Introduction

A wide variety of germline mutations in either BRCA1 or BRCA2 (BRCA1/BRCA2) genes are responsible for the majority of hereditary breast and ovarian cancers. The identification of mutations in the BRCA1/BRCA2 genes is necessary for genetic counseling and to consider risk-reduction strategies in high-risk family members.1

The incidence of breast cancer in Korea has continuously increased in recent years.2 Studies of breast cancer epidemiology in samples of Asian and European descent have found differences between populations, potentially due to the interactions between different lifestyle and genetic characteristics.3, 4 As genetic predispositions to breast cancer are increasingly understood, it has been suggested that there are differences between populations and that molecular diagnostic strategies should be important components of treatment. The discovery of founder mutations might lead to cost-effective options to screen high-risk families, as in Ashkenazi Jews.5

To date, only a few studies have examined the frequencies and spectra of mutations in large series of Korean breast cancer patients.6, 7, 8, 9, 10 Several mutations are considered to be founder mutations, as they account for the majority of detected BRCA1/BRCA2 mutations in Korea. Although some of these recurrent mutations are unique to Koreans, their effects vary across studies. In the present study, we investigated the frequencies and spectra of BRCA1/BRCA2 gene mutations in Korean breast cancer patients to identify common mutations and to establish an optimal molecular diagnostic strategy.

Materials and methods

A total of 277 patients who were diagnosed with breast and/or ovarian cancer in Samsung Medical Center were requested for BRCA1/BRCA2 mutation analysis during September 2001 to December 2009. Informed consent was obtained from all patients for clinical information and genetic analysis. The study was conducted in accordance with the Declaration of Helsinki of 1975, as revised in 2000. Selection of patients was based on the following criteria as previously described9, 10 and at least one of the following: (F) at least one first- or second-degree relative with breast and/or ovarian cancer diagnosed at any age, (E) early-onset breast cancer diagnosed at age 35 or before, (B) bilateral breast cancer and (M) multiple organ cancer, including breast cancer. The study subjects consisted of 134 unrelated Korean breast cancer patients who were identified as being at high risk of carrying BRCA1/BRCA2 mutations; 116 (87%) familial cases, 29 (22%) early-onset cancer cases, 13 (10%) bilateral cancer cases and 10 (7%) multiple organ cancer cases. There were no male breast cancer cases in our sample. The mean age at diagnosis was 43 years (range 22–66). A total of 96 healthy Korean subjects, who visited for regular health checkup, were screened as a control for novel sequence variations.

PCR and direct sequencing were performed covering whole exons and flanking intronic sequences of the BRCA1/BRCA2 genes with primers designed by the authors (available upon request). Reference sequences for the BRCA1 and BRCA2 genes were GenBank accession numbers NM_007294.2 and NM_000059.3, respectively. For the descriptions of the variations identified, we followed the nomenclature system of the Human Genome Variation Society (http://www.hgvs.org/mutnomen) and the conventional nomenclature system from the Breast Cancer Information Core (BIC; http://research.nhgri.nih.gov/bic/). In silico prediction of functional consequences of missense variation was performed using SIFT (Sorting Intolerant From Tolerant; http://sift.bii.a-star.edu.sg/),11 PolyPhen (Polymorphism Phenotyping, http://genetics.bwh.harvard.edu/pph2/),12 PANTHER (Protein analysis through Evolutionary Relationships, http://www.pantherdb.org)13 and SVM (Support Vector Machine).14

Results

A total of 26 mutations for the BRCA1 or BRCA2 genes were identified in 31 of 134 unrelated Korean patients (23.1%, 31/134) (Table 1). According to mutation type, frameshift mutations were most frequent (n=11), followed by nonsense mutations (n=10) and splicing mutations (n=5). Twelve of 26 distinct mutations were novel; they had not previously been reported to the BIC database or described in publications, to our knowledge. Five recurrent mutations (BRCA1: c.3627dupA, c.5445G>A and c.5496_5506del11insA; BRCA2: c.97G>T and c.6952C>T) were found in the present study, but were observed in no more than two patients and accounted for 32.3% (10/31) of the BRCA1/ BRCA2 mutations.

