SNV and Indel Identification and Assessment
Variants were extracted from VCF files if they were within a gene specified in a comprehensive list of 83 CPGs (Table 1) and had a predicted Sequence Ontology (SO) consequence indicating a deleterious effect on protein function. The gene list used for analysis was initially composed of all genes listed in a 2014 review of CPGs1 (n = 114; gene list in Table S3) and/or those sequenced by the Illumina TCP (n = 94). Two additional more recently described CPGs, namely NTHL1 (MIM: 602656)16 and CDKN2B (MIM: 600431),17 were also included (Table S3). We subsequently reviewed and filtered the genes to produce a list that would be applicable to referrals to clinical cancer genetic services. Genes were included if deleterious variants affecting them were associated with adult-onset tumors and if neoplastic lesions were likely to be a primary presenting feature. For example, SOS1 was not included because although Noonan syndrome is associated with increased neoplasia risk, other features of the condition are likely to prompt initial referral.
Table 1 Gene List Used for Analysis (n = 83)
AIP (MIM: 605555) EGFR (MIM: 131550)a NF1 (MIM: 613113) SDHB (MIM: 185470)
ALK (MIM: 105590)a EPCAM (MIM: 185535) NF2 (MIM: 607379) SDHC (MIM: 602413)
APC (MIM: 611731) ERCC2 (MIM: 126340)b NTHL1 (MIM: 602656)b SDHD (MIM: 602690)
ATM (MIM: 607585) ERCC3 (MIM: 133510)b PALB2 (MIM: 610355) SERPINA1 (MIM: 107400)b
AXIN2 (MIM: 604025) ERCC4 (MIM: 133520)b PDGFRA (MIM: 173490)a SMAD4 (MIM: 600993)
BAP1 (MIM: 603089) ERCC5 (MIM: 133530)b PHOX2B (MIM: 603851) SMARCA4 (MIM: 603254)
BMPR1A (MIM: 601299) EXT1 (MIM: 608177) PMS2 (MIM: 600259) SMARCB1 (MIM: 601607)
BRCA1 (MIM: 113705) EXT2 (MIM: 608210) POLD1 (MIM: 174761) SMARCE1 (MIM: 603111)
BRCA2 (MIM: 600185) FH (MIM: 136850) POLE (MIM: 174762) SRY (MIM: 480000)
BRIP1 (MIM: 605882) FLCN (MIM: 607273) POLH (MIM: 603968)b STK11 (MIM: 602216)
CDC73 (MIM: 607393) GATA2 (MIM: 137295) PRKAR1A (MIM: 188830) SUFU (MIM: 607035)
CDH1 (MIM: 192090) HFE (MIM: 613609)b PTCH1 (MIM: 601309) TGFBR1 (MIM: 190181)
CDK4 (MIM:123829)a HNF1A (MIM: 142410) PTEN (MIM: 601728) TMEM127 (MIM: 613403)
CDKN1B (MIM: 600778) KIT (MIM: 164920)a RAD51C (MIM: 602774) TP53 (MIM: 191170)
CDKN2A (MIM: 600160) MAX (MIM: 154950) RAD51D (MIM: 602954) TSC1 (MIM: 605284)
CDKN2B (MIM: 600431) MEN1 (MIM: 613733) RB1 (MIM: 614041) TSC2 (MIM: 191092)
CEBPA (MIM: 116897) MET (MIM: 164860)a RET (MIM: 164761)a VHL (MIM: 608537)
CHEK2 (MIM: 604373) MLH1 (MIM: 120436) RHBDF2 (MIM: 614404)a WT1 (MIM: 607102)
CYLD (MIM: 605018) MSH2 (MIM: 609309) RUNX1 (MIM: 151385) XPA (MIM: 611153)b
DDB2 (MIM: 600811) MSH6 (MIM: 600678) SDHA (MIM: 600857) XPC (MIM: 613208)b
DICER1 (MIM: 606241) MUTYH (MIM: 604933)b SDHAF2 (MIM: 613019)
a Considered to be proto-oncogenes.
b Considered to be associated with tumor predisposition in the homozygous or compound-heterozygous state only.
