1 Institute of Molecular Genetics, Consiglio Nazionale Ricerche, Alghero; 2 Institute of Pathology, University of Sassari; 3 Department of Radiotherapy and 4 Department of Medical Oncology, University of Sassari; 5 Department of Medical Oncology, Civil Hospital, Azienda Sanitaria Locale 1; 6 Laboratory II, Azienda Sanitaria Locale 1, Sassari, Italy; 7 Institute of Cancer Research, Sutton, Surrey; 8 Cancer Genome Project, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK
Received 4 February 2002; revised 22 April 2002; accepted 13 May 2002
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Abstract |
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The Sardinian population is genetically homogeneous and could be useful in understanding better the genetics of a complex disease like breast cancer (BC).
Patients and methods:
Using a screening assay based on a combination of single-strand conformation polymorphism, denaturing high-performance liquid chromatography and sequence analysis, 47 Sardinian families with three or more BC cases were screened for germline mutations in BRCA1 and BRCA2 genes.
Results:
Three BRCA1/2 germline sequence variants were identified. While BRCA2-Ile3412Val is a missense variant with unknown functional significance, BRCA2-8765delAG and BRCA1-Lys505ter are two deleterious mutations (due to their predicted effects on protein truncation), which were found in seven families (15%). BRCA2-8765delAG was found in six of eight (75%) BRCA1/2-positive families and seven of 501 (1.4%) unselected and consecutively collected BC patients. Prevalence of BRCA1/2 mutations in BC families was significantly correlated with the total number of female BCs (P <0.01) and increased by the presence of (i) at least one case of ovarian or male BC, or (ii) three generations affected, or (iii) bilateral BC.
Conclusions:
Identification of such features should address BC patients and their families to genetic counseling and BRCA1/2 mutational analysis. In addition, this is the first report of a detailed BRCA1/2 mutation screening in Sardinia, having immediate implications for the clinical management of BC families.
Key words: BRCA1/2 genes, breast cancer, genetic counseling, genetically homogeneous population, germline mutation, polymerase chain reaction-based screening
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Introduction |
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Previous studies using linkage analysis have suggested that germline mutations in BRCA1 and BRCA2 account for >90% of rigorously defined families with both breast cancer (BC) and ovarian cancer [13]. In a more comprehensive analysis presented by the Breast Cancer Linkage Consortium on about 200 families with at least four cases of BC and any number of ovarian cancers, 81% of the breastovarian cancer families presented with BRCA1 mutations whereas 14% of them were associated with BRCA2 [4]. Moreover, 76% of the families with male and female BC were associated with BRCA2 mutations [46]. Generally, the probability of finding a mutation within these two susceptibility genes in a BC patient increases with the number of related cases of early-onset BC or ovarian cancer in the family [7].
For BC, risk ratios have been demonstrated to increase with increasing numbers of affected first-degree relatives [7]. The cumulative lifetime risk, to age 70 years, of developing female BC in BRCA1/2 mutation carriers from multiple-case families has been estimated to be approximately 80% [4, 79]. Germline BRCA1/2 mutations have been also associated, in both sexes, with elevated risks for cancers at multiple organ sites, including prostate and gastrointestinal tumors [10, 11].
To date, several distinct mutations have been identified throughout the entire coding sequence of both BRCA1 and BRCA2 genes. Highly recurrent founder mutations have been described in genetically homogeneous populations or ethnic groups. While a single BRCA2 mutation accounts for the majority of hereditary BCs in Iceland [12], three different BRCA1/2 mutations have been described at high frequency among Ashkenazi Jews [13] and an even higher number (12) of recurrent founder mutations account for the vast majority of Finnish BRCA1/2 families [1416].
High frequencies of BRCA1 and/or BRCA2 mutations were also observed in BC patients unselected for family history from these genetically homogeneous populations, with the highest frequencies observed among the Ashkenazi Jews (2030%, depending on the age of onset) [17, 18]. Conversely, studies performed on unselected BC patients from different populations revealed that frequencies of BRCA1/2 mutations were considerably lower [1921].
In Sardinia, whose population is also genetically homogeneous (living for several hundred years in a roughly homogeneous environment), BC represents the principal death-causing malignancy as assessed by the Regional Tumor Registry [22], with an incidence of 93 per 100 000 inhabitants (standardized rate). In a previous study, we have identified a BRCA2 frameshift mutation at codon 2867 (8765delAG), causing a premature termination codon and showing a founder effect among seven unrelated BC families from North Sardinia [23]. To evaluate extensively the prevalence and spectrum of BRCA1 and BRCA2 mutations in the BC population from this region, BC families with at least three affected members were analyzed for mutations throughout the entire coding sequence of both BRCA1 and BRCA2 genes. The proportion of families in which BC is due to BRCA1/2 germline mutations has been estimated, and the cancer phenotype of families either positive or negative for such mutations has been defined.
