old) and 413 young subjects (Y, 22-37 years old, mean age 27.1 years old). The XPD gene, which encodes a DNA helicase involved in nucleotide excision repair and transcription, was analyzed for common polymorphisms that affect DNA repair efficiency and increase the risk of developing various cancers. The analysis was performed on blood mononuclear cells from people divided into three age groups (mean ages 28.7, 65.8 and 92.7 years old). The results showed that extreme longevity is associated with decreased expression of the XPD gene, and that the differences between young or middle-aged vs. extremely old group were significant. In addition, the methylation pattern of the XPD promoter was analyzed in 30 people divided into three age groups (29.5, 65.9, and 101.4 years old), and it was found that aging is associated with an increase in methylation level upstream of the transcription start site. The Lys751Gln genetic variant of the XPD gene was found to be associated with extreme longevity, while the Arg156Arg and Asp312Asn polymorphisms showed no such association in the Polish population. Genotyping of selected polymorphisms in the XPD gene was done by PCR amplification followed by digestion with an appropriate restriction enzyme.text were found (0.87%), in the M group the number increased to 6 (2.6%), and in the C group to 10 (4.3%). The difference between the Y and C groups was significant (P = 0.016), while the difference between the Y and M groups, as well as between the M and C groups, was not significant (P = 0.098, P = 0.202, respectively). No correlation was found between the level of XPD promoter methylation and age, sex, or smoking status.

Bisulfite treatment of DNA and DNA methylation analysis

Bisulfite modification of the genomic DNA was performed using EZ DNA Methylation-Gold Kit (Zymo Research Corporation, Orange, CA, USA). Five hundred nanograms of each genomic DNA was bisulfite-modified following the manufacturer's protocol with the only modification being the PCR incubation conditions; the samples were subjected to Table 1 PCR-RFLP conditions used for the analysis of the Arg156Arg, Asp312Asn, and Lys751Gln polymorphisms in the XPD gene. 

Primers T MgCl PCR Restriction Alleles m 2 ((cid:3)C) (mM) product (bp) enzyme F: 50CAAACATCCTGTCCCTACTGG30 62 1.5 309 HinfI C: 217 bp, 92 bp R: 50CTCCTGCGATTAAAGGCTGT30 A: 221 bp 

F: 50GCCCCTGTCTGACTTGTCC30 56.7 1.5 204 Eco103I G: 204 bp R: 50GCTCACCCTGCAGCACTTCCT30a (StyI) A: 181 bp, 23 bp 

F: 50GAGGTTCAAGTTGTCCAAAACC30 61 1.5 346 PstI A: 237 bp, 109 bp R: 50ACAAGAGTGCCAGGGGTCAG30 C: 174 bp, 109 bp, 93 bp 

a Underlined nucleotide is a mismatch introduced to create Eco103I (StyI) restriction T Melting temperature, F Forward, R Reverse, bp Base pairs m of a Arg156Arg b Asp312Asn c Lys751Gln

RNA isolation, reverse transcription, and real-time PCR

Bisulfite-treated DNA was purified and resuspended in 10 μl of the M-elution buffer supplied by the manufacturer. Fifty nanograms (1 μl) of this DNA was used as a template in a 12.5 μl PCR reaction performed with hot-start Platinum Taq polymerase (Invitrogen, Carlsbad, CA, USA) and with sense XPDsl 50GAGGATTAAATTTTAGTGAATGAGAT30 and antisense XPDasl 50ATAACCCCATCTTCAAAACCCCC30 primers. The PCR conditions were: initial denaturation at 94°C for 3 min, 40 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 1 min, final elongation at 72°C for 10 min. The PCR product of 377 bp, corresponding to the XPD promoter fragment located on both sides of the transcription start site, was gel-purified and cloned into the pGEM-T vector (Promega, Madison, WI, USA). Three clones from each person were sequenced.