and at least 1 living sibling were able to give informed consent and willing to participate in the in-person interview and examination, including providing blood for serum and for extraction of DNA; and either the proband or a sibling had a living offspring willing to participate. A minimum of 2 siblings and 1 offspring was required for the key triad. Additionally, we recruited and enrolled the spouses of interested individuals, primarily in the offspring gener- ation, to serve as a comparison group ascertained from the same source populations, but not selected for familial longevity. Spouse controls were employed to adjust for characteristics of individuals within a family which are likely to be correlated. Before examina- tion, written informed consent was obtained from all enrollees. The Institutional Review Boards at of all of the Field Centers and the Data Management and Coordinating Center (Washington Univer- sity, St. Louis) in the United States reviewed and approved this project and the regional Institutional Review Board in Denmark reviewed and approved this project.

2. Methods

Long-lived individuals, their siblings, and their offspring were recruited for an examination that characterized key intermediate phenotypes of longevity, including major chronic diseases, risk factors, and physical and cognitive function. A referent group con- sisting of the spouses, primarily of the offspring generation, was also recruited and examined. For this study, we restricted the analysis to the offspring generation and the examined sons, daughters, nieces, and nephews (n ¼ 2314) and their spouses (n ¼

2.3. Genotyping

Among offspring and their married-in controls, we were able to genotype APOE for 2307 of 2371 offspring (97.3%) of probands and their siblings and 764 of 793 spouse controls (96.3%). We were able to genotype the TOMM40 SNP rs2075650 for 2274 (95.9%) offspring and 746 married-in controls (94.1%). Of the 3071 participants in the analysis, 983 were Danish (32.0%).

2.1. Recruitment

Genotyping of APOE polymorphisms was based on SNPs rs7412 and rs429358 and was performed at the Biomedical Genomics Center at the University of Minnesota. Genotyping was performed using the Taqman genotyping platform and concordance among genotypes for both SNPs was 100% based on 51 blinded duplicates. We also sequenced DNA from 12 samples with different APOE genotypes to assure correct assignment of alleles. Genotyping of the TOMM40 SNP rs2075650 was performed as part of the LLFS genome wide association study and was typed via the Illumina HumanOmni 2.5 at the Center for Inherited Disease Research at Johns Hopkins University. Participants were classified by the presence or absence of the APOE ε4 and ε2 alleles, and as carrying 1 or more G alleles at rs2075650.

In the United States, each field center used Center for Medicare and Medicaid Services lists to mail a recruitment brochure to people who were at least 79 years old on January 1, 2005; had no recorded date of death; did not have end-stage renal disease or were not in hospice programs; and lived within 3 hours driving distance of 1 of the 3 US study centers (Boston University, Columbia University, and the University of Pittsburgh). Mailings were per- formed in collaboration with Center for Medicare and Medicaid Services and the National Institute on Aging via an Intra-Agency Agreement. Study participants were also recruited from the community using Web-based media and newspaper advertise- ments, mailing lists obtained through local government agencies, purchased public domain lists from various commercial vendors, and community presentations at churches and senior centers. The University of Southern Denmark study site first identified individ- uals who would be aged 90 and older during the study recruitment period through the Danish National Register of Persons, which contains current information on names, including past names such as maiden names for women, addresses, place of birth, marriages, and vital status (Pedersen et al., 2006). Second, using information on the place of birth and the names, parish registers available in regional archives were searched to locate the parents of the elderly individuals in order to identify sibships. Based on the above infor- mation, potentiallyeligible families were identified and contact was made with potential probands to further assess the family’s eligi- bility for and willingness to participate in the LLFS using criteria parallel to that used in the United States. Recruitment, informed consent, and study procedures were approved by the Institutional Review Boards of all participating sites.

2.4. Statistical analysis