BRCA1 Germ-Line Mutations and Tumor Characteristics in Eastern Chinese Women with Familial Breast Cancer



Although several studies detected the BRCA1 germ-line mutations in Chinese women with familial breast cancer, most of them did not employ conventional full gene sequencing, especially in eastern China. In addition, the clinicopathological features of BRCA1-associated breast cancer in Chinese women were not well investigated. In this study, we screened the complete coding regions and exon-intron boundaries of BRCA1 by polymerase chain reaction (PCR)-sequencing assay. Immunohistochemistry analyses were performed on tumor samples to detect the expression of estrogen receptor (ER), progesterone receptor (PR), P53, and human epidermal growth factor receptor-2 (HER-2). Breast cancer patients having one or more affected relatives referred from the Zhejiang Cancer Hospital, eastern China during 2008–2011 were selected for the study. A total of 62 familial breast cancer patients received the BRCA1 germ-line mutation screening. Five deleterious mutations were detected in this cohort. The mutation rate was 11.3% (7/62). We found two novel mutations (3414delC and 5,280 C > T) and two recurrent mutations (5,273 G > A and 5589del8). BRCA1 mutation tumors tended to be negative for ER, PR, and HER-2, and exhibited high histological grade compared with tumors without BRCA1 mutations. Our study suggests that recurrent mutations may exist in eastern Chinese women with familial breast cancer and PCR-sequencing assay is a useful tool to screen these mutations. It also suggests that BRCA1-associated breast cancers in Chinese women exhibit an aggressive phenotype. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.

BRCA1 is one of the major breast cancer susceptibility genes. The lifetime risk of developing breast cancer in BRCA1 mutation carrier is 60%–80% in a Caucasian population (Antoniou et al., 2003; Begg et al.,2008). Genetic testing for BRCA1 mutation is widely applied for high-risk women in North America and Europe. However, in China, especially in the mainland, not many genetic counseling and genetic testing services are available. Although several small size studies have been carried out on the prevalence and mutation spectrum of BRCA1 in Chinese population, little has been recognized in this field. It is therefore necessary to have an overview of this gene among Chinese high-risk breast cancer patients in order to develop genetic screening guidelines for Chinese women (Cao et al.,2010).

The incidence of breast cancer is increasing among Chinese women over the last several decades. In economically developed provinces and cities, it ranks first in incidence out of all cancers among females (Dai et al.,2012). The disease remains the second leading cause of cancer deaths in Chinese women (Lei et al.,2011). Therefore, it becomes imperative to determine the genetic background of breast cancer. Some earlier studies suggested lower rates of BRCA1 mutations in Chinese women as compared to Caucasian women. But, these studies employed low cost assays, such as single-strand conformation polymorphism or denaturing high-performance liquid chromatography (DHPLC) (Suter et al.,2004; Chen et al.,2009), which may underestimate the mutation rate. Although three recurrent mutations (1100delAT, 5589del8, and 4035delTT) have been found in Chinese women with high-risk breast cancer, the limited number of cases cannot confirm these mutations as the founder mutations (Li et al.,2008; Zhang et al.,2012). Therefore, the entire coding and splice-site regions of BRCA1 should be screened. In this study, we screened entire coding regions and exon-intron boundaries of BRCA1 in 62 familial breast cancer patients from eastern China. In addition, we compared the clinicopathological data of patients with BRCA1-associated and non-BRCA1-associated breast cancers.



All the cases were diagnosed between the years 2008 and 2011 in the Zhejiang Cancer Hospital, eastern China. The criterion of familial breast cancer is that patients have at least one first- or second-degree relatives affected with breast cancer, regardless of age. Written consent was obtained from all participated patients. This study was approved by the Research and Ethical Committee of Zhejiang Cancer Hospital, China.

