Genome-wide analyses have been performed to identify genomic regions linked to longevity followed by identification of the genetic variation causal to the trait. Both genome-wide linkage studies (GWLS) in family-based designs and genome-wide association studies (GWAS) in population-based designs confirmed the role of APOE alleles in human longevity. These studies suggest that centenarians are likely to harbor rare protective variants with strong effects (Deelen et al., 2011; Passtoors et al., 2012). Considering the frequency of centenarians is only approximately 1/5000 individuals in the population (Andersen et al., 2012), these alleles may not be detectable in general populations. Indeed, a. Introduction
Stability is crucial for longevity, and evidence suggests that genome maintenance plays a significant role in ensuring it (Vijg and Suh, 2005). Defects in DNA repair pathways, such as nucleotide excision repair, base excision repair, homologous recombination, nonhomologous end joining, mismatch repair, and telomere maintenance, as well as genes regulating these pathways, lead to premature aging and shorter life spans in humans and mice (Garinis et al., 2008; Martin, 2005; Thomson and Guerra-Rebollo, 2010). Recent technology advancements have made it possible to identify genetic variations in the entire genome, including rare causal variants with large effects in population-based studies. However, the high cost and bioinformatics challenges of analyzing whole genome sequencing data can be overcome by taking a candidate approach to comprehensively sequence hundreds to thousands of genes implicated in aging and longevity pathways. Here we used a target capture and next-generation sequencing to discover rare but potentially causal variants in 988 candidate genes in six Ashkenazi Jewish centenarians older than 105 years of age. We selected candidate genes from neurodegeneration/cognitive function, lipid metabolism, DNA damage repair, and genome maintenance pathways because they are involved in pathways that have demonstrated roles in aging and longevity. We report the discovery and validation of novel non-synonymous variants discovered in six centenarians and further genotyping and association analysis of these variants in 410 controls and 390 centenarians of Ashkenazi Jewish descent.

There is increasing evidence that longevity genes protect against cognitive decline, dementia, and neurodegeneration including AD (Barzilai et al., 2006; Christensen et al., 2006; Sanders et al., 2010), consistent with the epidemiologic finding that dementia is absent or markedly delayed among centenarians (age of onset >93 years) (Hitt et al., 1999). For example, a functional longevity allele (p.I405V) in the cholesteryl ester transfer protein (CETP) gene is significantly associated with slower memory decline and lower risk for dementia and AD (Sanders et al., 2010). In addition, centenarians are enriched with the AD-protective APOE e2 allele, while they are depleted of the AD-predisposing APOE e4 allele (Christensen et al., 2006). Remarkably, a recent GWAS has demonstrated that SNPs associated with longevity are most significantly enriched in genes related to AD, dementia, and tauopathies (Sebastiani et al., 2012). Furthermore, genetic variations associated with longevity have been associated with beneficial lipid profiles. The APOE protein functions as a lipid transporter, and the APOE e2 allele was associated with a beneficial lipid profile and lower risk of coronary heart disease (Bennet et al., 2007). Longevity-associated variation (p.I405V) in the lipid transfer protein CETP was associated with larger particle size of LDL and HDL (Barzilai et al., 2003), and lipoprotein particle size was significantly increased among centenarians and their offspring compared to controls (Barzilai et al., 2003; Vaarhorst et al., 2011). The frequency of homozygotes for the 641C allele in the apolipoprotein-C3 (APOC3) promoter was higher in centenarians and their offspring when compared to controls (Atzmon et al., 2006). Therefore, we selected genes involved in pathways implicated in neurodegeneration, cognitive function, and lipid metabolism as our candidate genes.

2. Materials and Methods
In total, we used 800 human samples in this study: 410 Ashkenazi Jewish controls and 390 Ashkenazi Jewish centenarians previously collected as part of a longevity study by Dr. Nir Barzilai of the Albert Einstein College of Medicine (Barzilai et al., 2003). We obtained informed written consent in accordance with the policy of the Committee on Clinical Investigations of the Albert Einstein College of Medicine. A centenarian is defined as a healthy individual living independently at 95 years of age or older, and a control is defined as an individual without a family history of unusual longevity; parents of controls survived to the age of 85 years or less. The control group largely consisted of spouses of the offspring of centenarians. For target capture and next-generation sequencing of our candidate genes, we used genomic DNA extracted from immortalized B-lymphocytes.