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Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil

Abstract

In Brazil, breast cancer is a public health care problem due to its high incidence and mortality rates. In this study, we investigated the prevalence of hereditary breast cancer syndromes (HBCS) in a population-based cohort in Brazils southernmost capital, Porto Alegre. All participants answered a questionnaire about family history (FH) of breast, ovarian and colorectal cancer and those with a positive FH were invited for genetic cancer risk assessment (GCRA). If pedigree analysis was suggestive of HBCS, genetic testing of the BRCA1, BRCA2, TP53, and CHEK2 genes was offered. Of 902 women submitted to GCRA, 214 had pedigrees suggestive of HBCS. Fifty of them underwent genetic testing: 18 and 40 for BRCA1/BRCA2 and TP53 mutation screening, respectively, and 7 for CHEK2 1100delC testing. A deleterious BRCA2 mutation was identified in one of the HBOC probands and the CHEK2 1100delC mutation occurred in one of the HBCC families. No deleterious germline alterations were identified in BRCA1 or TP53. Although strict inclusion criteria and a comprehensive testing approach were used, the suspected genetic risk in these families remains unexplained. Further studies in a larger cohort are necessary to better understand the genetic component of hereditary breast cancer in Southern Brazil.

Keywords
Breast cancer predisposition syndrome; hereditary breast cancer; genetic cancer risk assessment

Introduction

Breast cancer (BC) is a significant health care problem worldwide, and approximately 1.67 million new cases were diagnosed in 2012, representing 25% of all cancers (Globocan). In Latin America, BC is the most prevalent solid tumor diagnosed in women in the majority of countries (Goss et al., 2013Goss PE, Lee BL, Badovinac-Crnjevic T, Strasser-Weippl K, Chavarri-Guerra Y, St Louis J, Villarreal-Garza C, Unger-Saldaña K, Ferreyra M, Debiasi M, et al. (2013) Planning cancer control in Latin America and the Caribbean. Lancet Oncol 14:391-436.). In Brazil, 51,120 new BC diagnoses were estimated for 2014, and the disease is the first cause of death by cancer in women of all ages, especially in young women, under the age of 50 years (DataSUS, 2014DataSUS (2014) http://www.datasus.gov.br (accessed June 18, 2014).
http://www.datasus.gov.br...
; INCa, 2014INCa (2014) National Institute of Cancer - INCa, http://www.inca.gov.br (accessed November 2, 2014).
http://www.inca.gov.br...
). The South and Southeast regions of Brazil have the highest BC incidence rates: 70.98 and 71.98 cases in 100,000 women respectively (INCa, 2014INCa (2014) National Institute of Cancer - INCa, http://www.inca.gov.br (accessed November 2, 2014).
http://www.inca.gov.br...
). In comparison to national figures, Rio Grande do Sul, the southernmost State, presents high BC incidence (87.12:100,000) and mortality rates (13.18:100,000), adjusted rates in 2011 (INCa, 2014INCa (2014) National Institute of Cancer - INCa, http://www.inca.gov.br (accessed November 2, 2014).
http://www.inca.gov.br...
).

An estimated 5-10% of all BCs are hereditary, i.e. caused by germline mutations in high-penetrance cancer predisposition genes (King et al., 2003King MC, Marks JH, Mandell JB and Group, New York Breast Cancer Study (2003) Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science 302:643-646.). Of these, the more prevalent mutations are in BRCA1 (OMIM#113705) and BRCA2 (OMIM#600185) (Miki et al., 1994Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM and Ding W (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266:66-71.; Wooster et al., 1994Wooster R, Neuhausen SL, Mangion J, Quirk Y, Ford D, Collins N, Nguyen K, Seal S, Tran T and Averill D (1994) Localization of a breast cancer susceptibility gene, BRCA2, to chromosome 13q12-13. Science 265:2088-2090.), tumor suppressor genes which are associated with hereditary breast and ovarian cancer (HBOC) syndrome. Lifetime risks of breast and ovarian cancer are as high as 65-85% in BRCA1 and 45-85% in BRCA2 mutation carriers (Antoniou et al., 2006Antoniou AC, Pharoah PD, Easton DF and Evans DG (2006) BRCA1 and BRCA2 cancer risks. J Clin Oncol 24:3312-3313; author reply 3313-3314.; Cass et al., 2003Cass I, Baldwin RL, Varkey T, Moslehi R, Narod SA and Karlan BY (2003) Improved survival in women with BRCA-associated ovarian carcinoma. Cancer 97:2187-2195.; Euhus and Robinson, 2013Euhus DM and Robinson L (2013) Genetic predisposition syndromes and their management. Surg Clin North Am 93:341-362.). To date, over 3.000 distinct germline mutations, polymorphisms and sequence variants have been described in BRCA1 and BRCA2, spread throughout both genes (Breast Cancer Information Core, BIC, 2014BCI (2014) Breast Cancer International Core database, BIC, http://research.nhgri.nih.gov/bic. (accessed July10 14).
http://research.nhgri.nih.gov/bic...
). Most are point mutations, small insertions or deletions. However, large genomic deletions and duplications involving one or more exons of BRCA1, and less commonly, BRCA2, have been reported (Gutiérrez-Enríquez et al., 2007Gutiérrez-Enríquez S, de la Hoya M, Martínez-Bouzas C, Sanchez de Abajo A, Ramón y Cajal T, Llort G, Blanco I, Beristain E, Díaz-Rubio E, Alonso C, et al. (2007) Screening for large rearrangements of the BRCA2 gene in Spanish families with breast/ovarian cancer. Breast Cancer Res Treat 103:103-107.; Preisler-Adams et al., 2006Preisler-Adams S, Schönbuchner I, Fiebig B, Welling B, Dworniczak B and Weber BH (2006) Gross rearrangements in BRCA1 but not BRCA2 play a notable role in predisposition to breast and ovarian cancer in high-risk families of German origin. Cancer Genet Cytogenet 168:44-49.; Thomassen et al., 2006Thomassen M, Gerdes AM, Cruger D, Jensen PK and Kruse TA (2006) Low frequency of large genomic rearrangements of BRCA1 and BRCA2 in western Denmark. Cancer Genet Cytogenet 168:168-171.). Most of these mutations are caused by recombination events involving Alu repeats that are particularly numerous in the BRCA1 locus (Payne et al., 2000Payne SR, Newman B and King MC (2000) Complex germline rearrangement of BRCA1 associated with breast and ovarian cancer. Genes Chromosomes Cancer 29:58-62.). The proportion of genomic rearrangements over all BRCA1 gene mutations in HBOC families seems to be population-dependent varying from 2% in a series of American families (Hendrickson et al., 2005Hendrickson BC, Judkins T, Ward BD, Eliason K, Deffenbaugh AE, Burbidge LA, Pyne K, Leclair B, Ward BE and Scholl T (2005) Prevalence of five previously reported and recurrent BRCA1 genetic rearrangement mutations in 20,000 patients from hereditary breast/ovarian cancer families. Genes Chromosomes Cancer 43:309-313.) to 36% in Dutch patients (Petrij-Bosch et al., 1997Petrij-Bosch A, Peelen T, van Vliet M, van Eijk R, Olmer R, Drüsedau M, Hogervorst FB, Hageman S, Arts PJ, Ligtenberg MJ, et al. (1997) BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients. Nat Genet 17:341-345.). In Rio de Janeiro, (Moreira et al., 2012Moreira MA, Bobrovnitchaia IG, Lima MA, Santos AC, Ramos JP, Souza KR, Peixoto A, Teixeira MR and Vargas FR (2012) Portuguese c.156_157insAlu BRCA2 founder mutation: Gastrointestinal and tongue neoplasias may be part of the phenotype. Fam Cancer 11:657-660.) screened 168 BC affected women for BRCA2 c.156_157insAlu and found 3 unrelated carriers. Other studies have screened cohorts of Brazilian BC patients for specific mutations, or have focused on some subgroups (for example young women) (Carraro et al., 2013Carraro DM, Koike Folgueira MA, Garcia Lisboa BC, Ribeiro Olivieri EH, Vitorino Krepischi AC, de Carvalho AF, de Carvalho Mota LD, Puga RD, do Socorro Maciel M, Michelli RA, 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 8:e57581.; Ewald et al., 2011Ewald IP, Izetti P, Vargas FR, Moreira MA, Moreira AS, Moreira-Filho CA, Cunha DR, Hamaguchi S, Camey SA, Schmidt A., et al. (2011) Prevalence of the BRCA1 founder mutation c.5266dupin Brazilian individuals at-risk for the hereditary breast and ovarian cancer syndrome. Hered Cancer Clin Pract 9:12.; Gomes et al., 2007Gomes MC, Costa MM, Borojevic R, Monteiro AN, Vieira R, Koifman S, Koifman RJ, Li S, Royer R. Zhang S, et al. (2007) Prevalence of BRCA1 and BRCA2 mutations in breast cancer patients from Brazil. Breast Cancer Res Treat 103:349-353.) but the exact prevalence of any BRCA mutation remains largely unknown.