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Patients and methods |
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The 22 coding exons and flanking splice site regions of BRCA1 were amplified under standard polymerase chain reaction (PCR) conditions, using 32 sets of primers (11 sets of overlapping primers were used for amplification of exon 11). Similarly, the 26 coding exons of BRCA2 and their flanking intronexon junctions were amplified using 56 sets of primers (with overlapping primer sets for exons 10, 11, 14, 18 and 27). All primer sequences are available on request.
All PCR products were run in SSCP electrophoresis gels under different conditions as previously described [23]. Suspected variants were identified as having a mobility shift. For a large fraction of cases (32 probands; particularly from families with higher recurrence of BC) mutational screening was also performed by DHPLC (demonstrated to be more sensitive than SSCP analysis in detecting genetic variants [24]), using the WAVE Nucleic Acid Fragment Analysis System (Transgenomic, Omaha, NE, USA). Suspected variants are visualized as a characteristic pattern of peaks corresponding to the mixture of homo- and heteroduplexes formed when wild-type and mutant DNA are hybridized. All abnormal PCR products identified by both methods were directly sequenced using an automated fluorescence-based cycle sequencer (ABIPRISM 3100; Perkin Elmer, Foster City, CA, USA).
To confirm that each germline BRCA1/2 variant detected by sequencing was a real mutation and not a polymorphism, 53 unrelated normal individuals (corresponding to 106 chromosomes), originating from the same geographical area and with no family history of BC, were used as controls and screened for each gene variant identified.
Genomic DNA for mutational screening of 501 unselected BC patients was isolated from both peripheral blood samples (n = 51) and archival paraffin-embedded tissues (n = 450), using previously described procedures [23]. To avoid any bias, paraffin-embedded normal tissues from BC patients were consecutively collected at the University of Sassari during two entire years (1997 and 1998). All cases were thus included regardless of age at diagnosis, family history status and disease features. Sardinian origin was ascertained in all cases through genealogical studies.
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Results |
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As shown in Table 2, only three germline variants were detected, at exon 11 of BRCA1 and exons 20 and 27 of BRCA2. Although BRCA2-8765delAG, which we previously described as a frameshift mutation with a founder effect in North Sardinia [23], remained the only recurrent mutation among families from the same geographical area, its prevalence was demonstrated to be much lower than that which we reported for the first seven families [six of 47 (12.8%) versus four of seven (57%), respectively] (Table 2). The remaining two sequence variations, BRCA1-Lys505ter and BRCA2-Ile3412Val, were identified in two additional different families. BRCA2-Ile3412Val has been previously reported as a rare missense variant (Table 2), with uncertain significance on function [25] (a prevalence study of simple sequence variations in the BRCA2 gene using 332 chromosomes from worldwide populations strongly suggests that Ile3412Val cannot be considered as a disease-causing mutation [26]). BRCA1-Lys505ter is a nonsense mutation (Table 2) that was absent in blood DNA from 53 unrelated normal individuals (corresponding to 106 control chromosomes) and has not been reported before in databases. Interestingly, BRCA1-Lys505ter was detected in the family presenting the strongest association between breast and ovarian cancer in our cohort (Figure 1). Because of both their predicted effects on proteins and their absence in normal controls, BRCA2-8765delAG and BRCA1-Lys505ter were the disease-causing mutations identified in seven of 47 (15%) families. Figure 2 shows the nucleotide sequences corresponding to these mutations.
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Discussion |
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Also taking into consideration some lack of sensitivity of our screening assay to reduce the number of positive cases, the prevalence of BRCA1/2 mutations in Sardinian families with at least three affected members remains low (although quite similar to that reported in the Finnish population [14]). When we restricted the analysis to BC families selected towards higher risk (four cases or more affected by breast or ovarian cancer), the proportion of families due to BRCA1/2 mutations was markedly higher [seven of 26 (27%); Tables 3 and 4]. However, this prevalence was still lower than that reported in Ashkenazi Jews or in other genetically homogeneous populations (such as Icelandic and French-Canadian), where few founder mutations in BRCA1 and BRCA2 are present in more than half of high-risk BC families [2830]. Interestingly, the BRCA2 founder mutation observed in the French-Canadian population [30] happened to be identical to the BRCA2-8765delAG mutation found in our series (previously, we have demonstrated that such a mutation is associated with different haplotypes in Sardinian and French-Canadian families [23]).