Table 1 BRCA1 and BRCA2 mutations in Korean patients with breast cancer

During sequencing analysis, we also detected 13 other sequence variations (9 in BRCA1 and 4 in BRCA2), which were missense variations of unknown significance within the coding regions (Table 2). These variations have not been reported in the SNP database (http://www.ncbi.nlm.nih.gov/snp) and eight have been reported in the BIC database but are of unknown clinical importance. Among the 13 unclassified variations, 11 variations have been found in previous normal control studies8, 10 and/or in the present study, but there is not reliable functional assay at present. Four different protein prediction programs (PolyPhen, SIFT, PANTHER and SVM) were used to predict the functional consequences of the mutations and obtain diverse results.

Table 2 Missense variations of unknown significance in BRCA1 and BRCA2 genes

Discussion

There have been two reports of common mutations of the BRCA1/BRCA2 genes in Korean breast cancer patients who were identified as being at high risk of carrying BRCA1/BRCA2 mutations. Ahn et al.9 reported that the combination of seven common mutations (BRCA1: c.2433delC, c.3627dupA, c.4065_4068delTCAA, c.5470_5477del8 and c.5496_5506del11insA; BRCA2: c.1399A>T and c.7480C>T) accounted for 62.5% and this suggests the possibility of founder effect. Seong et al.10 analyzed data from previous two large studies7, 9 and reported that another set of seven common mutations (BRCA1: c.390C>A, c.2433delC, c.3627dupA, c.4065_4068delTCAA and c.5496_5506del11insA; BRCA2: c.3744_3747delTGAG and c.7480C>T) was recurrent and accounted for up to 43.9% in the pooled population. Adding the results of our series, the mutations with frequencies over 3.0% in the pooled dataset were as follows; BRCA1: c.390C>A (3.1%), c.3627dupA (6.9%) and c.5496_5506del11insA (4.6%); BRCA2: c.1399A>T (3.1%), c.3744_3747delTGAG (4.6%) and c.7480C>T (13.9%), and seven most common mutations accounted for 38.7% (50/129). Interestingly, the most common mutation (BRCA2: c.7480C>T) in the previous studies, where the proportion of the mutation ranged from 15 to 20%, was detected in only one patient (3.2%, 1/31). Considering the limited number of mutation-positive patients in an individual study or a pooled dataset, additional research is required to determine the founder effect on Korean population.

Most previous studies of BRCA1/BRCA2 genes in Korea used performed two-step approaches, in which mutations are first scanned in a rapid manner, and then subjected to sequencing analysis when sequence alterations are detected.6, 7, 8, 9, 10 Table 3 shows the mutation detection rates in the BRCA1/BRCA2 genes from each study in Korea. Compared with the two studies using similar inclusion criteria for the high-risk group of Korean breast cancer patients, mutation detection rates of whole-gene sequencing exceeded 20%. In contrast, a two-step approach with a combination of fluorescent-conformation sensitive gel electrophoresis and sequencing analysis resulted in a lower mutation detection rate (11.3%), although more strict inclusion criteria for family history (at least two patients with breast and/or ovarian cancer) was applied.

Table 3 BRCA1 and BRCA2 mutation detection rates in Koreans reported in previous studies

In the present study, we identified 26 mutations in BRCA1/BRCA2 genes in 31 of 134 (23.1%) unrelated Korean patients who were identified as being at high risk of carrying BRCA1/BRCA2 mutations. Given that the rates of mutation detection using whole-gene sequencing are much higher than those of the two-step screening method and that we did not observe founder mutations, whole-gene sequencing of both BRCA1 and BRCA2 genes should be the method of choice used to identify mutations in high-risk Korean patients.