3.2. Validation of novel nsSNPs discovered in 6 centenarians

To validate the 89 novel nsSNPs discovered in our candidate genes from 6 centenarians, we designed iPLEX genotyping assays which allowed us to detect 67 nsSNPs in 4 multiplexed assays (Fig. 1A). A total of 51 nsSNPs (76%) were confirmed by genotyping. Sixteen nsSNPs (24%) were considered not confirmed because the genotyping assays could not detect heterozygote calls in the same individuals that the novel nsSNPs were initially discovered and in a larger number of controls (n = 410) and centenarians (n = 390) (Fig. 1B).

3.3. Genotyping and association analysis of novel nsSNPs in 410 controls and 390 centenarians

2.5. Genotyping

To validate novel nsSNPs discovered in 6 centenarians, we designed iPLEX MassArray assays (Sequenom) using the web-based assay design suite program on mysequenom website (http://www.mysequenom.com). From a total of 89 novel nsSNPs, 67 nsSNPs were designed for iPLEX assays in 4 separate multiplexing groups based on the high quality of peaks visualized in the MassARRAY1 Typer software after performing MassArray. Using the iPLEX assays, genotyping was performed in 410 controls and 390 centenarians in the Genomics Shared Facility at Albert Einstein College of Medicine.

Using the established iPLEX assays, we performed genotyping analysis in 410 controls and 390 centenarians. The result from allelotypic analysis is shown in Table 2 with alleles ranked according to nominal p-values by Fisher’s exact test. We found a total of 13 nsSNPs that were detected only in centenarians but not in controls. These include 11 nsSNPs (c.1469G > T in PCDH10, c.10501A > G in DNAH1, c.768G > A in SHC1, c.484G > A in GRIA3, c.1343T > C in CHRNG, c.41G > C in IL1A, c.991G > A in OPRK1, c.1453C > T in PCDH12, c.1024A > G in USP24, c.634T > G in CHEK2 and c.109A > G in GRIK2) that were detected only in the initial discovery panel of 6 centenarians (minor allele frequency (MAF) = 0.1%) and 2 nsSNPs (c.1172C > T in ZMIZ1 and c.118C > T in PLCD4) for which additional centenarians were also found to be carriers. Most of the novel nsSNPs were rare with MAF less than 1%, and thus significance of rare alleles could not be estimated with the current sample size. Notably, MAF of the c.1394T > C in GABRR3 was significantly higher in controls as compared to centenarians (1.25% vs. 0.13%, p = 0.01). MAFs of some of the novel nsSNPs reached up to 5%, e.g. c.1021G > A in FOXO3, but none of them showed significant differences in frequencies between controls and centenarians. Genotypic analysis identified two novel nsSNPs (c.953A>G in PMS2 and c.1394T>C in GABRR3) that were significantly different between controls and centenar- ians (Table 3). Heterozygote genotype frequency for the c.953A>G variant in PMS2 was 0.5% in controls as compared to 2.64% in centenarians (5.3 fold difference, p = 0.02), while that of c.1394T>C variant in GABRR3 was higher in controls than in centenarians (2.02% and 0.27%, p = 0.04). No other genotypes showed significant differences in frequencies between controls and centenarians.

2.6. Statistical analysis

The association analyses were performed using SNP & Variation suite version 7.6.11 (Golden helix). For a basic allelic test, the genotypes dd, Dd, and DD were resolved into pairs of alleles d and d, D and d, or D and D. Both elements of each subject’sgenotypeareconsideredtocorrespondtothesamevalueofthedependent variable. The associations with these individual alleles are then tested. For a genotypic test, the genotypes dd, Dd, and DD were tested without regard to any ‘‘order’’, allelic count, or allelic pairing they might have. Statistical differences among groups were assessed by the Fisher’s exact test, for both allelotype and genotype distribution comparisons. A two-tailed p-value of <0.05 was considered significant.

3. Results

3.1. Discovery of novel nsSNPs in 988 candidate genes from 6 centenarians

Analysis of the results from target capture and next-generation sequencing of 6 centenarians (Table 1) identified a total of 7027 genetic variations (excluding variations annotated as ncRNAs) in our candidate genes, among which 1749 variations were located in exonic regions including 710 nsSNPs. In this study, we focused on novel nsSNPs that have not been discovered in other studies as they may exert strong effects leading to the rare phenotype of exceptional longevity in humans by directly affecting protein structures and/or functions. We identified 89 out of 710 nsSNPs as novel after comparing our sequence results to the dbSNP 132

4. Discussion

This is a pilot study to explore the hypothesis that individuals with exceptional longevity such as centenarians harbor individu- ally rare but collectively more frequent functional variants in major pathways of aging and longevity as compared to controls (Suh et al., 2008; Tazearslan et al., 2012). We capitalized on recent advances in next-generation sequencing technologies to identify novel, potentially causal variants in 988 candidate genes involved in major pathways of aging and longevity from 6 Ashkenazi Jewish centenarians who were 105 years or older at the time of sample collection. No such scale of a study has been reported thus far in genetics studies of human longevity. In this study, we focused on 89 novel nsSNPs discovered from 6 centenarians as potentially

Table 1

DemographicinformationofAJCentenarianstudysubjects forsequencinganalysis.

Gender Age of recruitment M 105 M 106 M 106 F 107 F 108 F 109

‘‘M’’ indicates male, ‘‘F’’ indicates

Please cite this article in press as: Han, J., et al., Discovery of novel non-synonymous SNP variants in 988 candidate genes from 6 centenarians by target capture and next-generation sequencing. Mech. Ageing Dev. (2013), http://dx.doi.org/10.1016/

J. Han et al./Mechanisms of Ageing and Development xxx (2013) xxx–xxx

Total SNPs (7,027)Exonic SNPs (1,749)nsSNPs (710)Novel SNPs y MassArray iPLEX