Besides BRCA1 and BRCA2, inherited mutations in other tumor suppressor genes also increase the risk for breast cancer and other tumors. Highly penetrant, hereditary breast cancer genes include PTEN (Cowden's syndrome), TP53 (Li-Fraumeni syndrome), STK11 (Peutz-Jeghers syndrome) and CDH1 (Hereditary diffuse gastric cancer. Germline mutations in CHEK2 gene are associated with a modest increase in the risk of breast (15-25% lifetime risk) and colorectal cancer in the hereditary breast and colon cancer syndrome (HBCC) (Euhus and Robinson, 2013Euhus DM and Robinson L (2013) Genetic predisposition syndromes and their management. Surg Clin North Am 93:341-362.; Meijers-Heijboer et al., 2003Meijers-Heijboer H, Wijnen J, Vasen H, Wasielewski M, Wagner A, Hollestelle A, Elstrodt F, van den Bos R, de Snoo A, Fat GT, et al. (2003) The CHEK2 1100delC mutation identifies families with a hereditary breast and colorectal cancer phenotype. Am J Hum Genet 72:1308-1314.).

In this study, we analyzed a population-based cohort of women recruited from primary health care units in Porto Alegre who were referred to genetic cancer risk assessment (GCRA) whenever they had a positive family history for breast, ovarian and colorectal cancer (Palmero et al., 2009Palmero EI, Caleffi M, Schüler-Faccini L, Roth FL, Kalakun L, Netto CB, Skonieski G, Giacomazzi J, Weber B, Giugliani R, et al. (2009) Population prevalence of hereditary breast cancer phenotypes and implementation of a genetic cancer risk assessment program in southern Brazil. Genet Mol Biol 32:447-455.). Those women with pedigrees suggestive of a hereditary breast cancer predisposition syndrome were offered genetic testing for germline mutations in one or more of the main breast cancer predisposition genes (BRCA1, BRCA2, TP53, PTEN and CHEK2). Our aim was to evaluate the prevalence of hereditary breast cancer phenotypes and breast cancer predisposition gene variations among at-risk women recruited from a population-based cohort in a region with high breast cancer incidence.

