Comparing the frequency of common genetic variants and haplotypes between carriers and non-carriers of BRCA1 and BRCA2deleterious mutations in Australian women diagnosed with breast cancer before 40 years of age

Mutations in BRCA1 and BRCA2 are found in a proportion of multiple case breast cancer families. The particular mutations that are present differ from family to family indicating the marked allelic heterogeneity of these genes. The only viable methods for the identification of mutations in genes prone to such variation are sequencing or extensive DNA screening techniques. Mutation screening of these genes has become widespread despite the costs involved. In fact, 890 and 975 non-protein truncating mutations have been identified in BRCA1 and BRCA2 respectively, making it difficult to identify causal mutations due to the large number of variants. Many such variants may appear deleterious but may nevertheless only be associated with disease because they are close to a causal mutation and not deleterious in their own right. An appealing strategy is therefore to avoid the large number of comparisons required to test each variant separately, but to instead use methods based on haplotypes, which are combinations of genetic variants or alleles (typically common polymorphisms) inherited together or in phase from a single parent. They result from the phenomenon of linkage disequilibrium (LD), where alleles at closely spaced markers do not segregate independently. Haplotypes can capture more information across genomic regions of interest on the human genome than is available by examining single genetic markers one at a time [1] however the generation of haplotypes is difficult, usually requiring intensive laboratory efforts or the collection and genotyping of several closely related relatives to infer phase. An alternative is to infer phase from genotype data using the statistical techniques that have been developed rapidly in the last few years, which is the approach adopted in this report.

Haplotypes are expected to play an important role in the fine mapping of complex diseases since disease-affected individuals with common haplotypes may have recent shared ancestry of chromosomal segments that harbour disease-causing variants [2]. By identifying "disease haplotypes" that are the hallmarks of deleterious BRCA1 and BRCA2 mutations, it may be possible to identify carriers implicitly rather than by screening of the entire gene. Some early work in this area has suggested that one haplotype in BRCA1 is over-represented in individuals carrying deleterious mutations [3] while another haplotype was associated with a 20% increased risk in breast cancer [4]. There is evidence that this scenario is particularly useful for common polymorphisms of low penetrance, where results show that an association can be detected via haplotype methods using single nucleotide polymorphisms surrounding the functional allele even if the functional allele is not typed [5]. We explored extending this approach to detecting rare deleterious mutations, which are likely to have arisen more recently and occur on extended, less common haplotypes.

Data was available on 680 participants in regard to BRCA1 and 245 participants for BRCA2 (table 1).

Analyses was restricted to the 392 (BRCA1) and 179 (BRCA2) population-based individuals diagnosed with a first primary invasive breast cancer before 40 years of age for whom BRCA1 and BRCA2 had been sequenced. Some sequencing was performed for cases in other age groups but the sample sizes were small and not sufficient to warrant separate analysis. The total number of variants found in the coding regions of BRCA1 and BRCA2 gene in cases under 40 were 22 (table 2) and 15 (table 3) respectively. Each of the deleterious mutations identified in our sample appeared only once, with the exception of 2800 del AAG which occurred once in each of two study participants.

Tables 4 and 5 list the details of the variants selected as tagSNPs by Haploview's Tagger program for BRCA1 and BRCA2 respectively. These tables display results of comparing the allele frequencies of these tagSNPs between individuals carrying and not carrying deleterious mutations. In both BRCA1 and BRCA2 there was some evidence that the tagSNPs allele frequency differed according to deleterious mutation status. Genotype frequencies for some variants generate small p-value for the test of HWE, most likely due to the fact that our analysis is restricted to breast cancer cases under age 40 years. The BRCA1 variant 3232A > G was found to occur more frequently in deleterious mutation carriers (p = 0.047) while BRCA2 variant 1342A > C was found to occur less frequently in deleterious mutation carriers (p = 0.043).

All four common BRCA1 variants used to form haplotypes occur more frequently in the deleterious mutation carriers when compared to the non-carrier group, but there was no evidence of a difference in the distribution between the two groups (p = 0.34). The opposite was true for BRCA2, where all four common variants were found to occur less frequently in the deleterious mutation carriers group when compared to non-carriers, but the evidence for difference in the distribution between the two groups was weak (p = 0.16).

