Total RNA was extracted from BMCs with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the procedure supplied by the manufacturer. One hundred nanogram of each RNA was used for reverse transcription that was performed with RevertAid First Strand cDNA Synthesis Kit (Fermentas, Lituania) according to the manufacturer’s protocol. The result- ing cDNA was diluted in DNAse, RNAse-free dH O 2 (Invitrogen, Carlsbad, CA, USA). Next, 1 ll of cDNA corresponding to 0.5 ng of total RNA was used as a template in real-time PCR. The reaction was performed in LightCycler 480 Instrument II (Roche, Mannheim, Germany) with LightCycler 480 Sybr Green I Master kit (Roche, Mannheim, Ger- many) and with XPD 50CATTTCCTGGGCTTCCTG AG30 sense and 50GCTCTCCTGCACCACATGCT30 antisense primers, as well as with sense 50CAGCCT GGATAGCAACGTACA30 and 50TTCTACAATGA GCTGCGTGTG30 antisense b-actin control gene primers. PCR conditions were: initial incubation at 95(cid:3)C for 10 min, 40 cycles of 95(cid:3)C for 12 s, 60(cid:3)C for 12 s, 72(cid:3)C for 12 s, then one melting curve cycle. The results obtained for the XPD gene were normal- ized against the results for the b-actin gene.

Blood mononuclear cells (BMCs) were isolated according to the method of Neitzel (Neitzel 1986) with modifications. Briefly, 4 ml of full blood were centrifuged at 25009g for 20 min at 4(cid:3)C. The bottom layer of approximately 2 ml, containing all types of blood cells, was diluted in 4 ml of phosphate- buffered saline (PBS) and the resulting suspension was overlayed onto 3 ml of lymphocyte separation medium LSM 1077 (PAA Laboratories GmbH, Pasching, Austria) or of LymphoSep (MP Biomed- icals, Irvine, CA, USA). The samples were centri- fuged at 6009g for 20 min at 19(cid:3)C. The layer of BMCs was collected and washed twice with PBS.

Statistical analysis

All analyses were performed using the Statistica software package (StatSoft, Tulsa OK). Hardy–Wein- berg equilibrium and differences in genotype frequen- cies between the analyzed groups were checked with the chi-square test. The distribution of genotypes was analyzed in three models of inheritance: dominant,

recessive and codominant using 2 9 2, 2 9 2, 3 9 2 contingency tables, respectively. The differences in quantitative parameters between groups were assessed using t-student/U Mann–Whitney test. For all tests the level of significance was established at 0.05.

heterozygotes were more frequent (57 vs. 44%, P = 0.0047, OR = 1.72, [95%CI: 1.18–2.51] than in the young controls. For the Arg156Arg and Asp312Asn polymorphisms no significant differences in the genotypes distribution were observed in the studied groups. Exclusion of the subjects suffering from cancers (n = 8) or myocardial infarction (n = 10) from the C group had no major influence on the genotypes frequencies in this group (data not shown).

Results

Genotyping results

XPD expression in blood mononuclear cells

The frequencies of each genotype in centenarian group of 149 subjects (C, 99.6–107.2 years old, mean age 101.1 years old, 130 females, 19 males) and in young control group of 413 subjects (Y, age range 18–45 years old, mean age 27.1 years old, 232 females, 181 males) are shown in Table 2. No significant difference in the distribution of genotypes in females and in males was observed. The distribu- tion of Lys751Gln genotypes differed significantly between the studied groups (P = 0.017). In cente- narians, the homozygous genotypes AA and CC were found less frequently (29 vs. 36%, OR = 0.71 [95%CI: 0.47–1.07], and 14 vs. 20%, OR = 0.652 [95%CI: 0.39–1.09], respectively), whereas the AC

XPD mRNA level in blood mononuclear cells was assessed in 54 people divided into three age groups: 22–37 years old (Y, 19 people, mean age 28.7 years old), 60–69 years old (M, 18 people, mean age 65.8 years old), and 90–100 years old (C, 17 people, mean age 92.7 years old), using real-time PCR. The results obtained for the XPD gene were normalized against the levels of the control b-actin mRNA. Real- time evaluation showed that the level of the specific XPD mRNA varied significantly between cases within each age group: the difference ranged up to 6.5-fold in the Y, 2.5-fold in the M, and twofold in

Table 2 Distribution of the XPD genotypes in centenarians and in young controls

Polymorphism Genotypes Centenarians Controls P n = 148 % n = 413 % Arg156Arg CC 53 36.0 137 33.0 [0.05 CA 69 47.0 191 46.0 AA 26 18.0 85 21.0 Polymorphism Genotypes Centenarians Controls P n = 148 % n = 409 % Asp312Asn GG 57 39.0 141 34.0 [0.05 GA 73 49.0 205 50.0 AA 18 12.0 63 15.0 Polymorphism Genotypes Centenarians Controls P n = 149 % n = 413 % Lys751Gln AA 43 29.0 150 36.0 0.017 AC 85 57.0 180 44.0 CC 21 14.0 83 20.0

n Number of subjects available for analysis * P value was calculated by a by chi-square (v2) test using a 3 9 2 table to

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