SNVs and Indels
Variant filters applied to annotated VCF files produced 89 unique variants in 119 individuals for further ACMG-guideline-based assessment. Of these, 22 (42 occurrences) could be classified as pathogenic, 23 (24 occurrences) could be classified as likely pathogenic, 24 (27 occurrences) could be classified as a variant of uncertain significance (VUS), and 20 (26 occurrences) could be classified as likely benign. Six occurrences of P/LP variants occurred in two members of the same family, and only three of these contributed to the detection rates quoted below. No pathogenic non-coding variants were identified.
Overall, 63 variants in 17 genes in 61 (13.9%) probands were assessed as P/LP (summary in Table 3; full description with phenotype and previous testing in Table S6). Most were nonsense or frameshift variants. Individuals with variants in moderate-risk CPGs CHEK2 (MIM: 604373; n = 14) and ATM (MIM: 607585; n = 10) were the most frequent; one homozygote was detected for CHEK2: c.1100delC (p.Thr367Metfs) (Ensembl: ENST00000328354; GenBank: NM_007194.3]; annotated in our data as c.1229delC [p.Thr410fs] [Ensembl: ENST00000382580; GenBank: NM_001005735.1]). Individuals with variants in BRCA2 (MIM: 600185; n = 6), PALB2 (MIM: 610355; n = 6), FH (MIM: 136850; n = 5), NF1 (MIM: 613113; n = 4), NTHL1 (MIM: 602656; homozygous, n = 3), MAX (MIM: 154950; n = 2), PTEN (MIM: 601728; n = 2), SDHB (MIM: 185470; n = 2), BMPR1A (MIM: 601299; n = 1), BRCA1 (MIM: 113705; n = 1), CDKN1B (MIM: 600778; n = 1), EXT2 (MIM: 608210; n = 1), MLH1 (MIM: 120436; n = 1), MSH2 (MIM: 609309; n = 1), and PMS2 (MIM: 600259; n = 1) were also noted.
Table 3 Summary of Filtered SNVs and Indels Deemed Pathogenic or Likely Pathogenic by ACMG Assessment
Gene RefSeq mRNA ID No. of Occurrences No. of Individuals with Associated Tumor Variant Description Consequence
ATM GenBank: NM_000051 1 1 c.193C>T (p.Gln65∗) stop gain
ATM GenBank: NM_000051 1 1 c.5623C>T (p.Arg1875∗) stop gain
ATM GenBank: NM_000051 1 0 c.6583+1G>A splice site (donor)
ATM GenBank: NM_000051 1 0 c.6866-6867insT (p.Ser2289Serfs) frameshift
ATM GenBank: NM_000051 1 1 c.748C>T (p.Arg250∗) stop gain
ATM GenBank: NM_000051 1 1 c.8147T>C (p.Val2716Ala) missense
ATM GenBank: NM_000051 1 0 c.8405delA (p.Gln2802fs) frameshift
ATM GenBank: NM_000051 1 0 c.5821G>C (p.Val1941Leu) missense
ATM GenBank: NM_000051 1 0 c.8122G>A (p.Asp2708Asn) missense
ATM GenBank: NM_000051 1 1 c.7775C>G (p.Ser2592Cys) missense
BMPR1Aa GenBank: NM_004329 1 1 c.730C>T (p.Arg244∗) stop gain
BRCA1 GenBank: NM_007300 1 1 c.1961−1962insA (p.Lys654fs) frameshift
BRCA2 GenBank: NM_000059 1 0 c.4525C>T (p.Gln1509∗) stop gain
BRCA2 GenBank: NM_000059 1 1 c.5682C>G (p.Tyr1894∗) stop gain
BRCA2 GenBank: NM_000059 1 0 c.6275−6276delTT (p.Leu2092fs) frameshift
BRCA2 GenBank: NM_000059 1 1 c.6402−6406delTAACT (p.Asn2135Leufs) frameshift
BRCA2 GenBank: NM_000059 1 0 c.6535−6536insA (p.Val2179fs) frameshift
BRCA2 GenBank: NM_000059 1 1 c.1805−1806insA (p.Gly602fs) frameshift
CDKN1B GenBank: NM_004064 1 0 c.148−149delAG (p.Arg50fs) frameshift
CHEK2 GenBank: NM_001005735 3 1 c.1392delT (p.Leu464fs) frameshift
CHEK2 GenBank: NM_001005735 10 6 c.1229delC (p.Thr410fs) frameshift
CHEK2 GenBank: NM_001005735 1 1 c.1051+1C>T splice site (donor)
CHEK2 GenBank: NM_001005735 1 0 c.784delG (p.Glu262fs) frameshift
CHEK2 GenBank: NM_001005735 1 1 c.562C>T (p.Arg188Trp) missense
EXT2 GenBank: NM_000401 1 0 c.613C>T (p.Gln205∗) stop gain
FH GenBank: NM_000143 3 0 c.1433−1434insAAA (p.Lys477_Asn478insLys) in-frame insertion
FH GenBank: NM_000143 1 1 c.320A>C (p.Asn107Thr) missense
FHb GenBank: NM_000143 1 0 c.521C>G (p.Pro174Arg) missense
