Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database

Abstract

The clinical classification of hereditary sequence variants identified in disease-related genes directly affects clinical management of patients and their relatives. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) undertook a collaborative effort to develop, test and apply a standardized classification scheme to constitutional variants in the Lynch syndrome-associated genes MLH1, MSH2, MSH6 and PMS2. Unpublished data submission was encouraged to assist in variant classification and was recognized through microattribution. The scheme was refined by multidisciplinary expert committee review of the clinical and functional data available for variants, applied to 2,360 sequence alterations, and disseminated online. Assessment using validated criteria altered classifications for 66% of 12,006 database entries. Clinical recommendations based on transparent evaluation are now possible for 1,370 variants that were not obviously protein truncating from nomenclature. This large-scale endeavor will facilitate the consistent management of families suspected to have Lynch syndrome and demonstrates the value of multidisciplinary collaboration in the curation and classification of variants in public locus-specific databases.

Figure 1

Overview of 5-tiered InSiGHT classification guidelines. (a) Simplified guidelines describing levels and types of evidence required to reach different classes. See the supplementary information for the full guidelines (Supplementary Note) and detailed rationale behind each criterion (Supplementary Table 3). The Lynch Syndrome molecular phenotype described in Classes 5 and 4 includes microsatellite instability and/or loss of expression of relevant protein(s) as determined by immunohistochemistry. In this study, variants resulting in a premature termination codon or large genomic deletions of functionally important domains, generally considered pathogenic on the basis of DNA sequence alone, are referred to as Class 5a “assumed pathogenic” variants. All other variants reaching class 5 are termed Class 5b. (b) Flowchart used to assist in interpretation of available functional assay data. Assays reviewed for classification are shown in Supplementary Table 4, and the values used to define abrogated or normal function are shown in Supplementary Table 5. The cut-offs <25% and >75% set for protein expression, as used in previous publications^,, are very conservative given reported abrogated function associated with MLH1 expression defects of ~50% or lower. For variants that had normal/inconclusive/intermediate MMR activity in 2 independent assays, but deficient protein function in 2 independent assays, abrogated function was assigned. AF – allele frequency; PP – posterior probability of pathogenicity derived by multifactorial likelihood analysis; CMMRD – constitutional mismatch repair deficiency (MIM 276300); LR – likelihood ratio; LS – Lynch Syndrome; MSS – microsatellite stable; CRC – colorectal cancer; IHC – immunohistochemistry; NMD – nonsense mediated decay.

Figure 2. Outcome of standardized 5-tiered InSiGHT classification of constitutional MMR gene variants

a) The plot represents the proportion of the 5-tiered classifications for all documented constitutional variants in the database, against the original LOVD database classifications assigned by submitters for each entry. Class 5a is a subset of Class 5 containing the assumed pathogenic nonsense mutations, small frameshift indels, and large deletions. Class 5b includes not-obviously truncating variants considered to be pathogenic on the basis of combined evidence (See Supplementary Note). Results show that standardized classification led to altered classifications for a considerable proportion of variant entries, including downgrading for variants submitted as pathogenic (24%), and upgrading of variants with unknown pathogenicity to likely pathogenic (5.6%) or pathogenic (48%). In addition, clinically important misclassifications were identified for unique variants initially submitted as not pathogenic (54 unique variants reclassified as Class 5b, and 25 reclassified as Class 4) and unique variants submitted as pathogenic (28 unique variants reclassified as Class 1, 16 reclassified as Class 2, and 218 reclassified as Class 3). b) Pie chart showing distribution of final InSiGHT VIC classifications.

Figure 3. Classifications of all documented unique variants by variant type

The plot represents the proportion of the different variant types within the 5 classes. Class 5a is a subset of Class 5 containing the assumed pathogenic mutations (nonsense mutations, small frameshift indels, and large deletions). All other variants reaching class 5 are termed Class 5b (see Supplementary Note). The different variant types are: PTC – variants that introduce premature terminating codons, i.e. nonsense mutations and small frameshift indels; LGDel – large genomic deletions or disrupting inversions; LGDup – large genomic duplications; SS – variants in the canonical splice site dinucleotides; NS – not obviously truncating non-synonymous variants outside the Kozak consensus sequence i.e. missense, small in-frame insertion/deletions, and read-through translation termination codon alterations; S – synonymous variants; I – intronic variants outside the canonical splice site dinucleotides; ATG/UTR – variants in the initiation codon, and the 5′ or 3′ untranslated regions. See Supplementary Figure 2 for further details of variant types, by MMR gene.

Figure 4. Contribution of microattribution to classification of “not obviously truncating” variants

Dark shading (YES) indicates the proportion of variants for each class, where the additional data obtained through microattribution contributed to their final classification.

Figure 5. Probabilities of pathogenicity for 481 Class 3 “uncertain” missense variants, derived by in silico analysis

Distribution of probabilities of pathogenicity as estimated from a calibrated algorithm based on customized MAPP and PolyPhen2 scores, for (a) MLH1, n=186; (b) MSH2, n=169; (c) MSH6, n=145; (d) PMS2, n=24; (e) all four MMR genes, showing stratification of variants with prior probabilities ≤20% or ≥80% to prioritize variants for further investigation and classification.