Recently, the gene encoding microsomal triglyceride transfer protein (MTP) has been reported as the first positional candidate for a heritable influence upon human life span (5). This link was drawn in an association-based fine-mapping experiment around microsatellite D4S1564, a genetic marker for longevity previ- ously identified via genome-wide linkage analysis (6). A two- marker haplotype within the MTP gene was found to be signif- icantly underrepresented in a sample of 190 long-lived Americans of European extraction, suggesting that it confers a higher mortality earlier in life (5). The ‘‘risk’’ haplotype was defined by the minor allele of rs2866164 (allele G) and the major y/I allele of MTP Q H 95 (allele Q95). Marker rs2866164 is a SNP in the MTP gene promoter known to be in perfect linkage disequilibrium with another promoter mutation, rs1800591 (also   aiiN?3/1 Meta)/E described as 493 G T; ref. 7). MTP Q H 95 is a nonsynony- mous single-base-pair substitution in exon 3 of the MTP gene, leading to an exchange of glutamine with histidine at amino acid

Table 1. Summary statistics for SNP rs2866164 in German, French, and U.S. Caucasian LLI and controls

Sample (no. of individuals) minor allele (G) P P Odds ratio (Wald 95% allele genotype Germans LLI (1,033) 0.269 0.34 0.13 1.18 (0.96–1.46) Centenarians (370) 0.276 0.28 0.30 1.21 (0.93–1.58) Controls (540) 0.253 French LLI (559) 0.253 0.54 NA NA Controls (552) 0.264 U.S. Caucasians LLI, sample 1 (175) 0.260 0.039 NA NA Controls, sample 1 (183) 0.331 LLI, sample 2 (244) 0.252 0.029 NA NA Controls, sample 2 (231) 0.316

P , P value from a (cid:1)2 test (1 df) for allele frequency difference; P , P value from a (cid:1)2 test (2 df) allele genotype genotype frequency difference; CI, confidence interval; NA, not available. Data of French and U.S. samples are as reported by Geesaman et al. (5). The second sample of the U.S. Caucasians represents the reported counts and differ slightly from those in the original publication.

control haplotype frequency estimates were used to calculate a pseudo-(cid:1)2 statistic (12). Significance was assessed in both in- stances by using 10,000 random permutations of cases and controls. Power calculations were also carried out by means of a Web-based tool, available at the University of California, Los ://¢ Angeles, Department of Statistics (http: calculators.stat.ucla. ‘etu/P edu pc).

inclusion in the study were good health, physical activity, and absence of major diseases. Furthermore, it was required that participants be mentally fit and able to answer a health history questionnaire routinely used in clinical studies. A total of 550 unrelated German control individuals aged between 60 and 75 years (mean age, 67.2 years) were recruited by the same means and in the same regions as the nonagenarians and centenarians. The controls matched the LLI by ancestry (all German), geo- graphic origin within the country, and sex.

Results

Both the entire collection of 1,039 German LLI and a subsample of 373 centenarians were subjected to a gender-matched case- r/I control analysis of markers rs1800591, rs2866164, and Q H 95, located in the MTP gene. All three SNPs were found to be in Hardy–Weinberg equilibrium. No evidence for association was detected between any of the tested SNPs and the longevity phenotype in either sample at the allele or at the genotype level. The association statistics for the most informative SNP, rs2866164 (for which comparable data on the U.S. Caucasian and French samples were also available), are presented in Table 1.

All participants gave written informed consent to participate in the study. Lifestyle and health-related and genetic data were completely anonymized before being logged into an electronic database. Approval for the project was received from the Ethics Committee of the University Hospital Schleswig–Holstein, Cam- pus Kiel, and from local data protection authorities.

d/E Genotyping. Markers rs1800591, rs2866164, and MTP Q H 95 were genotyped by using the Taqman Allelic Discrimination method with fluorescence-labeled minor groove-binding probes on an Applied Biosystems 7700 Sequence Detector. Genotype data were managed by means of an integrated database system (11). Additionally, positive controls on each genotyping plate were checked for consistency. All markers were tested for deviation from the Hardy–Weinberg equilibrium, as described below. The call rate for each marker exceeded 99%.

In all four LLI samples from Germany, France, and the U.S., the frequency of the minor rs2866164 allele (G) fell into the range between 0.252 and 0.269 (heterogeneity (cid:1)2 J itt 1.18, 3 df,   P 0.76). The allele frequency distribution in the German and French control groups was also found to be very similar ((cid:1)2 ceed  0.39, 1 df, P 0.53). Only the two U.S. control samples were characterized by a significantly increased frequency of the G allele of rs2866164 when compared with the two European controls ((cid:1)2   8.62, 2 df, P   0.013 for U.S. sample 1, (cid:1)2  6.76,   2 df, P 0.034 for U.S. sample 2).