Background
Breast cancer was the second most common cancer in females aged 15–64 in Korea [
1] and in 2013, a total of 17,292 incident breast cancer cases were reported in Korea Central Cancer Registry [
2]. Breast cancer incidence in Korea has markedly increased in recent years, and the crude incidence of breast cancer in Korea is the highest among Asian countries [
3]. Also, the average age at onset of breast cancer is 10 years earlier than Western populations. A younger age at diagnosis suggests that genetic susceptibility genes may be involved in a substantial proportion of breast cancer in Korea.
Recently, genetic counselling and genetic testing of
BRCA1 and
BRCA2 for the presence of germline inactivating mutations have been increasingly offered to identify individuals at elevated risk of breast and ovarian cancer in Korea. According to a large nationwide prospective Korean Hereditary Breast Cancer (KOHBRA) study, 153 distinct
BRCA1/2 mutations have been identified in Korean breast cancer patients with a family history of breast/ovarian cancer resulting in a prevalence of 22.3% [
3].
However, until recently, testing of
BRCA1 and
BRCA2 mutations has been focused on the identification of small-scale mutations (point mutations, small deletions and insertions). Such mutations occur throughout the whole coding sequence and at the splice junctions of both genes. They result in protein truncation, disruption of messenger RNA processing, or amino acid substitutions that have significant impact on protein function and are readily detectable by standard methods of Sanger sequencing of polymerase chain reaction (PCR)-amplified gene segments [
4]. Large genomic rearrangement (LGR) of
BRCA1 and
BRCA2, which is another mechanism of gene inactivation, is responsible for a variable but significant proportion of
BRCA mutations [
5]. High prevalence of LGRs in
BRCA1 have been demonstrated in several populations, including Dutch, Northern Italian, French, and Czech, and in such populations, LGR screening has been advocated as a cost-effective, initial phase screening test [
6]. In Korea, reported LGR cases are few, and routine PCR-based genetic testing methods are not capable of detecting LGRs. Therefore, an efficient genetic testing algorithm that incorporates LGR testing is necessary for accurate mutational screening of high-risk patients.
Herein, we performed multiplex ligation-dependent probe amplification (MLPA) for LGR analysis in a subset of small-scale mutation negative patients who were also stratified as high-risk for LGR based on previously published LGR risk criteria from other ethnicities [
5,
7]. Here we report the prevalence of the different type of
BRCA mutations according to risk stratification, to provide evidence for developing an effective and comprehensive
BRCA genetic screening strategy in Korean patients.
Methods
Patients and clinical diagnosis
A total of 106 patients at risk for hereditary breast and ovarian cancer (HBOC) and for whom mutation analysis was requested from January 2015 to November 2015 at Seoul St. Mary’s Hospital were included in this study. The family history, past medical history, and tumor pathology of the probands and their family members were detailed by their referring physicians and/or through review of patient’s medical records. All participants gave informed consent, and this study was approved by the Institutional Review Board (IRB)/Ethics Committee of Seoul St. Mary’s Hospital (IRB No.KC15RISI0915).
The referred patients all met the National Comprehensive Cancer Network genetic testing criteria for HBOC syndrome [
8] and high-risk subgroup for LGR was defined based on the previously published LGR risk criteria [
5,
7,
9]. The inclusion criteria for the high-risk subgroup were personal history of 1) early-onset breast cancer (diagnosed at ≤36 years); 2) two breast cancer primaries; 3) breast cancer diagnosed at any age, with ≥1 close blood relatives (includes first-, second-, or third-degree) with breast and/or epithelial ovarian cancer; 4) both breast and epithelial ovarian cancer diagnosed at any age; and 5) epithelial ovarian cancer with ≥1 close blood relatives with breast and/or epithelial ovarian cancer.
Sanger sequencing
Sanger sequencing was performed in all patients to detect small-scale mutations. Genomic DNA was isolated from the peripheral blood leukocytes, using the QIAmp DNA Mini Kit (Qiagen, Hamburg, Germany). Sanger sequencing was performed as described previously [
10]. Exon numbering and DNA sequence variant descriptions are based on NM_007294.3 and NM_000059.3 as reference sequences for
BRCA1 and
BRCA2. To classify variants, we followed the standards and guidelines of the American College of Medical Genetics and Genomics (ACMG) for the interpretation of sequence variants [
11], and all variants were scored and classified into five pathogenicity groups (class 1: benign; class 2: likely benign; class 3: uncertain significance (VUS); class 4: likely pathogenic; class 5; pathogenic).
