Prevalence of BRCA1 and BRCA2 Germline Mutations in Breast Cancer Women of Multiple Ethnic Region in Northwest China.

PURPOSE: The aim of this study is to further understand the status of BRCA1 and BRCA2 mutation among Chinese high-risk breast cancer patients in multiple-ethnic regions of China.
METHODS: A total of 79 blood samples of high-risk breast cancer patients from Xinjiang Uyghur autonomous region were analyzed by PCR-DHPLC sequencing analysis.
RESULTS: Analysis with full length of the two genes identified a total of 6 deleterious mutations (2073delA, 2394C-T [Q759X] and IVS16+1G>A in BRCA1; 1627A-T [K467X], 6873delCTCC and 9481delA in BRCA2) in this cohort. The prevalence of BRCA1/2 germline mutation was about 7.6% (6/79) in the Xinjiang multiple ethnic region of China. Among them, 3 novel deleterious mutations, 2073delA in BRCA1 (Han ethnic Chinese) and BRCA2 variants 6873delCTCC and 9481delA (both are Kazakh ethnic Chinese), were identified and they had never been reported in breast cancer information core (BIC) database before. 2394C-T (Q759X) and IVS16+1G>A, in BRCA1 and BRCA2 variants 1627A-T were previously reported in other populations but not Chinese. Among 6 of the BRCA-related tumors, three BRCA1- and one BRCA2-associated tumors were in triple negative (estrogen receptor, progesterone receptor, and HER2 negative expressed) status and exhibited a high tumor grade. So far none of these 6 deleterious mutations were reported in ethnic Han Chinese.
CONCLUSION: BRCA germline mutation in Chinese multiple ethnicity region may exhibit different genotypes compared to ethnic Han Chinese in other regions. These differences may arise from interaction of genetic background and environmental factors.

INTRODUCTION

China is a multiple ethnic country comprising 56 ethnic groups. As the majority of the population belongs to the Han ethnic group, China's other 55 ethnic groups are customarily referred to as the national minorities. Xinjiang is the largest administrative region located in the northwest of China, spanning over 1.6 million km2. It is home to a number of different ethnic groups including the Han, Uyghur, Kazakh, Hui, Kyrgyz, Mongol, etc. Three of the groups had a population of more than one million, including Han (8.75 million), Uyghur (8.8 million), and Kzakh (1.35 million) [1]. To date, only a few studies have been reported on BRCA-associated breast cancer in China; they included families with early-onset breast (under the age of 35 years) patients or with family history of breast/ovarian cancer or bilateral breast cancer [2-4]. These studies revealed some characteristics of BRCA-related cancer in Chinese: the prevalence in Chinese population was lower than those in Caucasian and other Asian patients. On the other hand, no characteristic mutation spots were reported in mainland China. All the above mentioned studies only involved ethnic Han Chinese. Up to now, few studies have investigated the prevalence of BRCA1 and BRCA2 germline mutation among patients in the multiple ethnic regions of China. We believe it is of great significance to have an overview of these two genes among the Chinese high-risk breast cancer patients in multiple ethnic regions of China, so as to develop genetic screening guidelines for the Chinese women.

METHODS

Subjects

All patient cases were recruited from the Xinjiang Cancer Institute and Hospital of Xinjiang Medical University, Xinjiang, China. The patients were included if one of the following items was met: 1) at least one first or second degree relative with breast cancer and/or ovarian cancer, regardless of age; 2) at least one first-degree relative with malignant tumors other than breast cancer and ovarian cancer; 3) breast cancer onset age below 35 years; 4) bi-lateral breast cancer; or 5) triple negative breast cancer (estrogen receptor [ER], progesterone receptor [PR], and HER2/neu negative) with onset age below 45 years of age. Seventy-nine breast cancer patients, who received the standard treatment at the Xinjiang Cancer Institute and Hospital during 2005 to 2011, were finally included. Their family histories were retrieved from the medical records and ascertained by the families and/or patients in person with standard questionnaires procedure. All the cases were from different families. The study was approved by the Ethics Committee (IRB approval number: XJYD1320) of the Cancer Hospital of Xinjiang Medical University and informed consents were obtained from all participants.

Mutation analysis

Genomic DNA was isolated from 5 mL peripheral blood and stored in 10 mM Tris (pH 8) EDTA at 4℃. Specific BRCA1 and BRCA2 coding regions and intron-exon boundaries, ranging from 206 to 639 bp in length, were amplified by polymerase chain reaction (PCR) (primer sequences and conditions available upon request). PCR was carried out with a 25 µL system containing 100 ng genomic DNA, 1.5 mM MgCl2, 50 mMKCl, 10 mM Tris-HCl (PH 8.3), 200 µM dNTPs, 0.5 µM of each primer, and 0.2 µL of Taq polyMerase (5 U/µL) (Clontech, Palo Alto, USA). The PCR conditions were: denaturing at 95℃ for 5 minutes followed by 35 cycles of denaturing at 94℃ for 30 seconds annealing at 55℃ for 45 seconds and extension at 72℃ for 1 minute; one final step at 72℃ for 5 minutes.

