Background
In 2009, the morbidity rate of breast cancer was 42.55
per 100,000 Chinese women, and breast cancer ranked first in cancer incidence and fifth in cancer-related deaths among females [
1]. The mean age at diagnosis of breast cancer is 45–55 years in Chinese women, which is considerably younger than that in western women [
2]. A significant proportion of breast cancer in Chinese women is caused by genetic alterations. Germline mutations in many genes, such as
BRCA1,
BRCA2,
ATM,
TP53,
RAD51C and XRCC2, have been identified to be associated with breast cancer [
3‐
5]. Several studies have investigated germline mutations in genes including
BRCA1,
BRCA2,
TP53,
BRIP1,
PALB2,
CHEK2,
RAD50,
NBS1 and
RAD51C in Chinese women with high risk breast cancer [
6‐
21]. We previously summarized the spectrum of the germline mutations in these genes and found that the
BRCA1 and
BRCA2 tumor suppressor genes are the two most important susceptibility genes and account for nearly 98 % of hereditary breast cancer in China [
22]. We found that the spectrum of
BRCA1 and
BRCA2 germline mutations in Chinese high risk breast cancer patients are much smaller than those in Caucasian patients, and little has been recognized in this field. The overall mutation frequencies in these two genes in Chinese high risk breast cancer patients ranged from 8.3 to 27.8 %, depending on the detection methods and patient inclusion criteria used. These frequencies are much lower than the 25–40 % in
BRCA1 and 6–15 % in
BRCA2 that have been observed in Caucasian populations [
22]. Because germline mutations in
BRCA1 and
BRCA2 greatly increase a woman’s risk of developing breast and/or ovarian cancer, and the prevalence and distribution of the germline mutations differ in different races/ethnicities, we were interested in identifying the full spectrum of these mutations in high-risk female breast cancer patients in the Chinese population.
In this study, we screened the entire coding regions and exon-intron boundaries of the BRCA1 and BRCA2 genes in 133 familial breast/ovarian cancer patients from eastern China. A total of 23 deleterious mutations, including 12 novel mutations (five in BRCA1 and seven in BRCA1), were detected in these two genes in 31 familial breast/ovarian cancer patients, and the total mutation frequency was 23.3 % (31/133). The highest frequency of 50.0 % (8/16) was found in the breast cancer patients with a history of ovarian cancer. Six recurrent mutations were found, including four in BRCA1 and two in BRCA2. We also found 11 unclassified variants (UVs), nine of which were novel. Additionally, using comparative evolutionary bioinformatic programs, we identified the non-synonymous amino acid changes that are likely to disrupt the functions of the BRCA1 and BRCA2 genes. Our study suggested that BRCA1 and BRCA2 mutations accounted for a considerable proportion of the hereditary breast/ovarian cancer patients in eastern China and that the spectrum of the mutations in these genes exhibited unique features.
Discussion
BRCA1 and BRCA2 are the most important genetic susceptibility genes for breast/ovarian cancer in both Caucasian and Chinese populations. The spectrum and frequencies of mutations in these two genes in Chinese women with familial breast/ovarian cancer have been insufficiently explored to date. Moreover, the penetrance has not yet been investigated. Due to the limited knowledge on hereditary breast/ovarian cancer, there is no genetic counseling or testing services available in Mainland China.
Our results demonstrated that the frequency of
BRCA1 and
BRCA2 mutations among Chinese women with familial breast/ovarian cancer was 23.3 %. Similar results have been reported in the Korean population [
23], Hispanic population [
24] and Africa American population [
25]. However, the frequency observed in the current study is lower than that reported in an Ashkenazi Jewish population, in which the frequency of
BRCA1 and
BRCA2 mutations was 69 % [
25]. Compared with other reports about Chinese populations, the frequency found in our cohort was the highest in patients with familial breast/ovarian cancer. Li et al. [
9] used PCR-DHPLC assay to screen for
BRCA1 and
BRCA2 mutations in 241 women with familial breast cancer from northern or southern China and found a frequency of 12.9 %. Although the PCR-DHPLC assay is cost-effective for screening for genetic mutations, a considerable number of disease-associated mutations may have been missed by this indirect detection method [
26]. Zhang et al. [
11] reported that the frequency of
BRCA1 and
BRCA2 mutations in northern Chinese familial breast cancer patients was 10.5 % (43/409) based on PCR-sequencing assay. The enrolment criteria and mutation detecting assay used in this were comparable with the criteria used in our study, but the reported frequency was much lower than that observed in the present study. In their subgroup analysis, the highest frequency was 23 % in the patients whose tumors had been diagnosed at or before the age of 40 years. However, the frequency reached 33.3 % in this group of patients in our cohort. Moreover, in the study conducted by Kwong et al., [
12] the frequency of
BRCA1 and
BRCA2 mutations in high-risk breast/ovarian cancer patients was 15.3 % (69/651). These authors also employed the conventional PCR-sequencing assay, and the patients were recruited from southern China. The proportion of high-risk breast/ovarian cancer patients, including familial breast cancer patients and early-onset cases and the frequency of two-gene mutations were much lower in the early-onset patients than in the familial breast cancer cases. Large genomic rearrangements account for 4–28 % of all
BRCA1 and
BRCA2 mutations [
27], and such mutations have been found in Chinese women at a high risk for breast cancer [
28‐
32]. Because the PCR-sequencing assay cannot detect these rearrangements, the frequency of mutations in our cohort might have been underestimated, and the frequency of
BRCA1 and
BRCA2 mutations in the eastern Chinese population could be significant.
