In the 250-kb region bracketing rs1553432, 60 SNPs were identified and assays were validated. Several of these densely spaced SNPs showed strong associations when analyzed in the set of 190 cases and controls used above; most of these markers were wae5! ¢ located near the 5 end of MTP or just upstream of this gene and were particularly dense near the promoter region. All of the identified associations were in strong linkage disequilibrium with rs1553432 (e.g., they fell on the same ‘‘long-range’’ haplotype). With interest narrowing in on a single gene, all known SNP polymorphisms for MTP and its promoter were genotyped in the original 190 cases and 190 controls. After haplotype reconstruc- tion of the area was completed, a single haplotype (see Fig. 1a, which is published as supporting information on the PNAS web >ae site), underrepresented in the LLI (P 0.0005, Table 1), could account for the majority of the variation at the locus. This risk haplotype was initially defined as the minor allele of rs2866164 p BAAS2/E paired with the major allele of MTP Q H 95. Both of these SNP markers had been previously identified by other groups; rs2866164 is an MTP promoter mutation and MTP Q(cid:2) H 95 is a semiconservative mutation in exon 3 of MTP (glutamine to histidine at amino acid 95). Evidence for association was present

Genetic Stratification and Controlling Type I Error. Genetic associ-

Genetic Stratification and Controlling Type I Error. Genetic associ- ation studies have been plagued with false positive or otherwise irreproducible results (24, 25). A recognized problem affecting genetic association studies is the failure to adequately match the genetic backgrounds of the cases and controls, a phenomenon called stratification. Our association studies comparing individ- uals born decades apart can be potentially vulnerable to this confounder because the geographic distribution of ethnicities has changed in the U.S. over the past 100 years. Specifically, our case population reflects the ethnic distribution of the U.S. near the beginning of the last century, whereas our control population was sampled from more recent generations. To minimize this problem, we only used DNA from people who identified them- selves as Caucasian, but this is obviously a diverse group.

Consequently, our cases and controls would differ not only with respect to the longevity phenotype but also have 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 back- ground) for the 190 cases and controls used for the first screening showed 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

Table 2. Risk SNP allele counts(cid:2)frequencies for U.S.-based collection

SNP allele Controls LLI rs2866164-C 245 (66%) 259 (74%) rs2866164-G 121 (34%) 91 (26%) MTP Q95 361 (95%) 343 (92%) MTP H95 19 (5%) 31 (8%)

Table 4. Allele counts and frequencies for U.S. confirmatory sample set: Two-SNP haplotype

MTP rs2866164-C rs2866164-G rs2866164-C rs2866164-G Q95 280 (77%) 63 (17%) 241 (67%) 98 (27%) H95 0 23 (6%) 0 21 (6%)

Shown are counts(cid:2)frequencies for rs2866164 and MTP Q(cid:2)H. Note that rs2866164 SNP has multiple ‘‘twins’’ displaying identical statistical including the (cid:4)493G(cid:2)T SNP. For rs2866164, P   0.034, and for MTP P   0.071.

P   0.0006. See Table 1 for details.

Table 5. Risk allele frequency differences in proactively matched LLI and controls (U.S.-based)

Table 7. rs2866164 frequency differences in French LLI cohort and controls

The minor allele of rs2866164 is underrepresented in LLI compared to controls(P(cid:2)0.0144).Thereisnoevidenceinthissamplesetthatthehaplotype explains the association better than rs2866164 alone.

The minor allele of rs2866164 is underrepresented in LLI compared to controls, but the effect is not statistically significant (P   0.54).

effort was made to collect geographically matched controls, stratification was not evident in this study. Testing 57 microsat- ellite markers, the (cid:1)2 mean between cases and controls was 1.00.

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.

ellite markers, the (cid:1)2 mean between cases and controls was 1.00. Tables 7 and 8 show the genotyping results for rs2866164 and the haplotype composed of rs2866164 and MTP Q(cid:2) H 95. Neither the minor allele of rs2866164 nor the rs2866164-G(cid:2) MTP-H95 haplotype is significantly underrepresented in the French LLI. tiv3/1 Interestingly, as shown in Table 9, the rs2866164-G MTP-H95 haplotype (HI) is out of HWE with the other haplotypes (HII,     collectively) in the LLI (P 0.0164) but not in controls (P 0.5923). The departure from HWE observed in the LLI can be entirely explained by an excess of HI(cid:2) HI homozygotes. Allele frequencies out of HWE are suggestive of either genotyping artifact or selective pressure (26). A genotyping artifact was   er3/1 ruled out by typing the 493G T polymorphism. As expected, this marker was perfectly correlated with rs2866164, arguing against marker-specific systematic error. The modest departure from HWE in the cases and lack of statistical distortion in the controls argue in favor of selective pressure at this locus that manifests itself as a population ages.