For the present study, data on families enrolled between the start of the study (September, 2002) and January 2004 were collected. We had available 379 nonagenarian partici- pants belonging to 164 sibships (mean age ¼ 93.3 6 3.1 standard deviation [SD] years), 525 offspring of the

Genotyping.—The 379 nonagenarian participants belong- ing to 164 sibships were genotyped for the same six microsatellite markers at which linkage was initially

manufacturer (Sequenom, San Diego). Briefly, after PCR, a primer extension reaction was performed to introduce mass differences between alleles and, after removing salts by adding a resin, »15 nl of the product was spotted onto a target chip with 384 patches containing matrix. Mass differences were detected using matrix-assisted laser de- sorption/ionization time-of-flight mass spectrometry (MALDI- TOF), and genotypes were assigned real-time using Typer 3.0 software (Sequenom). As quality control, cluster plots were made of the low- and high-mass allele signals as detected using mass spectrometry. Calls falling outside a cluster and no-calls falling inside a cluster were checked manually. In addition, 10% of samples were genotyped in duplicate and no inconsistencies were observed.

detected by Puca and colleagues (7): D4S2964, D4S1534, D4S414, D4S1572, D4S406, and D4S402 with an average intermarker spacing of 7.22 cM. They observed the highest LOD score between D4S1572 and D4S406. These markers were taken from the Human Linkage Set v2.5 MD10 and HD5, amplified using AB9700 polymerase chain reaction (PCR) machines (Applied Biosystems, Foster City), and measured using an ABI Prism DNA Analyzer 3700 (Applied Biosystems). Genotyping was performed accord- ing to the manufacturer protocols, except that the amount of primer pairs for the markers was reduced up to 5-fold and duplex PCRs were designed if possible to reduce costs, time, expense, and amount of genomic DNA used. Genemapper 3.0 (Applied Biosystems) was used for allele calling using the LIZ500((cid:1)250) size standard (Applied Biosystems). For the purpose of quality control, »5% of samples were genotyped twice and the results compared. The comparisons indicated that these markers could be genotyped reliably. Five Mendelian errors (0.2% of all genotypings) were detected using PedStats and no unlikely double recombinants using error detection in Merlin (both programs freely available at http://www.sph.umich.edu/csg/ abecasis/software.html). The errors were removed using the PEDWIPE (23) command in Merlin. The location of the markers was taken from the Marshfield integrated ge- netic map (http://research.marshfieldclinic.org/genetics/) us- ing Kosambi cM.

Statistical Analysis

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Cross-sectional association analysis.—For association analysis with the two MTP gene SNPs, z-tests (adjusted for the covariance between siblings in the case of the Leiden Longevity Study) were performed to compare allele fre- quencies in different age categories. In the Leiden Longevity Study, the 379 nonagenarian sib pairs were compared to the 251 unrelated partners of the offspring as a younger control group. The 525 offspring of the nonagenarians harboring potentially future long-lived cases were compared to their 251 partners as an age-matched and environment-matched control group. In the Leiden 85-Plus Study, the 655 octo- genarians born in the city of Leiden were compared to 244 young Leiden controls with parents and grandparents from the same geographic region.

and 251 of their partners from the Leiden Longevity Study, as well as the 1245 octogenarian participants from the Leiden 85-Plus Study and the 244 young Leiden controls, were genotyped for two SNPs in the MTP gene of which the two-SNP-haplotype associated with longevity (8) consisted. This haplotype was formed by the SNPs rs2866164/ rs1800591 and Q95H, where the genotypes of the MTP promoter SNPs rs2866164 and rs1800591 are perfectly correlated in the U.S. cases and controls as well as in the French cases and controls. Hence, these two SNPs provide exactly the same genetic information. Although Geesaman and colleagues (8) reported on the rs2866164, we genotyped rs1800591 (alias(cid:1)493G/T in the MTP gene) beside the SNP Q95H, the semiconservative mutation in exon 3 of the MTP gene. The minor allele G of rs28166164 described in the article by Geesaman and colleagues (8) corresponds with the minor allele T of (cid:1)493G/T described in this article.

Haplotype frequencies were estimated using THESIAS (freely available at http://ecgene.net/genecanvas/modules/ mydownloads/), which is designed for haplotype-based association analysis in unrelated individuals (24,25). These frequencies were also compared between the groups of the Leiden Longevity Study and the Leiden 85-Plus Study as described above, except that in the Leiden Longevity study only one individual of each sibship was analyzed to meet the requirement of analyzing unrelated individuals.

Genotyping of the (cid:1)493G/T SNP was performed using Taqman Assay by Design (Applied Biosystems). Assay by Design was used as recommended, except for the following modifications. A qPCR core kit was used (Eurogentec, Maastricht) with one third of the recommended amount of assay mix. PCRs were performed using an AB9700 (Applied Biosystems), and post-PCR fluorescence measure- ments were carried out on an ABI7900 (Applied Bio- systems). For genotyping, cluster plots were made of the fluorescent labels using SDS software (v2.2; Applied Biosystems). Spots falling outside a cluster were set to missing genotypes. As quality control, 10% of samples were genotyped in duplicate; no inconsistencies were observed. Genotyping of the Q95H SNP was performed using