BRCA1 Mutation Analysis

Blood samples were obtained from the participated patients in EDTA-tubes, and genomic DNA was extracted from peripheral blood leukocytes using the ZR Genomic DNA Kit (Zymo Research, Orange County, CA). The entire coding regions and exon-intron boundaries of BRCA1 [GenBank: U14680.1] were screened by polymerase chain reaction (PCR)-sequencing assay. A total of 32 pairs of primers were synthesized by Invitrogen. The primers and PCR conditions are available on request. The PCR productions were checked on standard agarose gels before mutation analysis. If no contaminating bands were confirmed, the fragments were sequenced using BigDye Termininator Cycle Sequencing Kit and ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). All mutations were confirmed by duplicate.

Functional Prediction of Variants of Uncertain Significance

To verify variants of uncertain significance likely to disrupt BRCA1 gene function, we used an evolutionary sequence conservation model (Align-GVGD; Align-GVGD is a program that combines the biophysical characteristics of amino acids and protein multiple sequence alignments to predict where missense substitutions in genes of interest fall in a spectrum from enriched deleterious to enriched neutral (Tavtigian et al.,2008).

Immunohistochemistry Assay

The expression of estrogen receptor (ER), progesterone receptor (PR), and P53 were detected by immunohistochemistry (IHC) assay. The following monoclonal antibodies were used: anti-ER (clone: SP1 from DAKO, Glostrup, DK; dilution 1:100), anti-PR (clone: EP2 from EPITOMICS, Burlingame, CA; dilution 1:200), and anti-P53 (clone: DO-7 from DAKO, Glostrup, DK; dilution 1:400). ER, PR or P53 was considered positive when ≥10% of tumor cells showed positive nuclear staining. Human epidermal growth factor receptor-2 (HER-2) expression was first detected by IHC assay using anti-HER-2 (polyclone from DAKO, Glostrup, DK; dilution 1:300). The membrane staining was scored and a score of 3+ was considered as HER-2 positive. The PathVysion HER-2 DNA Probe Kit (Vysis, Downers Grove, IL) was used for the fluorescence in situ hybridization (FISH) test for HER-2, when the score was 2+. HER-2 gene/chromosome 17 FISH ratio of >2.2 was considered positive.

Statistical Analysis

Differences in the mean age of onset between groups were compared by independent-samples t-test. The differences in clinicopathological characteristics between BRCA1-associated and non-BRCA1-associated breast cancers were evaluated by Chi-square test and Fisher exact test. P-value < 0.05 was considered statistically significant. The statistical analyses were performed using SPSS 17.0 software.


Patient Features

A total of 120 breast cancer patients having one or more affected relatives fulfilled the inclusion criterion. All of them were from eastern China. Finally, 62 breast cancer probands accepted BRCA1 germ-line mutation screening. In our cohort of 62 breast cancer families, there was an average of 2.8 ± 0.9 (average number ± standard deviation) breast cancers per family. The onset age of breast cancer ranged from 22 years to 74 years. The mean age (±standard deviation) of diagnosis was 45.0 (±10.4) years. In all of the 62 cases, 28 reported family history of other malignancies, including 7 stomach cancers, 7 colorectal cancers, 5 lung cancers, 4 ovarian cancers, 2 esophageal cancers, 2 endometrial cancers, 2 multiple myelomas, 2 cerebral gliomas, 1 hepatobiliary cancer, 1 pancreatic cancer, 1 thyroid cancer, 1 kidney cancer, 1 bladder cancer, and 1 mediastinal tumor.

BRCA1 Germ-Line Mutation

Five deleterious mutations in BRCA1 were found in this cohort (Table 1), including two novel mutations (3414delC and 5,280C > T) that have never been reported previously. Two recurrent mutations, 5,273 G > A and 5589del8 occurred twice in exons 19 and 24, respectively. There were three frame-shift mutations and two nonsense mutations that resulted in truncation of the BCRA1 protein. No splice-site mutation was found. The mutations were mainly located in exons 11, 19, and 24. The frequency of BRCA1 mutations was 11.3% (7/62) in familial breast cancer patients in this cohort. However, the frequency of BRCA1 mutations was as high as 26% (6/23) among the women in whom breast cancer were diagnosed before the age of 40.

