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

 67 SNPs successfully genotyped in multiplex assays(410 controls and 390 centenarians) 76% 24%51 SNPs 16 SNPsconfirmed not confirmed   Example: PMS2 c.953A>G Example: USP24 c.1024A>G Cent 910_AG call Ctrl 8596_GG call Ctrl 9301_AA call Cent 6959_AA call

Fig.1.Schematicoverviewofresultsfromsequencingandgenotypinganalysis.(A)DiscoveryofSNPsbytargetcaptureandnext-generationsequencingin6AJcentenarians. Total numbers of SNPs discovered in each category are indicated. (B) Genotyping and validation analysis of novel nsSNPs by MassArray iPLEX assays. Genotyping was performed for 410 controls and 390 centenarians. Total numbers of assayable, confirmed, and not confirmed nsSNPs are indicated. c.953A > G in PMS2 and c.1024A > G in USP24 represent specific examples of confirmed nsSNP and not confirmed calls, respectively.

functional rare variants, among which 67 nsSNPs were assayable for genotyping and 51 nsSNPs were validated to be real variants. Our genotyping analysis of these variants in 410 AJ controls and 390 AJ centenariansindicated that indeed mostof the novel nsSNPs are rare in frequency (Tables 2 and 3). Although we had a limited power to detect significant association of rare variants (MAF < 1.5%) with this sample size, we found that 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 (Table 3).

apoptosis in the case of major DNA damage (Kadyrov et al., 2006; Sacho et al., 2008). It is well reported that defects in this gene are involved in hereditary non-polyposis colorectal cancer type 4 (HNPCC4) (Gill et al., 2005). Mice defective in this gene (Pms2) have about a 100-fold elevated mutation frequency in all tissues, but they did not appear to age more rapidly than normal (Narayanan et al., 1997). However, there are recent reports showing that PMS2 deficiency prolonged the mean lifespan and median survival of telomerase-deficient mice concomitant with rescue of degenera- tive pathologies (Siegl-Cachedenier et al., 2007). PMS2 amino acid change (p.Y318C) was predicted to be possibly damaging or deleterious according to the in silico tools, SIFT (Kumar et al., 2009) and PolyPhen2 (Adzhubei et al., 2010), which predict the possible impact of an amino acid substitution on the structure or function of a protein. In addition, a protein structural modeling of the PMS2 nsSNP suggests that the side chain of Y318 (Fig. 2, red spheres) is exposed and could be mediating interactions with other factors.

PMS2 is a component of the post-replicative DNA repair (MMR) system. It heterodimerizes with MLH1 to form and introduces single-strand breaks near the mismatch generates new entry points for the exonuclease EXO1 to the strand containing the mismatch. It is also implicated in damage signaling which induces cell cycle arrest and can lead

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/

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Table 2

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