Identification of BRCA1/2 Founder Mutations in Southern Chinese Breast Cancer Patients Using Gene Sequencing and High Resolution DNA Melting Analysis

Background: Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China. Methodology/Principal Findings: A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM) analysis. Among the 451 probands analyzed, 69 (15.3%) deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations (c.470_471delCT, c.3342_3345delAGAA, c.5406+1_5406+3delGTA and c.981_982delAT) accounted for 34.5% (10/29) of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations (c.2808_2811delACAA, c.3109C>T, c.7436_7805del370 and c.9097_9098insA) accounted for 40% (16/40) of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated. Conclusion: In this study, our findings suggest that BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment. © 2012 Kwong et al.published_or_final_versio

High throughput germline mutation screening for hereditary breast cancer using Fluidgm Access Array and GS Junior System: a molecular diagnostic laboratory experience

Conference Theme: Sequence, sequence, and more sequenc

High throughput germline mutation screening for hereditary breast cancer using fluidigm access arrary & 454 GS junior

Open Access JournalAMP Abstracts: G50INTRODUCTION: Breast cancer is prevalent world-wide and has become the leading cause of cancer among the female population in Hong Kong. The Hong Kong Hereditary Breast Cancer Family Registry was established in 2007 and thus far 1194 patients with breast cancer were recruited from hospitals and clinics in Hong Kong. Mutational screening of susceptibility genes, BRCA1 and BRCA2, using Sanger full gene sequencing was performed and until now 706 cases have been analyzed. We sought to apply next-generation DNA sequencing to analyze the remaining samples at higher throughput. METHODS: We analyzed BRCA1, BRCA2 and also TP53 genes of 192 cases using 454 GS Junior System. Generation of barcoded amplicon libraries was streamlined by microfluidic PCR using Fluidigm Access Array System. Two Fluidigm runs can generate 41 BRAC1 and TP53 amplicons and 45 BRCA2 amplicons from each of 48 unique cases, and a total of 8 runs were performed. Each Fluidigm run was subject to one GS Junior run. Sequencing data (total length 213 Mbp) were automatically analyzed by an in-house developed bioinformatics pipeline, which mainly consists of GS Amplicon Variant Analyzer, SAMtools and Ensembl Variant Effect Predictor. To confirm the detected mutations, full BRCA1 and BRCA2 genes of 10 cases and individual deleterious mutations of remaining cases were subject to Sanger sequencing. RESULTS: On-going analysis revealed 3 novel deleterious mutations (1 BRCA1 frameshift insertion and 2 BRCA2 non-sense SNPs) among BRCA1, BRCA2 and TP53 mutations identified from 192 cases. Sensitivity of 454 sequencing approach was found to be comparable to Sanger sequencing, although detection of insertiondeletion polymorphisms around long homopolymers is challenging. Bioinformaticsbased filters were optimized based on Sanger sequencing data and were applied to minimize false positives. Signal intensity and the sequence of cyclic nucleotide flows were used in mutation prioritization to account for Carry Forward and Incomplete Extension errors around homopolymer sequences. CONCLUSIONS: Mutational screening of BRCA1, BRCA2, and TP53 genes from 192 cases were streamlined and performed at much higher throughput. The bioinformatics pipeline developed is expected to facilitate data analysis of similar diagnostic settings. In Hong Kong, our laboratory pioneered the application of both microfluidic PCR and next-generation sequencing systems for molecular diagnostics.link_to_OA_fulltex

BRCA1 and BRCA2 germline mutations and founder mutations in Chinese women with breast and ovarian cancer

Young Investigator Award - Biomedicine: abstract no. A

Male breast cancer: a comparison between BRCA mutation carriers and non-carriers in Hong Kong, Southern China

Poster Presentation: no. 0111Scientific Session AbstractsOBJECTIVES : Male breast cancer is suggested to be biologically different from female breast cancer. The differences in clinicopathology between male and female breast cancer raise the issues of establishing specific strategies and treatment regime for male breast cancer patients. The single most significant risk factor for male breast cancer is a mutation in the BRCA2 gene. The lack of information on hereditary breast cancer in males, particularly in Asians, leaves great but forgiven research area on epidemiological studies for this group of patients. METHOD: All male breast cancer patients and their family members from a Hong Kong Hereditary and High Risk Breast Cancer Program since year 2007 were recruited in this study. All received genetic counseling and BRCA mutation testing using DNA extracted from blood samples. A questionnaire was administered at their first visit which included questions on their demographics and socioeconomic status. Other information, including family history of breast cancer or other kinds of cancer, method of diagnosis, surgical strategies, pathological results, treatment regime, relapse, metastasis, and outcomes, were obtained from their medical records. Descriptive analysis was performed describing the background characteristics. Chi-square test and Student t test were applied to calculate the associations between BRCA mutation and risk factors. Survival analysis was performed to look for their survival patterns. RESULTS : Thirty-six male breast cancer patients were recruited between years 2007 and 2012, while 21 were diagnosed before year 2007 (range: 1996 to 2012). Mean, standard deviation, and median follow-up time were 5.75, 4.31, and 5.25 years. Seven were found to carry the BRCA mutation. All were BRCA2 mutation and the mutation rate was 19.4% (N = 7). Family history of cancer was found in 52.8% (N = 19).Male BRCA mutation carriers were found to have higher risk of secondary cancer, and their first- and second-degree family members had higher risk of either breast cancer or other kinds of cancers. T stage in BRCA patients was significantly higher than non-BRCA patients (p = 0.028). All BRCA mutation carriers had ER-positive cancers, compared with 96.2% who were noncarriers. Half of the male BRCA patients were PR positive, compared with higher percentage in non-BRCA patients (50% vs 80.8%, p = 0.117). Both groups had similar overall (p = 0.962) and disease-free survivals (p = 0.919). The means and standard deviations of 5-year overall survival between BRCA and non-BRCA patients were 2.08 0.25 and 4.24 0.12 years, respectively, and 2.08 3.03 and 4.41 1.46 years for disease-free survival. CONCLUSIONS : The prevalence of male breast cancer patients with BRCA2 mutation in Hong Kong is comparable with other similar studies. Male breast cancer patients with BRCA2 mutation are suspected to have higher chance of secondary cancer and familial cancer. Although percentage of ER-positive cancers are similar to the 2 groups, BRCA2 mutation carriers tend to have fewer PR-positive cancers, which may suggest a poorer prognosis although, due to a small sample size, this cannot be shown in this cohort. Further collaborative studies to better understand male breast cancer patients carrying the BRCA mutation are warranted.link_to_OA_fulltex

Enhancement of VUS interpretation by multiple algorithm in familial breast cancer

Conference Theme: Twenty Years of AdvancesSession: BRCA1/2 Mutations, variants of unknown clinical significance, and databasesPoster presentationlink_to_OA_fulltex

High throughput germline mutation screening for hereditary breast cancer in Southern Chinese patients by massively parallel DNA sequencing

Session: Genetics: Poster presentationlink_to_OA_fulltex

Epidemiology and survival patterns of triple negative breast cancer patients with or without BRCA germline mutation in Chinese

Abstract & poster presentatio