Although research on hereditary GC in China is limited, investigations on other neoplasms such as breast cancer, ovarian cancer, and prostate cancer have suggested that the genetic spectrum of Chinese patients with hereditary tumors may be different from that in Caucasians. The heterogeneous clinical features of hereditary tumor syndromes and the atypical presentation of cancer family history hampers attempts to summarize and cluster genotypes and phenotypes with a traditional single-gene resolution approach. In this study, patients with hereditary high-risk factors for cancer were enrolled and clinical information including age of diagnosis, special histologic types, family history of malignant tumors, and microsatellite status was used to explore the frequency and spectrum of germline variants of cancer-predisposing genes. This is the first multicenter research study in China aiming to reveal GC-related germline variants in CDH1 and other putative cancer susceptibility genes through targeted next-generation sequencing of high-risk GC patients. This prospective study indicates that one in every four GC patients with hereditary high-risk factors may bear pathogenic/likely pathogenic cancer susceptibility gene variants. We identified deleterious germline variants involving nine different genes: MLH1 (n = 1), MSH2 (n = 1), CDH1 (n = 1), BLM (n = 1), PALB2 (n = 1), EXT2 (n = 1), CHEK2 (n = 1), ERCC2 (n = 1), and SPINK1 (n = 2).
Compared with previous studies, the present spectrum of germline variants derived from Chinese gastric patients demonstrated a distinct pattern. Although genes such as CDH1, MSH2, and PALB2 were also identified in previous studies[6-9], there is a marked difference in the types of variants involved and other mutated loci were identified here for the first time.
We identified two pathogenic variants in the MMR genes, which are associated with Lynch syndrome (LS). First, the proband of the LZ2 family carried the MLH1 splicing variant (c.790+1G>A), which results in the loss of amino acids 227–295 in the MLH1 protein and has been shown functionally to render MLH1 defective in mismatch repair activity. This variant has been reported in individuals with LS and colorectal cancer[11, 12]. In addition, multiple clinical diagnostic laboratories/reputable databases classify this variant as pathogenic. Second, the proband of the BZ3 family carried the MSH2 nonsense mutation (c.610G>T, p.G204*). Sheng et al. detected this variant in one HNPCC family and classified it as a pathogenic mutation.
Both of the above families met the clinical criteria for LS. The lifetime risks for LS-associated cancers are highest for colorectal cancer at 52%–82%, followed by an endometrial cancer risk of 25%–60% in women, a 6%–13% risk for gastric cancer, and 4%–12% for ovarian cancer. Both probands had multiple primary malignant tumors.
Clinically defined HDGC is characterized by early-onset, multigenerational diffuse GC and lobular breast cancer. Clinical criteria for HDGC were established by the International Gastric Cancer Linkage Consortium (IGCLC). CDH1 is a cancer predisposition gene mutated in families meeting the criteria for clinically defined HDGC, with approximately 40% of HDGC families having germline mutations in CDH1. For example, Hansford et al. identified 47 distinct pathogenic mutations in 183 index cases meeting the clinical criteria for HDGC (25.7%), and among these, 31 cases carried pathogenic CDH1 mutations. The CDH1 germline mutation rate is negatively correlated with the morbidity of GC worldwide. In countries with low morbidity, such as Canada, the United States, the United Kingdom, and the Netherlands, the CDH1 mutation rate can be as high as 51.6% in patients meeting the HDGC clinical criteria. However, in Japan, which has the highest gastric cancer morbidity, the CDH1 mutation rate is 15.4%. There were 15 families meeting the HDGC clinical criteria of IGCLC2015 in our study but only one proband carried the CDH1 gene germline mutation (c.1475_1479delGAGTG, p.V493Sfs*42). Thus, the CDH1 mutation rate in our study was 6.7%.
The ERCC2 missense mutation (c.1532G>A, p.R511Q) is not described in any of the queried databases, but it was predicted in silico to be pathogenic when using the DANN, GERP, dbNSFP. FATHMM, LRT, MetaLR, MetaSVM, MutationAssessor, MutationTaster, PROVEAN, and SIFT bioinformatic tools. Therefore, this variant was classified as likely pathogenic.
The BLM gene is the causative gene of Bloom syndrome (BS). Bloom syndrome is an autosomal recessive disorder characterized by proportionate pre- and postnatal growth deficiency; sun sensitivity; telangiectatic, hypo- and hyperpigmented skin; predisposition to malignancy; and chromosomal instability. This variant was classified as likely pathogenic.
The EXT2 gene is the causative gene of hereditary multiple exostoses (HME). HME is an autosomal dominant disorder characterized by multiple exostoses most commonly arising from the juxtaepiphyseal region of the long bones. The EXT2 frameshift variant was classified as likely pathogenic.
