Dataset Information

A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes.

ABSTRACT: BACKGROUND: Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous. METHODS: Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput. RESULTS: 169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays. CONCLUSIONS: This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved.

SUBMITTER: Hondow HL

PROVIDER: S-EPMC3146935 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes.

Hondow Heather L HL Fox Stephen B SB Mitchell Gillian G Scott Rodney J RJ Beshay Victoria V Wong Stephen Q SQ Dobrovic Alexander A

BMC cancer 20110624

<h4>Background</h4>Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA ...[more]

PMID: 21702907

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Project description:INTRODUCTION:BRCA1/2 gene mutations increase risk of breast and/or ovarian cancer and may have implications for reproductive health. Indirect biomarkers of the ovarian primordial follicle pool (anti-Müllerian hormone (AMH)) and one small study in female cadavers suggest that ovarian reserve may be reduced in BRCA mutation carriers, but findings are conflicting and association between circulating AMH and primordial follicle number is not established. The aim of this study is to measure primordial follicle density in premenopausal ovarian tissue samples from women with BRCA1/2 gene mutations versus age-matched comparison group. METHODS AND ANALYSIS:Prospective observational study measuring associations between BRCA gene mutation status, premenopausal ovarian primordial follicle density and serum AMH concentrations versus age-matched premenopausal women from the general population. Primordial follicle density will be measured in cortical sections from ovarian tissue collected at the time of risk-reducing bilateral salpingo-oophorectomy (RRBSO) in 88 BRCA1 gene mutation carriers, 65 BRCA2 gene mutation carriers and 157 non-mutation carriers. Primordial follicle density will be determined by counting follicles in a known volume of ovarian cortical tissue using light microscopy. Follicles will be identified by immunohistochemical staining for oocyte marker mouse vasa homologue. To inform the mechanisms underlying reduced ovarian reserve, the proportion of follicles containing oocytes with DNA damage will be determined by immunohistochemical staining for phosphorylated histone H2AX and terminal deoxynucleotidyl transferase dUTP nick end labelling assay to identify apoptotic cells. Follicle density will be correlated with circulating AMH concentrations quantified in the same cohort, using an electrochemiluminescence immunoassay on an automated platform. ETHICS AND DISSEMINATION:Ethics approval has been granted by Peter MacCallum Cancer Centre to access biobanks, including; The Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab-HREC#97_27) and the What Happens after Menopause? (HREC12PMCC24-12/90) and Melbourne IVF.

Project description:BRCA testing is recommended for young women diagnosed as having breast cancer, but little is known about decisions surrounding testing and how results may influence treatment decisions in young patients.To describe the use of BRCA testing and to evaluate how concerns about genetic risk and use of genetic information affect subsequent treatment decisions in young women with breast cancer.Cross-sectional analysis of data collected following the opening of the study to accrual from October 10, 2006, through December 31, 2014, as part of the Helping Ourselves, Helping Others: Young Women's Breast Cancer Study, an ongoing prospective cohort study. Study participants included 897 women aged 40 years and younger at breast cancer diagnosis from 11 academic and community medical centers.Frequency and trends in the use of BRCA testing and how genetic information is used to make treatment decisions among women who test positive vs negative for a BRCA mutation.A total of 780 (87.0%) of 897 women reported BRCA testing by 1 year after breast cancer diagnosis (mean age at diagnosis, 35.3 vs 36.9 years for untested women; P?<?.001), with the frequency of testing increasing among women diagnosed from August 1, 2006, through December 31, 2013. Of 39 women who were diagnosed as having breast cancer in 2006, 30 (76.9%) reported testing. In 2007, a slightly lower percentage of women (87 of 124 [70.2%]) reported testing; however, the proportion tested increased each subsequent year, with 141 (96.6%) of 146 and 123 (95.3%) of 129 women diagnosed as having breast cancer in 2012 and 2013, respectively, reporting BRCA testing (P?<?.001). Among untested women, 37 (31.6%) of 117 did not report discussion of the possibility that they might have a mutation with a physician and/or genetic counselor, and 43 (36.8%) of 117 were thinking of testing in the future. A total of 248 (29.8%) of 831 women said that knowledge or concern about genetic risk influenced treatment decisions; among these women, 76 (86.4%) of 88 mutation carriers and 82 (51.2%) of 160 noncarriers chose bilateral mastectomy (P < .001). Fewer women reported that systemic treatment decisions were influenced by genetic risk concern.Rates of BRCA1 and BRCA2 mutation testing are increasing in young women with breast cancer. Given that knowledge and concern about genetic risk influence surgical decisions and may affect systemic therapy trial eligibility, all young women with breast cancer should be counseled and offered genetic testing, consistent with the National Comprehensive Cancer Network guidelines.

Dataset Information

A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes.

Publications

A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes.

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