Familial Cancer DOI 10.

1007/s10689-012-9551-5

SHORT COMMUNICATION

Portuguese c.156_157insAlu BRCA2 founder mutation: gastrointestinal and tongue neoplasias may be part of the phenotype
Miguel A. M. Moreira Irina G. Bobrovnitchaia Maria Angelica F. D. Lima Jesus P. Ramos Kelly R. L. Souza Ana Peixoto Anna Claudia E. Santos Manuel R. Teixeira Fernando R. Vargas

Springer Science+Business Media B.V. 2012

Abstract We have screened BRCA2 c.156_157insAlu founder mutation in a cohort of 168 women with diagnosis of breast cancer referred for genetic counseling because of risk of being carriers of hereditary breast and ovarian cancer syndrome. Portuguese founder mutation BRCA2 c.156_157insAlu was identied in three unrelated breast cancer probands. Genotyping identied a common haplotype between markers D13S260 and D13S171, and allele sizes were compatible to those described in the Portuguese families. Allele sizes of marker D13S1246, however, were concordant in two families, suggesting that the haplotype may be larger in a subset of families. Tumor phenotypes in Brazilian families seem to reinforce the high prevalence of breast cancer among affected males. However, an apparent excess of gastrointestinal and tongue neoplasias were also

observed in these families. Although these tumors are not part of the phenotypic spectrum of hereditary breast and ovarian cancer syndrome, they might be accounted for by other risk alleles contained in the founder haplotype region. Keywords Hereditary breast cancer BRCA2 Alu insertion c.156_157insAlu BRCA2 Founder mutation

Introduction It is well known that between 5 and 7 % of breast (BC) and ovarian (OC) cancers are associated with germline, highly penetrant, loss-of-function mutations in tumor suppressor genes [1]. Linkage studies show that up to 80 % of families with hereditary breast and ovarian cancer (HBOC) syndrome are associated with BRCA genes, but the actual disease-causing mutation identication rate is much lower, partly because some mutations are not easily identied by the usual PCR methods employed for direct sequencing. This is the case of the Portuguese BRCA2 c.156_157insAlu founder mutation. A number of founder mutations have been identied in different ethnic groups, and there may be slight differences in the tumor phenotype among carriers of a particular founder mutation [2]. The world distribution, age estimation, and BC risk associated with the Portuguese BRCA2 c.156_157insAlu mutation have been described by Peixoto et al. [3]. This Alu insertion within exon 3 of BRCA2 was previously identied by Teugels et al. [4] in a Portuguese family living in Belgium. Subsequently, Machado et al. [5] reported it in 20 families and Peixoto et al. [3, 6] reported it in 25 additional families, all Portuguese or with Portuguese ancestry, and sharing a

M. A. M. Moreira I. G. Bobrovnitchaia M. A. F. D. Lima A. C. E. Santos J. P. Ramos K. R. L. Souza F. R. Vargas (&) Genetics Program, Instituto Nacional de Cancer, Rio de Janeiro, Brazil e-mail: fvargas@inca.gov.br M. A. F. D. Lima A. C. E. Santos F. R. Vargas Departamento de Genetica e Biologia Molecular, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil A. C. E. Santos F. R. Vargas Departamento de Genetica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil J. P. Ramos Fundacao Oswaldo Cruz, Rio de Janeiro, Brazil A. Peixoto M. R. Teixeira Servico de Genetica, Instituto Portugues de Oncologia do Porto, Porto, Portugal

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common haplotype around the BRCA2 locus. Here we report on three Brazilian families with this mutation.

Patients and methods We have screened 168 unrelated BC patients from Rio de Janeiro, Brazil, referred for genetic counseling because of personal or familial history of breast and/or ovarian cancer. All patients included in the study had personal history of BC, and had been referred to genetic counseling clinic in the last 9 years. Median age at diagnosis was 44.4 years. Risk distribution according to three risk estimating tools (Myriad II [www.myriadpro.com/brca-risk-calculator], Penn 2 [www.afcri.upenn.edu/itacc/penn2], and BRCAPRO [7, 8]) is shown in Fig. 1. Screening of mutations with bidirectional automated sequencing was performed for selected exons of BRCA1 (exons 2, 6, 7, 11, 16, 23, and 24), and BRCA2 (exons 10, 11, 15, 16, 17, 20, 21, 22, 23, and 26). In the studied sample, 21 pathogenic mutations (17 frameshift, 2 nonsense, 2 splice site) were identied. Also, 18 sequence variants of unknown clinical signicance were observed (17 non-synonymous substitutions and 1 intronic mutation at position ?4). BRCA1 5382insC was the most frequently found pathogenic mutation, having been observed in four unrelated probands. Interestingly, none of these BRCA1 5382insC families reported Ashkenazi Jewish ancestry. Additionally, germline diseasecausing mutations in TP53 were observed in three BC probands, whose family histories also met Li-Fraumeni syndrome criteria. Screening of c.156_157insAlu mutation was accomplished with PCR primers and reaction

conditions described by Peixoto et al. [9], the amplied fragment was excised from gel, puried, and sequenced in an ABI 3130xl sequencer. Genotyping with markers D13S1246, D13S260, and D13S171 was performed in available patients and relatives, and estimation of allele sizes was performed with Genetic Proler (GE Healthcare).

