Elixir Pharmaceuticals, Howard Hughes Medical Institute, Genetics Division, Children's Hospital and Harvard Medical School, Boston Medical Center, Boston University School of Medicine, Foundation Jean Dausset Centre d'Etude du Polymorphisme Humain, and Whitehead Institute for Biomedical Research and Whitehead Massachusetts Institute of Technology Center for Genome Research were genotyped. The study aimed to identify genes impacting lifespan by performing a haplotype-based fine-mapping study of the interval containing (cid:1)50 putative genes. A haplotype marker within microsomal transfer protein (MTP) was identified as a modifier of human lifespan. The same variant was tested in a second cohort of long-lived individuals (LLI) from France, and although the association was not replicated, there was evidence for statistical distortion in the form of Hardy Weinberg disequilibrium. Microsomal transfer protein has been identified as the rate-limiting step in lipoprotein synthesis and may affect longevity by subtly modulating this pathway.

The study sample consisted of 653 individuals (197 males and 456 females) aged 98 years and older, recruited through various institutions. A follow-up study investigating LLI from France also demonstrated statistical distortion at MTP but did not completely confirm the observations from the U.S. cohort. The genetic influence of achieving extreme old age might be even greater than that of average life expectancy. Male and female siblings of centenarians have a 17- and 8-fold greater relative risk, respectively, of surviving to age 100.

Genotyping was performed on both cohorts using different platforms. Potential single-nucleotide polymorphisms (SNPs) were retrieved from the Human Genome Draft database, and assays were designed to be multiplexed up to five times. SNP genotyping on the U.S. cohort was performed using chip-based matrix-assisted laser desorption ionization time-of-flight MS (DNA MASSARRAY) on PCR-based extension products from individual DNA samples. SNP genotyping for the French cohort was performed on the TaqMan platform based on PCR sequence amplification of genomic DNA followed by fluorometric detection using allele-specific TaqMan Minor Groove Binder fluorescence-labeled probes.

Sixty random SNP markers were genotyped in 250 cases and 463 controls of the U.S. collection for proactive sample matching. 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. Sequencing was performed on PCR products purified using RapXtract, and quality scores were determined using PHRED program. SEQUENCER was used for sequence comparisons and SNP detection.

For haplotype reconstruction, 19 familial trios (mother, father, and offspring) were genotyped at densely spaced SNP markers to create a haplotype map of the 12 million-bp region of interest. A haplotype was defined as a contiguous region of DNA with little evidence (2.5%) for meiotic recombination within the common genetic history of the individuals genotyped.

In conclusion, this study provides proof of concept for the feasibility of using the genomes of LLI to identify genes impacting longevity.The Centre d'Etude du Polymorphisme Humain repository genotyped all selected markers. Of the 2,000 markers, 1,494 had high confidence calls on the MASSARRAY platform. Among those markers, 990 had a minor allele frequency of at least 5%. SNPs with lower heterozygosity were excluded as they have reduced power in mapping complex traits in association studies of limited sample sizes. Out of the remaining SNPs, 113 were eliminated because the frequency distribution of homozygotes 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 for 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 boundaries were ambiguous, a heuristic was applied that assigned boundaries to minimize the size (i.e., base pairs) within each block. Once haplotype boundaries were assigned, haplotype frequencies were estimated for each haplotype allele using a maximization algorithm (14). Any haplotype with a frequency of less than 2.5% was excluded from further analysis due to possible errors in either genotyping or estimation processes. 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 case-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 cases and controls. In cases where haplotype data could not be estimated with at least 95% confidence, the haplotype allele was treated as missing. Tests of association used the G test with Williams correction (a statistic following a chi-squared distribution) to test inferences about associating genetic markers (haplotype or SNP) with the longevity phenotype (15). For each allele, 2x2 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.