Gene-specific ACMG/AMP classification criteria for germline APC variants: Recommendations from the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel

Abstract

Purpose: The Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP) was established by the International Society for Gastrointestinal Hereditary Tumours and the Clinical Genome Resource, who set out to develop recommendations for the interpretation of germline APC variants underlying Familial Adenomatous Polyposis, the most frequent hereditary polyposis syndrome.

Methods: Through a rigorous process of database analysis, literature review, and expert elicitation, the APC VCEP derived gene-specific modifications to the ACMG/AMP (American College of Medical Genetics and Genomics and Association for Molecular Pathology) variant classification guidelines and validated such criteria through the pilot classification of 58 variants.

Results: The APC-specific criteria represented gene- and disease-informed specifications, including a quantitative approach to allele frequency thresholds, a stepwise decision tool for truncating variants, and semiquantitative evaluations of experimental and clinical data. Using the APC-specific criteria, 47% (27/58) of pilot variants were reclassified including 14 previous variants of uncertain significance (VUS).

Conclusion: The APC-specific ACMG/AMP criteria preserved the classification of well-characterized variants on ClinVar while substantially reducing the number of VUS by 56% (14/25). Moving forward, the APC VCEP will continue to interpret prioritized lists of VUS, the results of which will represent the most authoritative variant classification for widespread clinical use.

Keywords: ACMG/AMP variant classification guidelines; Adenomatous polyposis coli (APC); ClinGen; Familial adenomatous polyposis (FAP); InSiGHT.

Figure 1. APC gene, APC protein, and criteria boundaries and genotype-phenotype correlations

Representation of the APC gene and its main protein product (middle) on the reference sequence NM_000038.6 (non-coding exon 1 not shown). The figure shows the boundaries for the application of PVS1, BS3 and BP1 (top), and genotype-phenotype correlations (bottom). The APC protein comprises several domains and motifs as shown. The 15-aa repeats confer high affinity binding to β-catenin whereas the 20-aa repeats both bind and promote β-catenin phosphorylation, ubiquitination and subsequent proteolytic degradation by a cytoplasmic destruction complex. Abbreviations: AA – amino acid; SAMP motifs - Serine-Alanine-Methionine-Proline motifs; EB1 - end-binding protein; DLG domain: discs large domain.

Figure 2. PVS1 decision tree (A) and canonical splice variant modified weights (based on reference sequence NM_000038.6) (B)

(a) Splice variants must not have any detectable nearby (+/− 20 nucleotide) strong consensus splice sequence that may reconstitute in-frame splicing. (b) For details refer to Figure 2(B). PVS1_variable is applicable to listed variants only. (c) For Guanine to non-Guanine last nucleotide changes, evidence strengths are downgraded by one level. (d) For +2T>C changes where native splice site is not or weakly predicted, strengths are one level down from the other canonical ±1/2 splice variants at the same site. (e) For full gene deletions of a known haploinsufficient gene, a pathogenic classification is warranted in the absence of conflicting evidence with PVS1 alone. (f) Not applicable if promoter 1A and 1B are also deleted. Abbreviations: NT – nucleotide, Mod – moderate, Supp – supporting, N/A – not applicable.

Figure 3

Classification of the 58 selected pilot APC variants by the original ClinVar assertion (left) and the APC-specific ACMG/AMP guidelines (right).