Genotyping. Potential single-nucleotide polymorphisms (SNPs) were retrieved from the Human Genome Draft database. Assays were designed to be multiplexed up to five times with SPEC- TRODESIGNER software (Sequenom, San Diego).

SNP genotyping on the U.S. cohort was performed by Seque- vuen/ic nom’s chip-based matrix-assisted laser desorption ionization time-of-flight MS (DNA MASSARRAY) on PCR-based extension products from individual DNA samples. Cases and controls were always run on the same chip to avoid potential artifacts caused by chip-specific miss-calls.

Abbreviations: LLI, long-lived individuals; MTP, microsomal transfer protein; SNP, single- nucleotide polymorphism; HWE, Hardy–Weinberg equilibrium.

In the current study, a systematic exploration of the chromo- some 4 linkage peak has identified a single gene, microsomal transfer protein (MTP), as a modifier of lifespan in a cohort of

   Genotyping of the 493G T and 95H markers for the French cohort was performed on the TaqMan platform (Applied Bio- systems) based on PCR sequence amplification of genomic DNA followed by fluorometric detection using allele-specific TaqMan Minor Groove Binder fluorescence-labeled probes. »://b Complete details of the protocol are available at http: home. appliedbiosystems.com, protocol 4332856.

Proactive Sample Matching. Sixty random SNP markers (nonover- lapping with the stratification panel described above) were genotyped in 250 cases and 463 controls of the U.S. collection.  Homozygotes for the minor allele were assigned the value 1, heterozygotes 0, and major allele homozygotes 1. Based on the multivariate means calculated from this coded data, a subgroup of the 250 controls was selected that minimized the Mahalanobis distance with respect to the case samples. The Mahalanobis distance is a measure of distance between two multivariate means that normalizes each dimension based on the covariance matrix:

Sequencing. Sequencing was performed on the AB 3100 by using a Big Dye termination (version 3) chemistry on RapXtract (Prolinx, Bothell, WA)-purified PCR products. The PHRED program (Codoncode, Dedham, MA) was used for quality scores, and SEQUENCER (Gene Codes, Ann Arbor, MI) was used for sequence comparisons and SNP detection.

where V is a vector representing the mean genotyping values of 1 the cases, V(cid:1) is the mean vector for the controls, and S(cid:4)1 is the 2 inverse of the covariance matrix. No proactive sample matching was required for the French cohort as there was no detectable stratification between the cases and controls (see Results).

Haplotype Reconstruction. We genotyped 19 familial trios (mother, father, and offspring) at densely spaced SNP markers to create a haplotype map of the 12 million-bp region of interest. For each trio, we determined the parental origin of offspring alleles for all cases where phase could be resolved unambigu- ously. In cases where phase was ambiguous (i.e., triple heterozy- gotes), the data were treated as missing. By applying this method, four parental chromosomes were reconstructed, with intermit- tent missing allele data. A haplotype was defined as a contiguous  region of DNA with little evidence ( 2.5%) for meiotic recom- bination within the common genetic history of the individuals genotyped.

Results

We undertook fine-mapping of the chromosome 4 locus to identify specific gene variants associated with exceptional lon- gevity in a U.S. population. With this aim, 2,000 SNPs (an average of 1 every 6 kb) within the longevity linkage locus were selected from the SNP consortium database. Based on our experience with an earlier pilot study, we expected that only a fraction of these markers would be useful in an association study. To screen this initial set of SNPs, 19 familial trios (mother, father, and offspring) acquired from the Centre d’E´tude du Polymorphisme Humain repository were genotyped at all se- lected markers. Of these 2,000 markers, 1,494 had high confi- dence calls on the MASSARRAY platform. Of these markers, 990 had a minor allele frequency of at least 5%. SNPs of lower heterozygosity were excluded because of the reduced power of such markers with respect to mapping complex traits in associ- ation studies of limited sample size. Of the remaining SNPs, 113 were eliminated because the frequency distribution of the ho- mozygotes and heterozygotes was not statistically compatible with Hardy–Weinberg equilibrium (HWE). These failures were attributed to systematic artifacts introduced by the genotyping platform. The use of familial trios allowed a Mendelian check on the validity of each SNP assay. If more than one Mendelian inheritance error per assay was detected within the 19 trios, the assay was judged unreliable.

In situations where the boundaries were ambiguous, a heuristic was applied that assigned boundaries in such a way to minimize the size (i.e., base pairs) within each block. haplotype boundaries assigned, haplotype frequencies were timated for each haplotype allele by using an maximization algorithm (14). Any haplotype that had a quency of <2 2.5% was excluded from further analysis to possible errors in either the genotyping or the estimation cess. Within each haplotype block, between two and six SNP-based haplotypes were observed, and each of these types could be used as genetic markers tested for association.

To reconstruct haplotypes for the case(cid:2) control association studies, the haplotype boundaries and allele frequency estimates established in the trios were used as initial parameters to seed the haplotype allele frequency estimations for genotyping the cases and controls. In cases where haplotype data could not be estimated with at 95% confidence, the haplotype allele was treated as missing.