Familial Cancer

, Volume 15, Issue 2, pp 173–181 | Cite as

Screening of the BRCA1 gene in Brazilian patients with breast and/or ovarian cancer via high-resolution melting reaction analysis

  • Eneida Santos de Oliveira
  • Bárbara Luisa Soares
  • Sara Lemos
  • Reginaldo Cruz Alves Rosa
  • Angélica Nogueira Rodrigues
  • Leandro Augusto Barbosa
  • Débora de Oliveira Lopes
  • Luciana Lara dos Santos
Original Article


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.


BRCA1 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.

Ethical approval

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

Informed consent was obtained from all individual participants included in the study.


  1. 1.
    Lynch HT, Snyder C, Casey MJ (2013) Hereditary ovarian and breast cancer: what have we learned? Ann Oncol 24(Suppl 8):83–95CrossRefGoogle Scholar
  2. 2.
    Takano E, Mitchell G, Fox SB, Dobrovic A (2008) Rapid detection of carriers with BRCA1 and BRCA2 mutations using high resolution melting analysis. BMC Cancer 8:59CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    De Juan I, Esteban E, Palanca S, Barragán E, Bolufer P (2009) High-resolution melting analysis for rapid screening of BRCA1 and BRCA2 Spanish mutations. Breast Cancer Res Treat 115(2):405–414CrossRefPubMedGoogle Scholar
  4. 4.
    Druml B, Cichna-Markl M (2014) High resolution melting (HRM) analysis of DNA—its role and potential in food analysis. Food Chem 158:245–254CrossRefPubMedGoogle Scholar
  5. 5.
    Hodgson S, Caligo M, Chang-Claude J, Eccles D, Evans G, Moller P et al (2000) A survey of the current clinical facilities for the management of familial cancer in Europe. J Med Genet 37:605–607CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hondow HL, Fox SB, Mitchell G, Scott RJ, Beshay V, Wong SQ et al (2011) A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes. BMC Cancer 11(1):265CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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. 8.
    Hughes KS. Hughes RiskApps Express Entry. Accessed 10 Jan 2014
  9. 9.
    National Center for Biotechnology Information—NCBI. http://www.ncbi.nlm.nih.gov/gene/672. Accessed 20 Jan 2014
  10. 10.
    Primer3. http://frodo.wi.mit.edu/. Accessed 5 Mar 2014
  11. 11.
    OligoAnalyzer—IDT. http://www.idtdna.com/analyzer/applications/oligoanalyzer. Accessed 5 Mar 2014
  12. 12.
  13. 13.
    Breast Information Core—BIC. http://research.nhgri.nih.gov/bic. Accessed 27 Mar 2015
  14. 14.
    Leiden Open Variation Database—LOVD. http://www.lovd.nl/3.0/home. Accessed 27 Mar 2015
  15. 15.
    Dufloth RM, Carvalho S, Heinrich JK, Shinzato JY, dos Santos CC, Zeferino LC, Schmitt F (2005) Analysis of BRCA 1 and BRCA 2 mutations in Brazilian breast cancer patients with positive family history. Sao Paulo Med J 123(4):192–197CrossRefPubMedGoogle Scholar
  16. 16.
    Lourenço JJ, Vargas FR, Bines J, Santos EM, Lasmar CAP, Costa CH et al (2004) BRCA1 mutations in Brazilian patients. Genet Mol Biol 504:500–504CrossRefGoogle Scholar
  17. 17.
    Gomes MCB, Costa MM, Borojevic R, Monteiro ANA, Vieira ER, Koifman S, Koifman RJ, Li S, Royer R, Zhang S, Narod SA (2007) Prevalence of BRCA1 and BRCA2 mutations in breast cancer patients from Brazil. Breast Cancer Res Treat 103(3):349–353CrossRefPubMedGoogle Scholar
  18. 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
  19. 19.
    Esteves VF, Thuler LCS, Amêndola LC, Koifman RJ, Koifman S, Frankel PP et al (2009) Prevalence of BRCA1 and BRCA2 gene mutations in families with medium and high risk of breast and ovarian cancer in Brazil. Braz J Med Biol Res 42(5):453–457CrossRefPubMedGoogle Scholar
  20. 20.
    Ewald IP, Izetti P, Vargas FR, Moreira MA, Moreira AS, Moreira-Filho CA et al (2011) Prevalence of the BRCA1 founder mutation c.5266dup in Brazilian individuals at-risk for the hereditary breast and ovarian cancer syndrome. Hered Cancer Clin Pract 9(1):12CrossRefPubMedPubMedCentralGoogle Scholar
  21. 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
  22. 22.
    Johnson N, Fletcher O, Palles C, Rudd M, Webb E, Sellick G et al (2007) Counting potentially functional variants in BRCA1, BRCA2 and ATM predicts breast cancer susceptibility. Hum Mol Genet 16(9):1051–1057CrossRefPubMedGoogle Scholar
