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Cancer Epidemiology Biomarkers & Prevention 16, 2276, November 1, 2007. doi: 10.1158/1055-9965.EPI-07-0403
© 2007 American Association for Cancer Research
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BRCA1 and BRCA2 Mutations in an Asian Clinic-based Population Detected Using a Comprehensive Strategy

Peter Ang1, Irene H.K. Lim1, Tze-Chuen Lee3, Jie-Ting Luo2, Danny C.T. Ong2, Puay Hoon Tan4 and Ann S.G. Lee2,5

1 Department of Medical Oncology and 2 Laboratory of Molecular Oncology, Division of Medical Sciences, National Cancer Centre; 3 Singapore Biomedical Computing Resources, Bioinformatics Institute; 4 Department of Pathology, Singapore General Hospital; and 5 Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

Requests for reprints: Ann S.G. Lee, Division of Medical Sciences, National Cancer Center, Singapore 169610, Singapore. Phone: 65-6436-8313; Fax: 65-6372-0161. E-mail: dmslsg{at}nccs.com.sg


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background and objective: Genetic testing for germ line mutations in the BRCA1 and BRCA2 genes for some families at high risk for breast and/or ovarian cancer may yield negative results due to unidentified mutations or mutations with unknown clinical significance. We aimed to accurately determine the prevalence of mutations in these genes in an Asian clinic-based population by using a comprehensive testing strategy.

Materials and Methods: Ninety-four subjects from 90 families were accrued from risk assessment clinics. In addition to conventional mutational screening of BRCA1 and BRCA2, multiplex ligation-dependent probe amplification for the detection of large genomic rearrangements, evaluation of splice site alterations using transcript analysis and SpliceSiteFinder prediction, and analysis of missense mutations of unknown significance by multiple sequence alignment, PolyPhen analysis, and comparison of Protein Data Bank structures were incorporated into our testing strategy.

Results: The prevalence rates for clearly deleterious BRCA1 and BRCA2 mutations were 6.7% (6 of 90) and 8.9% (8 of 90), respectively, or 7.8% (7 of 90) and 11.1% (10 of 90), respectively, by including missense mutations predicted to be deleterious by computational analysis. In contrast to observations from European and American populations, deleterious mutations in BRCA2 (10 families) were more common than for BRCA1 (7 families). Overall, the frequency of mutations was 12.2% (n = 11) by conventional screening. However, by including deleterious mutations detected using multiplex ligation-dependent probe amplification (n = 1), transcript analysis (n = 2), and computational evaluation of missense mutations (n = 3), the frequency increased substantially to 18.9%. This suggests that the comprehensive strategy used is effective for identifying deleterious mutations in Asian individuals at high risk for breast and/or ovarian cancer. (Cancer Epidemiol Biomarkers Prev 2007;16(11):2276–84)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast cancer is the most frequent cancer among females in many Caucasian populations and in Singapore. Increased susceptibility for breast cancer is usually associated with deleterious mutations of the breast cancer susceptibility genes BRCA1 and BRCA2, particularly in families with both breast and ovarian cancer. Such germ line mutations in the BRCA1 and BRCA2 genes have been documented in most populations (1-3). Clinical genetic testing for BRCA1 and BRCA2 mutations after genetic counseling is the standard of care in North America and Europe but is not available in many countries in Asia.

Genetic testing of the BRCA1 and BRCA2 genes typically involves the DNA sequencing of all exons and intron-exon junctions. However, some high-risk pedigrees will have "negative" results, possibly due to unidentified mutations or mutations with unknown clinical significance, thus presenting a dilemma in risk assessment and genetic counseling (4).

Large genomic rearrangements, such as exon duplications or deletions found in high frequencies in European populations, are not detected by conventional genetic testing strategies (5). Recently, the multiplex ligation-dependent probe amplification (MLPA) assay has been used by many studies to detect large genomic rearrangements in the BRCA1 and BRCA2 genes (6, 7). In a large study of 300 families with four or more cases of breast or ovarian cancer but who were commercially tested negative for BRCA1 and BRCA2 mutations, MLPA detected genomic rearrangements in 12% of the probands (8), thus highlighting the importance of screening for such aberrations in addition to conventional PCR-sequencing protocols.

Intronic alterations that are located within or near to intron-exon junctions may affect mRNA splicing fidelity (9). These alterations are categorized as unclassified variants unless they have been evaluated by transcript analysis to determine their effect on mRNA splicing (10, 11). Other unclassified variants include missense mutations, which have unclear pathogenicity and which form between a third to half of all genetic variants documented in the Breast Cancer Information Core database. Pathogenicity of these variants may be established from family studies of cosegregation, absence in unaffected controls, or by using biochemical criteria, such as conservation of amino acid across species, severity of amino acid change, and involvement of an amino acid within a functional domain (12-14).