Subjects and Methods

Patient Recruitment

In 2004, a large population-based cohort study (the Núcleo Mama Porto Alegre – NMPOA-Cohort) was started in Porto Alegre, Southern Brazil, through a partnership between government, non-profit community-based organizations, universities and private entities. This prospective cohort intends to collect demographic and epidemiological data of a large sample of women and test a model for community- based breast cancer screening in an underserved population (Caleffi et al., 2010Caleffi M, Ribeiro RA, Bedin Jr. AJ, Viegas-Butzke JM, Baldisserotto FD, Skonieski GP, Giacomazzi J, Camey SA and Ashton-Prolla P (2010) Adherence to a breast cancer screening program and its predictors in underserved women in southern Brazil. Cancer Epidemiol Biomarkers perv 19:2673-2679.) (Smith et al., 2006Smith RA, Caleffi M, Albert US, Chen TH, Duffy SW, Franceschi D, Nyström L and Global Summit Early Detection and Access to Care Panel (2006) Breast cancer in limited-resource countries: Early detection and access to care. Breast J 12(Suppl 1) :S16-26.) in an attempt to ultimately decrease BC mortality rates in this region. The study recruited women above the age of 15 years who sought health care in 18 primary health care units (PCUs) located in specific city regions within a 24-month period. Family history (FH) of breast, ovarian and colorectal cancer was assessed in women (above age 15 years) seen in 18 primary health care units from the region by a brief questionnaire and is the basis for the study described elsewhere as the Genetic Cancer Risk Assessment Program of the NMPOA cohort (Palmero et al., 2009Palmero EI, Caleffi M, Schüler-Faccini L, Roth FL, Kalakun L, Netto CB, Skonieski G, Giacomazzi J, Weber B, Giugliani R, et al. (2009) Population prevalence of hereditary breast cancer phenotypes and implementation of a genetic cancer risk assessment program in southern Brazil. Genet Mol Biol 32:447-455.). The seven questions of this instrument refer to family history features that have been associated with an increased likelihood of clinically significant BRCA mutations and thus, these questions were designed primarily to identify patients at-risk for HBOC syndrome (Couch et al., 1997Couch FJ, DeShano ML, Blackwood MA, Calzone K, Stopfer J, Campeau L, Ganguly A, Rebbeck T and Weber BL (1997) BRCA1 mutations in women attending clinics that evaluate the risk of breast cancer. N Engl J Med 336:1409-1415.; Frank et al., 2002Frank TS, Deffenbaugh AM, Reid JE, Hulick M, Ward BE, Lingenfelter B, Gumpper KL, Scholl T, Tavtigian SV, Pruss DR, et al. (2002) Clinical characteristics of individuals with germline mutations in BRCA1 and BRCA2: Analysis of 10,000 individuals. J Clin Oncol 20:1480-1490.; Nelson et al., 2005Nelson HD, Huffman LH, Fu R, Harris EL and Force, U.S. Preventive Services Task Force (2005) Genetic risk assessment and BRCA mutation testing for breast and ovarian cancer susceptibility: Systematic evidence review for the U.S. Preventive Services Task Force. Ann Intern Med 143:362-379.; Shattuck-Eidens et al., 1997Shattuck-Eidens D, Oliphant A, McClure M, McBride C, Gupte J, Rubano T, Pruss D, Tavtigian SV, Teng DH, Adey N, et al. (1997) BRCA1 sequence analysis in women at high risk for susceptibility mutations. Risk factor analysis and implications for genetic testing. JAMA 278:1242-1250.; Srivastava et al., 2001Srivastava A, McKinnon W and Wood ME (2001) Risk of breast and ovarian cancer in women with strong family histories. Oncology (Williston Park) 15:889-902; discussion 902, 905-887, 911-813.). In addition, a question about family history (FH) of breast and/or colon cancer was included due to a previous suggestion of a higher than expected number of families with these tumors in cancer genetic clinics of Porto Alegre (Palmero et al., 2007Palmero EI, Ashton-Prolla P, da Rocha JC, Vargas FR, Kalakun L, Blom MB, Azevedo SJ, Caleffi M, Giugliani R and Schüler-Faccini L (2007) Clinical characterization and risk profile of individuals seeking genetic counseling for hereditary breast cancer in Brazil. J Genet Couns 16:363-371.). The questionnaire is presented in Table S1. Patients answering positively to at least one of the seven questions in the primary health care unit were referred to genetic risk assessment at NMPOA.

Genetic evaluation included medical and family histories recorded in detailed pedigrees with information traced as far backwards and laterally as possible, extending to paternal lines and including a minimum of three generations. Confirmation of the cancer FH was attempted in all cases and pathology and medical records, as well as death certificates, were obtained whenever possible upon specific consent from the patient and/or her family. Lifetime breast cancer risk estimates were obtained using the Claus, Gail, and Tyrer-Cuzick models. All pedigrees were reviewed by at least two clinical geneticists to assess presence of criteria for the diagnosis of LFS, LFL, HBCC, Cowden or other cancer predisposition syndromes. Patients fulfilling criteria for a breast cancer predisposition syndrome were candidates for the present study and offered genetic testing. For the clinical diagnosis of HBOC syndrome, the National Comprehensive Cancer Network (NCCN) and American Society of Clinical Oncology (ASCO) criteria were used (Ford et al., 1994Ford D, Easton DF, Bishop DT, Narod SA and Goldgar DE (1994) Risks of cancer in BRCA1-mutation carriers. Breast Cancer Linkage Consortium. Lancet 343:692-695.) (NCCN, 2014NCCN (2014) National Comprehensive Cancer Network - NCCN, http://www.nccn.org (accessed September 15, 2014).
http://www.nccn.org...
). In addition, prior probabilities of carrying a BRCA1 or BRCA2 mutations were determined for each patient using mutation prevalence tables and the modified Couch mutation prediction model (Domchek et al., 2003Domchek SM, Eisen A, Calzone K, Stopfer J, Blackwood A and Weber BL (2003) Application of breast cancer risk prediction models in clinical practice. J Clin Oncol 21:593-601.; Frank et al., 2002Frank TS, Deffenbaugh AM, Reid JE, Hulick M, Ward BE, Lingenfelter B, Gumpper KL, Scholl T, Tavtigian SV, Pruss DR, et al. (2002) Clinical characteristics of individuals with germline mutations in BRCA1 and BRCA2: Analysis of 10,000 individuals. J Clin Oncol 20:1480-1490.). For LFS, the original criteria described by Li and Fraumeni (Li et al., 1988Li FP, Fraumeni JF, Mulvihill JJ, Blattner WA, Dreyfus MG, Tucker MA and Miller RW (1988) A cancer family syndrome in twenty-four kindreds. Cancer Res 48:5358-5362.) were used; for LFL, pedigrees were classified according to the criteria of Birch (Birch et al., 1994Birch JM, Hartley AL, Tricker KJ, Prosser J, Condie A, Kelsey AM, Harris M, Jones PH, Binchy A and Crowther D (1994) Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families. Cancer Res 54:1298-1304.) and Eeles (Eeles, 1995Eeles RA (1995) Germline mutations in the TP53 gene. Cancer Surv 25:101-124.); for HBCC and Cowden's syndrome, the criteria described by (Meijers-Heijboer et al., 2003Meijers-Heijboer H, Wijnen J, Vasen H, Wasielewski M, Wagner A, Hollestelle A, Elstrodt F, van den Bos R, de Snoo A, Fat GT, et al. (2003) The CHEK2 1100delC mutation identifies families with a hereditary breast and colorectal cancer phenotype. Am J Hum Genet 72:1308-1314.) and (Nelen et al., 1996Nelen MR, Padberg GW, Peeters EA, Lin AY, van den Helm B, Frants RR, Coulon V, Goldstein AM, van Reen MM, Easton DF, et al. (1996) Localization of the gene for Cowden disease to chromosome 10q22-23. Nat Genet 13:114-116.), respectively, were used. Criteria to indicate genetic testing for HBOC were set so as not to miss any of the families at high risk for germline BRCA1 and BRCA2 mutations and included families fulfilling the ASCO criteria and/or who had a prior probability of mutation in a BRCA gene of 30% or more (Domchek et al., 2003Domchek SM, Eisen A, Calzone K, Stopfer J, Blackwood A and Weber BL (2003) Application of breast cancer risk prediction models in clinical practice. J Clin Oncol 21:593-601.; Frank et al., 2002Frank TS, Deffenbaugh AM, Reid JE, Hulick M, Ward BE, Lingenfelter B, Gumpper KL, Scholl T, Tavtigian SV, Pruss DR, et al. (2002) Clinical characteristics of individuals with germline mutations in BRCA1 and BRCA2: Analysis of 10,000 individuals. J Clin Oncol 20:1480-1490.). Genetic testing included mutation analysis of one or more of the four main breast cancer predisposition genes (BRCA1, BRCA2, TP53, and CHEK2). Initially, the index case was approached and informed of the study. Invitation to participate was made directly to all cancer-affected index-cases. In those unaffected by cancer an attempt was made to invite at-risk, cancer affected relatives. Blood samples were obtained from cancer-affected women and/or their family members after informed consent, depending on accessibility of individuals and willingness to participate in the study. The study was approved by the Institutional Review Board of the participating centers.

