Overview
The Long Life Family Study (LLFS), established in 2005 in response to an NIA RFA, enrolled families enriched for exceptional longevity (EL), to discover factors that contribute to healthy aging and survival. The “family longevity selection score” (FLoSS) was developed to select families with clustering of EL (Sebastiani et al.2009). From 2006 to 2009, LLFS enrolled 539 sibships (G1, probands), their offspring (G2, offspring) and spouses (total 4,953). . The probands were at least 78 years old in the US centers and 86 years old in Denmark. The families were selected to participate in the study based on The Family Longevity Selection Score (FLoSS), a score generated according to birth year cohort survival probabilities of the proband and their siblings. Probands and their families with FLoSS score of 7 or higher, at least one living sibling, and at least one living offspring (minimum family size of 3). Comparison with a referent cohort reveals that LLFS families have strong exceptional clustering of EL. The least exceptional LLFS G1 sibship shows more clustering of longevity than 99% of the Framingham Heart Study (FHS) sibships (Sebastiani et al. 2009). LLFS participants were comprehensively phenotyped by in-home visits (V1). The G2 offspring have a variety of Healthy Aging Phenotypes (HAPs), defined as an unusually low age-specific prevalence of one or more specific conditions or risk factors, compared to population-based cohorts suggesting enrichment of shared (possibly genetic) protective effects in LLFS families. There is considerable heterogeneity of HAPs in LLFS families: e.g. some show exceptional cognition (Barral et al. 2013); others show strong linkage to metabolic HAPs (An et al. 2013). Thus, LLFS is a unique resource for studying familial EL and its relationship to HAPs. In particular, G2 participants represent an unprecedented opportunity to prospectively study the progression of HAPs as related to survival in a cohort likely to show eventual higher EL prevalence.

The in-home visit consisted of demographic, anthropometric, cognitive, activities of daily living, ankle-brachial index, blood pressure, grip strength, pulmonary function measurements. Serum, plasma, lymphocytes, red cells, and DNA are stored for future studies. An array of biochemical assays were completed for Visit 1.

Establishment of the LLFS cohort
The original recruitment and examination for LLFS was conducted over a three-year period, April 2006 – May 2009. LLFS successfully enrolled and extensively phenotyped 4,953 individuals from 539 two-generational families that demonstrated clustering for exception longevity in the upper generation from three US field sites (Boston University, Boston, MA; Columbia University, New York, NY; and University of Pittsburgh, Pittsburgh, PA) and one field site in Denmark (University of Southern Denmark, Odense, Denmark), Table 1. These families included long-lived individuals (generally 90+), their siblings, spouses, offspring, and spouses of the offspring (a referent group). The LLFS has continued to recruit family members, growing to 5,097 participants (1,704 proband generation, ~1/3, and 3,393 offspring generation, ~2/3).

The U.S. field centers used Center for Medicare and Medicaid Services lists of Medicare enrollees to mail a recruitment brochure. The initial file included people who were at least 79 years old on January 1, 2005; had no recorded date of death; were not in the end-stage renal disease or hospice programs; lived in zip codes “near” (within 3 hours driving distance) one of the 3 U.S. study centers (Boston University Medical Center in Boston, MA, Columbia College of Physicians and Surgeons in New York City, NY, and the University of Pittsburgh in Pittsburgh, PA). A pilot mailing tested the yield of families recruited from mailing to individuals in their 80’s and higher age strata. Based on these yields, subsequent mailings targeted those age 89 and older. Study participants were also recruited from local communities using mailed brochures, posters, web-based media and newspaper advertisements as well as community-based presentations. Additional mailing lists were obtained through voter registries and purchased public domain lists from various commercial vendors (Newman et al., 2011). The University of Southern Denmark field center identified individuals who would be ages 90 and above 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, Gotzsche, Moller, & Mortensen, 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 information, potentially eligible families were identified and contact was made with potential probands to further assess the family’s eligibility for and willingness to participate in the LLFS using criteria parallel to that used in the United States.

The “family longevity selection score,” or FLoSS, was developed to select for families with sibships that were rare by virtue of the number of siblings achieving rare percentiles of survival, with an added bonus for being alive and therefore available for study (Sebastiani et al., 2009) The FLoSS was calculated for each family (Sebastiani et al., 2009), and included information on the current age or age at death of siblings, the sibship size and the number of living individuals available to study. Potential families were ranked by their FLoSS. To be eligible, a proband family had to have a FLoSS of 7 or greater and a minimum family size of 3 (specifically the proband, at least one living sibling, and one offspring), all willing and able to give informed consent and participate in the interview and in-

Interviews and examinations were conducted in the home setting with portable equipment by centrally trained and certified research assistants using a common protocol. The in-home visit consisted of demographic, anthropometric, cognitive, activities of daily living, ankle-brachial index, blood pressure, grip strength, pulmonary function measurements. All family members, regardless of age, undergo as much of the same assessment as possible. We developed proxy interview formats for individuals who are too ill cognitively and/or physically to participate in part or all of the in-person examination. If an in-home visit was not feasible, we conducted a comprehensive telephone interview and obtained biological specimens (blood or saliva sample) using an outside laboratory or physician’s office. Selected measures were designed to assess aspects of EL that: 1) have significant heritability, 2) are related to longevity and healthy aging, and 3) can be assessed in the home setting.

Generated resources include DNA, PAXgene tubes for RNA profiling, plasma and sera from whole blood, and banked lymphocytes. All participants provided informed consent approved by the field centers’ Institutional Review Boards (IRBs) for sharing genotype and phenotype on dbGaP. Genome-Wide Association data were generated using Illumina 2.5M Omni SNP array, which were imputed to 38 million SNPs using 1000 Genomes imputation, resulting in a number of publications (An et al., 2014; Bae et al., 2013; Barral et al., 2014; Feitosa et al., 2014; Lee et al., 2013; Minster et al., 2015; Thyagarajan et al., 2014) and which have been shared with the broader scientific community via dbGaP (dbGaP Study Accession: phs000397.v1.p1).

Annual follow-up has been performed annually by phone interview to update vital status and medical conditions. Expanded follow-up is conducted every year for the proband generation and includes reported physical function, activities of daily living and telephone assessed-cognitive function. Because they are generally so much younger these items are assessed only every three years for the offspring generation. However, once an offspring generation participant reaches age 70, they are given the expanded follow-up yearly.