In order to identify clinically relevant variants, we subjected the resulting data to a range of filters (Figure S1). First, variants were removed if they failed to satisfy the quality criteria of a genotype quality (GQ) ≥ 30 (a Phred-scaled probability that the called genotype is incorrect), read depth (DP) ≥ 10 (at least ten reads covering the variant base[s]), variant allele fraction (VAF) ≥ 33%, and filter PASS (quality criteria applied by the Isaac variant caller in the NIHR BioResource Rare Disease Project). Second, variants were excluded if they had an allele frequency above 0.01 in either the Exome Aggregation Consortium (ExAC) Browser18 (all populations) or the 1000 Genomes Project19 (all populations). Third, variants were retained for further review if the predicted consequence was among a list of SO terms indicating protein truncation, if there was evidence of pathogenicity in ClinVar20 (at least two-star evidence of pathogenic or likely pathogenic [P/LP] effect corresponding to multiple submissions with no conflicts as to the assertion of clinical significance), or if the variant was assigned a disease mutation (DM) status in the Human Gene Mutation Database (HGMD).21 In order to consider a subset of non-truncating variants that are predicted to be pathogenic by in silico tools but do not appear in public databases, we also retained variants exceeding a Phred-scaled CADD22 score threshold of 34 for further review. CADD was selected for this purpose given that it incorporates a range of tools and consequently a number of lines of evidence. The threshold was chosen as the median of scores assigned to other variants (affecting any gene) deemed pathogenic according to the criteria described below. Therefore, as a second variant filtering process, variants were identified for retention solely on the basis of CADD scores after variants retained for other reasons were assessed.
In the strategy described above, significant variants that are located in non-coding regions, such as introns, and affect genes in the gene list would not be extracted from the original VCF files because their SO consequence would not be in said list. Therefore, we used ClinVar to compile a list of known pathogenic variants falling outside of exons or splice sites and filtered VCFs on the basis of their genomic positions in a separate interrogation. Variants were incorporated in the list if they occurred in or near a gene in the list, were classified as near gene, non-coding RNA or untranslated region, and had at least two-star evidence of a P/LP effect. This process produced only three known pathogenic variants to search for in the WGS data. Distant non-coding variants affecting gene function (e.g., enhancers) were not considered in the current study.
Retained variants were subsequently excluded if their putative pathogenicity could be refuted by one of the following criteria: (1) a predicted protein-truncating variant for which there was at least two-star evidence of a benign or uncertain effect in ClinVar; (2) a predicted protein-truncating variant in a proto-oncogene in a list compiled on the basis of a literature review1 (constitutional cancer-predisposing variants in proto-oncogenes are associated with gain-of-function variants, so truncation of the protein product is unlikely to increase tumor risk), (3) a predicted protein-truncating variant affecting <5% of the canonical transcript (according to the LOFTEE VEP plugin), (4) a variant affecting a gene associated with only recessive tumor predisposition (as defined by a literature review1, 16, 23) unless an individual appeared to harbor two filtered variants in the same gene, and (5) a variant with HGMD DM status or that exceeded the CADD score threshold and had at least two-star evidence of a benign or uncertain clinical effect or one-star evidence if there were multiple submissions without a P/LP assertion.
We used the Integrated Genomics Viewer (IGV)24 to review variants that had passed filters to check for issues such as adjacent variants affecting the predicted consequence or variants being located at the end of sequencing reads. Pathogenicity was then assessed according to the American College of Medical Genetics (ACMG) criteria (Table S4),25 which provide a framework for compiling multiple weighted lines of evidence. Additionally, for each variant, it was noted whether the corresponding individual had previously been diagnosed with a tumor typically associated with pathogenic variants in that gene (according to Rahman,1 the Familial Cancer Database,23 or the original paper reporting the gene as a CPG). Validation of P/LP variants was carried out with data from the TCP or Sanger sequencing according to standard protocols if TCP data were unavailable. Primer sequences are available on request.