, 41 (25%) decreased the activity of NAGLU to levels consistent with known Sanfilippo Type B pathogenic alleles.…”