  23. 23.
    Tommasi S, Pilato B, Pinto R, Bruno M, Campana M, Digennaro M et al (2008) Mutation research/fundamental and molecular mechanisms of mutagenesis molecular and in silico analysis of BRCA1 and BRCA2 variants. Mutat Res 644:64–70CrossRefPubMedGoogle Scholar
  24. 24.
    Cox DG, Kraft P, Hankinson SE, Hunter DJ (2005) Haplotype analysis of common variants in the BRCA1 gene and risk of sporadic breast cancer. Breast Cancer Res 7(2):171–175CrossRefGoogle Scholar
  25. 25.
    Greenman J, Mohammed S, Ellis D, Watts S, Scott G, Izatt L et al (1998) Identification of missense and truncating mutations in the BRCA1 gene in sporadic and familial breast and ovarian cancer. Genes Chromosom Cancer 21:244–249CrossRefPubMedGoogle Scholar
  26. 26.
    Bergthorsson JT, Ejlertsen B, Olsen JH, Borg A, Nielsen KV, Barkardottir RB et al (2001) BRCA1 and BRCA2 mutation status and cancer family history of Danish women affected with multifocal or bilateral breast cancer at a young age. J Med Genet 38(6):361–368CrossRefPubMedPubMedCentralGoogle Scholar
  27. 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
  28. 28.
    McKean-Cowdin R, Spencer Feigelson H, Xia LY, Pearce CL, Thomas DC, Stram DO et al (2005) BRCA1 variants in a family study of African-American and Latina women. Hum Genet 116(6):497–506CrossRefPubMedGoogle Scholar
  29. 29.
    Savas S, Ozcelik H (2005) Phosphorylation states of cell cycle and DNA repair proteins can be altered by the nsSNPs. BMC Cancer 5:107CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Tavtigian SV, Deffenbaugh AM, Yin L, Judkins T, Scholl T, Samollow PB et al (2006) Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J Med Genet 43(4):295–305CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Calò V, Bruno L, la Paglia L, Perez M, Margarese N, di Gaudio F et al (2010) The clinical significance of unknown sequence variants in BRCA genes. Cancers (Basel) 2:1644–1660CrossRefGoogle Scholar
  32. 32.
    Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49(6):853–860CrossRefPubMedGoogle Scholar
  33. 33.
    Krypuy M, Ahmed AA, Etemadmoghadam D, Hyland SJ, DeFazio A, Fox SB et al (2007) High resolution melting for mutation scanning of TP53 exons 5–8. BMC Cancer 7:168CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    De Leeneer K, Coene I, Poppe B, De Paepe A, Claes K (2009) Genotyping of frequent BRCA1/2 SNPs with unlabeled probes: a supplement to HRMCA mutation scanning, allowing the strong reduction of sequencing burden. J Mol Diagn 11(5):415–419CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Chen D, Wang Y-Y, Chuai Z-R, Huang J-F, Wang Y-X, Liu K et al (2014) High-resolution melting analysis for accurate detection of BRAF mutations: a systematic review and meta-analysis. Sci Rep 4:4168PubMedPubMedCentralGoogle Scholar
  36. 36.
    Liu Y, Wu H, Yang X, Xu H, Chen D, Huang Q et al (2014) Diagnostic accuracy of high resolution melting analysis for detection of KRAS mutations: a systematic review and meta-analysis. Sci Rep. doi: 10.1038/srep07521 Google Scholar
  37. 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. 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
  39. 39.
    Erali M, Voelkerding KV, Wittwer CT (2009) High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 85(1):50–58CrossRefGoogle Scholar
  40. 40.
    Van der Groep P, van der Wall E, van Diest PJ (2011) Pathology of hereditary breast cancer. Cell Oncol (Dordr) 34(2):71–88CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Eneida Santos de Oliveira
    • 1
  • Bárbara Luisa Soares
    • 1
  • Sara Lemos
    • 2
  • Reginaldo Cruz Alves Rosa
    • 3
  • Angélica Nogueira Rodrigues
    • 2
  • Leandro Augusto Barbosa
    • 1
  • Débora de Oliveira Lopes
    • 1
  • Luciana Lara dos Santos
    • 1
  1. 1.Universidade Federal de São João del Rei (UFSJ)DivinópolisBrazil
  2. 2.Associação de Combate ao Câncer do Centro Oeste de Minas Gerais (ACCCOM)DivinópolisBrazil
  3. 3.Centro Universitário de Formiga (UNIFOR-MG)FormigaBrazil

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