* P value was calculated by a by chi-square (v2) test using a 3 9 2 table to compare

people, mean age 65.9 years old), and 100–102.4 (C, 10 people, mean age 101.4 years old) years old. This fragment contains 35 potential methylation sites: 23 upstream and 12 downstream of the transcription start site. Sequencing of three clones from each person (30 in total in each age group) showed that, overall, the analyzed promoter fragment was not heavily meth- ylated. In 3 Y, 2 M, and 2 C subjects it was methylation-free, while in the remaining persons 1–3 out of 35 potential sites were methylated. In total, out of 350 potential methylation sites (100%) in each age group, in the Y group methylation was present in 11 sites, in the M group in 16 sites, and in the C group in 15 sites (Fig. 3) which corresponds to 3.14, 4.57, and 4.29%, respectively. The differences between the Y and M, Y and C, as well as the M and C age groups were not significant (P = 0.345, P = 0.513, P = 0.857, respectively). However, older age was associated with the increase of methylation at the sites located upstream of the transcription start site (230 potential sites—100%—in each age group): while in the Y group only 2 methylated sites within this promoter fragment were detected, in the M group methylation number raised to 9, and in the C group to 7 which corresponds to 0.87, 3.91, 3.04%, respec- tively. The difference between Y and M groups was significant (P = 0.043), while the difference between Y and C, as well as M and C groups did not reach the level of significance (P = 0.1, P = 0.966, respec- tively). No clear correlations were noticed between methylated sites and the relative XPD mRNA level in subjects in which both analyses were performed.

the C group and tended to decrease with age, paralleling the decreasing levels of the XPD mRNA in older subjects. When the mean level of the XPD mRNA in young Y subjects was normalized to 100% (±45.16% SD), the relative mean amounts of this mRNA were 99.05 ± 31.21% in middle-aged M group and 60.30 ± 24.47% in extremely old C group. Statistical analysis showed that the mean level of the specific mRNA was significantly lower in the C group compared to the Y and to the M group (P \ 0.0001, P \ 0.0001, respectively). The differ- ence between the Y and M was not significant (P = 0.941, Fig. 2).

The methylation status of the 377 bp (-278 to ?99) fragment of the XPD promoter was assessed in 30 people divided into three age groups: 25–37 (Y, 10 people, mean age 29.5 years old), 65.3–66.5 (M, 10

p<0.0001 % p=0.941 150 p<0.0001 125 100 75 50 25 0 Y M C

Discussion

Recently, it has been shown that the levels of micronuclei and chromosome aberrations in the cells of the elderly are significantly higher than in the cells of young people (Kazim´ırova´ et al. 2009). This supports the notion that DNA repair capacity decreases with age (Burhans and Weinberger 2007; Slijepcevic 2008; Schumacher et al. 2008). XPD is involved in the maintenance of genomic stability. Its effectiveness depends both on the level of its expression and on its polymorphic variant. In this work we show that aging is associated with the progressive decrease of the amount of XPD mRNA and with the change of methylation pattern of the

Fig. 2 Mean amounts of XPD mRNA in blood mononuclear cells of young, middle-aged and extremely old human subjects. The mean amount of the XPD mRNA is significantly lower in blood mononuclear cells of extremely old persons than in younger age groups. Assessment of the XPD mRNA was performed using real-time PCR. Results obtained for the XPD mRNA were normalized against those of b-actin mRNA. Y (young) 22–37 years old subjects, n = 19; M (middle-aged) 60–69 years old subjects, n = 18; C (extremely old) 90– 100 years old subjects, n = 17

clear correlation of the detected methylation sites with the amount of XPD mRNA. There are two potential explanations of this observation. First, the pattern of methylation in clones isolated from the same donor usually was not identical and, since the number of analyzed clones was limited to three, it was impossible to judge which pattern dominates in a given donor’s BMCs and to correlate it with the mRNA level. Second, even though the pattern of methylation is different in young and in old cells, methylation might have no major impact on the XPD promoter’s transcriptional activity. Instead, other regulatory mechanisms such as microRNA might be responsible for controlling the XPD mRNA level. Which hypothesis is true should be verified by in vitro studies.

Y1 Y7 Y16 Y17 Y31 Y45 Y49 Y113 Y117 Y164 M422 M423 M439 M440 M443 M444 M445 M447 M449 M456 C218 C224 C407 C495 C524 C576 C580 C583 C625 C627