Denaturing high-performance liquid chromatography

PCR-DHPLC analysis was used to screen mutations from all coding exons and adjacent intronic splice junction regions of the BRCA1 and BRCA2 genes as previously described [5]. The DHPLC gradients and temperatures were determined by WAVE maker software and were properly adjusted.

Assessment of clinicopathologic features

ER, PR, histological grade (Bloom-Richardson Grading System) and HER2 status were collected from the pathology reports of the tumor samples. A triple negative breast cancer is characterized by the lack of ER, PR, and HER2.

RESULTS

Patient's characteristics

A total of 79 patients were recruited in this study and were analyzed for both BRCA1 and BRCA2 mutation. In all the included patients, 32 early onset cases were diagnosed below 35 years of age, 18 had at least one affected relative, 16 had bilateral breast cancer, and 3 had both bilateral breast cancer and affected relatives. Twenty-four were triple negative breast cancer with onset age below 45 years. Eight patients reported family history of malignancies of other organs, including the stomach, pancreas, gallbladder, bladder and uterus, liver and bone.

Pathogenic mutations

Six deleterious mutations in BRCA1/2 gene were found in the 79 patients (Table 1). Most of the BRCA1/2 mutations were either frameshift or nonsense mutations, which resulted in premature truncation of the BRCA1/2 protein (Table 1). Three novel deleterious mutations were identified: 2073delA in BRCA1 (Han ethnic Chinese) and BRCA2 variants 6873delCTCC and 9481delA (both are Kazakh ethnic Chinese). Among 6 of BRCA-related tumors, 3 BRCA1- and 1 BRCA2-associated tumors were all in triple negative status.

Table 1

Disease associated BRCA1 and BRCA2

A total of 6 disease-associated mutations were detected in this study. Three mutation in BRCA1 and 3 in BRCA2. GeneBank reference sequence BRCA1 version #U14680.1, BRCA2 version #U43746.1.

OC=ovarian cancer; FH=family history; TNBC=triple negative breast cancer; BIC=breast cancer information core; BI-BC=bilateral breast cancer.

No BRCA1/2 mutation was found in 18 patients with a family history of breast cancer. One pathogenic BRCA mutation (1/3, 33.3%) was detected in 3 breast cancer patients with family history of ovarian cancer. Two pathogenic BRCA mutations (2/8, 25%) were identified in 8 breast cancer patients with family history of other malignancies. In 32 breast cancer patients with onset under 35 years old, one pathogenic BRCA (1/32, 3.1%) mutation was detected. One pathogenic BRCA mutation (1/16, 6.25%) was detected in 16 patients with bilateral cancer. One BRCA pathogenic mutation (1/3, 33.3%) was detected in 3 cases with bilateral breast cancer and family history of breast cancer or other malignancies. Four pathogenic mutations (16.7%) (including 3 BRCA1 mutation and 1 BRCA2 mutation) were detected in 24 patients with triple negative breast cancer under 45 years old.

Variation of unknown significance

In our study, three unclassified variants were detected. These 3 novel variants were not previously reported in the breast cancer information core (BIC) database (Table 2).

Table 2

Variants of unknown significance

GeneBank reference sequence BRCA1 version #U14680.1, BRCA2 version #U43746.1.

FH=family history; BC=breast cancer; TNBC=triple negative breast cancer; BIC=breast cancer information core.

DISCUSSION

According to segregation analysis studies, 5% to 10% breast and/or ovarian cancer cases arise from an autosomal dominant pattern of inheritance [6,7]. Germline mutations in BRCA1 and BRCA2 genes are responsible for the genetic predisposition and increased risk for breast and ovarian cancer [8,9]. The prevalence of BRCA mutations varies among different populations due to founder mutation effects and other environmental and geographical factors [10,11]. Genetic cancer risk assessment guideline and genetic testing for inherited breast cancer susceptibility have become standard clinical management for selected patients in Western populations. But the situation in China is less managed, with only a very limited number of studies focusing on BRCA mutations among hereditary predisposition to breast cancer patients in China.

A few studies have reported BRCA1 and BRCA2 in high-risk cohorts among Chinese in Malaysia, Singapore, and Mainland China [12-15] with the majority of published studies in the Caucasian population. Ethnic Han Chinese patients were enrolled in most of these studies, with no study focusing on multiple ethnic groups in China. Moreover, up to now there have been no studies on the prevalence of BRCA mutations in triple negative breast cancer in the Chinese population.