In this study, we aimed to accurately determine the prevalence of mutations in the BRCA1 and BRCA2 genes in a cohort of Singaporean women accrued at risk assessment clinics by using a comprehensive testing strategy. In addition to conventional screening of the coding exons of the BRCA1 and BRCA2 genes and their intron-exon boundaries, MLPA analysis for large genomic rearrangements, RNA analysis, in silico prediction of intronic alterations, and evaluation of missense mutations of unknown clinical significance by computational analyses were done. Although these techniques have been used for studies to evaluate the prevalence of BRCA1/BRCA2 mutations, to our knowledge, there have not been any reports incorporating all of these techniques for a comprehensive testing strategy for BRCA1 and BRCA2 mutation detection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
A flow chart summarizing the methodology of the study, from patient accrual to validation of mutations, is shown in Fig. 1 .


Figure 1
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  		Figure 1. Flow chart of the methodology used for the screening of mutations in the BRCA1 and BRCA2 genes.

 
Subjects
All subjects were recruited from clinics at the National Cancer Center and KK Women's and Children's Hospital in Singapore under the Risk Evaluation and Prevention program. Detailed pedigree risk assessment was done with a combination of counselor assessment and the BRCAPRO model (15). Family histories of all cancers for three generations were recorded, including ages of diagnoses of all cancers. Subjects were eligible if there is a personal or family history of breast and ovarian cancer, if there are two primaries, if there is a personal and family history of breast (at least one premenopausal) or ovarian cancer(s) in a close relative from the same side of the family, or if the subjects have breast or ovarian cancer and were ages 39 years and below. One unaffected subject was tested as her sister had recent breast cancer at age 35 and was being evaluated for suspected ovarian cancer. Informed consent was obtained, and the research protocol was approved by the ethics review committee at both hospitals.

Between March 2002 and April 2006, 95 eligible subjects were accrued from 90 families. All subjects were of Asian descent. Subjects were predominantly Chinese (75.8%), followed by Malays (11.6%), Indians (4.2%), and others (8.4%), reflecting the ethnic composition of Singapore. The median age of participants at entry was 36 years, with a range from 19 to 72 years. Half of the subjects were below 40 years old.

Mutational Analysis of BRCA1 and BRCA2
Genomic DNA was isolated using standard techniques as previously reported (16). Purified DNA was amplified as described using published primers (17, 18) and primers described at the Breast Cancer Information Core Web site6 (16). Direct sequencing of the amplified products was done using the CEQ Dye Termination Cycle Sequencing quick start kit (Beckman Coulter), and the products were analyzed using the CEQ 8000 System (Beckman Coulter) according to the manufacturer's recommendations. The PCR products of exon 11 in BRCA1 and exons 10 and 11 in BRCA2 were in vitro transcribed/translated using the protein truncation test (TNT®T7 Quick for PCR DNA, Promega). The translation products were electrophoresed on 10% SDS-polyacrylamide gels. Any mutations detected were confirmed by separate PCR amplifications done at least once.

MLPA
MLPA was done with the MLPA P002-BRCA1 and P0045-BRCA2 test kits and the MLPA P087 confirmation kit (MRC Holland) according to the manufacturer's recommendations, using alternative protocol 2 for multiplex PCR. Fragment analysis was done on the Beckman CEQ 8000 System (Beckman Coulter). Peak profiles for each sample were compared with a normal control and with other samples within the same experimental batch.

RNA Analysis of Splice-Site Alterations
Total RNA was isolated from peripheral blood lymphocytes using Trizol reagent (Invitrogen). cDNA was then reverse transcribed from 150 ng of total RNA using the AMV reverse transcription system (Promega). cDNA (25 ng) was amplified using the following primers by standard procedures with HotStarTaq (Qiagen Gmbh). For amplification of exons 5 to 9 of BRCA1, the primers 5'-CTGAAACTTCTCAACCAGAAGAAA-3' (E6-8F) and 5'-GTAACAATTCTTGATCTCCCACAC-3' (E6-8R) were used. For amplification of exons 14 to 16 of BRCA2, the primers 5'-TCATGTTTCTTTAGAGCCGATTAC-3' (2E14-16F) and 5'-ATTTTAGTTGAAGAAGCACCCTTT-3' (2E14-16R) were used.

