Table 3

Subgroup analysis (females only) of EXO1 SNPs (German sample).

No. dbSNP ID Location Alleles MAF controls Corresponding AF P P P OR 95% CI CCA CCG APOE APOE (n=332) centenarians (n=313) 1 rs1776181 Promoter A/G 0.474 (A) 0.558 0.0032 0.0131 0.0014 1.45 1.16–1.83 2 rs1776180 Promoter C/G 0.427 (C) 0.513 0.0023 0.0060 0.0012 1.47 1.16–1.85 3 rs1776178 50-UTR A/G 0.409 (A) 0.495 0.0022 0.0047 0.0013 1.47 1.16–1.85 4 rs1635517 50-UTR G/A 0.423 (G) 0.515 0.0011 0.0025 5.31(cid:4)10(cid:3)4 1.52 1.19–1.92 5 rs1776177 50-UTR C/T 0.476 (T) 0.391 0.0026 0.0070 6.92(cid:4)10(cid:3)4 0.67 0.53–0.84 6 rs735943 Exon 10 A/G 0.405 (A) 0.502 5.90(cid:4)10(cid:3)4 4.75(cid:4)10(cid:3)4 1.66(cid:4)10(cid:3)4 1.56 1.23–2.00 7 rs4149965 Exon 11 G/A 0.273 (A) 0.226 0.0509 0.1495 0.0426 0.84 0.71–0.99

For abbreviations see legend to Table 2.

(P = 7.23 Bx  10(cid:3)7, OR = 0.42, 95% CI = 0.30–0.59, logistic sion with an additive model for the APOE allele e4).

Since allelic variants may potentially influence the longevity phenotype in men and women differently (Candore et al., 2006; Franceschi et al., 2000), we also performed a sex-stratified analysis. In men, no significant association was detected (data not shown). In women, all SNPs but one showed a stronger effect (Table 3) than in the analysis of both genders combined (Table 2).

In order to investigate the functional role of the two promoter SNPs rs1776181 and rs1776180, we established reporter gene constructs based on the four possible haplotypes (i.e. AG, GG, AC, GC, see Fig. 2A). Insertion of any promoter fragment increased reporter gene activity by a factor of up to 90 compared to the empty vector control. More importantly, our data indicate a

To evaluate the functional role of the seven associated SNPs in the context of the DNA repair capacity and expression regulation of EXO1, various in vitro assays were applied.

    (A) 30 ee**202=a100A G A G 181776181G G c Cc 1817761800.113 0.458 ~=—-0.369—=SS(0.060 __haplotypefrequency(B<2aon2axo<za€=5p6s cc ce 181776180(n=23) (n=10) (n=36)

Coding SNP variants do not affect DNA repair capacity of

Exo1 nuclease is involved in various DNA metabolic pathways, e.g. homologous recombination and mismatch repair. Since human EXO1 protein can complement its yeast homologue in preventing DNA damage induced by UV-radiation (Qiu et al., 1999), complementation studies using UV-sensitive knock-out yeast strains represent a well-established experimental model to study EXO1 function. To assess the effect of the two EXO1 cSNPs, we over- expressed all four variants of human EXO1 protein (H354R substitution corresponding to rs735943; V458M substitution corresponding to rs4149965) in exo1-deficient UV-sensitive FDER yeast cells (Dexo1/Drad51). All EXO1 proteins in our study were able to desensitize these yeast cells from DNA damage-induced cell death. We observed no quantitative differences between the four protein variants in terms of their ability to protect yeast cells from UV-induced DNA damage (Fig. S1A and B). We conclude that the EXO1 cSNP alleles do not influence the ability to repair UV-induced DNA damage. Given the multiple roles of EXO1 in the maintenance of genomic stability, we cannot exclude the possibility that the two analyzed cSNPs may be functionally relevant in a different context. Nevertheless, bioinformatic analyses of the variants revealed no evidence for functional consequences at the level of enzymatic activity, protein phosphorylation status or domain integrity (not shown).

Fig. 2. The C allele of EXO1 SNP rs1776180 contributes to increased promoter activity.(A)DualluciferaseassayswithreportergeneconstructsrepresentingEXO1 promoter haplotypes were used to quantify promoter activity. HeLa cells were transfected and used for quantification of constitutive promoter activity. As vector control (vc), empty vector pGL3Enhancer was used. Haplotypes are based on the SNP order rs1776181 and rs1776180. Haplotype frequencies as observed in the control individuals are given below each column. Data are expressed as relative light units (RLU) and represent mean values + SD (n = 3). Both haplotypes containing the C allele of rs1776180 showed a 6–9-fold higher activity than the corresponding haplotypes carrying the G allele (**P < 0.01 for both comparisons), irrespective of the allele at rs1776181. (B) Genotype-specific expression of EXO1 in female lymphoblastoid cells. Real-time PCR was used to quantify the relative expression level of EXO1 mRNA in human lymphoblastoid cells in relation to the rs1776180 genotypes. Data represent the mRNA expression level of EXO1, normalized to housekeeping gene b-actin, box-plots represent the 25th, 50th and 75th percentile, all relative expression values are displayed individually. In females, genotype CC (n = 10, diamond) of rs1776180 is associated with a 1.34-fold higher relative EXO1 expression than genotype GG (n = 23, filled circle) and with a 1.45-fold higher relative EXO1 expression than genotype CG (n = 36, filled triangle). Significance was tested using the Mann–Whitney U-test (*P < 0.05).

3.3. 50-UTR haplotypes of EXO1 do not differentially influence reporter gene activity

To analyze the effect of the three SNPs located in the EXO1 50- UTR (rs1776178, rs1635517 and rs1776177), we inserted a 584 bp 50-UTR fragment in front of luciferase mRNA to create a heterologous reporter gene system. Using site-directed mutagen- esis, we established constructs representing the common UTR haplotypes that show a frequency >1% in the German control individuals (i.e. AGC, GGC, AGT, GAC and GAT, see Table S4). Comparison of the five 50-UTR haplotypes by dual luciferase assay in HeLa cells did not reveal any differential reporter gene levels (Fig. S2), indicating that none of the analyzed 50-UTR haplotypes influences post-transcriptional processing or translation efficiency of EXO1.