Table 1. BRCA1 deleterious mutations in 62 familial breast cancers
MutationaExonAA changeAge at diagnosisFamily historyReferences
  • BC, Breast cancer; Bi-BC, Bilateral breast cancer; BIC, Breast Cancer Information Core; CG, Cerebral gliomas; EC, Endometrial cancer; OC, Ovarian cancer; RC, Rectal cancer.

  • a

    Genbank reference sequences: BRCA1 version # U14680.1

3414delCExon11Stop 110840Sister (BC)Novel
4184del4Exon11Stop 136438Mother (BC), maternal aunt (EC)BIC
5273G>AExon19W1718X40Sister (BC), father (CG)BIC
5273G>AExon19W1718X41Sister (BC), mother (EC), maternal aunt (RC)BIC
5280C>TExon19Q1721X32Sister (Bi-BC), mother (OC)Novel
5589del8Exon24Stop 182637, 39 (Bi-BC)Sister (BC)BIC
5589del8Exon24Stop 182640Mother (BC)BIC

Polymorphisms and Variants

Nineteen polymorphisms or variants were detected in this cohort (Table 2). Among them, 12 polymorphisms or variants did not change the coding of amino acid. The remaining seven polymorphisms or variants varied the amino acids of BRCA1 protein, five of the seven were polymorphisms reported in BIC database, whereas two were novel. The two novel variants were 1,656 C > G (H513D) and 2,405 A > T (R762S), occurring once in different breast cancer without any deleterious mutation in BRCA1. Using Align-GVGD algorithm, the two variants were classified as C0. Therefore, these variants were considered as benign.

Table 2. BRCA1 polymorphisms/variants in 62 familial breast cancers
Sequence variantsaPositionAA changeAllelic frequencyReferences
  • BIC, Breast Cancer Information Core.

  • a

    Genbank reference sequences: BRCA1 version # U14680.1

IVS1-7G > AIntron1Noncoding0.008Novel
IVS1-3A > GIntron1Noncoding0.008BIC
1656C > GExon11H513D0.008Novel
2201C > TExon11S694S0.331BIC
2405A > TExon11R762S0.008Novel
2430T > CExon11L771L0.331BIC
2685T > CExon11Y856H0.016BIC
2731C > TExon11P871L0.331BIC
3232A > GExon11E1038G0.323BIC
3667A > GExon11K1183R0.345BIC
4427T > CExon13S1436S0.254BIC
IVS14 + 13 A > GIntron14Noncoding0.024Novel
IVS14 + 14 A > GIntron14Noncoding0.008BIC
4787A > GExon15Q1556Q0.008Novel
4956A > GExon16S1613G0.331BIC
IVS18 + 66 G > AIntron18Noncoding0.317BIC
IVS18-27 A > GIntron19Noncoding0.008Novel
IVS19-124 A > GIntron19Noncoding0.016Novel

The Clinicopathological Characteristics of Patients with Deleterious BRCA1 Mutations

The clinicopathological characteristics of the BRCA1-associated and non-BRCA1-associated patients are summarized in Table 3. The mean onset age of breast cancer in BRCA1 carriers was significantly younger than that of non-carriers (mean age ± standard error: 38.3 ± 1.2 vs. 45.9 ± 1.4, P = 0.000). Compared with non-BRCA1-carriers, BRCA1-carriers were more likely to be triple negative (ER−, PR−, and HER-2−), and exhibited high histological grade (P < 0.05). No significant differences were found in the history of ovarian cancer, tumor size, lymph node status, clinical stage, P53 expression, vessel invasion, and pathological type between the two groups (Table 3).

Table 3. Different clinicopathological characteristics between BRCA1 carriers and noncarriers in 62 familial breast cancers
CharacteristicsnBRCA1 mutationP value
Carriers n (%)Noncarriers n (%)
  1. ER, Estrogen receptor; HER-2, Human epidermal growth factor receptor-2; IDC, Invasive ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor.