Results and discussion BRCA2 c.156_157insAlu was detected in three unrelated BC patients, and 11 individuals were studied in these families. All three families have Portuguese family names, and no family reported recent immigration, their ancestors have lived in Brazil for at least three generations, or as far as family memory reaches. Pedigrees of the three families, along with phenotypes and results of haplotype analyses, are illustrated in Fig. 1. A common haplotype for markers D13S260 and D13S171 was identied in mutation carriers, and allele sizes were compatible to those described by Peixoto et al. [3, 6]. Genotyping of marker D13S1246 in families 003 and 238 showed that mutation carriers share the same allele size (203) as reported by Machado et al. [5]. In family 115, two mutation carriers (II-2 and II-3) share the same allele (205), while III-1 showed two different alleles, 200 and 203. These ndings suggest that, if D13S1246 is segregating with the haplotype in this family, then at least two crossover events should have occurred, the rst one resulting in allele size 205, and the second event resulting in one of the observed alleles in individual III-1. In fact, D13S1246 is located 1.8 Mb telomeric to BRCA2 locus, while D13S260 and D13S171 are located much closer to BRCA2 (0.5 and 0.3 Mb, respectively) (Fig. 2). Peixoto et al. [6] estimated the cumulative incidence of BC in mutation carriers to be close to 90 %. Tumor phenotypes in Brazilian families seem to reinforce the high prevalence of BC among affected males. However, other tumors have been observed as well. Gastric cancer (GC) was present in one mutation carrier (I-2, family 003), and colorectal cancer (CRC) was diagnosed in one at-risk individual (II-1, family 115). Machado et al. [5] described two cases of GC in one affected family. Tongue cancer (TC) was observed in three individuals in family 115, one of them was an obligate mutation carrier (I-1 or I-2) and another was an at-risk individual (II-4). Interestingly, TC has also been observed in the family studied by Teugels et al. [4], who described the observation of several tongue cancers in the pedigree studied by them. Machado et al. [5] described three head and neck cancers in two carrier families. A search for these tumor phenotypes among 35 Portuguese BRCA2 c.156_157insAlu families studied by Peixoto et al. [3, 6, unpublished] revealed six individuals with GC from ve families, one of them an obligate carrier;

>20%

10-20%

<10%

36 59 74

64 36 88 68 21
Myriad II Penn II BRCAPro

58

Fig. 1 Distribution of probands risk of being carriers of BRCA1/2 germline mutation according to three risk estimating tools: Myriad II (a), Penn 2 (b), and BRCAPRO (c). Probands were classied into three risk categories: 010 % (green), 1020 % (blue), and [20 % (orange). Numbers within each box denote the total number of individuals in each risk category. (Color gure online)

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Portuguese c.156_157insAlu BRCA2 founder mutation

(A)
I

FAMILY 003 207/211 160/170 224/238 203/211 160/162 + 224/228

(B)
I 3
BC 40

FAMILY 115

GC 68

TC 68

TC 37 BC 49

AC 40

II II 1 211/211 160/160 224/224

BC 38 2 203/207 162/170 + 224/228

BC 48 CRC 40 TC 45

1 3
BC 30

203/211 160/162 + 228/238

III 1

200/205 162/172 + 222/228

202/205 3 162/172 + 224/228 202/214 162/172 224/226

(C)
I

FAMILY 238
BC 55

(D)
Centromere D13S1246 1.78 Mb 0.45 Mb

(E)

200/203 150/162 + 224/228

1 II 1 III 1 IV 201/203 162/162 + 228/238 1

BC 33 BC 42 BC 64

D13S260

BRCA2
D13S171 Telomere

0.36 Mb

203/207 162/164 + 228/238

Fig. 2 Pedigrees, phenotypes, and genotypes for markers D13S1246, D13S260, and D13S171. In a, b, and c are pedigrees of families 003, 115, and 238, respectively. Alleles that belong in the haplotype are marked in bold; d order of markers as shown in the pedigrees; e electropherogram showing c.156_157insAlu; AC = abdominal

cancer; BC = breast cancer; CRC = colorectal cancer; GC = gastric cancer; TC = tongue cancer; black square/circle = cancer affected individual; diagonal bar = deceased individual. Age at diagnosis (in years) is represented alongside tumor type

