To avoid correcting for the hundreds of partially independent hypotheses tested with the original sample set and to simulta- neously 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 underrep- resented in LLI. The association at rs2866164 was confirmed >( with this well-matched group of cases and controls (P 0.0144  by one-sided G test, relative risk 0.69, Table 5). The two-SNP pate ON  haplotype also demonstrated an association (P 0.0124, Table 6).

Multiple Associations? 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 geno- haatvy._s typed in the first tier of samples associated at P 0.05 was genotyped in the second tier of 190 cases and controls. None of these putative associations was replicated.

MTP Resequencing. Although the two-SNP haplotype was suffi- cient to account for all of the association at the locus, it is imprudent to conclude that this polymorphism was causative with respect to longevity. In particular, a few ‘‘twins’’ (SNPs   3/1 whose alleles are perfectly correlated) of 493 G T and rs2866164 were identified that could equally explain the data.

To search for additional SNPs that could explain the associ- ation as well as or better than rs2866164, we sequenced 12 DNA samples within the 12-kb risk block and the 72-kb block of DNA extending up to the initial rs1553432 SNP. In addition, all 18 exons of MTP were sequenced in a group of 50 LLI to search for rare functional polymorphisms that would not fall on well- defined haplotypes. Altogether, 104 SNPs were identified, al- though none explained the association better than rs2866164. Sixty-one of these SNPs (named MTP001, MTP002 etc.), were novel to our study (i.e., they were not included within the December 2001 Human Genome Draft). After adding the additional SNPs to the map, a new block structure was defined with significant changes (Fig. 1b).

How adequately did the maximally informative SNPs cover the area under the linkage peak? There were inevitable gaps in the haplotype map, because of incomplete sequences of the human genome in this region, areas with few documented SNPs, and regions where our genotyping was not successful. Attempting anything near 100% statistical coverage of the region would have been prohibitively expensive and time consuming given the extensive sequencing and genotyping required. As a compro- mise, we attempted to genotype, in 190 cases and controls (first tier), at least five SNPs near all of the well-characterized genes under the locus (including the alcohol dehydrogenase cluster and   NF- B), which involved assaying an additional 55 SNP markers. This effort yielded no additional associations, leaving MTP as the lone candidate to explain the original linkage result. We recog- nize that a study with denser genotyping, larger sample sizes, or more extreme ages may have produced additional candidates.

Testing in a New Cohort. Because of the potential unpredictable bias that affects association studies, we attempted to confirm the observation in a group of matched LLI from the Centre d’E´tude du Polymorphisme Humain centenarian cohort. Because an

Discussion

We have shown a statistically significant underrepresentation a risk allele in a group of LLI compared with younger

Table 6. Risk allele frequency differences in proactively matched LLI and controls (U.S.-based): Two-SNP haplotype

Table 8. Two-SNP haplotype frequency differences in French LLI cohort and controls

MTP rs2866164-C rs2866164-G rs2866164-C rs2866164-G Q95 365 (75%) 94 (19%) 316 (68%) 117 (25%) H95 0 29 (6%) 0 29 (6%)

MTP rs2866164-C rs2866164-G rs2866164-C rs2866164-G Q95 826 (73.2%) 228 (20.2%) 835 (74.0%) 221 (19.6%) H95 0 72 (6.3%) 0 65 (5.8%)

P(cid:2)0.0124.Thetwo-SNPhaplotypeisunderrepresentedinLLIcomparedto controls.

particles. Functionally, MTP is directly involved in the packaging of ApoB and triglyceride into these particles, and MTP and ApoB are thought to directly bind one another during this assembly (32). Rare humans with two nonfunctioning copies of the gene suffer from abetalipoproteinemia and are characterized by the near absence of ApoB particles in serum (33).

Table 9. Haplotype-based counts and frequencies from the French LLI cohort and controls

Haplotype-based genotype Controls LLI HII(cid:2)HII 360 369 HI(cid:2)HII 177 159 HI(cid:2)HI 24 33 HWE P((cid:1)2) value 0.93

Haplotype-based genotype

Drugs that inhibit MTP activity have been shown to improve lipoprotein profiles (34). Several food products have also been shown to reduce MTP activity, including garlic (35), ethanol (36), and citric flavanoids (37). One study found that the MTP atey/ od  promoter allele 493T up-regulated MTP expression by 2-fold mt tvst—4 (38), consistent with our finding that 493T is underrepresented in long-lived populations.

HI represents the rs2866164-G(cid:2)MTP-H95 haplotype, and HII collectively represents the other two haplotypes.