In this study based at our hospital, 79 patients with hereditary predisposition to breast cancer (including patients with early onset breast cancer, family history of breast, ovarian cancer or other malignant tumors, bilateral cancer, or triple negative breast cancer with onset age below 45 years old) were examined for both BRCA1 and BRCA2 mutation. Six (7.6%) of the patients were identified to harbor a deleterious BRCA mutation. Among the 79 patients, 3 (3.8%) had a BRCA1 mutation and 3 (3.8%) had a BRCA2 mutation. The results are consistent with the previous reports [3,15-17], showing BRCA2 mutation may not be less prevalent at least in some Asian populations. In contrast, in most published data from the Caucasian population, BRCA1 germline mutation is more prevalent than BRCA2 [18]. One pathogenic BRCA mutation (1/16, 6.25%) was detected among 16 bilateral cancer cases. One BRCA pathogenic mutation (1/3, 33.3%) was detected in 3 bilateral breast cancer cases with a family history of breast cancer or other malignancies. One pathogenic BRCA mutation (1/4, 25%) was detected in 4 breast cancer patients with an ovarian cancer family history. Due to our limited sample size, more comprehensive data need to be collected especially for bilateral breast cancer with a family history and breast cancer with a family history of ovarian cancer in multiple ethnic regions in Xinjiang, China.

Four pathogenic mutations (16.7%) were detected in 24 patients with triple negative breast cancer under the age of 45. All the 3 BRCA1 mutations are in triple negative breast cancer cases with higher histological grade of invasive ductal carcinoma (average score 7.7). It is consistent with the previous findings in the Asian populations, including Chinese [3,15-17] that BRCA1 mutated tumor conferred such features: ER or PR negative, higher histological grade, but with less medullary carcinoma compared to the Western population [19]. Therefore it is suggested that triple negative breast cancer of onset age below 45 may be good indicator for genetic screening in Chinese population. It is also supported in the recent National Comprehensive Cancer Network (NCCN) guidelines published in March 2011: 'woman with breast cancer who was diagnosed up to age 45 years with or without triple negative breast cancer' (http://www.jnccn.org/content/8/5/562.full).

Three BRCA1- and three BRCA2-disease-associated mutations were found in our study. Among the 6 BRCA mutations, two BRCA1 and one BRCA2 sequence variants were found in BIC or published literature. BRCA1 2394 C-T (Q759X) was previously described in an Indian family and European population (BIC) [20], but not identified in the Chinese population before. In this study this mutation was found in triple negative breast cancer patients with onset age 44 in ethnic Han Chinese group without the family history. BRCA1 IVS 16+1 G>A was previously reported in European and Latin American populations (BIC); in this study, it was found in an ethnic Uyghur triple negative breast cancer patient in China. BRCA2 1627A-T (K467X) was previously reported in a Korean population and identified as recurrent mutation in the Korean population [21-25]. In this study it was found in a triple negative breast cancer patient (onset age 42) of Han Chinese ethnicity; and it was also the first in China. Three novel deleterious mutations were firstly identified in this study, which had never been reported in BIC or published literatures. Three of the 6 deleterious mutations in this study were found in ethnic Han Chinese for the first time, 1 was from Uyghur Ethnic group (never before reported in the Uyghur population outside China) [20, 26-29], and 2 from Ethnic Kazak group (never before reported in the Kazak population outside China). It suggested that the BRCA mutation prevalence and spectrum in the Xinjiang multiple ethnic group region may arise from interaction of genetic background and environmental factors.

We detected three novel mutations of unknown significance (Table 2). All these mutations are from 3 independent Han Chinese families. One early onset breast cancer case (onset age: 23) harbors -7 C-T BRCA1 mutation, which is located in 5'UTR with CG-TG substitution and may influence CpG island function. Patient MGL harbors one BRCA1 missense mutation and in frame deletion in BRCA2; it is unknown whether these alterations influences the protein folding structure. Patient GZR harbors 3 missense mutation clustered in a small region of Exon 16 in BRCA1. Interestingly, D1582G changed aspartic acid (side-chain negative charge polarity) into Glycine (side chain neutral). Further functional assay should be done to classify the clinical significance of these variants. Some novel techniques could be considered for early breast cancer screening in China [30].

Our study only provides some preliminary information on the prevalence of BRCA in the Xinjiang multiple ethnic regions in China. Larger sample genetic testing should be done among the breast cancer patients with hereditary predisposition, especially bilateral breast cancer with a family history, breast cancer with a family history of ovarian cancer and triple negative breast cancer with onset age below 45 years. We did find some potentially 'hot' BRCA mutation regions or spots in the Xinjiang women, but a conclusion can not be made on larger sample study. No founder mutation has been found in Xinjiang and other parts of Mainland China as yet. More data should be collected on BRCA distribution in Chinese breast cancer patients, especially in multiple ethnic regions of China. China is a developing country with multiple ethnic groups. Due to variations in the breast cancer prevalence, molecular subtypes and onset age among the different races and ethnicities, caution must be exercised in the clinical guidelines of the genetic and high familial risk assessment of breast cancer in the NCCN data, mostly of the Western populations, in the Chinese genetic testing as modified for the Chinese population.