Amplification consisted of 35 cycles, each of 1 min, at 94°C, 56°C, and 72°C. PCR products were electrophoresed on a 3% NuSieve GTG agarose gel (Cambrex; for exons 5-9 of BRCA1) or 2% agarose gel (for exons 14-16 of BRCA2), and PCR fragments were purified by gel extraction with the PureLink Quick Gel extraction kit (Invitrogen). Direct sequencing was done as described above.

In silico Splice Site Analysis
SpliceSiteFinder7 was used to determine the presence and relative efficiencies of donor, acceptor, and branch point sites.

Computational Analyses of Missense Mutations
BRCA1 and BRCA2 sequences from various organisms that contain the region that encompasses the site of missense mutation were obtained from Swiss-Prot. Multiple sequence alignment was then conducted using ClustalX 1.83. The Swiss PDB-Viewer was used for rendering and viewing of protein database structures of mutation sites that were available for analysis. When a mutation occurred at the vicinity of putative phosphorylation sites as reported in the literature, NetPhos 2.0 was used to predict possible sites of phosphorylation (19). Comparison of relevant protein structures deposited at the Protein Data Bank8 was done. These Protein Data Bank structures are protein structures which are derived by X-ray crystallography or by nuclear magnetic resonance. Deductions from these analyses were compared with the predictions of the effects of the mutation made by PolyPhen (20). PolyPhen (polymorphism phenotyping) combines information on sequence features, structural variables, and contacts to characterize nucleotide substitutions (20).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The clinicopathologic features of the breast and ovarian tumors are summarized in Table 1 . As expected, BRCA1 or BRCA2 mutations were more likely to be present in patients from hereditary breast and ovarian cancer (HBOC) families than in those with no family history. All of the patients with BRCA1 or BRCA2 mutations were Her2 negative. All but one BRCA1 mutation carrier had estrogen, progesterone, and Her2-negative breast cancer.


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  		Table 1. Clinicopathologic features of cases with and without BRCA1 or BRCA2 mutation

 
Prevalence and Mutational Spectrum of BRCA1 and BRCA2 Mutations
Deleterious mutations are indicated in bold in Table 2 . All frameshift and nonsense mutations generating premature termination codons were classified as deleterious. Missense mutations that were absent in at least 50 normal individuals and which involved a highly conserved amino acid were classified as deleterious. An exon 13 duplication in BRCA1 (21) and the IVS7-15del10 in BRCA1 were deemed deleterious, as there was linkage with breast and/or ovarian cancer within the respective families.


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  		Table 2. Mutations in BRCA1 and BRCA2

 
Fifteen novel genetic alterations were detected in the BRCA1 and BRCA2 genes, with 11 classified as deleterious mutations and four as polymorphisms (Tables 2 and 3 ).


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  		Table 3. Polymorphisms in BRCA1 and BRCA2

 
Frameshift, nonsense, and intronic mutations and an exon duplication were identified in 16 of 95 subjects (16.8%) or in 14 of 90 families (15.6%). The 90 families composed of 26 HBOC families, 34 breast cancer only families, one ovarian cancer only family, and 29 unrelated subjects with early-onset breast cancer and no family history (Table 1). The prevalence of clearly deleterious BRCA mutations in these groups were 35% (9 of 26) in HBOC families, 8.8% (3 of 34) in breast cancer only families, and 6.9% (2 of 29) in subjects with early-onset breast cancer and no family history (Table 2). The prevalence rates for clearly deleterious BRCA1 and BRCA2 mutations were 6.7% (6 of 90) and 8.9% (8 of 90), respectively, or 7.8% (7 of 90) and 11.1% (10 of 90), respectively, by including probably deleterious missense mutations. Deleterious mutations in BRCA1 and BRCA2 were observed in 7 and 10 families, respectively.

By ethnicity, these mutations were detected in 12 of 67 Chinese families (17.9%), 1 of 11 Malay families (9.1%), and 1 of 4 Indian families (25.0%; Table 2). The mutation in the Indian family was the 185delAG mutation frequently found in Ashkenazi Jews and in studies from India (22-28). The Malay founder mutation previously identified in Singapore was not identified in this cohort (16, 29).

Six different BRCA1 deleterious mutations were identified in six families (Table 2). Five of these have been previously reported either in the Breast Cancer Information Core or in the literature and comprised three frameshift mutations, one nonsense mutation, and an intronic deletion within intron 7, causing a frameshift with a predicted stop at codon 182 (Fig. 2 ; ref. 30). A novel genomic rearrangement resulting in the duplication of exon 13 of BRCA1 was identified by MLPA and confirmed by DNA sequencing of the breakpoint (21).


