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

Table 3

Genotypic analysis of novel nsSNPs in 410 controls and 390 centenarians.

Gene Transcript Nucleotide changea Amino acid change Homb count (%)c Hetd count (%) p-valuee Controls Centenarians Controls Centenarians PMS2 NM_000535 c.953A>G p.Y318C 1 (0.25) 0 (0.00) 2 (0.50) 10 (2.64) 0.02* GABRR3 NM_001105580 c.1394T>C p.V465A 1 (0.25) 0 (0.00) 8 (2.02) 1 (0.27) 0.04* ZMIZ1 NM_020338 c.1172C>T p.P391L 0 (0.00) 0 (0.00) 0 (0.00) 3 (0.82) 0.11 EPHB2 NM_017449 c.1106G>A p.R369Q 0 (0.00) 2 (0.52) 26 (6.45) 34 (8.90) 0.14 GRM1 NM_001114329 c.2581G>A p.G861S 0 (0.00) 2 (0.54) 14 (3.52) 18 (4.85) 0.23 PLCD4 NM_032726 c.118C>T p.L40F 0 (0.00) 0 (0.00) 0 (0.00) 2 (0.53) 0.23 AFTPH NM_203437 c.1796G>A p.R599Q 0 (0.00) 0 (0.00) 12 (2.96) 6 (1.58) 0.24 RELN NM_173054 c.9340A>G p.I3114V 0 (0.00) 0 (0.00) 9 (2.26) 4 (1.08) 0.27 PCDH11X NM_032968 c.3140G>A p.G1047E 0 (0.00) 0 (0.00) 5 (1.23) 9 (2.37) 0.28 RAD52 NM_134424 c.1186C>T p.R396C 0 (0.00) 1 (0.27) 14 (3.53) 18 (4.85) 0.32 CDC25A NM_001789 c.391A>G p.I131V 0 (0.00) 2 (0.53) 10 (2.50) 12 (3.18) 0.36 UBE3C NM_014671 c.1406G>A p.R469Q 0 (0.00) 0 (0.00) 1 (0.25) 3 (0.81) 0.36 OPRM1 NM_001008505 c.1256A>G p.Y419C 0 (0.00) 0 (0.00) 13 (3.23) 18 (4.75) 0.36 SENP6 NM_001100409 c.1447G>C p.D483H 0 (0.00) 0 (0.00) 1 (0.25) 3 (0.79) 0.36 ANAPC5 NM_016237 c.2027C>T p.A676V 0 (0.00) 0 (0.00) 9 (2.27) 13 (3.50) 0.39 ANAPC7 NM_001137664 c.96G>C p.Q32H 1 (0.25) 3 (0.79) 23 (5.68) 17 (4.50) 0.41 MLH3 NM_014381 c.3145G>A p.D1049N 0 (0.00) 0 (0.00) 11 (2.77) 14 (3.88) 0.42 FANCI NM_018193 c.1963G>A p.G655R 0 (0.00) 0 (0.00) 10 (2.51) 6 (1.60) 0.45 FOXO3 NM_001455 c.1021G>A p.A341T 4 (1.01) 1 (0.27) 36 (9.07) 30 (8.04) 0.46 PCDH10 NM_032961 c.1469G>T p.R490L 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.33) 0.47 DNAH1 NM_015512 c.10501A>G p.I3501V 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.27) 0.48 SHC1 NM_003029 c.768G>A p.M256I 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.27) 0.48 GRIA3 NM_000828 c.484G>A p.V162M 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.26) 0.48 CHRNG NM_005199 c.1343T>C p.I448T 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.26) 0.48 IL1A NM_000575 c.41G>C p.C14S 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.26) 0.48 OPRK1 NM_000912 c.991G>A p.A331T 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.26) 0.49 PCDH12 NM_016580 c.1453C>T p.H485Y 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.26) 0.49 USP24 NM_015306 c.1024A>G p.I342V 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.26) 0.49 CHEK2 NM_001005735 c.634T>G p.F212V 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.27) 0.49 GRIK2 NM_175768 c.109A>G p.R37G 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.26) 0.49 LTF NM_002343 c.1841C>T p.P614L 0 (0.00) 0 (0.00) 16 (3.99) 19 (5.19) 0.49 ATM NM_000051 c.4388T>G p.F1463C 0 (0.00) 1 (0.27) 21 (5.26) 24 (6.37) 0.49 FARP1 NM_005766 c.692C>T p.P231L 0 (0.00) 0 (0.00) 17 (4.19) 20 (5.28) 0.50 PPIL2 NM_148175 c.655G>A p.E219K 0 (0.00) 1 (0.27) 12 (3.02) 14 (3.76) 0.55 THBS1 NM_003246 c.583G>A p.A195T 2 (0.53) 1 (0.29) 10 (2.65) 14 (4.02) 0.56 POLD1 NM_002691 c.2275G>A p.V759I 0 (0.00) 0 (0.00) 14 (3.54) 17 (4.57) 0.58 HERC1 NM_003922 c.1130G>T p.W377L 0 (0.00) 0 (0.00) 1 (0.25) 2 (0.53) 0.61 PCDH15 NM_001142769 c.4669A>G p.K1557E 0 (0.00) 0 (0.00) 3 (0.75) 1 (0.27) 0.62 POLI NM_007195 c.1169C>G p.P390R 0 (0.00) 0 (0.00) 10 (2.51) 7 (1.87) 0.63 MAPK8IP3 NM_015133 c.1772G>A p.R591H 3 (0.75) 1 (0.27) 23 (5.78) 21 (5.74) 0.79 NF1 NM_001042492 c.7595C>T p.A2532V 0 (0.00) 0 (0.00) 8 (1.98) 6 (1.59) 0.79 HIF1A NM_181054 c.2080G>A p.A694T 0 (0.00) 0 (0.00) 13 (3.26) 11 (2.96) 0.84 SIRT2 NM_012237 c.347G>A p.R116H 2 (0.49) 1 (0.27) 25 (6.17) 24 (6.38) 1.00 POLE NM_006231 c.2131T>C p.S711P 0 (0.00) 0 (0.00) 2 (0.50) 1 (0.27) 1.00 MNAT1 NM_001177963 c.521T>C p.I174T 0 (0.00) 0 (0.00) 4 (0.99) 4 (1.06) 1.00 RAD23A NM_005053 c.434G>A p.G145D 0 (0.00) 0 (0.00) 1 (0.26) 1 (0.27) 1.00 MLH3 NM_014381 c.3145G>A p.D1049N 0 (0.00) 0 (0.00) 1 (0.25) 1 (0.26) 1.00 GOLM1 NM_177937 c.179G>A p.R60K 0 (0.00) 0 (0.00) 7 (1.75) 6 (1.61) 1.00 ABCA1 NM_005502 c.3053A>G p.D1018G 0 (0.00) 0 (0.00) 8 (1.98) 8 (2.12) 1.00 F13A1 NM_000129 c.1861G>T p.A621S 0 (0.00) 0 (0.00) 2 (0.52) 2 (0.57) 1.00 FARP1 NM_005766 c.2200A>G p.M734V 0 (0.00) 0 (0.00) 10 (2.51) 9 (2.45) 1.00

