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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. vol.45 no.2 Santiago  2012

http://dx.doi.org/10.4067/S0716-97602012000200003 

Biol Res 45: 117-130, 2012

ARTÍCULOS DE INVESTIGACIÓN

 

BRCA1 and BRCA2 mutations in breast cancer patients from Venezuela

 

Karlena Lara1, Nigmet Consigliere1, Jorge Pérez2, Antonietta Porco1*

1 Universidad Simón Bolívar, Departamento de Biología Celular, Laboratorio de Genética Molecular Humana B, Caracas;
2 Centro Clínico de Estereotaxia CECLINES, Caracas.


ABSTRACT

A sample of 58 familial breast cancer patients from Venezuela were screened for germline mutations in the coding sequences and exon-intron boundaries of BRCA1 (MIM no. 113705) and BRCA2 (MIM no. 600185) genes by using conformation-sensitive gel electrophoresis. Ashkenazi Jewish founder mutations were not found in any of the samples. We identified 6 (10.3%) and 4 (6.9%) patients carrying germline mutations in BRCA1 and BRCA2, respectively. Four pathogenic mutations were found in BRCA1, one is a novel mutation (c.951_952insA), while the other three had been previously reported (c.1129_1135insA, c.4603G>T and IVS20+1G>A). We also found 4 pathogenic mutations in BRCA2, two novel mutations (c.2732_2733insA and c.3870_3873delG) and two that have been already reported (c.3036_3039delACAA and c.6024_6025_delTA). In addition, 17 variants of unknown significance (6 BRCA1 variants and 11 BRCA2 variants), 5 BRCA2 variants with no clinical importance and 22 polymorphisms (12 in BRCA1 and10 in BRCA2) were also identified. This is the first genetic study on BRCA gene mutations conducted in breast cancer patients from Venezuela. The ethnicity of our population, as well as the heterogeneous and broad spectrum of BRCA genes mutations, must be considered to optimize genetic counseling and disease prevention in affected families.

Key words: BRCA genes, Breast cancer, screening mutations.


 

INTRODUCTION

Deleterious germline mutations in the BRCA1 (MIM no. 113705) and BRCA2 (MIM no. 600185) tumor suppressor genes have been significantly associated with elevated risk of developing breast and ovarian cancer. Lifetime risk of breast cancer is as high as 80% among women with mutations in these genes, and for ovarian cancer the risk is greater than 40% and 20% for carriers of BRCA1 and BRCA2 mutations, respectively (King et al., 2003).

The BRCA1 gene is mapped to chromosome 17q21, spanning more than 80 kb distributed in 22 exons, and encodes for a protein of 1,863 amino acids. The BRCA2 gene is located at locus 13q12, comprises 10.4 kb organized in 27 exons, and encodes for a protein of 3,418 amino acids (Wooster et al., 1994; Wooster et al., 1995). More than 1600 mutations in BRCA1 and 1800 mutations in BRCA2 have been described throughout both genes according to the Breast Cancer Information Core website (BIC).

Little is known about the contribution of BRCA1 and BRCA2 mutations to hereditary breast and/or ovarian cancer in Hispanic populations. In the last few years, important contributions have been made by different groups that have reported the prevalence of BRCA1 and BRCA2 mutations in different populations from South America, such as Chile (Jara et al., 2006; Gallardo et al., 2006), Colombia (Torres et al., 2007), Brazil (Dufloth et al., 2005; Gomes et al., 2007) and Mexico (Ruiz-Flores et al., 2002). All these studies have in common the finding that the spectrum of mutations is quite different among the Latin-American populations investigated.

Ethnicity plays an important role in hereditary breast cancer given that specific founder mutations have been associated with the development of this pathology in various ethnic groups, such as the mutations found in Ashkenazi Jews (Warner et al., 1999). More recently, a study conducted in 53 breast/ovarian cancer families from Colombia reported three recurrent mutations (two in BRCA1 and one in BRCA2) with possible founder effects (Torres et al., 2007). These findings highlight the importance of molecular diagnosis of mutations in the BRCA1 and BRCA2 genes in different populations. In addition, the identification of mutations in these genes allows the screening, counseling, testing, and clinical management of high risk families and also contributes to estimating the prevalence of these mutations in a specific population.