CRC was present in 12 individuals from eight families, two of them obligate carriers; and head and neck cancer was observed in three individuals, two of them obligate carriers. Gastrointestinal (GI) and head and neck (HN) neoplasias have not been reported in association with germline mutations in BRCA1/2, and are not considered part of HBOC syndrome phenotype. Familial history of GI and HN neoplasias in our sample was, respectively, 27 and 16 %, rates similar to those observed among sporadic cancer patients. The apparently recurrent pattern of GI and HN tumors in some BRCA2 c.156_157insAlu carrier families might be associated with the founder haplotype. The chromosomal region involved in the haplotype might harbor unidentied alleles that could be responsible for these variant phenotypes. It is noteworthy that these variant tumors seem to be concentrated in a few families, suggesting that a slightly different version of the founder haplotype might be shared by a subset of BRCA2 c.156_157insAlu carrier families. If, as suggested by the ndings in our families, marker D13S1246 is located within the founder haplotype, this region is larger than previously estimated, increasing the possibility that it could

harbor (so far unidentied) allele(s) that could increase the risk of development of the variant neoplasias recurrently observed in a subset of these families.
Acknowledgments This work was partially funded by Fundacao de Amparo a Pesquisa no Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Cientco e Tecnologico (CNPq), Brazil. MAMM is recipient of CNPq grant 304403/2008-3. FRV is recipient of CNPq grants 401966/2010-0 and 476808/2010-3. Ethical standards This study has been approved by the local Institutional Review Board.

References
1. Paradiso A, Formenti S (2011) Hereditary breast cancer: clinical features and risk reduction strategies. Ann Oncol 22(Suppl 1):i31 i36 ` 2. Ferla R, Calo V, Cascio S, Rinaldi G, Badalamenti G, Carreca I, Surmacz E, Colucci G, Bazan V, Russo A (2007) Founder mutations in BRCA1 and BRCA2 genes. Ann Oncol 18(Suppl 6):vi93vi98 3. Peixoto A, Santos C, Pinheiro M, Pinto P, Soares MJ, Rocha P, Gusmao L, Amorim A, van der Hout A, Gerdes AM, Thomassen

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M. A. M. Moreira et al. M, Kruse TA, Cruger D, Sunde L, Bignon YJ, Uhrhammer N, Cornil L, Rouleau E, Lidereau R, Yannoukakos D, Pertesi M, Narod S, Royer R, Costa MM, Lazaro C, Feliubadalo L, Grana B, Blanco I, de la Hoya M, Caldes T, Maillet P, Benais-Pont G, Pardo B, Laitman Y, Friedman E, Velasco EA, Duran M, Miramar MD, Valle AR, Calvo MT, Vega A, Blanco A, Diez O, Gutierrez Enrquez S, Balmana J, Ramon Y, Cajal T, Alonso C, Baiget M, Foulkes W, Tischkowitz M, Kyle R, Sabbaghian N, Ashton-Prolla P, Ewald IP, Rajkumar T, Mota-Vieira L, Giannini G, Gulino A, Achatz MI, Carraro DM, de Paillerets BB, Remenieras A, Benson C, Casadei S, King MC, Teugels E, Teixeira MR (2011) International distribution and age estimation of the Portuguese BRCA2 c.156_157insAlu founder mutation. Breast Cancer Res Treat 127(3):671679 4. Teugels E, De Brakeleer S, Goelen G, Lissens W, Sermijn E, De ` Greve J (2005) De novo Alu element insertions targeted to a sequence common to the BRCA1 and BRCA2 genes. Hum Mutat 26(3):284 5. Machado PM, Branda RD, Cavaco BM, Eugenio J, Bento S, Nave M, Rodrigues P, Fernandes A, Vaz F (2007) Screening for a brca2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes. J Clin Oncol 25(15):20272034 Peixoto A, Santos C, Rocha P, Pinheiro M, Prncipe S, Pereira D, Rodrigues H, Castro F, Abreu J, Gusmao L, Amorim A, Teixeira MR (2009) The c.156_157insAlu BRCA2 rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal. Breast Cancer Res Treat 114(1):3138 Berry DA, Iversen ESJ, Gudbjartsson DF, Hiller EH, Garber JE, Peshkin BN, Lerman C, Watson P, Lynch HT, Hilsenbeck SG, Rubinstein WS, Hughes KS, Parmigiani G (2002) BRCAPRO validation, sensitivity of genetic testing of BRCA1/BRCA2, and prevalence of other breast cancer susceptibility genes. J Clin Oncol 20(11):27012712 Parmigiani G, Berry D, Aguilar O (1998) Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2. Am J Hum Genet 62(1):145158 Peixoto A, Santos C, Rocha P, Pinto P, Bizarro S, Teixeira MR (2009) Molecular diagnosis of the Portuguese founder mutation BRCA2 c.156_157insAlu. Breast Cancer Res Treat 117(1): 215217

6.

7.

8.

9.

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