Figure 2
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  		Figure 2. IVS7-15del10 in BRCA1. Ethidium bromide–stained gel of reverse transcription–PCR products with the corresponding sequencing chromatogram of the aberrant transcript, and the gene, mRNA, and amino acid sequences for the aberrant transcript. The branch and acceptor sites (a and ag) are indicated by asterisks. Lanes 5 and 5a of the gel correspond to cases 5 and 5a listed in Table 2.

 
Of the eight deleterious mutations in BRCA2, six were novel (Table 2). There were seven frameshift mutations and a GT to AT 5' splice site mutation in intron 15, resulting in skipping of exon 15 detected in one subject (Fig. 2; ref. 31).

Eight missense mutations of unknown significance were detected in both BRCA1 and BRCA2, and these were evaluated by computational analyses to determine if they were deleterious (Tables 2 and 4 ).


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  		Table 4. Alignment of BRCA1 and BRCA2 amino acids across species at sites of missense mutations, percentages of conservation, PolyPhen, and overall predictions and pedigree diagnosis

 
None of the mutations listed in Table 2 were detected in 50 normal individuals, matched for ethnicity. Several polymorphisms were detected and are listed in Table 3.

Evaluation of Splice Site Alterations
All cases with BRCA1 and BRCA2 intronic splice site alterations were subjected to RNA analysis. Of the 15 intronic alterations detected, two variants, IVS7-15del10 in BRCA1 and IVS15+1G>A in BRCA2, showed aberrant splicing and were classified as deleterious (Table 2; Fig. 2). The remaining 13 variants with normal transcripts were classified as benign polymorphic alterations (Table 3).

IVS7-15del10 in BRCA1, 10-bp deletion in intron 7 of BRCA1, was identified in two subjects who were sisters (Table 2; Fig. 2). Reverse transcription–PCR of the region spanning exons 5 to 9 amplified two fragments, a fragment of the expected size of 472 bp and another fragment of 531 bp of reduced intensity possibly due to nonsense-mediated mRNA decay, which occurs if an alternative transcript has a premature termination codon >55 nucleotides upstream of the last exon-exon junction (32). Sequencing of both fragments revealed that the aberrant fragment had a 59-bp insertion between exons 7 and 8 in the mRNA sequence. Based on this, a possible mechanism for the partial inclusion of intron 7 is shown in Fig. 2. The deletion of the original branch site within the 10-bp intronic deletion causes the utilization of a new branch site upstream with the insertion of the 59-bp intronic sequence, causing a frameshift and a predicted stop at codon 182. SpliceSiteFinder predicted that the strength of the new 3' splice acceptor site (score, 64.4) and new branch point (maximum score, 100) are at least comparable with or higher than the original scores of 67.8 and 83.9, respectively. Both the putative normal and new branch points are based on the consensus sequence YTRAY (33).

The BRCA2 IVS15+1G>A mutation is located in the consensus sequence of the 5' donor splice site and resulted in the generation of an altered transcript by skipping of exon 15.

Evaluation of Missense Mutations of Unknown Clinical Significance by Computational Analyses
Table 4 shows the amino acid sequence comparison for sites of missense mutations used to establish the degree of conservation and the results of computational analyses for assessing the significance of the missense mutations. Of the eight missense mutations, three are predicted to be damaging mutations (BRCA1 P58A, BRCA2 C161W, and BRCA2 G2544D), two are predicted to be benign (BRCA1 M1652I and BRCA2 A2351G), and the remaining three are of unclear status (BRCA1 V191I, BRCA1 K1690Q, and BRCA2 K2729N; Table 4).

Codon 58 of BRCA1 lies within the RING domain, which is a highly conserved region among the nine mammalian sequences examined. The domain is implicated in interactions with at least five different proteins (34, 35).

Codon 161 of BRCA2 has polar residues conserved in all species represented at that site. A mutation to the large hydrophobic Tryptophan residue is likely to be damaging, as it may remove possible covalent or polar bonding.

The G2544D mutation involves a conserved glycine that lies in a highly conserved region, which plays a structural role in forming a loop turn and is likely to affect the binding affinity of BRCA2 with DSS1. The residues involved in the BRCA2-DSS1 interaction are highly conserved, and two DSS1-interacting BRCA2 residues (Ala2564 and Arg2580) were previously found to be mutated in cancer (36). This BRCA2-DSS1 complex directly functions in RAD51-mediated recombination double-stranded break repair (36). However, PolyPhen, which did not examine the Protein Data Bank structure 1mje, classified this mutation as benign.