Screening for germline mutations

DNA samples were obtained from peripheral blood, using a commercial DNA extraction kit, Gentra Puregene Blood kit, according manufacturer's instructions (Qiagen, Valencia, CA, USA). All DNA samples were screened for BRCA1/2 and TP53 mutations using Denaturing High Performance Liquid Chromatography (DHPLC) or High Resolution Melting (HRM), and samples showing variants were submitted to Sanger sequencing.

BRCA1/BRCA2 genes

All samples were amplified by PCR reactions using oligonucleotide primers and corresponding annealing temperatures previously described in the literature (Friedman et al., 1997Friedman LS, Gayther SA, Kurosaki T, Gordon D, Noble B, Casey G, Ponder BA and Anton-Culver H (1997) Mutation analysis of BRCA1 and BRCA2 in a male breast cancer population. Am J Hum Genet 60:313-319.; Friedman et al., 1994Friedman LS, Ostermeyer EA, Szabo CI, Dowd P, Lynch ED, Rowell SE and King MC (1994) Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families. Nat Genet 8:399-404.). Mutation screening by DHPLC was carried out using a WAVE MD 4000 DNA Fragment Analysis System equipped with a DNASep Cartridge (Transgenomic Inc., Omaha NE, USA) as described elsewhere (Fackenthal et al., 2005Fackenthal DL, Chen PX and Das S (2005) Denaturing high-performance liquid chromatography for mutation detection and genotyping. Methods Mol Biol 311:73-96.). The HRM curve analysis was performed in a LightScanner instrument (Idaho Tecnology Inc.) using the Light Scanner Mastermix with LCGreen dye (Idaho Tecnology Inc.). Heterozygous profiles were identified by visual inspection of the chromatograms/melting curves and putative sequence variants were re-analyzed by bi-directional sequencing on a MegaBACE 1000 (Amersham Biosciences, Buckinghamshire, UK) or an ABI-PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, USA) using an independent PCR product. Sequence alterations were classified based on data available in the Breast Cancer Information Core (BIC, 2014), ClinVar (Landrum et al., 2014Landrum MJ, Lee JM, Riley GR, Jang W, Rubinstein WS, Church DM and Maglott DR (2014) ClinVar: Public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res 42:D980-985.), Universal Mutation database - UMD (Caputo et al., 2012Caputo S, Benboudjema L, Sinilnikova O, Rouleau E, Béroud C, Lidereau R and French BRCA GGC Consortium (2012) Description and analysis of genetic variants in French hereditary breast and ovarian cancer families recorded in the UMD-BRCA1/BRCA2 databases. Nucleic Acids Res 40:D992-1002.) and AlignGVGD (Tavtigian et al., 2006Tavtigian SV, Deffenbaugh AM, Yin L, Judkins T, Scholl T, Samollow PB, de Silva D, Zharkikh A and Thomas A (2006) Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J Med Genet 43:295-305.). New or pathogenic mutations were also searched in The Human Gene Mutation Database (HGMD, 2014HGMD (2014) The Human Gene Mutation Database (HGMD), http://www.hgmd.cf.ac.uk/ (accessed November 30, 2014).
http://www.hgmd.cf.ac.uk/...
,) and LOVD (Vallée et al., 2012Vallée MP, Francy TC, Judkins MK, Babikyan D, Lesueur F, Gammon A, Goldgar DE, Couch FJ and Tavtigian SV (2012) Classification of missense substitutions in the BRCA genes: A database dedicated to Ex-UVs. Hum Mutat 33:22-28.). All the 18 HBOC families were screened for BRCA1 genomic rearrangements and 10 of these families were screened for BRCA2 genomic rearrangements using Multiplex Ligation-dependent Probe Amplification (MLPA) methodology using the SALSA P002B BRCA1 and SALSA P045 BRCA2 MLPA probe mix assays (MRC-Holland, Amsterdam, The Netherlands) as recommended by the manufacturer (MRC-Holland) and information on copy number was extracted with Coffalyser V9.4 Software (MRC-Holland). All analyses were performed in duplicate and in at least two independent experiments.

