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Spanish family study on hereditary breast and/or ovarian cancer: Analysis of theBRCA1 gene

2000; Wiley; Volume: 91; Issue: 1 Linguagem: Inglês

10.1002/1097-0215(20010101)91

1097-0215

Miguel de la Hoya, Pedro P�rez-Segura, Nathalie van Orsouw, Eduardo Díaz‐Rubio, Trinidad Cald�s,

Ovarian cancer diagnosis and treatment

Breast and ovarian cancer often segregate in families. One criterion for defining hereditary disease is that the patient has at least 3 close relatives (first- and second-degree relatives) with breast and/or ovarian cancer. BRCA1 is a breast/ovarian cancer susceptibility gene identified in 19941. BRCA1 codifies for a large 220 kDa cell cycle regulated nuclear phosphoprotein2. Many studies suggest a role for BRCA1 in DNA repair and recombination3. Other evidence supports a role for BRCA1 in transcription regulation4) and apoptosis induction5. Unfortunately, a clear picture of BRCA1's function has not yet emerged. Mutations in the BRCA1 gene account for approximately 45% of breast cancer families and almost 100% of breast/ovarian cancer families6. The recent BCLC study analyzing 237 breast and/or ovarian cancer families confirms a high prevalence of BRCA1 deleterious mutations (90%) in breast/ovarian cancer families and a somewhat lower prevalence (30%) than previously suggested in breast cancer families7. However, other studies suggest a lower prevalence8. For instance, 40% of French, 29% of Italian and 21% of British breast/ovarian families demonstrated to harbor BRCA1 deleterious mutations9-11. Very few studies have been published concerning Spanish breast and breast/ovarian families. One study determined that only 3.5% of Spanish breast cancer families harbor deleterious BRCA1 mutations but did not study breast/ovarian families12. Another study13) indicated very low prevalence of BRCA1 mutations in both Spanish breast cancer families (2.6%) and Spanish breast/ovarian cancer families (11.5%). Clearly, additional BRCA1 studies are needed to determine which Spanish women interested in genetic counseling should be offered the opportunity of a test for mutations in BRCA1. In order to clarify this issue, here we report the nature and prevalence of BRCA1 deleterious mutations, unclassified variants and polymorphism in 90 Spanish families suspected to be at risk of harboring BRCA1 mutations. The Oncology Service of our hospital selected 90 families for genetic counseling. Families were grouped as follows: Group 1 (Breast/ovarian cancer families), this group included 12 families, the median age at diagnoses was 40 years (range 23–59); Group 2 (Breast cancer families), this group included 25 families, the median age at diagnoses was 50 years (range 29–69) and Group 3 (Low risk families), a case of early-onset breast or ovarian cancer (<45 years) without family history (36 patients) or 2 cases of breast cancer in the family (17 families) where the mean age at diagnosis was 51 years (range 27–67). Characteristics of the families in terms of the total number of cancer cases are given in Table I. All information concerning the family history of cancers were obtained by personal interviews of the patients. Diagnoses were verified on the basis of clinical and pathological records. After appropriate written consent was obtained, a blood sample was collected from each individual who decided to participate in the study. DNA was extracted from peripheral blood lymphocytes according to standard methods. The entire BRCA1 coding region and the splice-junctions were amplified from genomic DNA using 37 primers sets, the sequences of the primers have been described elsewhere14. PCR was performed in 25 μl containing 100 ng DNA, 1× PCR buffer (Ecogen SRL, Spain), 0.4 μM each primer, 200 μM each deoxynucleotide triphosphates (Promega, Madison, WI), 5% deionized Formamide (Sigma Chemicals Co., St. Louis, MO) and 1 U EcoTaq DNA polymerase (Ecogen SRL, Spain). Amplification was carried out in a PTC-100 thermocycler (MJ Research) at 95°C for 5 min, followed by 10 cycles at 94°C for 40 sec, 43°C for 1 min minus 0,5°C per cycle, 72°C for 1 minute 30 sec, 30 cycles at 94°C for 40 sec, 40°C for 1 min, 72°C for 1 min 30 seconds with an increase of 1 second per cycle and 1 cycle at 72°C for 10 min. Subsequently, PCR fragments were subjected to 1 round of complete denaturation and renaturation, that is, 98°C for 10 min, 55°C for 30 min and 37°C for 30 min to create heteroduplex molecules. DGGE analysis was performed in a