Codon 1652 of BRCA1 lies in the hydrophobic core of the protein, and its hydrophobicity is shown to be conserved in the alignment (Table 4). Because the mutation to isoleucine is a conservative mutation, it is most probably a benign mutation. PolyPhen, however, has not evaluated the similarity between the leucine found in rhesus monkey in the alignment and isoleucine, and predicted the mutation to be probably damaging (Table 4).

The A2351G mutation of BRCA2 is a conservative mutation in a nonconserved region and hence is likely to be a benign mutation.

Codon 191 of BRCA1 is within a putative site of interaction with oncogene and cell cycle regulators, such as c-myc and estrogen receptors (37). Although V191I is conserved in hydrophobicity and PolyPhen predicted this mutation to be benign, it is still possible that the V191I mutation may affect the phosphorylation state of BRCA1 because NetPhos 2.0 predicted its neighbor T190 to be a likely site of phosphorylation.

The K1690Q mutation in BRCA1 is predicted to be benign by PolyPhen and unclear by our analysis (Table 4). The Protein Data Bank structure 1jnx of BRCA1's BRCT domain showed a salt-bridge interaction between the lysine residue and the glutamic acid residue at codon 1661 (38). A mutation from lysine to glutamine will replace the salt-bridge interaction with a hydrogen bond between the mutated glutamine residue at codon 1690 with the glutamic acid residue at codon 1661. However, the effect of this mutation on the structure and dynamics of the protein is unclear.

The K2729N mutation of BRCA2 is a relatively conservative mutation in a conserved region. Hence, the effect of the mutation on the structure and function of the protein remains unclear.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first comprehensive study of both the BRCA1 and BRCA2 genes, involving complete mutation screening, evaluation for large genomic rearrangements and splice site variants, and analysis of missense mutations by computational analysis, in an Asian population. In our cohort of 90 families, the combined frequency of frameshift/nonsense mutations in the BRCA1 and BRCA2 genes was 12.2% by conventional mutation screening. However, by including deleterious mutations detected using MLPA analysis (n = 1), RNA analysis (n = 2), and computational evaluation of missense mutations (n = 3), the overall frequency of deleterious mutations increased substantially to 18.9%. The comprehensive mutation testing strategy used was most successful in families with both breast and ovarian cancer.

The prevalence rates for BRCA1 and BRCA2 mutations that were observed in this study were 6.7% (6 of 90) and 8.9% (8 of 90), respectively, or 7.8% (7 of 90) and 11.1% (10 of 90), respectively, by including missense mutations predicted to be deleterious by computational analysis. These frequencies are comparable with data from other studies on Chinese women, the majority of which have been small studies with fewer than 50 subjects. In women with early-onset breast cancer, prevalence rates ranging from 8.0% to 9.5% in BRCA1 (39-42) and 2.4% in BRCA2 (40) have been observed, although one study reported no mutations in BRCA1 among 35 Singaporean-Chinese women (43). It should be noted, however, that the age limits set for each of these studies differ and range from <35 to <45 years, which may account for differences in prevalence rates between studies. In addition, a cancer genetics clinic with risk assessment allows risk stratification for genetic testing of a higher risk cohort.

In Chinese cases with a family history of breast or ovarian cancer, mutations in BRCA1 were detected between 8.1% and 12.5% (30, 40, 44, 45) and between 2.7% and 16.7% for BRCA2 (30, 40, 44, 45). In one study on 25 HBOC families, 40% of the women had mutations in BRCA1 (46).

The majority of the BRCA2 frameshift mutations detected were novel mutations. This may be because there is a paucity of information on BRCA2 mutations among the Chinese, Malay, and Indian ethnic groups. Furthermore, we found that in our cohort of Singaporean women, deleterious mutations in BRCA2 (10 families) were more common than for BRCA1 (seven families). This contrasts with observations from European and American populations, wherein the frequency of BRCA1 mutations is higher than that of BRCA2 (1, 3). In a large U.S. study of 1948 families, BRCA1 and BRCA2 mutations were detected in 14.6% and 7.4% of the families, respectively (3). However, in Chinese women from Shanghai, a 1:1 ratio for BRCA1/BRCA2 mutations was observed (44). Thus, our findings underscore the importance of screening for BRCA2 mutations in individuals at high risk of developing breast and/or ovarian cancer.