TP53 gene

Patients fulfilling Li-Fraumeni and Li-Fraumeni-like syndrome criteria (Birch et al., 1994Birch JM, Hartley AL, Tricker KJ, Prosser J, Condie A, Kelsey AM, Harris M, Jones PH, Binchy A and Crowther D (1994) Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families. Cancer Res 54:1298-1304.; Eeles, 1995Eeles RA (1995) Germline mutations in the TP53 gene. Cancer Surv 25:101-124.; Li et al., 1988Li FP, Fraumeni JF, Mulvihill JJ, Blattner WA, Dreyfus MG, Tucker MA and Miller RW (1988) A cancer family syndrome in twenty-four kindreds. Cancer Res 48:5358-5362.) were screened for TP53 germline mutations as follows: exons 2-11 were screened by HRM (as described by (Garritano et al., 2009Garritano S, Gemignani F, Voegele C, Nguyen-Dumont T, Le Calvez-Kelm F, De Silva D, Lesueur F, Landi S and Tavtigian SV (2009) Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus. BMC Genet 10:5.) followed by bi-directional sequencing of altered regions. Sequence variations were classified according to data submitted to the TP53 database at the International Agency for Research on Cancer - IARC version R17 (Petitjean et al., 2007Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P and Olivier M (2007) Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: Lessons from recent developments in the IARC TP53 database. Hum Mutat 28:622-629.). All possible deleterious mutations were confirmed by a second and independent analysis.

CHEK2 gene

Families with a history of breast and colorectal cancer consistent with HBCC syndrome were screened for a specific CHEK2 mutation (1100delC), located in exon 10 by PCR amplification followed by direct sequencing. To ensure amplification of the functional copy of CHEK2 and exclusion of CHEK2 pseudogenes, a strategy of long-range PCR amplifications with primers designed outside the pseudogene sequences was used as described by (Vahteristo et al., 2001Vahteristo P, Tamminen A, Karvinen P, Eerola H, Eklund C, Aaltonen LA, Blomqvist C, Aittomäki K and Nevanlinna H (2001) p53, CHK2, and CHK1 genes in Finnish families with Li-Fraumeni syndrome: Further evidence of CHK2 in inherited cancer predisposition. Cancer Res 61:5718-5722.).

Results

Sample characteristics

Of all 9,234 women included in the NMPOA cohort (Porto Alegre, Brazil), 1,286 (13.9%) answered positively to at least one of the seven questions about FH. Those above 18 years (n = 1,247) were invited for GCRA. Of the 1,247 patients referred to GCRA, 902 (72.3%) effectively participated in the assessment and of these, 214 (23.7%) women from 183 families fulfilled criteria for one or more of the breast cancer predisposition syndromes (BCPS) considered in our study: 65 fulfilled criteria for HBOC, 122 for LFL and 22 for HBCC syndromes.

None of the patients assessed reported a personal and/or family history suggestive of classic Li-Fraumeni and Cowden's Syndrome. Detailed information on study design, patient recruitment, and demographic data of the 902 patients seen for GCRA is described elsewhere (Palmero et al., 2009Palmero EI, Caleffi M, Schüler-Faccini L, Roth FL, Kalakun L, Netto CB, Skonieski G, Giacomazzi J, Weber B, Giugliani R, et al. (2009) Population prevalence of hereditary breast cancer phenotypes and implementation of a genetic cancer risk assessment program in southern Brazil. Genet Mol Biol 32:447-455.).

Of the 214 women with criteria for a BCPS, 64 (29.9%; corresponding to 50 families) decided to continue with the genetic investigation and proceeded to germline mutation testing. An additional 54 cancer-unaffected and at-risk patients (25.2%) attempted contact with their cancer-affected relatives to invite them for GCRA but they did not schedule an appointment. Among the 50 probands tested, the most frequent cancer site was breast, and as expected, the majority of these diagnoses were made before the age of 50 years. Only two probands were diagnosed with ovarian cancer (one HBOC and one LFL family). Moreover, six probands had colorectal cancer (3 LFL and 3 HBCC families). Ten of the 50 probands were cancer unaffected (from 7 LFL families and three from families with both HBOC and LFL criteria). However, four of them were supposedly obligate carriers by family history. Additional information on cancer site and phenotype among the 50 families studied is summarized in Tables 1 and 2. At least one tumor diagnosis was confirmed by pathology reports, medical records, or death certificates in 45/50 (90%) families. However, confirmation of a sufficient number of cancer diagnoses to affirm with certainty the BCPS phenotype was only possible in 13 families (26%). Due to the high frequency of breast cancer diagnoses in the 50 families studied, 14 (28%) of them fulfilled criteria for more than one BCPS when testing was indicated and therefore, these families were screened for germline mutations in more than one predisposition gene. Thus, eight families were tested for BRCA1/BRCA2 and TP53 germline mutations, two families for BRCA1/BRCA2 and CHEK2 mutations, three families for TP53 and CHEK2 mutations, and one family was screened for mutations in all four genes (BRCA1/BRCA2, CHEK2 and TP53). During the process of genetic testing, three families presented additional information of the presumed cancer diagnoses and one of the phenotypes was excluded (HBOC in family 681, HBCC in family 284, and LFL in two families, 163 and 186) (Table 2).

Table 1
Cancer phenotype of the 50 BCPS families submitted to genetic testing.
Table 2
Detailed description of the 14 families that at inclusion were identified as having criteria for more than one BCPS.

Mutation detection studies

HBOC

Nineteen individuals from 18 families with HBOC criteria underwent genetic testing. After DHPLC or HRM screening, the PCR products showing variant or dubious profiles were sequenced for confirmation. A total of 183 of 646 (28.3%) and 278 of 798 (34.8%) BRCA1 and BRCA2 amplicons were sequenced, respectively. There was a complete agreement between the results from DHPLC and sequencing for both BRCA genes.

