IGF-1 signaling have been shown to delay aging and extend life span in model organisms (Bartke, 2011). In humans, genetic variations in this pathway are implicated in longevity through genetic association studies (Pawlikowska et al., 2009; Suh et al., 2008; van Heemst et al., 2005). DNA repair pathways counteract the deleterious effects of DNA damage, maintaining genomic stability. There is increasing evidence that genome maintenance is a major longevity assurance pathway (Vijg and Suh, 2005) and defects in this pathway lead to a shorter life span and premature aging in humans and mice (Garinis et al., 2008; Martin, 2005). Thus, for our study we included genes involved in major DNA repair pathways, including nucleotide excision repair, base excision repair, homologous recombination, nonhomologous end joining, mismatch repair, and telomere maintenance, as well as genes involved in regulation of these pathways such as ubiquityla- tion (Thomson and Guerra-Rebollo, 2010).

has opened up the door to discoverall possible genetic variations in the entire genome, including not only common alleles with small effects typically detected by GWAS, but also rare, causal variants with large effects in population-based studies. Currently whole genome sequencing results from one female and one male supercentenarian have been reported (Sebastiani et al., 2011). The bioinformatics challenge in analysis of whole genome sequencing data and high cost can be overcome by taking a candidate approach for comprehensive sequencing analysis of 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 6 Ashkenazi Jewish centenarians older than 105 years of age. Candidate genes from neurodegeneration/cognitive function, lipid metabolism, DNA damage repair and genome maintenance were selected because they are involved in pathways which have demonstrated roles in aging and longevity.

From our candidate aging and longevity pathways, we selected a total of 988 candidate genes spanning 5.69 Mb for target capture and next generation sequencing analysis (see SupplementaryTable S1 for entire gene list). We report discovery and validation of novel non-synonymous variants discovered in 6 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 amongst centenar- ians (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 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 offsprings compared to controls (Barzilai et al., 2003; Vaarhorst et al., 2011). The frequency of homozygotes for the (cid:3)641C allele in the apolipoprotein-C3 (APOC3) promoter was higher in centenarians and their offspring when compared to controls (Atzmon et al., 2006). Taken together, we selected genes involved in pathways implicated in neurodegeneration, cognitive function, and lipid metabolism as our candidate genes.

2. Materials and Methods

In total, 800 human samples were used in this study: 410 Ashkenazi Jewish controls and 390 Ashkenazi Jewish centenarians that were previously collected as partofalongevitystudybyDr.NirBarzilaioftheAlbertEinsteinCollegeofMedicine (Barzilaietal.,2003).AllbloodsampleswererapidlyprocessedtoobtainDNAatthe General Clinical Research Center of the Albert Einstein College of Medicine. Informed written consent was obtained 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 consisted largely 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.

2.2. Target selection and generation of customized target capture