Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing

Abstract

Inherited loss-of-function mutations in BRCA1 and BRCA2 and other tumor suppressor genes predispose to ovarian carcinomas, but the overall burden of disease due to inherited mutations is not known. Using targeted capture and massively parallel genomic sequencing, we screened for germ-line mutations in 21 tumor suppressor genes in genomic DNA from women with primary ovarian, peritoneal, or fallopian tube carcinoma. Subjects were consecutively enrolled at diagnosis and not selected for age or family history. All classes of mutations, including point mutations and large genomic deletions and insertions, were detected. Of 360 subjects, 24% carried germ-line loss-of-function mutations: 18% in BRCA1 or BRCA2 and 6% in BARD1, BRIP1, CHEK2, MRE11A, MSH6, NBN, PALB2, RAD50, RAD51C, or TP53. Six of these genes were not previously implicated in inherited ovarian carcinoma. Primary carcinomas were generally characterized by genomic loss of normal alleles of the mutant genes. Of women with inherited mutations, >30% had no family history of breast or ovarian carcinoma, and >35% were 60 y or older at diagnosis. More patients with ovarian carcinoma carry cancer-predisposing mutations and in more genes than previously appreciated. Comprehensive genetic testing for inherited carcinoma is warranted for all women with ovarian, peritoneal, or fallopian tube carcinoma, regardless of age or family history. Clinical genetic testing is currently done gene by gene, with each test costing thousands of dollars. In contrast, massively parallel sequencing allows such testing for many genes simultaneously at low cost.

Fig. 1.

Proportions of patients with primary ovarian, fallopian tube, or peritoneal cancers with germ-line loss-of-function mutations in BRCA1 (red); BRCA2 (blue); BARD1, BRIP1, CHEK2, MRE11, NBN, PALB2, RAD50, or RAD51C (green); MSH6 (purple); or p53 (yellow).

Fig. 2.

Genes harboring germ-line loss-of-function mutations in patients with primary ovarian, fallopian tube, or peritoneal cancers. Coding regions of all genes are on the same scale. Untranslated regions are at 1:10 scale and introns are at 1:200 scale relative to coding exons. Mutations occurring in multiple individuals are indicated with the number of affected individuals above the small balloon.

Fig. 3.

Pedigrees of subjects with germ-line TP53 mutations. Subjects with carcinomas are indicated with solid symbols. Sites of carcinoma are breast (Br), Colon (Col), leukemia (Leuk), lung (Lung), peritoneum (Perit), ovary (Ov),and uterus (Ut). Ages under symbols indicate age at death (d.) for deceased individuals, age at cancer diagnosis for affected individuals, or present age for unaffected individuals. Probands are indicated with arrows. Other family members have not been tested. (A) CF1265.01 carries TP53 p.R175C. (B) CF1373.01 carries TP53 p.Y126D. (C) CF 1560.01 carries TP53 p.R175H as a mosaic mutation. (D) Sequences at the TP53 c.524G > A (p.R175H) mutant site for lymphocytes of CF1560.01. Electropherogram peaks indicate that DNA from pooled lymphocytes included ∼80% WT and 20% mutant sequences. The amplicon from lymphocyte DNA was subcloned and 65 clones were sequenced, with 51 subclones containing WT sequence and 14 subclones containing mutant sequence. Representative subclones are shown. (E) Sequences at the TP53 c.524G > A (p.R175H) mutant site for two distinct cancers of CF1560.01. DNA sequences from the fallopian tube carcinoma and from the retroperitoneal leimyosarcoma revealed primarily mutant sequence, consistent with loss of the WT allele in neoplastic tissue.