Denaturing Gradient Gel Electrophoresis System DGGE-2000 (C.B.S. Scientific Co., California, USA); 5μl aliquot of control PCR was mixed with 2 μl of standard Dye Loading Buffer and was loaded onto a 10% AA/Bis-Acrilamida (37.5:1) gel (0% to 60% or 20% to 80% urea-formamida chemical gradient according to melting profiles of each PCR fragment) in 1XTAE ( 40 mM Tris-base, 20 mM NaAC, 1 mM EDTA, pH 8) for 16 hr at 80V and 58°C. The gel was stained in a solution of ethidium bromide, and the DNA was photographed under ultraviolet light. When either fresh or paraffin-embedded breast or ovarian tumor tissue was available Hematoxyline-Eosine stain was done in order to know the proportion of tumor cells, only samples with more than 95% of tumor cells were selected, DNA was obtained from microdisected tumor cells and DGGE-LOH analysis of individuals harboring germ-line BRCA1 deleterious or unclassified variants was performed as previously described15. For each heterozygous pattern identified by DGGE, cycling sequencing reaction was performed with ABI Prism dRhodamine Terminator sequencing kit (Perkin Elmer, Norwalk, CT) and analyzed in the ABI Prism 310 Genetic Analyzer (Perkin Elmer). All mutations were confirmed by 2 independent sequencing PCR in both DNA strands. After screening 90 Spanish families for BRCA1 mutations, 7 families harboring deleterious BRCA1 germ-line mutations were identified (Table I). The proportion of families with detectable mutations varied according to selection criteria and are summarized in Table I. The prevalence of BRCA1 deleterious mutations in Spanish breast/ovarian cancer families (Group 1) was among the highest prevalence described in European countries8. In total 6/12 (50%) of this group had detectable mutations. By contrast, we have found only 1 family harboring a BRCA1 deleterious mutation of 25 breast cancer families screened (Group 2). This family (Number 7), with 5 breast cancer patients (2 of them being bilateral breast cancer patients) reported in 3 generations, harbor the missense mutation A 1708 E. We did not find any deleterious mutation in 53 families considered to be at low risk of harboring BRCA1 deleterious mutations (Group 3). We identified 5 different deleterious BRCA1 germ-line mutations. These mutations were unique in 3 occasions and observed twice on 2 occasions. (These results are summarized in Table II). All mutations but 1 are located in the 3′ third of the gene (exon12–24). Interestingly, we did not identify any deleterious mutation located in the exon 11 (which represents more than 50% of the coding sequence). Mutations identified included 1 deletion (589del CT), 1 insertion (4229ins ATCT) and 2 splicing mutations (IVS20+1G/A, IVS18-1 G/C) leading to frameshift and also 1 missense mutation (A 1708 E) reported to suppress BRCA1 trans-activating activity4. Mutations 589delCT and A 1708 E were detected twice in non-related families. Mutations 4229insATCT and IVS18-1 GC represent 2 new BRCA1 deleterious mutations not previously reported16. Tumor DNA was available from women harboring BRCA1 deleterious mutations 589delCT, 1370insATCT and A1708E. In all 3 cases a DGGE-LOH analysis indicated that the deleterious allele, but not the wild-type, was retained in tumor DNA, confirming a tumor suppressor role for BRCA1 (Table II). Five missense mutations were identified in 9 families (Table II). Four of these missense mutations had been previously reported16. One novel missense mutation I89T was identified in a patient with breast cancer at 34 years (Group 3). Mutation S1040 N was found in 5 non-related families: 8, 26, 97 (Group 1 and 2) and 32, 55 (Group 3). Mutations N1236K, S1516I and T1720A were found in 3 different families: 8, 23, 90 (Group1 and 2). Determination of the potential pathogenicity of missense mutations S1040N, N1236K and S1512I was studied by DGGE-LOH analysis of DNA from blood and tumor of the proband. The wild type allele and the variant allele were equally retained in the tumor DNA, suggesting a non-pathogenic role for these missense mutations. There was no available tumor DNA to perform DGGE-LOH analysis for mutations I89T and T1720A. Hence, we do not have data enough to suggest a pathogenic or nonpathogenic role for these missense mutations. In addition to these mutations, 14 BRCA1 polymorphic variants were detected. They are shown in Table III. Interestingly, 11 polymorphic variants are clustered in the central coding region of the gene (exons 9–16). Most of them are located in the exon 11. All this