Although several studies have reported that the
BRCA2 mutations are more frequent than
BRCA1 mutations in Asian population [
11,
12,
33,
34],
BRCA1 mutations seemed to be more prevalent in our cohort. This finding might be attributable to two points. First, most studies have reported that
BRCA2 mutations predominantly occur in relatively late-onset breast cancer patients compared with
BRCA1 mutations [
11,
35], but the patients enrolled in our study were much younger than those in other studies, which might have resulted in an underestimation of the contribution of
BRCA2 mutations. Second, a greater number of recurrent mutations were found in
BRCA1 than in
BRCA2 in our study, which elevated the frequency of
BRCA1 mutations.
In the present study, we found that 52.2 % (12/23) of the deleterious mutations were novel; these mutations included five mutations in
BRCA1 and seven mutations in
BRCA2. In our previous systemic analysis of the spectrum of
BRCA1 and
BRCA2 mutations in Han Chinese women, we reported that 56.3 % (40/71) and 47.9 % (35/73) of the
BRCA1 and
BRCA2 mutations were novel, respectively [
22]. It seems that the spectrum of
BRCA1 and
BRCA2 mutations in Chinese women exhibit unique features. The
BRCA2 mutation c.1-40delGA in our cohort was novel. Bakker et al. [
36] found a
BRCA2 c.1-40 G > A mutation in a Japanese Fanconi anemia family. The functional analysis of these authors used a mouse embryonic stem cell-based assay that revealed that this mutation caused aberrant splicing, reduced transcript levels and hypersensitivity to DNA damaging agents, suggesting that this mutation was likely pathogenic. These authors thought that this finding was relevant for mutation analysis in hereditary breast and ovarian cancer syndrome families in a diagnostic setting. The mutation c.1-40delGA, which deletes a guanine in intron 1 and an adenine in exon 2 and causes the loss of the donor site of intron 1, should also be pathogenic.
Six
BRCA1 and
BRCA2 recurrent mutations were identified in multiple patients, and these accounted for 45.2 % (14/31) of the total patients with mutations. Of these mutations, one (c.3780_3781delAG) was novel, another (c.5468-1del8) was recently reported in Chinese women [
11], and the remaining four had been reported in the BIC database. Founder mutations provide population-specific genetic risk assessment, and facilitate genetic mutation screening. Thus far, few studies have suggested that putative founder mutations of
BRCA1 and
BRCA2 might exist in Chinese women at a high risk for breast cancer, such as the c.981delAT and c.5470_5477del8 mutations in
BRCA1 and the c.3109C > T, c.7436_7805del370 and c.9097_9098insA mutations in
BRCA2 [
9,
10,
12]. In our cohort, the
BRCA1 c.5470_5477del8 mutation and
BRCA2 c.3109C > T mutation were both recurrent, but no other three putative founder mutations was found. Our haplotype analysis revealed that
BRCA1 c.5154G > A and c.5468-1del8 mutations were the two putative founder mutations. Since there are only two patients reported for each of the putative founder mutation, the founder effects are needed to be investigated by larger sample size of patients. In our previous study, we reported that the most common recurrent mutations in Chinese women at high risk for breast cancer are c.5470_5477del8 in
BRCA1 and c.3109C > T in
BRCA2 [
22], which were reported to be the putative founder mutations. However, the study that enrolled the greatest number of familial breast cancer patients from northern China did not find these six putative founder mutations except the
BRCA1 c.5468-1del8 mutation [
11]. The discrepancy regarding the founder mutations in Chinese familial breast cancer patients may be due to geographic differences. The characterization of
BRCA1 and
BRCA2 founder mutations and association between the founder mutations and breast cancer risk should be studied in a large-scale Chinese population size.
Although, elevated mutation rates of
BRCA1 and
BRCA2 were found in patients who had been diagnosed at or before 40 years of age, no significant differences were found between the
BRCA1 mutation carriers,
BRCA2 mutation carriers and non-carriers when compared to a mean age at diagnosis. The inconsistent results implied that these observations did not withstand multiple comparisons in our cohort. Breast cancer patients with family histories of ovarian cancer exhibited the highest overall mutation rate of
BRCA1 and
BRCA2, which implied that
BRCA1 and
BRCA2 mutations are more likely to occur in families with a history of both breast and ovarian cancer. This result is consistent with those of other studies [
9,
11].
Eleven UVs were found in our study, and the potentials for these variants to disrupt the functions of
BRCA1 and
BRCA2 varied according to the algorithm program used. The UVs accounted for nearly 1/3 of the total mutations/variants in this study. The risks of breast and ovarian cancer in the UVs carriers might be as high as those in the carriers of the classical pathogenic mutations. A variety of approaches have been used to investigate the clinical relevance of these UVs. Co-segregation analysis is regarded as a robust approach because it is directly related to the disease risk and is not affected by selection bias [
37]. The absence of co-segregation provides strong evidence against pathogenicity. Unfortunately, the samples required for us to perform co-segregation analysis of UVs and the deleterious mutations in the multi-tumor families were not available.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
WMC: designed the study, analyzed the mutational data, performed haplotype analysis and drafted the manuscript. YG and ZWP: performed PCR and sequencing studies. HJY, SNX and XWD: collected the clinical and pathological data. WWY: performed the statistical analysis. XJW: conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.