Total627 (11.3)55 (88.7) 
 ≤40 years306 (85.7)17 (30.9) 
 >40 years321 (14.3)38 (69.1) 
Had ovarian cancer in family0.39
 Yes41 (14.3)3 (5.5) 
 No586 (85.7)52 (94.5) 
Tumor size   0.68
 ≤2 cm203 (42.9)17 (32.7) 
 >2 cm394 (57.1)35 (67.3) 
Lymph node status0.45
 Positive353 (42.9)32 (59.3) 
 Negative264 (57.1)22 (40.7) 
Clinical stage0.77
 I112 (28.6)9 (17.3) 
 II303 (42.9)27 (51.9) 
 III182 (28.6)16 (30.8) 
ER status0.005
 Positive330 (0.0)33 (62.3) 
 Negative266 (100.0)20 (37.7) 
PR status0.004
 Positive340 (0.0)34 (64.2) 
 Negative256 (100.0)19 (35.8) 
HER-2 status0.07
 Positive220 (0.0)22 (42.3) 
 Negative366 (100.0)30 (57.7) 
p53 status0.38
 Positive305 (83.3)25 (55.6) 
 Negative211 (16.7)20 (44.4) 
Triple negative0.00
 Yes116 (100.0)5 (9.6) 
 No470 (0.0)47 (90.4) 
Histological grade0.03
 I–II352 (40.0)40 (87.0) 
 III163 (60.0)6 (13.0) 
Vessel invasion1.00
 Yes141 (14.3)13 (23.6) 
 No486 (85.7)42 (76.4) 
Pathological type0.23
 IDC457 (100.0)38 (95.0) 
 ILC20 (0.0)2 (5.0) 


In this study, we employed the PCR-sequencing assay to screen BRCA1 germ-line mutations in 62 women with familial breast cancer from eastern China. Deleterious germ-line BRCA1 mutations were detected in 7 out of 62 cases. The mutation rate of BRCA1 was 11.3% in this cohort, which is lower than that reported in Caucasian patients (Nathanson et al.,2001), but higher than some studies in Chinese patients. Li et al. (2008) used PCR-DHPLC-sequencing assay to detect BRCA1 germ-line mutations in familial breast cancers from eastern and northern China and found that the frequency of BRCA1 mutation was 6.1% (16/261). The largest study conducted to screen germ-line mutations in BRCA1 gene by using PCR-sequencing assay, in Chinese women from northern China showed that the frequency of BRCA1 mutations was only 3.9% (16/409) in familial breast cancers (Zhang et al.,2012). Furthermore, two studies from Hong Kong (Kwong et al.,2009a,b) and Malaysia (Thirthagiri et al.,2008) also adopted PCR-sequencing assay for the BRCA1 mutation screening in Chinese familial breast cancers. The frequencies of BRCA1 mutations were found to be 6.7% (8/119) and 6.8% (8/118), respectively. In our study, the frequency of BRCA1 mutation tended to be higher as compared with the above mentioned reports. There may be several possible reasons for these differences. First, PCR-sequencing assay is the gold standard for the gene mutation screening and it is very unlikely to underestimate the mutation frequency using this assay. However, the DHPLC assay may well have missed some deleterious mutations in other studies (Klein et al.,2001). Second, the frequency of BRCA1 mutations may vary in different areas of China, being the highest in eastern China. Third, large genomic rearrangements of BRCA1 mutations were found in both Caucasian (Hogervorst et al.,2003; Montagna et al.,2003; Mazoyer et al.,2005) and Chinese populations (Yap et al.,2006; Lim et al.,2007; Kang et al.,2010; Kwong et al.,2011) by using the multiplex ligation-dependent probe amplification (MLPA) or other assays. Therefore, DHPLC or direct sequencing assay may result in the underestimation of overall frequency of BRCA1 mutations. Finally, the small sample size in our study may reflect the existence of bias.

