Whole Exome Sequencing Identifies Candidate Genes Associated with Hereditary Predisposition to Uveal Melanoma

Abstract

Purpose: To identify susceptibility genes associated with hereditary predisposition to uveal melanoma (UM) in patients with no detectable germline BAP1 alterations.

Design: Retrospective case series from academic referral centers.

Participants: Cohort of 154 UM patients with high risk of hereditary cancer defined as patients with 1 or more of the following: (1) familial UM, (2) young age (<35 years) at diagnosis, (3) personal history of other primary cancers, and (4) family history of 2 or more primary cancers with no detectable mutation or deletion in BAP1 gene.

Methods: Whole exome sequencing, a cancer gene panel, or both were carried out. Probands included 27 patients with familial UM, 1 patient with bilateral UM, 1 patient with congenital UM, and 125 UM patients with strong personal or family histories, or both, of cancer. Functional validation of variants was carried out by immunohistochemistry, reverse-transcriptase polymerase chain reaction, and genotyping.

Main outcome measures: Clinical characterization of UM patients with germline alterations in known cancer genes.

Results: We identified actionable pathogenic variants in 8 known hereditary cancer predisposition genes (PALB2, MLH1, MSH6, CHEK2, SMARCE1, ATM, BRCA1, and CTNNA1) in 9 patients, including 3 of 27 patients (11%) with familial UM and 6 of 127 patients (4.7%) with a high risk for cancer. Two patients showed pathogenic variants in CHEK2 and PALB2, whereas variants in the other genes each occurred in 1 patient. Biallelic inactivation of PALB2 and MLH1 was observed in tumors from the respective patients. The frequencies of pathogenic variants in PALB2, MLH1, and SMARCE1 in UM patients were significantly higher than the observed frequencies in noncancer controls (PALB2: P = 0.02; odds ratio, 8.9; 95% confidence interval, 1.5-30.6; MLH1: P = 0.04; odds ratio, 25.4; 95% confidence interval, 1.2-143; SMARCE1: P = 0.001; odds ratio, 2047; 95% confidence interval, 52-4.5e15, respectively).

Conclusions: The study provided moderate evidence of gene and disease association of germline mutations in PALB2 and MLH1 with hereditary predisposition to UM. It also identified several other candidate susceptibility genes. The results suggest locus heterogeneity in predisposition to UM. Genetic testing for hereditary predisposition to cancer is warranted in UM patients with strong personal or family history of cancers, or both.

Figure 1.. UM patient with germline PALB2 mutation with evidence of biallelic inactivation of PALB2 in the tumor

A) Whole exome sequencing of germline DNA (PB) and tumor tissue (T) identified a germline canonical splice site variant c.3201+1G>C with a somatic frameshift mutationc.3279delT:p.Ile1093fs in PALB2. B) Variants were confirmed by direct sequencing. C), Mutation signatures from the patient’s tumor were significant for SBS39 and SBS1 which are commonly reported in sporadic UM. D) The expression of PALB2 mRNA was significantly lower in the tumor tissue from the patient compared to the matching non-tumor choroid.

Figure 2:. Assessment of biallelic inactivation in tumors from patients with germline variants in MLH1, SMARCE1 and MSH6

A) Direct sequencing confirmedthe germline variant c.200G>A, p.Gly67Glu and showed allelic imbalance in the tumor tissue. The variant is reported as pathogenic in ClinVar (rs63749939). B) Immunohistochemistry of tumor and tumor with adjacent non-tumor tissue show loss of nuclear expression of MLH1 protein in all tumor cells with preserved expression in the nuclei in non-tumor tissue. C) and D) A patient showed a null variant in SMARCE1 with variant of uncertain significance in MSH6. Immunohistochemistry showed preserved nuclear expression of SMARCE1 (C) and MSH6 (D) in the tumor.