Screening of the BRCA1 gene in Brazilian patients with breast and/or ovarian cancer via high-resolution melting reaction analysis
The aim of this study was to evaluate the profile of BRCA1 mutations among cancer-affected Brazilian women from the Midwest region of Minas Gerais state with clearly defined risk factors for hereditary breast and ovarian cancer (HBOC) syndrome. In this Brazilian region, the first Center for Hereditary Cancer Control began operation in 2011, and 90 % of patients receive assistance from the public health service. Eighteen patients at high risk for HBOC were subjected to molecular analysis. Primers were designed for 22 coding exons of the gene; DNA was extracted; and real-time PCR followed by high-resolution melting reaction was performed. The amplicons were sequenced to confirm the identified profiles. Only exon 11 was directly sequenced due its length. Multiplex ligation-dependent probe amplification (MLPA) was performed for those patients in whom no pathogenic mutations were found. Among the 14 alterations identified in this study, the c.5263_5264insC pathogenic mutation was present in two patients (11.1 %). Four alterations showed no clinical relevance; one exhibited inconclusive clinical relevance according to the examined databases; and eight alterations presented a divergent classification between the databases. No deletions or duplications were found using the MLPA technique. The HRM methodology was highly sensitive in identifying variants in the BRCA1 gene and can dramatically reduce the amount of sequencing required to identify germline mutations in BRCA genes, enabling cheaper tests and increasing their availability to Brazilian women assisted by the public health service.
KeywordsBRCA1 Breast and ovarian cancer High-resolution melting Brazil
This study was funded by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, APQ-02863-11), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 401989/2010-0) and Associação de Combate ao Câncer do Centro Oeste de Minas Gerais (ACCCOM).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee.
Informed consent was obtained from all individual participants included in the study.
- 7.De Juan Jiménez I, García Casado Z, Palanca Suela S, Esteban Cardeñosa E, López Guerrero JA, Segura Huerta Á et al (2013) Novel and recurrent BRCA1/BRCA2 mutations in early onset and familial breast and ovarian cancer detected in the Program of Genetic Counseling in Cancer of Valencian Community (eastern Spain). Relationship of family phenotypes with mutation prevalence. Fam Cancer 12(4):767–777CrossRefGoogle Scholar
- 8.Hughes KS. Hughes RiskApps Express Entry. http://22.214.171.124/HRAExpressEntry/(S(k2fucwgitmeepopnkz5ptd50))/Default.aspx. Accessed 10 Jan 2014
- 9.National Center for Biotechnology Information—NCBI. http://www.ncbi.nlm.nih.gov/gene/672. Accessed 20 Jan 2014
- 10.Primer3. http://frodo.wi.mit.edu/. Accessed 5 Mar 2014
- 11.OligoAnalyzer—IDT. http://www.idtdna.com/analyzer/applications/oligoanalyzer. Accessed 5 Mar 2014
- 12.Poland Software. http://www.biophys.uni-duesseldorf.de/html/local/POLAND/poland.html. Accessed 12 June 2014
- 13.Breast Information Core—BIC. http://research.nhgri.nih.gov/bic. Accessed 27 Mar 2015
- 14.Leiden Open Variation Database—LOVD. http://www.lovd.nl/3.0/home. Accessed 27 Mar 2015
- 18.Carraro DM, Koike Folgueira MAA, Garcia Lisboa BC, Ribeiro Olivieri EH, Vitorino Krepischi AC, de Carvalho AF et al (2013) Comprehensive analysis of BRCA1, BRCA2 and TP53 germline mutation and tumor characterization: a portrait of early-onset breast cancer in Brazil. PLoS ONE. doi: 10.1371/journal.pone.0057581 Google Scholar
- 21.Felix GES, Abe-Sandes C, Machado-Lopes TMB, Bomfim TF, Guindalini RSC, Santos VCSAR, Meyer L, Oliveira PC, Neiva JC, Meyer R, Romeo M, Toralles MB, Nascimento I, Abe-Sandes K (2014) Germline mutations in BRCA1, BRCA2, CHEK2 and TP53 in patients at high-risk for HBOC: characterizing a Northeast Brazilian population. Hum Genome Var 1:14012. doi: 10.1038/hgv.2014.12 CrossRefGoogle Scholar
- 27.Arnold N, Peper H, Bandick K, Kreikemeier M, Karow D, Teegen B et al (2002) Establishing a control population to screen for the occurrence of nineteen unclassified variants in the BRCA1 gene by denaturing high-performance liquid chromatography. J Chromatogr, B: Anal Technol Biomed Life Sci 782(1–2):99–104CrossRefGoogle Scholar
- 37.Van Der Stoep N, Van Paridon ÃCDM, Janssens T, Krenkova P, Stambergova A, Macek M et al (2009) Diagnostic guidelines for high-resolution melting curve (HRM) analysis: an interlaboratory validation of BRCA1 mutation scanning using the 96-Well Lightscanner. Hum Mutat 30(6):899–909CrossRefGoogle Scholar
- 38.Abtahi H, Sadeghi MR, Shabani M, Edalatkhah H, Akhondi MM, Talebi S (2011) Causes of bimodal melting curve: asymmetric guanine-cytosine (GC) distribution causing two peaks in melting curve and affecting their shapes. Afr J Biotechnol 10(50):10196–10203Google Scholar