BRCA1 polymorphic variants have been previously identified in other populations16. A breast cancer genetic counseling service represents an attractive offer for many families. However, both the cost and the time needed for BRCA1 and BRCA2 testing implies that a previous selection criteria must be established. In this study we have screened for BRCA1 mutations a total of 90 families divided in 3 clinically selected groups (1, 2 and 3) from whom testing might be indicated. We find 6 BRCA1 germ-line deleterious mutations in 12 breast/ovarian cancer families (50% of Group 1). This prevalence, lower than expected from the BCLC study7), is similar to that described for French and Netherlands families9,17) and it is higher than that described for Italian and British families10,10. Interestingly, it is much higher than the prevalence previously described for Spanish breast/ovarian families13. This discrepancy may be due to different technical approach (DGGE vs. SSCP) and/or to different regional origin of families studied (Central Spain versus the North-East). We only find 1 BRCA1 germ-line deleterious mutation in 25 breast cancer families (4% Group 2). The mutation was found in a family with 5 members affected in 3 generations. The BCLC study indicates that 30% of breast cancer families harbor BRCA1 mutations7. Our study suggest a very low prevalence of deleterious mutations in Spanish breast cancer families and this is in agreement with 2 previous Spanish studies12,13. Other studies from Iceland, Hungary and Stockholm also suggested very low prevalence of BRCA1 germ-line mutations in breast cancer families8,13. It is not likely that we have underestimated BRCA1 mutations due to low sensitivity of our screening protocol as DGGE is considered to be nearly 100% sensitive and specific19. Moreover, the high prevalence found in breast/ovarian families suggests that in our hands the DGGE method is highly sensitive. Our screening method does not detect gross chromosomal rearrangements, which have been described to account for 10% of germ-line BRCA1 mutations20. It might be possible that this rate would be increased in the Spanish population. Another explanation for the low incidence of mutations in our group of breast cancer families could be the number of affected members in the families, because the high prevalence of BRCA1 mutations has been given from studies in big families with many members affected or it could be due to the high proportion of late onset breast cancer families in our group because the median age was 50 years. We did not find any mutation in Group 3, which includes 36 women diagnosed with breast cancer before 45 years (of this group, 16 women were diagnosed before 35 years). Whether BRCA1 deleterious mutations are involved in early-onset breast cancers without family history or not remains a controversial issue. Although we need to study a larger cohort of Spanish women, our data suggests that BRCA1 related early-onset breast cancer might be a rare event in Spanish population. Similar results have been reported for other countries. In a large study, 9/254 women diagnosed with breast cancer before age 35 years carried mutations21. BRCA1 missense mutations represent a serious dilemma for genetic counseling since very often it is not known whether this variant represents a true cancer associate BRCA1 mutation or just a rare polymorphism without relationship with breast/ovarian cancer. This problem should be resolved with a functional assay which unfortunately is not yet available. Here we report several data that suggest a non-pathogenic role for 3 BRCA1 missense mutations: Ser 1040 Asn, Asn 1236 Lys and Ser 1512 Ile. This data can be of importance for a genetic counseling service. Another highly penetrant gene, BRCA2, has been identified. We are currently screening the same families for mutations in the BRCA2 gene, and preliminary results indicate that the involvement of BRCA2 is similar to that of BRCA1.The present study indicates that when BRCA1 mutation analysis is performed in women from families with breast-ovarian cancer (Group 1), there is a high likelihood of finding a BRCA1 mutation. The majority of women counseled at our hospital are from breast cancer-only families (Group 2). The low frequency of BRCA1 mutation carriers in this group suggests further studies to determine whether other genes are involved in these families. Miguel de la Hoya*, Pedro Pérez-Segura*, Nathalie Van Orsouw , Eduardo Díaz-Rubio*, Trinidad Caldés [email protected]*