reached statistical significance due to the small sample size. In particular, we could detect additional centenarians harboring the p.P391L variant in ZMIZ1 or the p.L40F variant in PLCD4. Both SIFT and PolyPhen2 predicts those two amino acid substitutions to be ‘‘damaging’’ or ‘‘deleterious’’. Additionally, there were 11 variants that were detected only in the discovery panel of 6 centenarians. Interestingly, all but the p.I342V variantin USP24 were predicted to be functional by the SIFT or PolyPhen2 analysis. These variants could be good candidates for further genotyping analysis in a larger sample size and for functional analysis to assess the relevance of the genetic variations to longevity.

centenarians, including 710 nsSNPs. In this study, we followed up on 89 novel nsSNPs (1.3% of total SNPs and 12.5% of total nsSNPs), among which 67 nsSNPs (9.4% of total nsSNPs) were assayed for a validation study and genotyping analysis by iPLEX MassArray method. As expected, these novel nsSNPs were found to be rare in our populations (Table 2). Nevertheless, we could validate 51 novel nsSNPs (76%) to be true positives. We concluded that our customized target capture and next-generation sequencing can reproducibly detect genetic variation in candidate genes, licensing for a large scale genetic discovery study to identify all possible variations including rare variants in a cost-effective manner. Since our targets contain regulatory regions including 2 kb proximal promoter, exon–intron junctions, and 50 and 30 UTRs, we hope to

We identified a total of 7027 genetic variations (excluding variations annotated as ncRNAs) in our 988 candidate genes from 6