Breast cancer is the second most common cancer and second most common cause of cancer death among women in Venezuela (Capote, 2006). By 2008, breast cancer had an incidence rate of 42.5 cases every 100,000 women (incidence cases of 5,404) and a mortality rate of 13.7% (http://globocan.iarc.fr/factsheets/cancers/breast.asp).To date, there is no data on the participation of germline BRCA mutations in breast cancer patients from Venezuela. In this study, we performed a mutational analysis of BRCA1 and BRCA2 genes using conformation-sensitive gel electrophoresis (CSGE), in order to establish a genetic profile in a sample of Venezuelan breast cancer patients.

METHODS

Patients

The study included 58 breast cancer patients diagnosed at the "Centro Clínico de Estereotaxia" (CECLINES), Caracas, Venezuela. Peripheral blood was acquired from the subjects after obtaining their signed consent. Blood collection was carried out between May 2006 and November 2009. The patients were identified as high risk individuals with BRCA gene mutations because they met at least one of the following criteria according to the Breast Cancer Linkage Consortium (1997): early onset (less than 45 years of age) and/or bilaterality; more than three cases of breast cancer and more than one case of ovarian cancer in the family; more than two first-degree relatives affected; and male breast cancer.

DNA Extraction

Genomic DNA was extracted from peripheral blood samples according to Bowen and Keeney (2003). Ethanol precipitated DNA samples were resuspended in sterile water and frozen at -20°C until further use.

Multiplex PCR amplification and screening for Ashkenazi Jewish founder mutations

Fragments possibly containing the Ashkenazi Jewish founder mutations c.185_186delAG and c.5382insC in BRCA1 and c.6174delT in BRCA2 were amplified by multiplex PCR using three sets of primers and the conditions previously described by Kuperstein et al. (2000), with the exception that the primers lacked the 5'-Cy5 modification. Products were diluted 1:1 in formamide buffer (98% formamide, 10 mM EDTA and 0.05% bromophenol blue) and screened for mutations using a modification of the fluorescent multiplex-PCR analysis (FMPA) technique (Kuperstein et al., 2000). The fragments were analyzed on an 8% denaturing polyacrylamide gel containing 7 M urea, and separated at 10° C, 2700 V, 100 mA and 80 Watts for 90 minutes in 1 X TBE buffer. Gels were revealed by silver staining.

Multiplex amplification and screening of the BRCA1 and BRCA2 genes by CSGE

BRCA1 and BRCA2 screening was performed by PCR amplification of the entire coding regions, including the flanking splice sites and 3' UTR, using 88 sets of primers (38 for BRCA1 and 50 for BRCA2). Tables I and II summarized the sequences of all primers that were employed. The primers were grouped into 35 multiplex sets (15 for BRCA1 and 20 for BRCA2), according to the PCR product size and annealing temperature (Table III). Some exons were amplified as uniplexes and then mixed for further analysis. Individual and Multiplex PCR (MPCR) amplifications included an initial amplification for 5 min at 95°C, followed by 35 cycles at 95°C for 1 min, annealing at temperatures indicated in Table III for 1 min and 1 min at 72°C. A final elongation step was done at 72°C for 10 min. The PCR reactions were carried out on a Mastercycler Gradiente Thermal Cycler (Eppendorf) using 0.2, 0.4, 0.6 and 0.8 mM of dNTP's (according to primers conforming the MPCR: 1, 2, 3 and 4 pairs, respectively), 0.075 U of Platinum ®Taq DNA Polymerase (Invitrogen, São Paulo, Brazil), 1 X of Platinum ®Taq DNA Polymerase Buffer (200 mM Tris-HCl pH 8.4 and 500 mM KCl), 0.75 μΜ of each primer and approximately 200 ng of genomic DNA. The final concentration of MgCl2 used for these amplification reactions are also indicated in Table III. The MPCR products were screened for mutations in a mildly denaturing CSGE gel containing 12.5% polyacrylamide as previously described (Ganguly et al., 1993).







DNA sequencing

Samples displaying abnormal CSGE patterns were sequenced using an automated ABI 377 genetic analyzer (Applied Biosystems, Foster City, CA, USA) at the UEGF-IVIC (Caracas, Venezuela). All the nucleotide changes identified were confirmed by repeating the PCR and sequencing reaction using the corresponding forward and reverse primers. Mutation nomenclature was according to den Dunnen y Antonarakis (2001).

RESULTS

In the 58 breast cancer patients that were analyzed in the present study (Table IV), no Ashkenazi Jewish founder mutations were identified. Ten out of these 58 breast cancer patients (17.2%) carried BRCA mutations, six (10.3%) in BRCA1 and four (6.9%) in BRCA2 (Table V).