MLPA analyses
MLPA was performed for all Sanger sequencing-negative patients in the LGR high-risk subgroup. MLPA probe mixes P002 and P045 were used for screening of LGRs in
BRCA1 and
BRCA2, respectively, and P087 and P077 were used for confirmation, according to the manufacturer’s recommendations (MRC-Holland, Amsterdam, Netherlands). MLPA data were analyzed using Genemarker v1.91 (Softgenetics, State College, PA). Peak heights were normalized and a deletion or duplication was defined as recommended by the manufacturer. Direct sequencing of the probe binding and ligation sites was performed in relevant exons to detect if any polymorphism was located close to the ligation site, which may lead to a false decrease in peak signal [
4].
Statistical analyses
Categorical variables were compared using the Chi-square test. Continuous variables were compared using the independent samples t-test or Mann–Whitney-Wilcoxon rank sum tests. MedCalc version 12.1.4 (MedCalc Software, Mariakerke, Belgium) was used and P < 0.05 was considered statistically significant.
Discussion
In this report of 106 consecutive Korean patients at risk for HBOC from a single center cohort, we identified 2 LGRs in Sanger-negative, high-risk patients. Also, 11 BRCA1 and 6 BRCA2 small-scale mutations were identified and our report extends the spectrum of BRCA mutations by detecting two novel frame-shift mutations in BRCA1/2. The overall prevalence of BRCA mutations was 20% for all patients, 30% for breast cancer, and 18% for ovarian cancer patients.
The frequency of mutations was related to the type as well as the number of risk factors. Strong predictors of the likelihood of carrying a
BRCA mutation in patients with personal history of breast cancer were the occurrence of both breast and ovarian cancer (43%), bilateral breast cancer (38%), and positive family history (37%), and the presence of positive family history (55%) in patients with personal history of ovarian cancer. However, there was no difference between the proportion of early-onset breast cancer patients between the mutation positive group and the mutation negative group. Furthermore, all of the
BRCA mutation-positive patients with early-onset breast cancer, had multiple risk factors other than early-onset. This is in agreement with earlier reports that in Korean, non-familial, early-onset breast cancer patients without other risk factors, the prevalence of
BRCA mutation is low [
3,
18].
The most important finding of this study is that selective screening of high-risk, Sanger-negative Korean patients for LGRs using MLPA analysis, identified 2 patients with LGRs in
BRCA1. The LGRs comprised 2% of all enrolled patients but accounted for 7% of Sanger-negative, high-risk patients and 12% of all identified
BRCA mutations in high-risk patients. The frequency of LGR varies considerably among populations, with LGRs accounting for one third of
BRCA1 mutations in northern Italy, and 27–36% of
BRCA1 mutations in Netherlands, but less common in other populations [
9]. LGRs in
BRCA are considered to be rare in Korea, with a reported frequency of 0.45% in familial breast cancer patients [
12] and 2.1% in Sanger-negative, familial breast cancer patients [
14]. And, only 5 LGR cases have been reported so far in Korea [
3]. However, the characterization of risk characteristics of LGRs in any given population allows a more efficient and cost-effective mutational screening approach. Our results suggest that risk stratification and selectively screening Sanger-negative, high-risk patients for LGRs is an effective screening strategy for LGR detection in Korean patients. And with emerging therapies, such as poly ADP ribose polymerase inhibitors in combination with conventional treatment [
19], a comprehensive genetic evaluation strategy encompassing LGRs as well as small-scale mutations has become even more critical.
This study has certain limitations. We did not perform MLPA for all Sanger-negative patients, but only in high-risk patients, and the number of LGR cases is limited. And since, Sanger-negative, non-high-risk patients were not included in the MLPA analysis, the true frequency of LGRs may be greater than the reported 2% of all enrolled patients. However, previous reports have indicated that patients harboring LGR appears to be at the highest end of the range of risks associated with
BRCA mutation [
5,
9], and the probability of an LGR among non-high-risk patients can be regarded as significantly low. Whereas the present study is limited by small sample size and was not designed to provide a comprehensive survey of the frequency of LGRs, our data suggest that in high-risk families with a negative Sanger sequencing result, LGRs represent a significant source of
BRCA mutations.
Conclusions
In summary, we have applied a simple LGR risk criteria, and have shown that screening Sanger-negative high-risk patients for LGR is an effective and necessary genetic testing strategy in Korean patients. Based on the results of this study, risk stratification of at risk patients for HBOC and selective screening for LGRs in BRCA1 is recommended for Korean patients.
Acknowledgements
We are grateful to the patients and their treating physicians who participated in this study, and also acknowledge the support of Catholic Genetic Laboratory Center for carrying out this study.