MAX GenBank: NM_002382 1 1 c.289C>T (p.Gln97∗) stop gain
MAXb GenBank: NM_002382 1 1 c.1A>G (p.Met1Val) start loss
MLH1 GenBank: NM_000249, NM_001258273 1 1 c.1884−1G>A splice site (acceptor)
MSH2 GenBank: NM_000251 1 0 c.1452−1455insAATG (p.Leu484-Met485fs) frameshift
NF1 GenBank: NM_001042492 1 1 c.1541−1542delAG (p.Gln514fs) frameshift
NF1 GenBank: NM_001042492 1 1 c.4620delA (p.Ala1540fs) frameshift
NF1 GenBank: NM_001042492 1 1 c.5831delT (p.Leu1944fs) frameshift
NF1 GenBank: NM_001042492 1 1 c.7768-7769insA (p.His2590fs) frameshift
NTHL1c GenBank: NM_002528 3 3 c.268C>T (p.Gln90∗) stop gain
PALB2 GenBank: NM_024675 4 3 c.3113G>A (p.Trp1038∗) stop gain
PALB2 GenBank: NM_024675 1 1 c.3116delA (p.Asn1039fs) frameshift
PALB2 GenBank: NM_024675 1 1 c.62T>G (p.Leu21∗) stop gain
PMS2a GenBank: NM_000535 1 1 c.741−742insTGAAG (p.Pro247_S248fs) frameshift
PTEN GenBank: NM_000314 1 1 c.1003C>T (p.Arg335∗) stop gain
PTEN GenBank: NM_000314 1 1 c.697C>T (p.Arg233∗) stop gain
SDHB GenBank: NM_003000 1 1 c.223+1C>A splice site (donor)
SDHB GenBank: NM_003000 1 1 c.689G>A (p.Arg230His) missense
This list incorporates one individual per family. See Table S6 for more comprehensive description.
a Occurring in the same individual.
b Occurring in the same individual.
c Homozygous.
The 61 P/LP SNV and indels detected by WGS were confirmed by a second analysis (TCP for 51 variants and Sanger sequencing for ten variants).
Pre-testing information was available for 57/63 P/LP variants, 41/57 (71.9%) of which occurred in an individual who had at least one previous genetic test and 7/57 (12.3%) of which were eventually detected by clinical services. No P/LP variants were observed in genes that had previously been tested in a sample from the relevant individual by diagnostic services (Figure 2). The mean number of genes tested in those with a P/LP variant was 5.3, which was not significantly different from that in probands without such variants detected (Student’s t test p = 0.396).
Of the 61 probands identified with a P/LP variant, 36 (59%; 8.2% of all probands) had previously been diagnosed with a tumor typically associated with the relevant CPG. A further eight (1.8%) probands were found to harbor a VUS and had been diagnosed with an associated tumor.
Two probands harbored two P/LP variants in multiple CPGs. One individual with colorectal adenocarcinoma at age 50 years and breast cancer at 57 years carried a PMS2 frameshift variant (c.741−742insTGAAG [p.Pro247_Ser248fs] [Ensembl: ENST00000265849; GenBank: NM_000535.6]) and a BMPR1A nonsense variant (c.730C>T [p.Arg244∗] [Ensembl: ENST00000372037; GenBank: NM_004329.2]). Immunohistochemistry of the bowel tumor showed loss of PMS2; MSI was also demonstrated, leading to diagnostic sequencing of PMS2, although there was no family history of neoplasia other than an ovarian cancer in a second-degree relative after age 70 years. The proband had previously undergone surveillance colonoscopy for inflammatory bowel disease, resulting in the identification of a number of polyps; however, there was no evidence from histology reports that these were juvenile polyps. Additionally, an individual with bilateral pheochromocytoma at ages 16 and 35 years and no reported family history of neoplasia was identified with variants in FH (c.521C>G [p.Pro174Arg] [Ensembl: ENST00000366560; GenBank: NM_000143.3]) and MAX (c.1A>G [p.Met1Val] [Ensembl: ENST00000358664; GenBank: NM_002382.4]).29 The latter variant is predicted to abolish the MAX initiation codon, and previous analysis of tumor tissue from an individual carrying it demonstrated loss of the wild-type allele and a lack of full-length MAX protein product.30