Several authors reported that mutations of BRCA1 and BRCA2 genes in the breast site-specific cancer families would account for 2040% of the cases, strictly depending on the total number of BC cases in the family [3, 9] (it has been even suggested that most of the families with five or fewer cases of women affected with BC and without any case of ovarian or male BC would be attributable to mutations in other low-penetrance susceptibility genes distinct from BRCA1 and BRCA2 [4]). In addition, the majority of the studies previously mentioned presented a higher proportion of breastovarian cancer families or male BC families. The low prevalence of mutations found in our study should be due to the fact that either the majority of our families [35 of 47 (74%)] contained three or four cases of female BC or a very small fraction of them were associated with ovarian and male BC. In line with this hypothesis, the percentages of BRCA1/2 mutations among families with at least five cases of BC or association with ovarian/male BC in our series were 50% or higher (Table 4).
Considering the familial cases, BRCA2 mutations were notably more recurrent than BRCA1 mutations in the BC families from Sardinia (the very low proportion of BRCA1 mutations might be explained by the very limited number of families with association with ovarian cancer). This is in contrast to the mutation frequencies among BC families from most other populations, in which BRCA1 has been reported to play the major role in BC predisposition [1720] (BRCA2 mutations are instead prevalent in Iceland [29]).
Generally, BRCA2 mutations have been associated with early-onset BC [31]. The median age of BC onset among all affected BRCA2 carriers from this study is similar to that reported in other studies (49 years [12, 25, 29]). However, only 13 of 59 (22%) familial BC patients who underwent BRCA1/2 mutational analysis were diagnosed at 40 years of age (Table 1), and only two (15%) of them presented the BRCA2-8765delAG mutation. Thus, our findings indicate an even lower proportion of BRCA2 mutations in very early-onset BC families.
Considering the incidence of the identified BRCA1/2 mutations among the unselected patients from the same population, an extensive screening was performed for the most prevalent BRCA2 germline mutation only (for the BRCA1 mutation, analysis was limited to a smaller fraction of unselected cases). Although the proportion of hereditary BC in Sardinia is estimated to be comparable with that reported for other populations [22], the fraction of 1.4% BRCA2 mutation carriers is very low in Sardinian BC patients (although a similar prevalence was observed in the Finnish population [16]). This is in a quite sharp contrast with frequencies in unselected cases from either the Ashkenazi Jewish population, where two BRCA1 founder mutations occur at a frequency 510 times higher [17, 18], or other isolated populations [16, 20, 21] (in Iceland, a single BRCA2 founder mutation was observed in 10% of unselected BC patients [12, 29]).
The association with other tumors in BC families positive for BRCA1/2 germline mutations has been widely described [10, 29, 30] and also observed in our families. In addition, patients with prostate cancer or bladder cancer from families positive for BRCA2-8765delAG (families 2, 4, 12 and 48; Table 3) were found to carry the same BRCA2 mutation ([23] and G. Palomba, unpublished observations), strongly suggesting that germline alterations in such a susceptibility gene may predispose to other types of cancer.
Our study indicates that the prevalence of BRCA1 and BRCA2 germline mutations in Sardinian BC families is low. However, the presence in the family of (i) at least one case of ovarian cancer, or (ii) at least one case of male BC, or (iii) five or more cases of female BC, or (iv) three generations affected, or (v) synchronous or asynchronous bilateral BC, is a strong predictor for the presence of mutations in the BRCA1 and BRCA2 genes, which makes these kinds of family specially eligible for the mutational screening. In addition, construction of deeply rooted pedigrees in Sardinia, like that reported from Thiesi (Figure 3), could allow us to relate a higher number of individuals, apparently unrelated at present, in very large families. This would greatly increase the number of familial members who could benefit from referral to genetic counseling.
To evaluate the biological impact of BRCA1/2 alterations in BC pathogenesis as well as to assess the existence of any correlation with the prognosis and clinical outcome, we have already started a mutational screening of these two genes in a larger subset of archival tissues from Sardinian BC patients for whom both clinical and pathological records are available (G. Palomba, unpublished data).
Finally, the majority of families studied here have remained negative for mutations in the BRCA1/2 genes, even though many showed a clear predisposition to BC. Further work is required to investigate in more details other BRCA1/2 inactivation mechanisms (i.e. mutations in the promoter regions) or mutations in other, as yet unidentified, low-penetrance susceptibility genes. This would determine more accurately the mutation spectrum in Sardinian BC families.
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Acknowledgements |
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Footnotes |
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These authors contributed equally
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References |
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