Sequencing results were compared to data deposited in the BIC, ClinVar, UMD, HGMD and LOVD databases. We identified 12 and 31 sequence variants in BRCA1 and BRCA2, respectively (Table 3). Most of them were previously described and deposited in one or more databases as variants with no clinical significance. However, databases diverged about classification of some variants. In BRCA1, four variants were classified as variants of unknown significance (VUS) in at least one database, but none was classified as VUS in all three databases. In BRCA2, two variants were consistently classified as VUS in all databases: c.9004G > A (p.E3002K) and c.9581C > A (p.P3194Q). However, in HGMD the variant p.E3002K is described as a deleterious mutation, and its pathogenicity was demonstrated (Biswas et al., 2012Biswas K, Das R, Eggington JM, Qiao H, North SL, Stauffer S, Burkett SS, Martin BK, Southon E, Sizemore SC, et al. (2012) Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay. Hum Mol Genet 21:3993-4006.; Cote et al., 2012Cote S, Arcand SL, Royer R, Nolet S, Mes-Masson AM, Ghadirian P, Foulkes WD, Tischkowitz M, Narod SA, Provencher D, et al. (2012) The BRCA2 c.9004G > A (E2002K) [corrected] variant is likely pathogenic and recurs in breast and/or ovarian cancer families of French Canadian descent. Breast Cancer Res Treat 131:333-340.; Karchin et al., 2008Karchin R, Agarwal M, Sali A, Couch F and Beattie MS (2008) Classifying Variants of Undetermined Significance in BRCA2 with protein likelihood ratios. Cancer Inform 6:203-216.). The pedigree from this family can be seen in Figure 1. We also found three new variants in BRCA2, not described in BIC, ClinVar, UMD, HGMD or LOVD: c.1402A > G (p.R468G), c.2842G > A (p.V948I) and c.7017G > A (p.K2339K). More detailed results can be found in Table 3. Screening for large gene rearrangements in all 18 HBOC probands for BRCA1 and in 10 for BRCA2 did not show any detectable abnormalities.

Table 3
BRCA1 and BRCA2 sequence variants identified in the 18 families fulfilling HBOC syndrome criteria.
Figure 1
Pedigree of a family with p.E3002K mutation in BRCA2 gene.

LFL

One hundred twenty two families fulfilled the criteria for LFL syndrome (13.5% of the entire initial sample), being 13 and 119 families fulfilling Birch and Eeles criteria, respectively. Ten families had both Birch and Eeles criteria for LFL. Of the 122 families, 40 probands (from 40 unrelated families) underwent genetic testing Twelve of these 40 families (30%) also fulfilled criteria for at least on other BCPS (Table 2).

No deleterious mutation was found among the sequenced individuals, and the polymorphisms detected are shown in Table 4.

Table 4
TP53 sequence variants identified in the 40 families fulfilling LFL syndrome criteria.

HBCC

Among the 183 families with a hereditary breast cancer phenotype, 22 fulfilled criteria for HBCC (Meijers-Heijboer et al., 2003Meijers-Heijboer H, Wijnen J, Vasen H, Wasielewski M, Wagner A, Hollestelle A, Elstrodt F, van den Bos R, de Snoo A, Fat GT, et al. (2003) The CHEK2 1100delC mutation identifies families with a hereditary breast and colorectal cancer phenotype. Am J Hum Genet 72:1308-1314.), but only seven of them underwent genetic testing. Interestingly, all of the seven families also fulfilled criteria for a BCPS other than HBCC at inclusion in the study. The common CHEK2 1100delC mutation was identified in one of these seven families (14.3%) with multiple breast cancer diagnoses (proband had multiple breast cancers, first at age 52), colorectal cancer (ages 50 and 68 years), lung cancer and endometrial cancer (two cases) (Figure 2).

Figure 2
CHEK2 1100delC mutation family. Pedigree (A), forward (B) and reverse (C) sequencing of germline DNA. WT=wild type; dx=age at diagnosis; d=age of death; red arrows indicate the last readable base.

Discussion

The identification and characterization of genetic alterations in families at high risk for breast cancer predisposition syndromes enables carriers to undertake individualized cancer screening and prevention strategies, thus increasing the likelihood of increased disease-free survival rates.

For HBOC, although we aimed at selecting patients at a somewhat higher prior probability of mutation than in most studies (average mutation probability between 20-30% using different criteria and prediction models) we identified a known deleterious germline mutation in only one of the 18 families studied. Using a strategy of screening for four common alterations in BRCA1 and BRCA2 (185delAG, 5382insC and exon 13 6kb duplication in BRCA1 and 6174delT in BRCA2), Gomes (Gomes et al., 2007Gomes MC, Costa MM, Borojevic R, Monteiro AN, Vieira R, Koifman S, Koifman RJ, Li S, Royer R. Zhang S, et al. (2007) Prevalence of BRCA1 and BRCA2 mutations in breast cancer patients from Brazil. Breast Cancer Res Treat 103:349-353.) showed a mutation frequency of 2.3% of the 5382insC mutation in 402 Brazilian breast cancer patients unselected for family history. Although the average prior probability of mutation in their sample is not clear, one would expect that it was likely less (unselected sample) than the cutoff probability used for offering genetic testing in our report. In another Brazilian study that assessed BRCA1 mutation prevalence in a group of 47 women from Rio de Janeiro, Lourenço et al. (Lourenço JJ VF, 2004Lourenço JJ, Vargas FR, Bines J, Santos EM, Lasmar CAP, Costa CH, Teixeira EMB, Maia MCM, Coura F, Silva CHD, et al. (2004) BRCA1 mutations in Brazilian patients. Genet Mol Biol 27:500-504.), using more strict inclusion criteria, found 7 (15.0%) mutation carriers; again, 5382insC was one of the most common mutations encountered (4/7). Analyzing germline mutations in all coding regions of BRCA1 and in common founder mutations in the BRCA2, CHEK2 and TP53 genes in a cohort of 106 high-risk HBOC patients, Felix et al. (Felix et al., 2014Felix GE, Abe-Sandes C, Machado-Lopes TM, Bomfim TF, Guindalini RS, Santos VC, Meyer L, Oliveira PC, Claudio Neiva J, Meyer R, et al. (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.) found 10 mutation carriers, and of them 9 harbored mutations in BRCA1 (and none in BRCA2), performing a mutation frequency of 8,49%. A recent study of our group accessed the prevalence of three founder mutations (BRCA1 c.68_69del, BRCA1 c.5266dupC - former named as 5382insC, and BRCA2 c.5946del) commonly identified in Ashkenazi individuals in a sample of 137 non-Ashkenazi cancer-affected women from Rio de Janeiro and Porto Alegre (all of them fulfilled clinical criteria for HBOC). The only mutation found was BRCA1 c.5266dupC (5382insC), present in 7/137 women, a prevalence of 5.10% (Ewald et al., 2011Ewald IP, Izetti P, Vargas FR, Moreira MA, Moreira AS, Moreira-Filho CA, Cunha DR, Hamaguchi S, Camey SA, Schmidt A., et al. (2011) Prevalence of the BRCA1 founder mutation c.5266dupin Brazilian individuals at-risk for the hereditary breast and ovarian cancer syndrome. Hered Cancer Clin Pract 9:12.). A posterior study also conducted in Porto Alegre evaluated the prevalence of three Ashkenazi founder mutations (BRCA1 185delAG, BRCA1 5382insC and BRCA2 6174delT) in a group of 255 Ashkenazi Jewish women, non selected for personal or familial cancer history, and found a carrier frequency of 2/255 for 185delAG (0,78%), 1/255 for 6174delT (0,4%) and no mutated alleles for 5382insC, reveling a carrier frequency lower than expected for this ethnic group (Dillenburg et al., 2012Dillenburg CV, Bandeira IC, Tubino TV, Rossato LG, Dias ES, Bittelbrunn AC and Leistner-Segal S (2012) Prevalence of 185delAG and 5382insC mutations in BRCA1, and 6174delT in BRCA2 in women of Ashkenazi Jewish origin in southern Brazil. Genet Mol Biol 35:599-602.).

