Prospective association analysis.—For prospective asso- ciation analyses in the Leiden 85-Plus Study, Kaplan–Meier estimates of the survival curves for the various genotype groups were calculated. A sex-adjusted hazard ratio was estimated using a Cox proportional hazards model in all 1245 octogenarians from the Leiden area. For these ana- lyses, we took delayed entry into account using STATA/SE 8 (Cosinus Computing, Waalwijk).

Meta analysis.—The meta-analysis was carried out on the MTP T-Q haplotype estimating random and fixed effect using the ‘‘meta’’ library in the free software R version 2.1.1 (26,27) in a total of 4915 long-lived cases older than 85 years and 3002 Caucasian controls from the United States first tier (190 cases and 190 controls) and second tier

Genotyping of the Q95H SNP was performed using the MassARRAY platform according to protocols of the

 F SIBCSHT+ zorsva.+ uisiseaF 90rSe0.“74r rebigna‘N‘Nbt bivsra 7F zzsisva. \+ 986zS¢0} virsra.} resisba.  31098 GO7   1251201151109590Position in cM from pter

Table 1. Nonparametric LOD Scores Obtained in 164 Nonagenarian Sibships of the Leiden Longevity Study in the Chromosome 4 Linkage Region of Puca and Colleagues (7) and in 21 Sibships in Which at

Location in Kosambi LOD Score LOD Score Marker cM from pter 90þ 164 Sibships 98þ 21 Sibships D4S2964 88.4 (cid:1)0.02 0.48 D4S1534 95.1 (cid:1)0.02 0.24 D4S414 100.8 (cid:1)0.02 0.06 D4S1572 108.0 (cid:1)0.17 0.00 D4S406 117.1 (cid:1)0.40 (cid:1)0.42 D4S402 124.5 (cid:1)0.37 (cid:1)0.48

Note: LOD ¼ Logarithm of odds.

offspring, and the partners of the offspring (Table 2). In addition, the octogenarians of the Leiden 85-Plus Study born in the city of Leiden were compared to the young Leiden control group (Table 2). The genotype distributions of all groups were in Hardy–Weinberg equilibrium.

Figure 1. Nonparametric linkage analysis of chromosome 4q25 in 164 nonagenarian sibships of the Leiden Longevity Study (—) and the 137 nonagenarian sibships described by Puca and colleagues (7) (- - -). *Indicates markers from the initial marker set used by Puca and colleagues with an average spacing of 7.22 cM, resulting in a logarithm of odds (LOD) score of 2.67, and markers genotyped in the Leiden Longevity Study.

Table 3 shows the frequencies of the (cid:1)493 T and 95 Q alleles (the alleles of the previously associated haplotype) of the MTP gene polymorphisms in all study groups. The allele frequencies did not differ between the case and control groups in the Leiden Longevity Study or in the Leiden 85-Plus Study (p . .32). Also, no association of these polymorphisms was found in sex-specific analyses (data not shown).

matched (488 cases and 462 controls) (8), French [564 cases and 564 controls; (8)], German [1039 cases and 550 controls; (28)], Danish [1625 cases and 551 controls; (29)], the Leiden Longevity Study [164 cases and 251 controls; this article), and the Leiden 85-Plus Study (655 cases and 244 controls; this article). For fixed effects, the in- verse variance weighting was used for pooling and the DerSimonian–Laird estimator was used in the random- effects model.

To investigate whether an MTP haplotype is associated with longevity, the MTP haplotype frequencies are estimated in the age groups of the Leiden Longevity Study and the Leiden 85-Plus Study (Table 3). The two MTP poly- morphisms are in complete linkage disequilibrium (D9¼1.0) and the haplotype(cid:1)493G/95H is extremely rare. Because the THESIAS program was designed for haplotype-based association analysis in unrelated individuals, we investigated just one sibling per sibship to perform association analysis of the MTP haplotype in the Leiden Longevity Study. We found no association of an MTP haplotype with longevity in either of our Leiden Studies ( p . .46) (Table 3).

R ESULTS

Linkage Analysis at Chromosome 4q25

In total, 379 nonagenarian participants belonging to 164 sibships of the Leiden Longevity Study were genotyped for the same set of markers at 4q25 with which Puca and colleagues (7) initially detected linkage with the lon- gevity phenotype. We found no evidence for linkage at this chromosome 4 region in the Leiden Longevity Study (Figure 1). Markedly, in the 164 nonagenarian sibships the LOD score is for the whole region even little less than zero (Table 1), suggesting support against linkage. Also, when repeating the linkage analyses on the more stringent pheno- type as investigated by Puca and colleagues, in which one sibling is at least 98 years old (21 sibships), we observed no significant effect of this 4q25 locus on lon- gevity (Table 1).

Prospective Association Analyses

Using the follow-up data of the two combined cohorts of the Leiden 85-Plus Study (N ¼ 1245) from the Leiden area, the effect of the genotypes of the two MTP SNPs on sur- vival after the age of 85 years was tested. The proportional hazard ratios of the heterozygous carriers of the minor alleles of the (cid:1)493G/T and Q95H SNPs compared to the homozygous wild-type carriers were 0.98 (confidence inter- val [CI], 0.85–1.13) and 1.13 (CI, 0.89–1.42), respectively. Furthermore, the proportional hazard ratios of the homozy- gous carriers of the minor alleles of the (cid:1)493G/T and Q95H SNPs compared to the homozygous wild-type carriers were 0.94 (CI, 0.70-1.26) and 1.18 (CI, 0.29–4.74), respectively. Because these proportional hazard ratios do not differ from 1.0 and the survival curves of the different genotypes overlaid (Figure 2) for both polymorphisms, these MTP polymorphisms were associated with neither overall mor- tality risk nor specific causes of death (data not shown).

Cross-Sectional Association Analyses

To test for association of the MTP gene with longevity, the two SNPs forming the previously associated haplotype, i.e., (cid:1)493G/T and Q95H, were genotyped in the nonage- narian sib pairs of the Leiden Longevity Study, their