Smaller genomic regions have been successfully demonstrated by others (16-18), and advantages over single markers have been shown (19-21). In addition, the diversity of the genome can be effectively captured by reducing it to sequential segments of limited diversity (18, 22, 23). Once the common haplotypes within a block have been defined, SNPs within the same block redundant for discriminating between the different haplotypes can be omitted from subsequent typing and analysis. After removing the redundant SNPs, 875 validated SNPs and 700 maximally informative SNPs remained for the association studies. Testing for stratification was performed in the U.S. cohort using 60 random SNP markers genotyped in all cases and controls, and chi-squared values were calculated from the allele counts. Because these SNPs were selected at random, any differences in allele frequencies were inferred as representative of the differences in genetic backgrounds between cases and controls. If the genetic backgrounds of the two-armed study were perfectly matched, the mean chi-squared of the G test statistics for these markers has an expected value.

By densely genotyping across the 12 million-bp region previously identified through the linkage study, we were able to reconstruct a rough draft of the underlying haplotype structure. A total of 195 blocks were defined within the 12 million-bp region, with 2 to 15 SNPs per block. Approximately 75% of the mapped region was within regions of strong linkage disequilibrium. Using this carefully reconstructed assortment of SNP-based haplotype markers, we initiated a case-control association study between groups of unrelated LLI (age). For the French cohort, 57 microsatellites from the Applied Biosystems LMS-MD10 panel located on six different chromosomes (chromosomes 2, 9, 10, 11, 17, and 18) were tested on all cases and controls and checked for stratification by the above method.

Table 1 shows haplotype allele counts/frequencies for the U.S. cohort. Table 3 shows allele counts and frequencies for the U.S. confirmatory sample set. Shown are counts/frequencies for the two-SNP haplotype formed by combining alleles of these SNPs in cases (LLI) and controls. Only three of the four haplotypes were observed, fulfilling the criteria of no historic recombination between the two SNPs. As discussed in the text, the haplotype composed of the rs2866164-G allele and Q95 allele is underrepresented in LLI, suggesting this variant confers mortality risk (P < 0.0005Diverse group. Consequently, our cases and controls would differ not only with respect to the longevity phenotype but also with ethnicity as an uncontrolled confounder. If the effect is strong enough, associations will be found that reflect these ethnic differences rather than differences in lifespan. The mean (cid:1)2 for randomly selected SNP markers (representing differences in genetic background) for the 190 cases and controls used for the first screening showed the absence of stratification ((cid:1)2 mean of 1.07 compared with an expected value of 1.0). Unfortunately, the second set of 190 LLI and controls used to replicate the hypothesis showed moderate stratification ((cid:1)2 mean of 1.7). Although modest, any amount of stratification is undesirable, and the methods of correcting for this potential confounder have not been well validated empirically.

Tables 2 and 4 show the genotyping results for U.S.-based collection confirming the presence of risk SNP allele counts. Note that rs2866164 SNP has multiple twins displaying identical statistical including the (cid:4)493G(cid:2)T SNP. For rs2866164, P is 0.034, and for MTP P is 0.071. P is 0.0006. See Table 1 for details.

Table 5 shows risk allele frequency differences in proactively matched LLI and controls (U.S.-based). The minor allele of rs2866164 is underrepresented in LLI compared to controls (P is 0.0144). There is no evidence in this sample set that the haplotype explains the association better than rs2866164 alone.

Table 7 shows rs2866164 frequency differences in French LLI cohort and controls. The minor allele of rs2866164 is underrepresented in LLI compared to controls, but the effect is not statistically significant (P is 0.54).

To avoid correcting for the hundreds of partially independent hypotheses tested with the original sample set and to simultaneously eliminate stratification as a problem, 250 cases were proactively matched (see Methods) against a new set of 463 controls. Using the approach discussed in Methods, a subgroup of 250 controls was selected that best matched the cases with respect to genetic background. The mean (cid:1)2 for this group of samples (measured using a second, independent set of 60 SNPs) was 0.92, indicating a very high level of genetic balance. It should be emphasized that none of these samples were used in tier I to generate the single inference that the risk allele was underrepresented in LLI. The association at rs2866164 was confirmed with this well-matched group of cases and controls (P is 0.0144 by one-sided G test, relative risk 0.69, Table 5). The two-SNP haplotype also demonstrated an association (P is 0.0124, Table 6).

After fully characterizing the MTP finding, there remained the possibility that one or more additional genes associated with longevity could be contributing to the linkage peak. To address this, any SNP or SNP-based haplotype genotyped in the first tier of samples associated at P is 0.05 was genotyped in the second tier of 190 cases and controls. None of these putative associations was replicated.

MTP resequencing showed that none of the identified SNPs explained the association better than rs2866164. Sixty-one of these SNPs (named MTP001, MTP002 etc.) were novel to our study. After adding the additional SNPs to the map, a new block structure was defined with significant changes (Fig. 1b).