A total of 117 patients with breast cancer with a familial history of cancer, all of whom were diagnosed by experienced pathologists at The Xijing Hospital (Shaanxi, China) were recruited for the present study between July 2015 and December 2016. The inclusion criteria were as follows: i) Women aged between 18 and 74 years old with primary breast cancer, regardless of histological type or stage; and ii) patients had at least one first-degree, or two second-degree relatives with cancer (no limitation on tumor type). Exclusion criteria were as follows: i) Patients with breast cancer with no familial history of cancer; and ii) patients with breast cancer without informed consent forms. Within the same time period, a total of 250 healthy volunteers were included in the study as a control group. The mean ages of the case and control groups were 52.5±9.7 and 52.1±9.9 years, respectively. All participants enrolled in our research were from unrelated families and provided written informed consent. The Ethics Committee of Xijing Hospital approved the study protocol. Peripheral blood samples were drawn and stored in EDTA-coated tubes at −80°C.

DNA was extracted from peripheral blood using commercially available kits, in accordance with the manufacturer's protocol (E.Z.N.A.^® Blood DNA Mini kit, Omega Bio-Tek, Norcross, GA, USA). A 300 bp fragment within the extracellular domain of ERBB2 was amplified using PCR. Primer sequences were: Forward 5′-ACGTGCTCATCGCTCACAAC-3′ and reverse 5′-CCCAGAAGGGACACCATTTC-3′. The PCR product was confirmed using electrophoresis in a 1.5% agarose gel and subsequently purified using the E.Z.N.A.^® Gel Extraction kit (Omega Bio-Tek). The purified PCR product sequencing was performed with a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) on the 3730×l DNA Analyzer system (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the data obtained were analyzed with the 3730×l Genetic Analyzer Data Collection Software v3.0 (Applied Biosystems; Thermo Fisher Scientific, Inc.).

The scores of SIFT (13), PolyPhen v2 (14), MutationTaster (15), and GEPR++ (16) was collected from dbNSFP (version 2.6) (17) to predict the possible impact of an amino acid substitution on the structure and the function of HER2. PyMOL 2.0 software (https://pymol.org/2/) (18) was used for the analysis of the 3D structure of HER2 encoded by rs185670819. Conservation analysis among a range of vertebrate species was performed using data obtained from University of California Santa Cruz Genome Browser [Human Dec. 2013 (GRCh38/hg38) assembly; http://genome.ucsc.edu] (19).

A χ² test was used to assess the differences in genotype frequencies between cases and controls. A one-sided P-value and odds ratios (OR) with 95% confidence interval (95% CI) were calculated to demonstrate an association between the allelic ERBB2 and breast cancer risk. P<0.05 was considered to indicate a statistically significant difference. The Hardy-Weinberg equilibrium (HWE) test was performed to confirm the samples are representative of the population according to a method previously described (20).

To examine whether ERBB2 c.338G>A: p.R113Q was a potential risk factor for patients with breast cancer with a familial history of malignant tumors, ERBB2 c.338G>A was sequenced using the DNA isolated from peripheral blood samples of 117 Chinese women with breast cancer. Both the case and control groups consisted of individuals from the Han ethnic community of China, and there were no relationships between the recruited patients. The clinical and pathological characteristics of the patients with breast cancer are presented in Table I. The majority of the subjects had early-stage breast cancer (stage I, stage IIA and stage IIB); accounting for 78.6% of the overall participants, and 99.1% of the patients had no distant metastasis. The histological type of invasive ductal carcinoma accounted for 88%, which was the most common among the recruited patients. Almost one-half of the patients with breast cancer were negative for HER2 expression (n=54, 46.2%).

The R113Q variant that was detected in the present study (Table II; Fig. 1) resulted in an amino acid alteration from arginine to glutamine in the receptor L domain of ERBB2. All the R113Q carriers had heterozygous genotypes (Table II). The frequency of R113Q occurrence was significantly higher in patients with breast cancer with a family history of tumors (8/117, 6.8%) compared with the healthy group (3/250, 1.2%; χ²=8.712, P=0.003; Table II). The risk of breast cancer increased significantly with the occurrence of R113Q carriers (OR=6.04; 95% CI 1.573–23.214, P=0.009; Table II). There was no deviation in the HWE of R113Q (P=0.70), which indicated the population was representative.

Additionally, the results of the 1,000 Genomes Project indicated that the frequency of occurrence of this allele accounts for 1.9% of the Han people from Beijing and the south of China (Table III), a figure similar to our finding (1.2%). Furthermore, the R113Q allele was present at a lower frequency in Japanese populations and was not detected at all in Europeans (Table III) (21).

Out of the eight carriers with family histories of cancer, six of the patients had at least one relative with tumors of the digestive system. The rate of R113Q-associated breast cancer with a family history of tumors of the digestive system was 75% (Table IV). In the present study, the R113Q was more prevalent in patients with breast cancer who did not present with amplification of ERBB2 (Table IV).

The R113Q variant in the ERBB2 gene was predicted to be involved in the extracellular ligand-binding domain, a domain that is highly conserved in biological evolution. The mutation-induced structural change of the extracellular domain was thus hypothesized to cause a deficit in function. The arginine at residue 113 of ERBB2 was highly conserved among different vertebrates (Fig. 2) where a red box indicates residue 113 of HER2 in each of the vertebrates. Although the predicted effects of the R113Q mutation from four different programs were inconsistent, two of the in silico tools predicted its pathogenicity, although specific details were not provided (Table V). The results of the 3D structure suggested that the alteration of the amino acid at position 113 (Fig. 3) led to a structural change in the side chain of ERBB2. Accordingly, the R113Q variant was predicted to have a potentially damaging effect on the function of the protein, the mechanism of which will be investigated in future studies.