The search for genes having a major influence on longevity could be started by performing a genome scan

We therefore performed linkage analysis in the chromosome 4q25 linkage region in 379 nonagenarian participants belonging to 164 sibships from the Leiden Longevity Study (19). Recently, we have reported excessive survival in three generations of relatives of these nonagenarian sibling pairs (19), demonstrating that our selection criteria result in individuals carrying familial longevity effects. Additionally, we performed association analysis of the two-SNP haplo- type of the MTP gene in the nonagenarian sib pairs, the offspring of these sib pairs (assuming that they are predis- posed to be long-lived), and the partners of the offspring (assuming that they represent the general population). Furthermore, we investigated the first and second cohorts of the prospective population-based Leiden 85-Plus Study (20,21) for association with the two-SNP haplotype of the MTP gene. Association of the two MTP SNPs were tested cross-sectionally, by comparing allele frequencies in the octogenarians with those in a young control group from the same Leiden area, as well as prospectively, by testing (using the follow-up data) the effects of the MTP genotypes on survival after the age of 85 (22). Finally, we performed a meta-analysis of all known data on the MTP T-Q haplotype and longevity to estimate the odds ratio of this putative risk haplotype on survival in Caucasian populations.

nonagenarian sib pairs belonging to 238 sibships (mean age ¼ 60.6 6 6.8 SD years), and 251 of their partners (mean age ¼ 59.6 6 7.3 SD years).

Participants: Leiden 85-Plus Study

As a confirmation study for the cross-sectional associa- tion analysis, we investigated the population-based Leiden 85-Plus Study that consists of two cohorts of inhabitants of Leiden, The Netherlands (20–22). In total, 655 participants (mean age ¼ 87.5 6 3.1 SD years) born in the city of Leiden were compared to 244 control participants (mean age ¼ 31.0 6 5.6 SD years), with parents and grandparents from the same geographic region.

For the prospective association analyses, we investigated the follow-up data available for all participants of the Leiden 85-Plus Study. For cohort ’87, participants aged 85 years or older were enrolled between 1987 and 1989 and were followed for mortality until May 2001. For cohort ’97, participants who were exactly 85 years old were enrolled between 1997 and 1999 and were followed for mortality until April 2004. In cohort ’87, almost 14 years after the start of the study, 704 participants (98%) had died. In cohort ’97, almost 7 years after the start of the study, 308 partici- pants (51%) had died. For the participants in both cohorts, the cause of death was registered and subdivided into death by cardiovascular disease, by cancer, by infectious disease, or by other causes. From the combined cohorts, a total of 1245 participants who originated from the Leiden area were genotyped for the prospective association analysis.

P M ARTICIPANTS AND ETHODS

Participants: Leiden Longevity Study

The Leiden 85-Plus Study was approved by the medical ethical committee of the Leiden University Medical Centre. Written informed consent was obtained from all participants after the nature and possible consequences of the studies were explained.

The individuals investigated in the present study partici- pate in the ongoing Leiden Longevity Study (19). Families participating in the Leiden Longevity Study have at least two siblings meeting four inclusion criteria: (i) men are at least 89 years old and women are at least 91 years old, (ii) participants have at least one living brother or sister who fulfils the first criterion and is willing to participate, (iii) the nonagenarian sibship has an identical mother and father, and (iv) the parents of the nonagenarian sibship are Dutch and Caucasian.

Methods

Longevity Study, blood was drawn in a 10-ml coagulation Vacutainer (BD Alphen a/d Rijn), 8-ml and 4.5-ml EDTA Vacutainers, an 8-ml citrate Vacutainer, and a 2.5-ml PAXgene tube (PreAnalytiX, Hombrechtikon). Serum, EDTA, and citrate plasma were stored at (cid:1)808C, and EDTA and citrate buffy coats at (cid:1)208C. DNA was isolated from the EDTA buffy coats. From generation 2, the whole buffy coat was isolated using a QIAamp DNA Blood Maxi Kit (Qiagen, Venlo) according to the manufacturer’s protocol. DNA isolation from generation 3 was outsourced to BaseClear (Leiden, The Netherlands), where 200 ll of the buffy coat was isolated using the Chemagic DNA Blood 100 Kit (Chemagen, Baesweiler) and carried out according to the manufacturer’s protocol on a Magnetic Separation Module I (Chemagen), equipped with a 96-rod head. All DNA concentrations were determined using OD260 measurement. In the Leiden 85-Plus Study, DNA was available for 1245 participants of the combined cohorts.

The offspring of nonagenarian sib pairs, representing individuals susceptible to longevity, are also enrolled. In addition, their partners were included as the age-matched and environment-matched control group for the offspring cases and as the young control group for the nonagenarian sib pairs. It was previously found that the parents, the sib- lings, as well as the offspring of the nonagenarian sib pairs show lower mortality ratios as compared to the general population (19). In this Leiden Longevity Study we thus include families enriched for longevity.

In accordance with the Declaration of Helsinki, written informed consent was obtained from all participants prior to entering the study. Good clinical practice guidelines were maintained. The study protocol was approved by the medi- cal ethical committee of the Leiden University Medical Center before the start of the study.