Statistical Analysis. Markers were assessed for significant devia- tions from the Hardy–Weinberg equilibrium by using an exact test with 1,000 permutations, as implemented in ARLEQUIN, Ver. wen I://I   2.001 (http: lgb.unige.ch arlequin). The significance of allele and genotype frequency differences between samples was tested by using a (cid:1)2 statistic with the appropriate degrees of freedom. Genotype-based odds ratios were computed, combining carriers of the minor allele of each marker in one genotype class, and 95% confidence intervals were calculated by using the Web- based Simple Interactive Statistical Analysis (SISA) tool, avail- )://b nh aSt/s: able at http: home.clara.net sisa. Haplotype frequencies were estimated from nonphased genotype data by using an expecta- tion maximization (EM) algorithm. To evaluate the possible association with longevity of individual haplotypes, two different approaches were followed. First, EM-inferred haplotype fre- quencies were subjected to a regression-based haplotype asso-    ciation test with WHAP (www.genome.wi.mit.edu shaun whap). In the second approach (called HAPRAND), case and

As expected, markers rs1800591 and rs2866164 were found to be in perfect linkage disequilibrium when all three SNPs were jointly analyzed in the German samples. Haplotype reconstruc- tion yielded only three of the eight possible haplotypes (Table 2). For both the German LLI and centenarians, the permutation tests did not indicate any significant haplotype frequency dif- ferences between cases and controls. ‘‘Risk’’ haplotype rs2866164-G, MTP Q95 was found to be overrepresented in the U.S. control samples (Table 3) instead of being underrepre- sented in any of the LLI, as suggested by Geesaman et al. (5).

by important demographic differences. For instance, the Amer- ican controls were on average much younger (38.6 years) than the German (67.2 years) and French (51.2 years) controls. To

Table 2. MTP haplotype frequencies in German LLI, centenarians, and controls

Haplotype* Controls LLI P P Centenarians P P WHAP HAPRAND WHAP HAPRAND G-C–Q 0.749 0.732 0.35 0.31 0.724 0.29 0.24 T-G–Q 0.195 0.217 0.16 0.15 0.224 0.20 0.15 T-G–H 0.055 0.051 0.58 0.64 0.052 0.81 0.76 Global test 0.36 0.25 0.41 0.41

P , permutation P value from a regression-based test for a haplotype frequency difference between LLI or WHAP respectively, and controls; P , permutation P value for a pseudo-(cid:1)2 statistic. HAPRAND are given in marker order rs1800591, rs2866164, MTP Q/H 95.

association. Yet, no statistically significant frequency differences were observed between German LLI and controls at the allele, genotype, or haplotype level.

rule out an age effect masking the putative MTP association, we also typed 359 younger German controls (mean age, 39.8 years) for SNP rs2866164. The G allele of rs2866164 had a frequency of 0.231 in the younger German control group, which did not differ significantly from the 0.253 observed in the older controls ((cid:1)2  1.09, 1 df, P aa 0.30). Consequently, no age-related trend toward a higher frequency of G in younger individuals, as suggested by the American samples, exists among the Germans. The American LLI also comprised a larger proportion of males than the European collections. However, stratification of the case-control comparison by gender did not reveal any gender- specific association in the German sample (data not shown).

Geesaman et al. (5) reported that the minor allele (G) of rs2866164 and the rs2866164-G, MTP Q95 haplotype of the MTP gene were significantly underrepresented in long-lived U.S. Caucasians, suggesting this allele or haplotype conferred a higher mortality earlier in life. However, comparison with all other samples analyzed reveals that the MTP ‘‘risk’’ haplotype is in fact consistently overrepresented in U.S. controls. Thus, the question arises as to which factors could have contributed to this discrepancy.

First, the higher frequencies of the rs2866164-G allele and of the ‘‘risk’’ haplotype in the U.S. control samples could have been false-positive results due to population stratification. U.S. Cau- casians represent a considerably admixed population to which immigrants from a number of different European countries have contributed over the last 400 years. Because their origin has changed with time, the ethnic and therefore genetic composition of the LLI sample (going back to the beginning of the 20th century) in the U.S. study (5) is likely to differ from that of the control population (comprising more recent generations of U.S. citizens). Geesaman et al. (5) went to great lengths to limit the likelihood of a stratification artifact by (i) replicating the asso- ciation in independent U.S. Caucasian LLI and control samples, (ii) correcting for population structure using the genomic control approach based on 60 random SNPs, and (iii) undertaking proactive matching of their cases and controls. However, it has recently been shown that, in studies designed to identify modest genetic risk factors, the use of a few dozen SNPs may in general lack sufficient power to reveal even moderate levels of stratifi- cation (13–15). Thus, that proactive matching was deemed necessary in the original study at all implies that, from the outset, the U.S. LLI and controls may not have been well matched. The genomic control method that was subsequently applied, although state of the art at the time, was perhaps insufficient to detect the residual stratification. In this case, differential gene flow and admixture could have contributed to the MTP haplotype fre- quency difference between the two U.S. Caucasian control samples and all other samples analyzed.

Discussion