Although there have been hospital-based genetic testing studies for BRCA1/BRCA2, there have been no Asian risk assessment clinic-based reports with predominantly Asian individuals seeking counseling and genetic testing (22, 47, 48). The advantages of clinic-based ascertainment is, firstly, the use of risk stratification to allow for genetic testing of a higher risk cohort and, secondly, the exclusion of breast cancer families related to other predisposition genes. We excluded one family with Cowden's syndrome and another with Li-Fraumeni syndrome (data not shown). Additionally, we screened for the CHEK2*1100delC mutation, which also predisposes to early-onset breast cancer, and all our subjects were negative (unpublished data).

The technology to sequence BRCA1 and BRCA2 is increasingly available in Asian countries, and although genetic testing is often done as part of research studies, the set up of formal assessment clinics could increase the scope of hereditary cancers detected.

To ascertain the pathologic effect of the intronic alterations on mRNA splicing fidelity and expression, RNA analysis using reverse transcription–PCR and sequencing and in silico splice prediction methods were used (9, 10). This current study identified two deleterious intronic aberrations, IVS7-15del10 in BRCA1 and IVS15+1G>A in BRCA2. The occurrence of these splicing aberrations emphasizes the importance of studying mutations both at the genomic DNA and RNA levels to determine the pathogenic effect of the mutations (49) which would effect on genetic counseling. However, if RNA is not available, then another option would be to use theoretical splicing prediction approaches, such as SpliceSiteFinder, to determine if the BRCA1 and BRCA2 splice site variants identified from genomic sequence would cause aberrant splicing (10).

Missense mutations of unknown clinical significance or unclassified variants pose a challenge in genetic counseling. By using bioinformatic tools to assess such mutations, deleterious mutations may be identified in high-risk individuals, providing information for risk reduction decision making. Such computational tools provide a feasible alternative to the evaluation of missense mutations by segregation analysis, which is particularly difficult in small families or in families for which pedigree information is unknown or limited.

This study has shown that intronic alterations, large genomic rearrangements, and missense mutations in combination contribute significantly to deleterious mutations in our study population. Improved mutation detection by a comprehensive screening approach for the BRCA1 and BRCA2 genes, as shown here, may be effective for the identification of deleterious mutations, with implications for genetic counseling and risk assessment of individuals at high-risk for breast and/or ovarian cancer.


   Acknowledgments
 
We thank Geraldine Low Hui Min and Dr. Karen P.L. Yap for excellent technical assistance, the referring doctors, patients, and their families who have participated in this study, and WHO Immunology Center, Singapore for providing control samples for this study.


   Footnotes
 
Grant support: SingHealth Cluster Research grant EX020/2001 (P. Ang), National Medical Research Council of Singapore grants NMRC/0711/2002 (P. Ang) and NMRC/0763/2003 (A. Lee), and from the National Cancer Centre, Singapore Cancer Research and Education fund (A. Lee).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