The patients analyzed in this study fell into 5 subsets (Table VI). All patients belonging to the subset of families with male breast cancer were carriers of BRCA2 gene mutations (100%), whereas all those patients belonging to the subset of families with breast/ovarian cancer presented BRCA1 mutations (100%). Lower frequencies of BRCA mutations were found in patients belonging to subsets of families with bilateral breast cancer (37.5%), early age of onset (18.1%) and multiple breast cancer cases in the family (10%).

BRCA1 mutations

Four BRCA1 truncating mutations were identified (Table V). The c.951_952insA mutation was identified in two unrelated patients and represented a novel mutation with no previous report on the BIC database. The c.4603G>T mutations on exon 14 of BRCA1 gene, was previously reported and identified on the BIC database as a missense mutation (p.R1495M). However, Ozcelik et al (1999) and Yang et al (2002) determined that this mutation affects the splicing process by altering the -1 position of the exon 14 donor splice site, which causes the skipping of exon 14 resulting in a frame shift and in consequence leading to a stop codon at amino acid 1462 of the BRCA1 protein.

In addition, six BRCA1 variants of unknown significance were detected in eight patients, five were missenses and one was an inframe deletion (Table VII). From these variants, the c.179A>C, IVS20-22C>T and g.6002C>T mutations had no precedents on the BIC data base. It has not yet been demonstrated whether these variants have pathogenic consequences.

All of the 58 patients presented at least one polymorphism in BRCA1 (Table VIII). In particular, the IVS1+101C>G and c.4427T>C, c.6998C>T polymorphisms were reported here for the first time. Another polymorphism detected (IVS7+16(TTC) nTTTTC) was a triplet deletion (TTC) in a (TTC)7TTTTC region on intron 7 of BRCA1 . Although this polymorphism is not included in the BIC database, it has been previously reported in a Spanish population (Salgado et al., 2008). Three genotypes were identified: (TTC)7/7, (TTC)7/6 y (TTC)6/6 with frequencies of 41.4%, 48.3% and 10.3%, respectively.

BRCA2 mutations

Four different truncating mutations in the BRCA2 gene were identified, as shown in Table V. Two of these mutations (c.2732_2733insA and c.3870_3873delG) had not been previously reported in the BIC database. In addition, eleven BRCA2 mutations of unknown significance were detected in fourteen different patients (Table VII), which included eight missense mutations, one intervening sequence and two 3'UTR variants. From these, mutations c.1282T>C, c.3479G>A, c.3875T>A, c.9799T>C, IVS12-63A>C, c.10594G>T and c.11323T>C had not been previously reported in the BIC database.

In BRCA2, ten different polymorphisms (Table VIII) were identified in 55 of the 58 patients. The c.10590A>C and c.11314_11323insT polymorphisms were reported here for the first time.

DISCUSSION

Since the identification of BRCA1 (Friedman et al., 1994; Miki et al., 1994) and BRCA2 (Wooster et al., 1995) genes, the major genes known to confer high risk of breast and ovarian cancer, several mutations have been identified throughout the entire gene sequence, most of which are nonsense or frame shift mutations that produce truncated proteins (BIC database). The identification of mutations with founder effect in some specific ethnic groups has highlighted the importance of genetic testing in different populations. Despite the high prevalence of breast cancer in the Venezuelan population, studies related to the identification of BRCA1 and BRCA2 mutations, among patients with either breast cancer or a high-risk family history, have not been previously conducted.

Mutation screening was performed using the combined approach of CSGE and sequencing analysis. In a previous study developed in our laboratory, this method showed a sensitivity of over 90% for mutation detection (Albánez et al., 2011), besides being simple, rapid and cost-effective for this purpose. In addition, this technique has been shown to detect almost every unique single-base mismatch when it was compared to DGGE and SSCP for BRCA mutations detection (Ganguly, 1997). Indeed, we discarded the SSCP method during this investigation since we found only 33% sensitivity to detect the BRCA mutations (data not shown), which is in agreement with a previous study where SSCP showed 35% sensitivity to detect mutations in the coagulation factor IX gene (Sarkar et al., 1992)

As previously mentioned, eight distinct germline mutations, 4 in BRCA1 and 4 in BRCA2, were detected in 10 of the 58 Venezuelan breast cancer patients, representing a frequency of 17.2%. This frequency was similar to that previously reported in the Chilean population (15.6% and 20.3%, Jara et al., 2006; Gallardo et al., 2006, respectively), but higher than that found in the Brazilian population (13%; Dufloth et al., 2005) and lower than that found in 53 families from Colombia (24.5%; Torres et al., 2007). One of the BRCA mutations detected in our study was found in two unrelated patients, another was found in two related patients, and the rest were detected in 6 different patients. These findings suggest that the BRCA1 /2 gene mutation spectrum is rather broad in the Venezuelan population. All the disease-causing mutations were small deletions, insertions or missenses that cause premature stop codons.

