Tests of Association. The G test with Williams correction (a statistic following a (cid:1)2 distribution) was used to test inferences about associating genetic markers (haplotype or SNP) with the longevity phenotype (15). For each allele, 2  2 contingency tables were constructed as  allele vs.  longevity. For tests where only one direction of allele frequency difference was tested, P values were divided by two.

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. For the U.S. cohort, 60 random SNP markers were genotyped in all cases and controls, and (cid:1)2 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 back- grounds between cases and controls. If the genetic backgrounds of the two-armed study were perfectly matched, the mean (cid:1)2 of the G test statistics for these markers has an expected value

Testing for Association. 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. Approx- imately 75% of the mapped region was within regions of strong linkage disequilibrium. Using this carefully reconstructed as- e/ sortment 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 chromo- somes (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. Haplotype allele counts(cid:2)frequencies for U.S. cohort

Table 3. Allele counts and frequencies for U.S. confirmatory sample set

Allele rs2866164-C rs2866164-G rs2866164-C rs2866164-G Q95 256 (74%) 61 (18%) 244 (67%) 104 (29%) H95 0 29 (8%) 0 16 (4%)

SNP allele Controls LLI rs2866164-C 241 (67%) 282 (76%) rs2866164-G 119 (33%) 88 (24%) MTP Q95 275 (94%) 348 (94%) MTP H95 19 (6%) 24 (6%)

Shown are counts(cid:2)frequencies for the two-SNP haplotype formed by com- bining 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   0.0005.

See Table 2 for details. For rs2866164, P    0.0027, and for MTP Q(cid:2)H, P 0.55.

  when each of these SNPs was analyzed separately (P 0.034 for ra RAR>  rs2866164, P 0.071 for MTP 95Q H, Table 2). Goodness-of-fit analysis indicated that neither SNP alone was likely to explain the statistical distortion between cases and controls as well as the wl,|  two-SNP haplotype (P 0.007). RS2866164 is perfectly corre- lated with another MTP promoter SNP, rs1800591 (also known we / al3/1 as 493 G T) that has been previously associated with several phenotypes including lipoprotein profiles, central obesity, and insulin resistance (see Discussion).

To reduce genotyping costs and increase the power by con- firming the hypothesis in independent populations, the study was divided in two sequential tiers of samples, with the first tier comparing 190 LLI with 190 controls at SNP-based haplotype markers. The first tier was intended as a screen to generate the hypotheses tested in the second tier, thereby greatly reducing the statistical penalty for testing a large number of associations. Although several SNPs and haplotype markers were significant   om at P 0.05, the marker showing the strongest association (P 0.0005) was the SNP rs1553432, located 72 kb upstream from MTP. Other SNPs demonstrating statistical distortion include vi ws>  a rs750032 (P 0.0029), rs951085 (P 0.0041), and rs752766 (P 0.0058). The rs1553432 association provided a potentially inter- esting first hypothesis to pursue with dense genotyping and haplotype mapping of the nearby genes, although this result was

Genotyping an additional set of 190 cases and controls (re- ferred to hereafter as the second tier; see Tables 3 and 4) strengthened the evidence for an association with longevity at  the two-SNP haplotype (P 0.0006, Table 4, one-sided G test). Because a single hypothesis was tested with this independent set of samples, there was no need to correct for multiple tested hypotheses. In the second tier of samples, rs2866164 was mar-   ginally less associated with lifespan (P 0.0027, Table 3) than when analyzed as part of the two-SNP risk haplotype identified D/E  in the first tier. MTP Q H 95 alone showed no association (P 0.55, Table 3).