Two novel deleterious mutations (3414delC in exon11 and 5,280 C > T in exon19) were identified in our study, and have never been reported previously. Two recurrent mutations (5,273 G > A and 5589del8), both occurring twice, were also identified (28.6% of total mutations). BRCA1 5589del8 had been reported previously in Chinese population (Suter et al.,2004; Lin et al.,2006; Li et al.,2008). Li et al. (2008) analyzed the haplotype of 5 cases with BRCA1 5589del8, and found that they had same or similar allelotypes. These results indicate that there may be some degree of shared ancestry for this recurrent BRCA1 mutation in the Chinese population. BRCA1 5273G>A was first reported in America, but it had never been reported in the Chinese population previously. BRCA1 4184del4 has been frequently reported in the Caucasian population (Evans et al.,2004; Saxena et al.,2006). To our knowledge, our study is the first to report such mutation in Chinese women with familial breast cancer. In this study, we also found two missense mutations (H513D and R762S), which had never been reported previously. Using the Align-GVGD algorithm, the two missense mutations were considered benign.

The mean age of onset of breast cancer in BRCA1 carriers was 7.6 years younger than that of non-BRCA1 carriers. It is similar to that of the other studies (Frank et al.,2002; Li et al.,2008; Zhang et al.,2012). Compared with non-BRCA1 carriers, BRCA1 carriers tended to be triple negative, and exhibit high histological grade. These phenotypes are similar to that in Caucasian women (Armes and Venter,2002; Kwong et al.,2009a,b; Zhang et al.,2012), suggesting that BRCA1-associated breast cancer may tend to be more aggressive. However, further studies are needed to describe whether these phenotypes contribute to the differences in clinical outcome. Although not significant (P = 0.07), there was a tendency towards HER-2 negativity and the frequency of HER-2 negativity was 100% in BRCA1-carriers. The frequency of HER-2 positive was 42.3% in non-BRCA1 carriers, which was 18% to 20% higher than that of general breast cancer patients (Wolff et al.,2007; Ross et al.,2009). This might be due to the small sample size of this study. Some studies reported that the history of ovarian cancer might increase the frequency of BRCA1 mutations. However, we did not find such characteristics, which may be due to our limited sample size. All of the BRCA1-associated breast cancers were invasive ductal carcinomas in this cohort, and it was similar to that of other studies in the Chinese population (Li et al.,2008; Zhang et al.,2012). However, in Caucasian women, 35%–60% of BRCA1-associated breast cancers were shown to be medullary carcinomas (Ames et al.,1998). The difference between the two races may be due to the different genetic background. In BRCA1-associated breast cancer, the frequency of TP53 mutations ranges from 56% to 100% (Holstege et al.,2009), and the expression of P53 increase (Lakhani et al.,2002). However, we did not find any significant association between the expression of P53 and the status of BRCA1 mutation. This is the first study to analyze the expression of P53 in BRCA1-associated breast cancer in a Chinese population. However, because of the limited sample size, we were not able to determine the relationship between the two factors.

In conclusion, we found that the frequency of BRCA1 germ-line mutations in eastern Chinese women with familial breast cancer is relatively higher than those in other areas of China, and the PCR-sequencing assay is a useful tool for detection BRCA1 mutations. Some typical mutations in Caucasian populations may also exist in Chinese populations. However, further studies are needed to determine whether the spectrums of BRCA1 mutations between Caucasian and Chinese are the same. BRCA1 recurrent mutations may exist in Chinese women, and a larger sample size is required to determine whether these mutations are founder mutations. The clinicopathological features of BRCA1-associated breast cancers in Chinese women are similar to Caucasian women. Therefore, inclusion of these features into risk assessment will be helpful to identify patients, who are most likely to harbor BRCA1 mutations.


The authors sincerely thank Dr. Wenyong Sun, Dr. Bo Chen, and Dr. Guo Zhang for contributing to the immunoassays.