The CHEK2 variant (c.1553_1554insG, p.S518Rfs*7) is a well-described, lower penetrance mutation that is mainly associated with breast cancer but also colorectal cancer and prostate cancer. This frameshift variant results in the loss of almost 10% of the protein sequence and a functional study reported that the missing region includes amino acid residues Pro515–Pro522, which is a nuclear localization signal (NLS) Thus, this variant was classified as likely pathogenic.
PALB2 colocalizes with BRCA2 in nuclear foci, promoting its localization and stability in nuclear structures, and enabling its recombinational repair and checkpoint functions. A previous study showed that PALB2 is a breast cancer susceptibility gene. The PALB2 splicing mutation (c.1684+1G>A) results in abnormal splicing of the mRNA, which affects protein function. This variant was detected in one patient with high-risk neuroblastoma and classified as likely pathogenic in accordance with a previous report.
The SPINK1 splicing mutation (c.194+2T>C) affects a donor splice site in intron 4 of the SPINK1 gene. It is predicted to affect mRNA splicing, resulting in a significantly altered protein due to either exon skipping, shortening, or the inclusion of intronic material. Experimental studies have shown that this splice site variant completely abolishes the expression of SPINK1 mRNA and protein in cell cultureThis variant is recurrent in individuals of Asian descent with chronic pancreatitis[22, 23]. Loss-of-function variants of SPINK1 are known to be pathogenic. Multiple clinical diagnostic laboratories have, therefore, classified this variant as pathogenic.
In summary, of the nine pathogenic/likely pathogenic mutations found in this study, four mutations have been reported in previous studies, while the other five mutations are considered novel mutations.
We found a total of 129 variants of uncertain significance (VUS) in 27 of the patients. Most of these VUS were missense mutations, while 27 VUS were predicted in silico to be high-risk variants. The pathogenic classification of two of these VUS is controversial as described below. We used Sanger sequencing to validate these VUS in the two patients. The familial pedigrees of these two patients are shown in Fig. 3.
MUTYH-associated polyposis (MAP) is an autosomal recessive disease that usually appears in patients with an attenuated polyposis phenotype. This syndrome is associated with biallelic mutations in the MUTYH gene. MAP typically presents with multiple colorectal adenomas and an increased risk for colorectal cancers. Gastric cancer among these patients is uncommon; it is reported in only 2% of cases. The proband of the BZ1 family carried the MUTYH splicing mutation (c.934-2A>G), which alters a conserved intronic nucleotide and causes aberrant splicing based on in vitro studies. However, whether this alteration causes a biological loss of function of the MUTYH protein in humans is uncertain. This variant has been widely studied in East Asian populations and is frequently reported in individuals with colorectal cancer. Only one homozygous patient with gastric cancer has been described with this mutation, while the rest of the reported patients harbor this variant in the heterozygous state[26-28]. Multiple clinical diagnostic laboratories/reputable databases have classified this variant as a VUS or likely pathogenic. This conflicting evidence has prevented the pathogenicity or neutrality of this variant from being established with certainty, and it was, therefore, classified here as a VUS. Additional studies are needed to clarify the significance of this variant.
The proband of the BZ14 family carried the CHEK2 missense mutation (c.1111C>T, p.H371Y). Liu et al. have reported that the c.1111C>T variant confers a significantly increased risk of breast cancer in the Chinese population but the clinical significance of this association has not been established. Additional, smaller studies in Asian populations have identified this variant in breast cancer cases as well as the controls[30, 31]. In vitro functional studies have shown that this missense change causes a decrease in phosphorylation and enzymatic activity compared with the wild-type CHEK2 protein. However, the decreased activity caused by this variant is not as pronounced as the effect caused by a known kinase-disruptive variant. In silico analyses support that this variant does not alter protein structure or function. Although there is some indication that this variant could cause disease, the evidence is insufficient at this time to prove this conclusively. Therefore, based on these data and the proband’s pedigree, we classified it as a VUS.
There are many differences in ethnicity, diet, and living habits between China and Western countries. Therefore, we cannot simply refer to the relevant foreign screening criteria for hereditary gastric cancer. One of the purposes of this study was to establish screening criteria for hereditary gastric cancer in China. This study referenced the clinical criteria for HDGC and LS as screening criteria. We enrolled patients with hereditary high-risk factors including the age of diagnosis, special histologic types, family history of malignant tumors, and microsatellite status. We observed that patients who had multiple onset primary malignancies seemed to be more likely to have pathogenic germline mutations(50% vs 7.69%, Fisher Exact P = 0.0105; see Table 3).
Our study has several limitations that should be noted. The size of the cohort recruited in this study was limited; thus, we were unable to unequivocally define the disease-causing variants and, therefore, the GC-predisposing genes. A large number of VUS were disclosed with the application of next-generation sequencing. Several candidate VUS were considered to be potentially pathogenic based on certain ACMG criteria and bioinformatic prediction. Thus, further functional studies in vitro and in vivo should be performed to correctly classify these variants.