6 http://research.nhgri.nih.gov/bic/ Back

7 http://www.genet.sickkids.on.ca/~ali/splicesitefinder.html Back

8 http://www.rcsb.org/pdb Back

Received 5/ 3/07; revised 8/ 7/07; accepted 8/16/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Szabo CI, King MC. Population genetics of BRCA1 and BRCA2. Am J Hum Genet 1997;60:1013–20.[Medline]
  2. Neuhausen SL. Ethnic differences in cancer risk resulting from genetic variation. Cancer 1999;86:2575–82.[CrossRef][Medline]
  3. Chen S, Iversen ES, Friebel T, et al. Characterization of BRCA1 and BRCA2 mutations in a large United States sample. J Clin Oncol 2006;24:863–71.[Abstract/Free Full Text]
  4. Ang P, Garber JE. Genetic susceptibility for breast cancer-risk assessment and counseling. Semin Oncol 2001;28:419–33.[Medline]
  5. Petrij-Bosch A, Peelen T, van Vliet M, et al. BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients. Nat Genet 1997;17:341–5.[CrossRef][Medline]
  6. Hogervorst FB, Nederlof PM, Gille JJ, et al. Large genomic deletions and duplications in the BRCA1 gene identified by a novel quantitative method. Cancer Res 2003;63:1449–53.[Abstract/Free Full Text]
  7. Mazoyer S. Genomic rearrangements in the BRCA1 and BRCA2 genes. Hum Mutat 2005;25:415–22.[CrossRef][Medline]
  8. Walsh T, Casadei S, Coats KH, et al. Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer. JAMA 2006;295:1379–88.[Abstract/Free Full Text]
  9. Chen X, Truong TT, Weaver J, et al. Intronic alterations in BRCA1 and BRCA2: effect on mRNA splicing fidelity and expression. Hum Mutat 2006;27:427–35.[CrossRef][Medline]
  10. Tesoriero AA, Wong EM, Jenkins MA, et al. Molecular characterization and cancer risk associated with BRCA1 and BRCA2 splice site variants identified in multiple-case breast cancer families. Hum Mutat 2005;26:495.[Medline]
  11. Campos B, Diez O, Domenech M, et al. RNA analysis of eight BRCA1 and BRCA2 unclassified variants identified in breast/ovarian cancer families from Spain. Hum Mutat 2003;22:337.[Medline]
  12. Billack B, Monteiro AN. Methods to classify BRCA1 variants of uncertain clinical significance: the more the merrier. Cancer Biol Ther 2004;3:458–9.[Medline]
  13. Goldgar DE, Easton DF, Deffenbaugh AM, Monteiro AN, Tavtigian SV, Couch FJ. Integrated evaluation of DNA sequence variants of unknown clinical significance: application to BRCA1 and BRCA2. Am J Hum Genet 2004;75:535–44.[CrossRef][Medline]
  14. Fleming MA, Potter JD, Ramirez CJ, Ostrander GK, Ostrander EA. Understanding missense mutations in the BRCA1 gene: an evolutionary approach. Proc Natl Acad Sci U S A 2003;100:1151–6.[Abstract/Free Full Text]
  15. Parmigiani G, Berry D, Aguilar O. Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2. Am J Hum Genet 1998;62:145–58.[CrossRef][Medline]
  16. Lee AS, Ho GH, Oh PC, et al. Founder mutation in the BRCA1 gene in Malay breast cancer patients from Singapore. Hum Mutat 2003;22:178.[Medline]
  17. Friedman LS, Ostermeyer EA, Szabo CI, et al. Confirmation of BRCA1 by analysis of germ line mutations linked to breast and ovarian cancer in ten families. Nat Genet 1994;8:399–404.[CrossRef][Medline]
  18. Ottini L, D'Amico C, Noviello C, et al. BRCA1 and BRCA2 mutations in central and southern Italian patients. Breast Cancer Res 2000;2:307–10.[CrossRef][Medline]
  19. Blom N, Gammeltoft S, Brunak S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 1999;294:1351–62.[CrossRef][Medline]
  20. Ramensky V, Bork P, Sunyaev S. Human non-synonymous SNPs: server and survey. Nucleic Acids Res 2002;30:3894–900.[Abstract/Free Full Text]
  21. Yap KP, Ang P, Lim IH, Ho GH, Lee AS. Detection of a novel Alu-mediated BRCA1 exon 13 duplication in Chinese breast cancer patients and implications for genetic testing. Clin Genet 2006;70:80–2.[CrossRef][Medline]
  22. Liede A, Narod SA. Hereditary breast and ovarian cancer in Asia: genetic epidemiology of BRCA1 and BRCA2. Hum Mutat 2002;20:413–24.[CrossRef][Medline]
  23. Hedau S, Jain N, Husain SA, et al. Novel germ line mutations in breast cancer susceptibility genes BRCA1, BRCA2 and p53 gene in breast cancer patients from India. Breast Cancer Res Treat 2004;88:177–86.[CrossRef][Medline]
  24. Kumar BV, Lakhotia S, Ankathil R, et al. Germline BRCA1 mutation analysis in Indian breast/ovarian cancer families. Cancer Biol Ther 2002;1:18–21.[Medline]
  25. Saxena S, Szabo CI, Chopin S, et al. BRCA1 and BRCA2 in Indian breast cancer patients. Hum Mutat 2002;20:473–4.[Medline]