There are no reports of the BRCA1 mutation c.951_952insA on exon 11 in the BIC database and therefore its possible pathological effect is suggested by the absence of important sequences in the altered protein, such as the NLS and BRCT domains, and could be supported by the fact that this was the only mutation identified in two unrelated patients with breast cancer. One of these patients developed breast cancer at 29 years of age, while the other has three second-degree and one third-degree relatives with breast cancer; one of the second-degree relatives was also affected with ovarian cancer. All these are characteristics of the presence of mutations in the BRCA1 gene (Ford et al., 1998). The other three BRCA1 mutations (c.1129_1135insA, c.4603G>T and IVS20+1G>A) found in this study have been previously reported in the BIC database as mutations with clinical importance. The c.1129_1135insA was identified in two related patients (sisters) with family history of breast/ovarian cancer, indicating that this mutation may be segregating with the pathology in this family. The mutation c.4603G>T was identified in a patient with early-onset of breast cancer (35 year) and with a sister affected with this pathology. These two mutations (c.1129_1135insA and c.4603G>T) have been reported mostly in populations from Western Europe (BIC database), and it is not surprising to find these mutations in the Venezuelan population, which is characterized by a high ethnic heterogeneity and possesses an important European influence (Rodríguez-Larralde et al., 2001). Finally, the IVS20+1G>A mutation was identified in a patient that developed breast cancer at the age of 34. This variant can affect the splicing process in two ways, by promoting the skip of exon 20 generating a truncated protein of 1737 amino acids or by promoting the retention of part of intron 20, which in this case generates a truncated protein of 1767 residues (Tesoriero et al., 2005). In both cases the resulted protein lacks important functional domains and therefore appears to produce loss of BRCA1 function and is expected to be associated with the same breast cancer risk as other protein truncating mutations (Brose et al., 2004).

Of the BRCA2 mutations, the c.3036_3039delACAA mutation has 105 records in the BIC database, most of which correspond to European populations, mainly Italian and Spanish, both having influence on the ethnic background of the Venezuelan population (Rodríguez-Larralde et al., 2001). This mutation was detected in a patient with early-onset of breast cancer (35 years) whose father was affected with the pathology as well. Previous investigations have suggested that male breast cancer is one of the hallmarks for the presence of BRCA2 mutations (Honrado et al., 2005). The other previously described mutation detected in BRCA2 (c.6024_6025_delTA), which was identified in a patient who developed breast cancer at an early age (38 years), has bilaterality and a family history of the pathology in three male relatives. Although we were not able to test the male relatives to determine if they were carriers of this mutation, the evidence of the relation of BRCA2 mutation and male breast cancer (Honrado et al., 2005) indicate that this mutation is responsible for the increased risk of developing breast cancer in this family. One of the novel mutations in BRCA2, c.2732_2733insA was found in a bilateral breast cancer patient whose sister was affected with this pathology. The other novel mutation (c.3870_3873delG) was identified in the only male breast cancer patient included in this study. We considered these novel mutations as pathogenic because they generate truncated proteins of 837 and 1227 amino acids, respectively, that affect regions before and inside the conserved BRC repeats in the BRCA2 protein, which is well known to participate in the interaction between BRCA2 and RAD51 (Mitchell, 2002). Therefore, considering the role in the maintenance of genomic integrity of BRCA2 and RAD51, the carriers of these mutations could display a diminished capacity in the signaling and/or repair of certain forms of DNA damage.

It has been established that tumors arising in carries of BRCA1 and BRCA2 gene mutations differ morphologically and histopathologically from sporadic breast cancer of age-matched controls (Honrado et al., 2005). Breast cancers in patients with BRCA1 germline mutations are more often negative for estrogen receptor (ER-), progesterone receptor (PR-), and HER-2 (HER-2-) compared to controls, whereas BRCA2 tumors do not show a significant difference in the expression of any of these proteins (Lakhani et al., 2002). In agreement with this (Table IV), the tumors from the c.951_952insA, c.1129_1135insA (BRCA1) and c.6024_6025delTA (BRCA2) mutations carriers have been shown to be ER-, ER-/PR- and ER-, respectively. On the other hand, the tumor developed in the carrier of the c.3036_3039delACAA BRCA2 mutation was an invasive ductal carcinoma, which is the most common histological type in all forms of hereditary breast cancer and seems to be significantly more frequent in BRCA1- and BRCA2-mutation carriers than in non-carriers (Chappuis et al., 2000).

