Results so far obtained indicate that there are no significant differences in BRCA1 allele (haplotype) frequencies among the three populations (FIG. 3). This also ap- plied to the APOE4 variant that was included as a positive control. However, in keep- ing with earlier studies the frequency of the APOE4 variant was lower in the centenarians as compared to controls (FIG. 3). Comparison to previously published results obtained from 795 breast- and/or ovarian cancer probands sampled from the US and Europe40 confirmed that at the allele frequency level only minor differences are observed.

Although allele frequencies might give an early indication of possible of the population-specific effect of the different haplotypes and disease, actual ciation should be carried out using the frequencies of allele combinations (i.e., + otype frequencies). As shown in FIGURE 4, the BRCA1 genotype a a showed to more frequent in both centenarian groups compared to the control = + (p 0.1473), whereas genotype “a b” was less frequent (p = 0.0526). In this + the A4 A4 APOE genotype was not found for any of the 99 New England ians and scored 2 times for the 27 control individuals in which this was (FIG. 4).

The high number of genotypes and the low frequency of some of the alleles re- quire the analysis of large numbers of samples. Only when the sample repository is sufficiently high, reliable conclusions can be drawn regarding the frequencies of all genotypes including some of the rare ones. In this respect, especially haplotype “f” is of interest because of the presence of Arg356 instead of Gln356. There is evidence

Frequency (%)201510                    ~OP 7= = Hi Centenarians NE (n=102)AS entenarians It (n=84)L x BG] Controls It (n=97)s v y —___=~] ControlsSchacher eval, 1994) (=161)4> >LL ==— T Tata atb APOE4/E4Genotype 

FIGURE 4. Genotype frequency of two of the most frequent BRCA1 allele combina- tions in the two centenarian populations and the control population. The APOE4/E4 frequen- cy has been included as control and compared with Schächter et al.22 as in FIGURE 3.

that Arg356 homozygosity is less frequent in breast cancer patients than in controls44 indicating that it may be protective against breast cancer. Although in our present study the frequency of allele f with the Arg356 present was found to be slightly higher in centenarians, the number of individuals analyzed is still too low to draw any definite conclusions.

SUMMARY

One way to test possible causal relationships between various functional path- ways and the aging process is to comparatively analyze polymorphic variation in genes participating in these pathways in elderly populations with different aging- related characteristics. The results of the Human Genome Project now make it fea- sible to identify most if not all genes involved in the candidate pathways that drive the aging process or confer longevity. For this purpose, efficient genotyping methods are needed that allow the comprehensive sequence analysis of multiple genes at the population level. Using our recently developed two-dimensional gene scanning (TDGS) technology platform we demonstrated the feasibility of this approach by screening two different populations of centenarians for polymorphic variation in the BRCA1 breast cancer susceptibility gene. The results indicate that using this tech- nology relatively large numbers of individuals, i.e., hundreds, can be screened in a relatively short time for all possible sequence variants of this very large gene. Al- though the number of individuals analyzed is still insufficient to draw definite con- clusions, a clear trend towards a difference in genotype frequency involving two of the most common alleles became apparent. Based on these results we conclude that rather than studying individual polymorphisms in groups of elderly with different

VIJG et al.: BRCA1 GENE SEQUENCE VARIATION IN CENTENARIANS

aging-related characteristics, it should be feasible to analyze multiple genes partici- pating in functional pathways that are possibly involved in aging and longevity for differences in the frequency of potentially functional alleles.

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