  26. Saxena S, Chakraborty A, Kaushal M, et al. Contribution of germ line BRCA1 and BRCA2 sequence alterations to breast cancer in Northern India. BMC Med Genet 2006;7:75.[Medline]
  27. Valarmathi MTAA, Deo SS, Shukla NK, Das SN. BRCA1 germ line mutations in Indian familial breast cancer. Hum Mutat 2003;21:98–9.[Medline]
  28. Valarmathi MT, Sawhney M, Deo SS, Shukla NK, Das SN. Novel germ line mutations in the BRCA1 and BRCA2 genes in Indian breast and breast-ovarian cancer families. Hum Mutat 2004;23:205.[Medline]
  29. Sng JH, Ali AB, Lee SC, et al. BRCA1 c.2845insA is a recurring mutation with a founder effect in Singapore Malay women with early onset breast/ovarian cancer. J Med Genet 2003;40:e117.[Free Full Text]
  30. Li SS, Tseng HM, Yang TP, et al. Molecular characterization of germ line mutations in the BRCA1 and BRCA2 genes from breast cancer families in Taiwan. Hum Genet 1999;104:201–4.[CrossRef][Medline]
  31. Bergthorsson JT, Ejlertsen B, Olsen JH, et al. BRCA1 and BRCA2 mutation status and cancer family history of Danish women affected with multifocal or bilateral breast cancer at a young age. J Med Genet 2001;38:361–8.[Abstract/Free Full Text]
  32. Nagy E, Maquat LE. A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance. Trends Biochem Sci 1998;23:198–9.[CrossRef][Medline]
  33. Maquat LE. Defects in RNA splicing and the consequence of shortened translational reading frames. Am J Hum Genet 1996;59:279–86.[Medline]
  34. Johnson NC, Kruk PA. BRCA1 Zinc RING Finger Domain Disruption Alters Caspase Response in Ovarian Surface Epithelial Cells. Cancer Cell Int 2002;2:7.[CrossRef][Medline]
  35. Narod SA, Foulkes WD. BRCA1 and BRCA2:1994 and beyond. Nat Rev Cancer 2004;4:665–76.[CrossRef][Medline]
  36. Yang H, Jeffrey PD, Miller J, et al. BRCA2 function in DNA binding and recombination from a BRCA2-1-ssDNA structure. Science 2002;297:1837–48.[Abstract/Free Full Text]
  37. Wang Q, Zhang H, Kajino K, Greene MI. BRCA1 binds c-Myc and inhibits its transcriptional and transforming activity in cells. Oncogene 1998;17:1939–48.[CrossRef][Medline]
  38. Williams RS, Green R, Glover JN. Crystal structure of the BRCT repeat region from the breast cancer-associated protein BRCA1. Nat Struct Biol 2001;8:838–42.[CrossRef][Medline]
  39. Tang NL, Pang CP, Yeo W, et al. Prevalence of mutations in the BRCA1 gene among Chinese patients with breast cancer. J Natl Cancer Inst 1999;91:882–5.[Free Full Text]
  40. Song CG, Hu Z, Wu J, et al. The prevalence of BRCA1 and BRCA2 mutations in eastern Chinese women with breast cancer. J Cancer Res Clin Oncol 2006;132:617–26.[Medline]
  41. Hu Z, Wu J, Liu CH, et al. The analysis of BRCA1 mutations in eastern Chinese patients with early onset breast cancer and affected relatives. Hum Mutat 2003;22:104.[Medline]
  42. Sng JH, Chang J, Feroze F, et al. The prevalence of BRCA1 mutations in Chinese patients with early onset breast cancer and affected relatives. Br J Cancer 2000;82:538–42.[CrossRef][Medline]
  43. Ho GH, Phang BH, Ng IS, Law HY, Soo KC, Ng EH. Novel germ line BRCA1 mutations detected in women in singapore who developed breast carcinoma before the age of 36 years. Cancer 2000;89:811–6.[CrossRef][Medline]
  44. Suter NM, Ray RM, Hu YW, et al. BRCA1 and BRCA2 mutations in women from Shanghai China. Cancer Epidemiol Biomarkers Prev 2004;13:181–9.[Abstract/Free Full Text]
  45. Zhi X, Szabo C, Chopin S, et al. BRCA1 and BRCA2 sequence variants in Chinese breast cancer families. Hum Mutat 2002;20:474.[Medline]
  46. Li N, Zhang X, Cai Y, et al. BRCA1 germ line mutations in Chinese patients with hereditary breast and ovarian cancer. Int J Gynecol Cancer 2006;16 Suppl 1:172–8.[Medline]
  47. Takeda M, Ishida T, Ohnuki K, et al. Collaboration of breast cancer clinic and genetic counseling division for BRCA1 and BRCA2 mutation family in Japan. Breast Cancer 2004;11:30–2.[Medline]
  48. Choi DH, Lee MH, Bale AE, Carter D, Haffty BG. Incidence of BRCA1 and BRCA2 mutations in young Korean breast cancer patients. J Clin Oncol 2004;22:1638–45.[Abstract/Free Full Text]
  49. Claes K, Poppe B, Machackova E, et al. Differentiating pathogenic mutations from polymorphic alterations in the splice sites of BRCA1 and BRCA2. Genes Chromosomes Cancer 2003;37:314–20.[CrossRef][Medline]



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