To prevent complete melting of a DNA fragment consisting of one melting domain upon denaturation, GC-clamps (a stretch of 30 to 50 G and C bases) are introduced to the target DNA fragments.32 A convenient way of attaching a GC-clamp to the target fragment is by making it part of one of the primers.33 Without GC-clamping, a DNA fragment consisting of one melting domain will become completely single-stranded upon denaturation and run off the gel. By adding a GC-clamp, a single high melting domain is artificially created at one end of the target fragment. This will prevent complete melting and ensure that the fragment halts at the melting temperature of the target sequence. However, when the target DNA fragment consists of multiple melting domains, only mutations in the lowest melting domain are readily detected. To facilitate detection of all possible mutations, it is imperative that the target fragment represents only one melting domain. Fortunately, since the addition of a GC-clamp allows for stacking interactions with neighboring bases, the entire target fragment will often behave as one melting domain. However, this is not always the case, and in practice, the target fragment needs to be designed through the strategic positioning ofMore valuable than the study of single polymorphisms in candidate genes, as has been done so far. So far, a total of 7 polymorphic sites in 6 TDGS fragments in BRCA1 have been identified that give rise to an amino acid change. Owing to strong linkage disequilibrium, these 7 polymorphic sites generate only 6 different haplotypes, i.e., alleles (AF; TABLE 2). The frequencies of these alleles were compared between groups of centenarians and controls. Centenarians were selected for this study because breast cancer is extremely rare in such individuals of highly advanced age. Hence, it is possible that optimal sequence variants of this gene contribute to this extreme resistance, possibly as a consequence of its increased proficiency in the process of recombinational repair. Two different centenarian populations were studied, i.e., one population consisted of 102 centenarians from the New England area42 and one population consisted of 84 centenarians from Italy.43 The control population consisted of 97 individuals from Italy. All three populations were mainly white.

Frequency (%)
70
60
50
40
20
10

(HH Centenarians It (n=168)
[Hl Controls tt (n=194)
[17 Breast ovarian cancer probands(Shattuck-Eidens et al, 1997) (n=1590)
                   arians NE (n=198)1994) (n=650)= (n=54)1994) (n=322)

Haplotype/allele
APOE4

FIGURE 3: BRCA1 allele frequencies in two different centenarian populations and one control population (NE = New England; IT = Italy). The allele frequency of APOE4 has been determined in the same populations as a control and was compared with the results of Schchter et al.22. Results so far obtained indicate that there are no significant differences in BRCA1 allele (haplotype) frequencies among the three populations (FIG. 3). This also applied to the APOE4 variant that was included as a positive control. However, in keeping 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 population-specific effects of the different haplotypes and disease, actual association should be carried out using the frequencies of allele combinations (i.e., haplotype frequencies). As shown in FIGURE 4, the BRCA1 genotype aa showed to be more frequent in both centenarian groups compared to the control group (p = 0.1473), whereas genotype ab was less frequent (p = 0.0526). In this figure, the APOE4/E4 genotype was not found for any of the 99 New England centenarians and scored 2 times for the 27 control individuals in which this was present (FIG. 4).

The high number of genotypes and the low frequency of some of the alleles require 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 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.