Among the variants of unknown significance indentified in the present study, c.179A>C (p.K20N), c.3238G>A (p.S1040N) and c.3827T>G (p.N1236K) in BRCA1 and c.1282T>C (p.Y352H), c.3479G>A (p.S1084N) and c.6328C>T (p.R2034C) in BRCA2 generate biochemically similar changes in amino acids. Therefore these variants are probably not the main cause of the disease in the carrier patients. The novel c.4182_4184delAAT variant found in BRCA1 was identified in the two previously mentioned patients that were related and also carried the pathogenic mutations c.1129_1135insA. Thus, it is not possible to establish whether the c.4182_4184delAAT variant is also pathological. However, if it is determined that this variant is not in the same chromosome as the c.1129_1135insA mutation, any clinical significance could be discarded, since it is well known that an individual with both BRCA alleles mutated is not viable (McCarthy et al., 2003; Reid et al., 2008). This could be established by analyzing the genotype for both mutations in each parent. If each of these variants is identified in a different progenitor, it will mean that both variants are indeed in different BRCA alleles in these patients. Other novel variants with unknown significance found in this study are IVS-22C>T in intron 20 of BRCA1 and c.IVS12-63A>C in intron 12 of BRCA2. According to the BIC database, approximately 4% of the genetic variants are reported as splice-site alterations and the knowledge about their effect at the cDNA level is scarce. It is well known that accurate RNA splicing requires that the conserved sequence motifs at the intron-exon junctions and the branch point must be mutation free (Shapiro and Senapathy, 1987). Among these conserved sequences are a highly conserved eight-nucleotide sequence at the exon-intron boundary, the splice donor or 5' splice site sequence [(A/C) AG//gta/g)agt] and the acceptor or 3' splice site, preceded by a pyrimidine-rich region (tyttytytyyyyncag//G, where y represents any pyrimidine and n represents any nucleotide). Therefore, the IVS-22C>T mutation could lead to an aberrant transcript since it is located inside the pyrimidine-rich region. Similarly, the c.IVS12-63A>C variant can affect the splicing process by altering important sequences. The variants c.7697T>C (p.I2496T) and c.8237C>T (p.S2670L) in BRCA2 lay in a region of the gene through which it has been shown that BRCA2 interacts with DSS1 (amino acids 2378 to 3115) (Yang et al., 2002), a protein that stabilizes BRCA2 and regulates its function during DNA repair, acting as a co-factor (Kojic and Holloman, 2004). Both of these mutations change polar for nonpolar amino acids and therefore may affect the interaction between these two proteins. Finally, three novel variants, g.6002C>T in BRCA1 and c.10594G>T and c.11323T>C in BRCA2, were found in the 3'-UTR region. These variants could affect the stability, localization or transportation of the mRNA given the important participation of the 3'-UTR in the fate of this molecule.

In conclusion, the prevalence of mutations of the BRCA1 and BRCA2 genes found in this study among patients with breast cancer was 17.2% (10/58), which is similar to what has been reported in other countries for breast cancer families. This study represents the first analysis of BRCA disease-associated mutations in Venezuela. The ethnicity of our population, as well as the heterogeneous and broad spectrum of BRCA genes mutations, must be considered to optimize genetic counseling and disease prevention in affected families.

ACKNOWLEDGEMENTS

We thank J. Bubis and D. Ajami for their critical reading of the manuscript. This study was supported by a research grant from LOCTI N° 5662-08

ELECTRONIC-DATABASE INFORMATION

The URLs for data presented in this article are as follows:

BIC database, (http://research.nhgri.nih.gov/bic/) last viewed April, 2011

ChartsBin statistics collector team 2010, Current Worldwide Breast Cancer Incidence Rate, ChartsBin.com, (http://globocan.iarc.fr/factsheets/cancers/breast.asp) last viewed September, 2011

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* Corresponding author: Prof. Dr. A. Porco, Laboratorio de Genética Molecular Humana B. Departamento de Biología Celular. Universidad Simón Bolívar. Apartado 89000. Caracas 1080-A, Venezuela. e-mail: aporco@usb.ve.; Tel/fax: +58-212-9064217.

Received: April 18, 2011. In Revised Form: August 5, 2011. Accepted: September 15, 2011.

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