Indeed, BRCA mutation prevalence is probably heterogeneous, not only depending on criteria adopted for inclusion in a given study, but also related to testing methodology and specific features of different populations. For example, in a Finnish study of 128 HBOC patients, (Laurila et al., 2005Laurila E, Syrjäkoski K, Holli K, Kallioniemi A and Karhu R (2005) Search for large genomic alterations of the BRCA1 gene in a Finnish population. Cancer Genet Cytogenet 163:57-61.) did not identify any BRCA1 mutation after sequencing the entire coding region of the gene. Finally, the effects of a small sample size and less than optimal confirmation of the cancer diagnoses in many of the families in our cohort must also be considered.

The negative findings of gene rearrangements in BRCA1 and BRCA2 could also be related to small sample size and, again, to the large variability of rearrangement prevalence. One complicating factor is that the knowledge about prevalence of such rearrangements in South American HBOC patients is limited and studies in other populations report a highly variable prevalence of such rearrangements. A Brazilian study conducted with 120 women fulfilling criteria for HBOC and screened for mutations, CNVs and rearrangements in BRCA and other genes found rearrangements of BRCA1 in only two cases (exon 24 amplification and exon 16-17 deletion) (Silva et al., 2014Silva FC, Lisboa BC, Figueiredo MC, Torrezan GT, Santos EM, Krepischi AC, Rossi BM, Achatz MI and Carraro DM (2014) Hereditary breast and ovarian cancer: Assessment of point mutations and copy number variations in Brazilian patients. BMC Med Genet 15:55.). In the Dutch, for example, large genomic rearrangements constitute 36% of the mutations detected in BRCA1 (Petrij-Bosch et al., 1997Petrij-Bosch A, Peelen T, van Vliet M, van Eijk R, Olmer R, Drüsedau M, Hogervorst FB, Hageman S, Arts PJ, Ligtenberg MJ, et al. (1997) BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients. Nat Genet 17:341-345.).

In spite of the large number of sequence variants identified in the BRCA1/BRCA2 genes worldwide, many of them are still classified as VUS, and the available databases diverge about classification of variants. In this study, we found four BRCA1 variants classified as VUS in at least one database, and two BRCA2 variants that were consistently classified as VUS in all three analyzed databases, although one of them is, indeed, pathogenic (E3002K) (Biswas et al., 2012Biswas K, Das R, Eggington JM, Qiao H, North SL, Stauffer S, Burkett SS, Martin BK, Southon E, Sizemore SC, et al. (2012) Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay. Hum Mol Genet 21:3993-4006.; Cote et al., 2012Cote S, Arcand SL, Royer R, Nolet S, Mes-Masson AM, Ghadirian P, Foulkes WD, Tischkowitz M, Narod SA, Provencher D, et al. (2012) The BRCA2 c.9004G > A (E2002K) [corrected] variant is likely pathogenic and recurs in breast and/or ovarian cancer families of French Canadian descent. Breast Cancer Res Treat 131:333-340.; Karchin et al., 2008Karchin R, Agarwal M, Sali A, Couch F and Beattie MS (2008) Classifying Variants of Undetermined Significance in BRCA2 with protein likelihood ratios. Cancer Inform 6:203-216.). Although the results are conflicting between the databases, functional studies done by Biswas et al. (2012)Biswas K, Das R, Eggington JM, Qiao H, North SL, Stauffer S, Burkett SS, Martin BK, Southon E, Sizemore SC, et al. (2012) Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay. Hum Mol Genet 21:3993-4006. point to the fact that the mutation p.E3002K negatively impacts ssDNA binding and function, resulting in a deleterious phenotype. The findings on the likely pathogenicity of this mutation were corroborated by work published by Cote et al. (2012)Cote S, Arcand SL, Royer R, Nolet S, Mes-Masson AM, Ghadirian P, Foulkes WD, Tischkowitz M, Narod SA, Provencher D, et al. (2012) The BRCA2 c.9004G > A (E2002K) [corrected] variant is likely pathogenic and recurs in breast and/or ovarian cancer families of French Canadian descent. Breast Cancer Res Treat 131:333-340. in a recent study of 58 French Canadian families with breast and/or ovarian cancer and 960 cases not selected for family history of cancer, which found this variant in seven of the 58 families with a family history of cancer and in none of those not selected for family history. Additionally the AlignGVGD score for pathogenicity for this specific mutation is C55, pointing to a possibly/probably pathogenic alteration. Three novel sequence variants were found in BRCA2, two of them in the same individual. Further characterization of these novel variants is imperative and is underway. All efforts will be made to further characterize variants of unknown significance, especially if they were not described previously.

