Germline mutations in MAP3K6 are associated with familial gastric cancer

Abstract

Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.

Figure 1. Pedigree of the Maritime Canadian family.

Clinically affected individuals are indicated with shaded symbols. Individuals for whom DNA samples were collected are indicated by a number. Individuals shaded within 2 quarter sections were affected but were negative for the MAP3K6 mutation. Individuals shaded on one half had another, non-gastric, cancer. Generations I–VI are indicated.

Figure 2. Histology of two gastric cancer patients from the Maritime Canadian family.

A) Patient 1826. Section shows a tumor composed of solid and glandular components (H&E, 40×). B) The tumor cells show signet ring cell morphology in solid areas (H&E, 200×). C) Patient 1884. Section shows sheet of tumor cells infiltrated lamina propria and submucosa (H&E, 40×). D) The tumor entirely consists of signet ring cells (H&E 200×).

Figure 3. A) Pedigree of the Portuguese Familial Gastric Cancer family.

Affected individuals are shaded in black with the sequenced proband indicated with a triangle. Deceased individuals are marked with a strike-through. Generations I–III are indicated. B) Tumor cells showing signet ring cell morphology (H&E, 200×). C) Tumor cells retaining E-cadherin protein expression (IHC analysis performed with the rabbit anti-E-cadherin Antibody (24E10 Cell Signaling, MA, USA), according to manufacturer's instructions, 200×).

Figure 4. Summary of SNVs observed in the Maritime sub-pedigree (Proband, Mother, Maternal Aunts) and in 115 probands from an additional screened cohort, including a frameshift-truncating mutation Portuguese pedigree (F849Sfs*142).

All SNVs are shown in relation to the predicted functional domains of MAP3K6.

Figure 5. Methylation analysis of the Portuguese family.

Left panel: Schematic representation of the MAP3K6 gene adapted from Ensembl genome browser (release 75). The two CpG islands analyzed are represented. CpG island 1 is mainly non-methylated for several normal tissues and cells lines represented in the scheme, while CpG island 2 displays low methylation frequency (light green) in normal tissues such as B-cells, Colon, Liver and Whole Blood, and high methylation (blue) in colon (HCT116), liver (HepG2) and blood (Jurkat) cancer cell lines. A DNase HSS predicted to harbor a promoter-associated regulatory element overlapping with CpG island 2. Right panel: For the CpG island 1, no hypermethylation was detected (white circles). For the CpG island 2, we observed complete methylation in the proband's tumor DNA (black circles) and no methylation in the PBLs' DNA. The DNA of normal gastric mucosa from controls displayed partial methylation (grey circles). All gastric cancer cell lines mimicked the full methylation observed for the tumor DNA (black circles).