variants hypothesised to be associated with in- creased longevity (reviewed in De Benedictis et al. 2001; Bessenyei et al. 2004; Lewis and Brunner 2004). These are typically either genes conferring susceptibility to age-related diseases or genes in- volved in cellular maintenance and repair (Lewis and Brunner 2004). Most studies have considered genes of the first type and, although studies have been made of polymorphisms in cellular detoxifi- cation genes (De Benedictis et al. 1998; Taioli et al. 2001; Christiansen et al. 2004; Pesch et al. 2004), with the exception of the investigation of polymorphisms within the BER protein poly (ADP-ribose) polymerase-1 (PARP) (De Benedic- tis et al. 1998; Muiras et al. 1998; Cottet et al. 2000), none of these have been DNA repair gene variants. Although PARP activity is greater in elderly individuals, no association has been found for single nucleotide polymorphisms within the PARP gene and longevity (De Benedictis et al. 1998; Cottet et al. 2000) or for an [AC] repeat in n the promotor sequence (Muiras et al. 1998).

DNA was extracted from blood using standard methods. Ethical approval was obtained from the West Cumbria Local Research Ethics Committee.

Genotyping analysis

Seven polymorphisms in four DNA repair genes were genotyped: 2 SNPs and 1 microsatellite in XRCC1 of the BER pathway, 1 SNP and 1 mi- crosatellite within XRCC3 of the HR pathway, 1 SNP in XRCC4 and 1 microsatellite in XRCC5 both components of the NHEJ pathway. Meth- ods for the R194W and R399Q SNPs in XRCC1 followed the method of Lunn et al. (1999), XRCC3 T241M was as described by Shen et al. (1998) and the I134T SNP in XRCC4 was

Here we compare DNA repair genotype frequencies of seven polymorphisms within four

Results

genotyped utilising the method of Relton et al. (2004). An [AC] repeat in the 3¢ UTR of n XRCC1, an intronic [AC] repeat in XRCC3 and n a [GAPyA] repeat situated 120 kb 5¢ of XRCC5 n were amplified using the primers of Price et al. (1997) with fluorescently tagged primers as described by Wilding et al. (2005). Briefly, fluor- escently tagged multiplex PCR products were separated on an ABI Prism 310 Genetic Analyser and allele sizes converted to repeat lengths based on allele size as described by Price et al. (1997) At least 10% of all genotyping was randomly under- taken in duplicate with no discrepancies found.

Genotype and allele frequencies are given in Table 1 for the four SNPs. Genotypes for two newborn samples and four retired workers were not available due to failure to amplify PCR product in any reaction. All four SNP polymor- phisms were found to be in Hardy–Weinberg equilibrium.

SNP genotype frequencies were not different between the male and female newborns (the gen- der of one individual was not available and therefore this individual was excluded from this analysis) thus, these were combined for assessing the association with age (data not shown). There is no evidence for differing gene frequencies between the newborn cohort and the retired worker population as evidenced by the v2 test results (Table 1).

Statistical analysis

undertaken according to standard methods. Comparison of newborn and retired worker genotype frequencies at the 4 SNP loci was undertaken using v2 analysis. The distributions of microsatellite repeat length frequencies between the newborn population and the aged populations were first compared using the log likelihood ratio test utilising S-plus. Where sig- nificance was encountered, numbers of individual repeats were compared using Fisher’s exact test. Due to multiple comparisons, significance levels were adjusted using the sequential Bonferonni

Distributions of repeat lengths at the three microsatellite loci are shown in Figure 1. Repeat lengths in the XRCC1 microsatellite ranged from 9 to 24, XRCC3 repeat lengths ranged from 11 to 22 and XRCC5 repeat lengths ranged from 14 to 18+2. The XRCC5 microsatellite is a tetrameric repeat, however, a small proportion of alleles (designated n+2) had additional 2 bp repeats (Price et al., 1997). Log likelihood ratio tests identified significant differences in the repeat dis- tribution between newborns and aged individuals

Table 1. Genotype frequencies, allele frequencies, tests for conformation to Hardy–Weinberg equilibrium and v2 comparison genotype frequencies between newborn and retired workers at four DNA repair loci.

Genotype n (%) Allele frequency H–W P XRCC1 R194W RR RW WW R W Newborns (n=288) 252 (87.5) 35 (12.2) 1 (0.3) 0.936 0.064 0.854 Retired workers (n=426) 382 (89.7) 43 (10.1) 1 (0.2) 0.947 0.053 0.855 v2=0.836; P=0.658 XRCC1 R399Q RR RQ QQ R Q Newborns 110 (38.2) 140 (48.6) 38 (13.2) 0.625 0.375 0.530 Retired workers 162 (38.0) 195 (45.8) 69 (16.2) 0.609 0.391 0.425 v2=1.33; P=0.514 XRCC3 T241M TT TM MM T M Newborns 112 (38.9) 135 (46.9) 41 (14.2) 0.623 0.377 0.976 Retired workers 168 (39.4) 200 (46.9) 58 (13.6) 0.629 0.371 0.901 v2=0.061; P=0.970 XRCC4 I134T II IT TT I T Newborns 262 (91.0) 26 (9.0) 0 (0.0) 0955 0.045 0.422 Retired workers 398 (93.4) 27 (6.3) 1 (0.2) 0.966 0.034 0.456 v2=2.463; P=0.292

0.5 XRCC1 0.4 0.3 0.2 0.1 * 0.0 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 0.5 XRCC3 0.4 0.3 * 0.2 * 0.1 0.0 11 12 13 14 15 16 17 18 19 20 21 22 0.5 XRCC5 0.4 * 0.3 0.2 * 0.1 0.0 14 14+2 15 15+2 16 16+2 17 17+2 18 18+2 ycneuqerF

Figure 1. Distribution of microsatellite repeat lengths in newborns (unfilled bars) and retired workers (solid bars) for XRCC1, XRCC3 and XRCC5. *P<0.0005 (Fisher’s exact test).

within all three microsatellite loci (XRCC1 LLR=17.12, P=0.05; XRCC3 LLR=58.35, P £ 0.0005; XRCC5 LLR=49.70, P £ 0.0005). Through the use of Fisher’s exact test corrected for multiple comparisons significant differences in repeat length distribution were identified for XRCC1 [AC] , XRCC3 [AC] and [AC] and 16 13 15 XRCC5 [GAPyA] and [GAPyA] . 15 16+2