When comparing haplotype frequencies between the two groups (tables 6 and 7), there was very weak evidence that haplotype AGGT in BRCA1 was over-represented in individuals carrying deleterious mutations (p = 0.151). Overall, there was no evidence of difference between haplotype distributions between deleterious mutation carriers and non-carriers in BRCA1 (with 6.d.f., p = 0.717).The most common haplotype in BRCA2, AATA, was also found to occur slightly more frequently in deleterious mutation carriers (p = 0.158). Again, there was no evidence of difference in BRCA2 haplotype distributions between the two groups (with 6.d.f., p = 0.851).

In this paper we used BRCA1 and BRCA2 sequence data from Australian breast cancer cases less than 40 years of age at the time of diagnosis to classify individuals according to their deleterious mutation status, and resolved haplotypes of common polymorphisms separately in the groups that did and did not carry deleterious mutations.

The sample size of the deleterious mutation group was small for both genes, with only 13 and 11 individuals carrying deleterious mutations for BRCA1 and BRCA2 respectively. The power to detect differences in haplotype frequencies is therefore quite low, which might explain some of the high p-values obtained from permutation testing.

Our selection of tagSNPs for BRCA1 gene has one in common with tagSNPs selection of Osorio et al. [3] (where they have used 4427T- > C as a tagSNP) and Cox et al. [4] (Q356R as a tagSNP). In Osorio et al. their class II haplotype occurs more frequently among BRCA1 mutation carriers. This haplotype is essentially characterized by the 4427C- > T variant allele which was used as a tagSNP in our study. We found that minor allele occurred more frequently in deleterious mutation carriers compared to non-carriers (32% vs 23%) but the evidence for this at the population level was weak p = 0.25. Cox et al. found slight increase in risk of breast cancer with the Q356R polymorphism, contradicting an earlier result showing an inverse association [15]. We found that Q356R occurred more frequently among deleterious mutation carriers but again the evidence at the population level was weak.

There have been several case control studies seeking BRCA1 and BRCA2 variants associated with an increased risk of breast cancer. Freedman at al. [16] investigated if common BRCA2 variants contribute to the more common forms of breast cancer in a large multiethnic cohort. Twenty one tagging SNPs were selected to predict common BRCA2 haplotypes. A number of haplotypes were found to be associated with increased risk of breast cancer, all of which could be attributed to a single marker (intron 24: rs206340) that was not selected as a tag SNP for analysis in our study. Freedman at al. [17] repeated similar analysis on BRCA1 gene. Specifically, they have used 28 variants to define patterns of common variation (5 in common with variants used in our study: Q356R, P871L, K1183R, S1613G and E1038G). They found no evidence for significant association between common variation in BRCA1 and risk of breast cancer.

The suggestive associations that we have observed do not imply a physical association on the same chromosome (as would be the case if the rare, deleterious mutation was in cis phase with a haplotype consisting of, for example, the minor alleles of several common variants) or a functional association (as might be the case even if the rare, deleterious mutation was in trans phase with a common variant haplotype, since it may still act to modify the penetrance of the disease causing variant). Establishing the phase of rare, deleterious mutations and the common variants we used to define haplotypes for both BRCA1 and BRCA2 would require either a much larger sample size than was available for this study, genetic data from extended pedigrees or expensive laboratory investigation.

We found some evidence that a single common BRCA1 variant occurs more frequently in deleterious mutation carriers. All four common variants used to form BRCA1 haplotypes are over represented in deleterious mutation carriers so the frequency of less common haplotypes is also greater in this group but there is no evidence for this at the population level. In BRCA2, there is some evidence that a single common variant is under-represented in deleterious mutation carriers so the most common BRCA2 haplotype occurs more frequently in this group. All four common variants used to form BRCA2 haplotypes occur less frequently in deleterious mutation carriers. We found no evidence of difference in haplotype distributions between the two groups in both genes, which concords with previous research. These findings are unlikely to have implications for screening at the population level for BRCA1 or BRCA2.