The finding of a high number of pedigrees suggestive of the LFL phenotype in our sample was somewhat surprising. First, because the questionnaire used to screen women from the general population for GCRA, was originally designed to identify the HBOC phenotype, and second because Li-Fraumeni syndrome and its variants have not been considered common cancer predisposition syndromes in most countries. However, Eeles I criteria, the only ones present in the majority of the 40 probands studied, are very relaxed and mutation prevalence in these families has been described as low (under 10%), which is in agreement with the absence of deleterious TP53 mutations identified in our study. Interestingly, in other published mutation study of Brazilian LFL families (including 10 families from the State of Rio Grande do Sul), the mutation prevalence for LFL Eeles 1 and Birch families was much higher (23.1% and 61.5%, respectively). Again, our findings may be related to relatively small sample sizes and to the presence of lower-risk LFL families, some of them not having a sufficient number of cancer diagnoses confirmed to allow certainty about the BCPS phenotype. Finally, screening for the common CHEK2-1100delC mutation in seven HBCC families resulted in the identification of one mutation-positive family corresponding to a mutation prevalence of about 14%, (comparable to the 18% previously described in the literature) (Petrij-Bosch et al., 1997Petrij-Bosch A, Peelen T, van Vliet M, van Eijk R, Olmer R, Drüsedau M, Hogervorst FB, Hageman S, Arts PJ, Ligtenberg MJ, et al. (1997) BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients. Nat Genet 17:341-345.). With the small sample size analyzed, these results could merely be related to chance, however they go along with other previous observations of a higher than expected number of colon cancers in the families breast-cancer affected women undergoing GCRA (Palmero et al., 2009Palmero EI, Caleffi M, Schüler-Faccini L, Roth FL, Kalakun L, Netto CB, Skonieski G, Giacomazzi J, Weber B, Giugliani R, et al. (2009) Population prevalence of hereditary breast cancer phenotypes and implementation of a genetic cancer risk assessment program in southern Brazil. Genet Mol Biol 32:447-455.).

Two major limitations can be identified in our study. First, the relatively small sample size, which is not entirely unexpected. The limited acceptance of genetic testing that we faced throughout this study may be explained by cultural aspects of the population, low literacy of the majority of women counseled and this in turn could be associated with limited understanding of the benefits and implications of testing. Study design may also have influenced genetic testing acceptance. The women included in this study were recruited from their primary health care units and were not originally concerned about their genetic risk for cancer; or, if they were concerned, they did not seek genetic counseling directly. Furthermore, since our cohort was originated from a population-based sample, some of our probands were cancer-unaffected individuals from at-risk families for whom genetic testing required participation of at least one willing and alive cancer-affected relative. Another potential concern is that only a proportion of cancers in probands and/or relatives were confirmed, and therefore, there may be misclassifications of cancer site, tumor type as well as age at tumor diagnosis. Confirmation of cancer diagnoses in relatives, often distant ones, is usually challenging and in this population it may be particularly difficult, since many of them have lost contact with their families. All of these factors together may also contribute to poor understanding about increased risk and the existence and benefits of risk reduction strategies and consequent reduced motivation for genetic testing. In spite of the use of relatively strict inclusion criteria for testing of some BCPS (such as HBOC) and less strict criteria for others (LFL) and the comprehensive testing methodology used, the high risk posed to most of the families described here remains unexplained. Even considering the limitations highlighted before, this study raises several questions about the importance of genetic factors in determining the high breast cancer incidence and mortality rates in Southern Brazil and the acceptability of genetic testing in this population. In order to validate the low prevalence of germline mutations found in this work, a larger cohort should be analyzed. If indeed it is, we may have to revise criteria for genetic testing in this population and thoroughly investigate the contribution of different and/or novel high penetrance genes or the influence of multiple, more prevalent genetic variants of lower penetrance.

Acknowledgments

The Núcleo Mama (NMPOA) Cohort, from which the patients derive, is maintained by Associação Hospitalar Moinhos de Vento, in a partnership with Instituto da Mama do Rio Grande do Sul and the Municipal Health Agency from Porto Alegre. The authors are indebted to Danielle Renzoni da Cunha, Ghyslaine Martel-Planche, Matias Eliseo Melendez, Patricia Schneider, Hermides Pinto Junior and Andre Muller for their expert technical assistance; to Karen Barbosa and Diego Pasetto for their assistance with data managing, patient recruitment and pedigree organization and to Ana Cecília Mano de Azevedo, Ademar Bedin Júnior, Luciane Poletto Antunes, Juliana Zignani, Fávio Marcel Telis Gonzalez, Luciano Artico, and the NMPOA team for their help with the recruitment, evaluation and follow-up of the patients included in this study. This study was supported by a grant from Susan G. Komen for the Cure (POP0403033), and in part by grants from Fundo de Incentivo à Pesquisa – FIPE, Hospital de Clínicas de Porto Alegre (# 04-170) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES - PRODOC grant number 00202/03-7). EI Palmero was supported in part by grants from The International Agency for Research in Cancer (IARC) and CNPq (process number 203732/2005-7).

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Internet Resources

Supplementary Material

The following online material mis available for this article: -

Table S1 - The Family History Syndrome questionnaire

This material is available as part of the online version of this article from http://www.scielo.br/gmb.

  • Associate Editor: Héctor Seuanez
  • Erratum

    In the article “Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil”, with DOI number: http://dx.doi.org/10.1590/1678-4685-GMB-2014-0363, published in the journal Genetics and Molecular Biology, 39(2):210-222, on page 210 the author name spelled as Florence Le Calvez Kelm should read Florence Le Calvez-Kelm.

Publication Dates

  • Publication in this collection
    24 May 2016
  • Date of issue
    Apr-Jun 2016

History

  • Received
    12 Dec 2014
  • Accepted
    24 Nov 2015
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