Saturation-scale functional evidence supports clinical variant interpretation in Lynch syndrome

Abstract

Background: Lynch syndrome (LS) is a cancer predisposition syndrome affecting more than 1 in every 300 individuals worldwide. Clinical genetic testing for LS can be life-saving but is complicated by the heavy burden of variants of uncertain significance (VUS), especially missense changes.

Result: To address this challenge, we leverage a multiplexed analysis of variant effect (MAVE) map covering >94% of the 17,746 possible missense variants in the key LS gene MSH2. To establish this map's utility in large-scale variant reclassification, we overlay it on clinical databases of >15,000 individuals with LS gene variants uncovered during clinical genetic testing. We validate these functional measurements in a cohort of individuals with paired tumor-normal test results and find that MAVE-based function scores agree with the clinical interpretation for every one of the MSH2 missense variants with an available classification. We use these scores to attempt reclassification for 682 unique missense VUS, among which 34 scored as deleterious by our function map, in line with previously published rates for other cancer predisposition genes. Combining functional data and other evidence, ten missense VUS are reclassified as pathogenic/likely pathogenic, and another 497 could be moved to benign/likely benign. Finally, we apply these functional scores to paired tumor-normal genetic tests and identify a subset of patients with biallelic somatic loss of function, reflecting a sporadic Lynch-like Syndrome with distinct implications for treatment and relatives' risk.

Conclusion: This study demonstrates how high-throughput functional assays can empower scalable VUS resolution and prospectively generate strong evidence for variant classification.

Fig. 1

Validation of MSH2 function scores across 48 previously classified MSH2 missense variants. LoF scores for known pathogenic/likely pathogenic (red, at left) and known benign/likely benign variants (blue, at right) are plotted against codon position. Gray shaded interval denotes intermediate score range

Fig. 2

Function scores and variant reclassification for MSH2 missense variants, for A all single nucleotide missense variants, B missense VUSs, and C missense variants previously classified as pathogenic or likely pathogenic, including those used for validation. For each group of variants, splicing status was scored by SpliceAI (bar charts at left), and for splice-neutral variants (SpliceAI score<0.2), a histogram of LoF scores from deep mutational scanning are displayed to the right

Fig. 3

Reclassification outcomes for 718 MSH2 missense variants. Flow diagram showing starting and final variant classifications; in total, 74% of the missense VUSs have sufficient evidence to enable potential reclassification to benign (B), likely benign (LB), likely pathogenic (LP) or pathogenic (P). A subset of remaining VUSs had intermediate function scores (n=19) or had abnormal function scores but lacked sufficient lines of evidence (or had conflicting evidence) and so remain as VUS (n=12)

Fig. 4

Cancer associations by variant type. Association between colorectal cancer (blue) or uterine/endometrial cancer (female) and missense variants in MSH2 (missense, separated by DMS+SpliceAI function score), or P/LP variants in other Lynch syndrome genes; odds ratios shown from logistic regression

Fig. 5

Joint analysis of germline and tumor mutations. A Patterns of germline and somatic mutations among LS genes, among germline carriers of MSH2 missense variants, separated into those scored as functionally neutral (top) or deleterious (bottom) by deep mutational scanning LoF score. B Fraction of individuals with a somatic P/LP mutation in MSH2, by MSH2 germline missense functional status. C Tumor microsatellite status, by MSH2 germline missense variant functional status. ***, P<0.001; **, P<0.01

Fig. 6

Mutational patterns in patients with somatic MSH2 missense variants. Tumor and germline mutations in LS genes are shown in patients who carry functionally neutral somatic MSH2 missense variants (upper track, n=38), or those with a functionally abnormal somatic MSH2 variant (lower track, n=46). Mutations and tumor characteristics are denoted as in Fig. 5A