Using the established iPLEX assays, we performed genotyping analysis in 410 controls and 390 centenarians. The result from allelic 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 six centenarians (minor allele frequency (MAF) = 0.1%) and two 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 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 centenarians (Table 3). Heterozygote genotype frequency for the c.953A>G variant in PMS2 was 0.5% in controls 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's genotype are considered to correspond to the same value of the dependent 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 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 six centenarians 

Analysis of the results from target capture and next-generation sequencing of six centenSNPs were successfully genotyped in multiplex assays for 410 controls and 390 centenarians. Out of 67 nsSNPs that were assayable for genotyping, 51 were validated as real variants. The genotyping analysis of these variants indicated that most of the novel nsSNPs had low frequency. A novel nsSNP in PMS2 (p.Y318C) was significantly enriched in centenarians (5.3 fold difference, p = 0.02), while a novel nsSNP in GABRR3 (p.V465A) was significantly depleted in centenarians (7.5 fold difference, p = 0.04), as compared to controls.

PMS2 is a component of the post-replicative DNA repair (MMR) system and is involved in apoptosis in the case of major DNA damage. Defects in this gene are involved in hereditary non-polyposis colorectal cancer type 4 (HNPCC4). Mice defective in this gene have a high mutation frequency but do not appear to age more rapidly than normal. Recent reports show that PMS2 deficiency prolonged the mean lifespan and median survival of telomerase-deficient mice concomitant with rescue of degenerative pathologies.

The PMS2 amino acid change (p.Y318C) was predicted to be possibly damaging or deleterious according to the in silico tools, SIFT and PolyPhen2, which predict the possible impact of an amino acid substitution on the structure or function of a protein. In addition, protein structural modeling of the PMS2 nsSNP suggests that the side chain of Y318 is exposed and could be mediating interactions with other factors.

Table 2 shows the allelotypic analysis of novel nsSNPs in 410 controls and 390 centenarians. Out of the 67 nsSNPs that were assayable for genotyping, 29 had a minor allele count of less than 1% in both controls and centenarians.NM_001142769 c.4669A>G p.K1557E 3 (0.38) 1 (0.13) 0.63 POLI 
NM_007195 c.1169C>G p.P390R 10 (1.26) 7 (0.94) 0.63 MAPK8IP3 
NM_015133 c.1772G>A p.R591H 29 (3.64) 23 (3.14) 0.67 NF1 
NM_001042492 c.7595C>T p.A2532V 8 (0.99) 6 (0.80) 0.79 HIF1A 
NM_181054 c.2080G>A p.A694T 13 (1.63) 11 (1.48) 0.84 SIRT2 
NM_012237 c.347G>A p.R116H 29 (3.58) 26 (3.46) 1.00 ANAPC7 
NM_001137664 c.96G>C p.Q32H 25 (3.09) 23 (3.04) 1.00 ABCA1 
NM_005502 c.3053A>G p.D1018G 8 (0.99) 8 (1.06) 1.00 GOLM1 
NM_177937 c.179G>A p.R60K 7 (0.88) 6 (0.81) 1.00 MNAT1 
NM_014381 c.3145G>A p.D1049N 1 (0.13) 1 (0.13) 1.00 POLE 
NM_006231 c.2131T>C p.S711P 2 (0.25) 1 (0.13) 1.00 RAD23A 
NM_000129 c.1861G>T p.A621S 2 (0.26) 2 (0.28) 1.00 F13A1 
NM_005766 c.2200A>G p.M734V 10 (1.25) 9 (1.23) 1.00 

a cDNA change.
b % minor allele frequency.
c Fisher's exact test.
* p<0.05.