depletion of heterozygotes, whereas the frequencies of CASP5 genotypes in the control group perfectly respected Hardy–Weinberg distribution (Table 2). In the pooled sample of elderly subjects, only 205 (40%) genotypes were heterozygous, while the expected number of heterozygotes was equal to 254 (50%) (p=0.000014). When we compared CASP5 allelism in the pooled group of elderly subjects against healthy controls, it was evident that despite nearly identical minor allele frequency (0.470 vs. 0.475) there was a depletion of heterozygotes in the aged group (40% vs. 49%) at the expense of the increase of both AA and GG homozygous genotypes (33% vs. 28% and 27% vs. 23%, respectively). Furthermore, when the studied subjects were stratified according to smoking status and gender, this deviation retained statistical significance both in males and females as well as both in smokers and non-smokers (Table 3). On the contrary, this stratification did not reveal deviations from Hardy–Weinberg equi- librium in the reference subjects (Table 3). Given convincing statistical values and consistency of all performed comparisons (Tables 2 and 3), one may conclude that heterozygosity for CASP5 Ala90Thr polymorphism is negatively associated with life expectancy. We also considered differences in the genotype distribution

2. Materials and methods

While analyzing genetic predisposition to lung cancer (LC), we screened 111 LC patients with apparent characteristics of “extreme” disease susceptibility such as early onset and either non-smoking status or relatively modest smoking history. “Supercontrol” for lung cancer cases consisted of 110 elderly tumor-free heavy smokers (Ulybina et al., 2009). Similarly designed, yet unpublished study has been carried out for breast cancer (BC) predisposition. A group of “extreme” BC susceptibility consisted of 121 so-called “high-risk” BC patients characterized by early onset of the disease (=/b40 years), and/or first-degree-family history of BC, and/or bilateral cancer lesion. “Supercontrol” for BC cases included 142 elderly tumor-free females. When the depletion of CASP5 Ala90Thr heterozygotes was noticed in both of the above elderly groups, a validation study has been designed. Additional group of elderly subjects consisted of 258 elderly tumor-free individuals. 549 population controls were represented by middle-aged blood donation volunteers. All study participants were recruited in St.-Petersburg residential area, belonged to white race and had predominantly Slavic origin. The exact characteristics of the study groups are presented in Table 1.

We also considered differences in the genotype between cancer patients and non-affected subjects. However, LC nor BC patients demonstrated statistically significant from the comparison groups (Table 2).

4. Discussion

There are some hypotheses suggesting negative impact of genome heterozygosity on longevity. It is reasoned that heterozygous organisms have increased intensity of metabolic processes resulting in higher rate of development, and, consequently, in accelerated ageing (Altukhov and Moskaleichik, 2006). In support of this assumption, Bonafè et al. (2001) observed decreased heterozygosity in centenarians using inter-Alu PCR. However, other evidence for negative impact of “general” heterozygosity on life expectancy are lacking. Furthermore, while analyzing the distribution of 19 coding polymorphisms of apoptotic gene SNPs in elderly tumor-free subjects, we did not observe general trend for increased homozygosity in aged population (Ulybina et al., 2009 and unpublished observations). Therefore, it can be suggested that the depletion of heterozygous genotypes in geriatric patients is specific for the CASP5 Ala90Thr polymorphism.

Genotyping for CASP5 Ala90Thr polymorphism was performed by real-time allele-specific PCR using variant primers 5′-CCTTGTCTTCAA- TTTTGGT-3′ and 5′-CCTTGTCTTCAATTTTGGC′-3 and common primer 5′- CAGTTAAGATGTTGGAATAC-3′ (Ulybina et al., 2009). Statistical analysis was done using chi-square test.

3. Results

While analyzing the distribution of CASP5 Ala90Thr polymorphism in LC cases vs. elderly tumor-free smokers and in BC patients vs. elderly tumor-free women, we observed a statistically significant reduction of frequency of heterozygotes in both aged groups (Table 2). In order to validate this unusual finding, we considered the rest of our DNA collection of tumor-free elderly subjects, and selected all those who were 85 years or older (n=258). DNA samples from 549 middle-aged donors served as control. Similarly to the initial observation, aged subjects demonstrated statistically significant

Possible explanation for age-related differences in the frequency of the CASP5 heterozygotes can be drawn from the inflammatory function of this caspase. It is commonly accepted that chronic inflammation is a substantial component of pathogenesis of ageing (Ostan et al., 2008; Pawelec and Larbi, 2008). One may speculate, that the presence of both alternative alleles of the CASP5 enhances propensity to inflammation thus accelerating ageing-related process- es. Similar observations for immune-related genes have been reported

Table 1

Characteristics of the studied subjects.

Study groups Number of Males/ Mean age Smokers/ Mean tobacco exposure for smokers (range), subjects females (range), years non-smokers pack-years Elderly subjects Elderly tumor-free smokers (N/=75 years) 110 110/0 79 (75–89) 110/0 55 (30–126) Elderly tumor-free women (N/=75 years) 142 0/142 80 (75–91) 14/127a 10 (1–48) Elderly tumor-free subjects (N/=85 years) 258 86/172 88 (85–103) 41/217 24 (5–50) Comparison groups Lung cancer patients, young-onset and/or 111 96/15 54 (32–64) 94/17 30 (10–40) non-smokers “High-risk” breast cancer patients 121 0/121 45 (25–78) Not recorded Not recorded Middle-aged healthy subjects 549 207/342 35 (18–50) 327/222 8 (1–36)