Contents lists are available at the Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero Evidence for Depletion of CASP5 Ala90Thr Heterozygous Genotype in Aged Subjects Yulia M. Ulybina a, Ekaterina Sh. Kuligina a, Nathalia V. Mitiushkina a, Nathalia Yu. Sherina a, Grigoriy A. Yanus a,b, Tatiana V. Gorodnova a,b, Anna S. Katanugina a, Andrey V. Koloskov c, Alexandr V. Togo a, Evgeny N. Imyanitov a,b,d, a N.N. Petrov Institute of Oncology, Pesochny-2, St.-Petersburg, Russia b St.-Petersburg Pediatric Medical Academy, Russia c 26th City Hospital, St.-Petersburg, Russia d St.-Petersburg Medical Academy for Postgraduate Studies, Russia Our previous studies included genotyping of multiple coding apoptotic gene polymorphisms and unexpectedly demonstrated a depletion of heterozygous CASP5 Ala90Thr (rs507879, c.268 GNA) genotypes in elderly subjects. The present investigation aimed to validate this trend. An analysis of 510 subjects aged 75-103 years revealed 205 (40%) CASP5 Ala90Thr heterozygotes compared to 254 (50%) expected from the minor allele frequency of 0.470 (p=0.000014). This deviation was not observed in 549 middle-aged (18-50 years) controls (270 (49%) heterozygotes observed vs. 274 (50%) expected; minor allele frequency 0.475; p=0.743). The unfavorable significance of the CASP5 heterozygous genotype may be explained by the role of caspase-5 in inflammation-related processes. Almost all prior gene-longevity association studies focused on discriminating between good and bad gene variants. Here we present a distinct situation where the combination of alternative alleles (i.e., heterozygosity) appears to be unfavorable compared to the single case-control comparison. Genome-wide association studies already led to the identification of new genetic determinants for common age-related diseases (Kronenberg, 2008). However, their immediate use in genuine longevity research is compromised by the requirement for an extremely large study size (Franceschi et al., 2007; Lunetta et al., 2007). Introduction Life expectancy is beyond any reasonable doubt. Several allelic variants related to either longevity per se or resistance to common age-related diseases have been identified within the recent decade, and many more remain to be discovered in future years (Franceschi et al., 2007; Lunetta et al., 2007). The contribution of ApoE polymorphism in the risk of Alzheimer's disease, cardiovascular diseases, and overall lifespan is frequently quoted as the most reproducible observation in molecular epidemiology of aging (Lewis and Brunner, 2004). Some studies demonstrated age-related differences in p53 Arg/Pro allele distribution (Bojesen and Nordestgaard, 2008). The analysis of IGF1 signaling pathway genes led to the identification of highly reproducible association between FOXO3A gene polymorphism and survival chances (Flachsbart et al., 2009). Previous research in this field was based on a candidate gene approach and included participants from lipid metabolism, energy production and storage, DNA repair, oxidative stress, inflammation, and other relevant pathways. Recent invention of so-called genome-wide association studies (GWAS) provided a possibility to assess the distribution of nearly all known SNPs within almost all positive gene-longevity association studies reported distinct frequencies of at-risk allele-containing genotypes in elderly subjects vs. controls, i.e., they discriminated between good and bad gene variants (Franceschi et al., 2007; Lunetta et al., 2007). Here we present an unusual observation where the presence of either of alternative homozygous genotypes appears to be neutral or favorable, whereas the combination of the variant alleles (i.e., heterozygosity) shows a reduced frequency in aged subjects. This study originated from our activities in cancer-associated gene polymorphisms. While comparing allele frequencies in cancer patients vs. non-affected subjects, we used elderly tumor-free individuals as a super-control, i.e., an example of pronounced cancer tolerance (Imyanitov, 2009). Previously, we performed a systematic analysis of cancer-predisposing relevance of coding polymorphisms in apoptotic genes. We initially analyzed 19 coding apoptotic gene SNPs in lung cancer patients vs. elderly tumor-free smokers and in breast cancer cases vs. elderly tumor-free females. The groupsDepletion of heterozygous genotypes for the CASP5 Ala90Thr polymorphism was observed in elderly subjects. The study groups consisted of elderly tumor-free smokers (N/=75 years), elderly tumor-free women (N/= 75 years), and elderly tumor-free subjects (N/= 85 years). The number of observed minor alleles and expected numbers of genotypes and frequency of heterozygotes were recorded. The p-value for deviation from Hardy-Weinberg equilibrium was also calculated. Notably, the middle-aged cancer patients and healthy donors showed a similar frequency of the Ala90Thr heterozygotes, indicating that the association of this genotype with decreased survival cannot be explained by premature mortality from malignant disease. However, possible impacts of the CASP5 polymorphism on other age-related maladies cannot be analyzed within our data sets because only cancer-related status was documented with a sufficient level of comprehension and reliability. 

The Ala90Thr polymorphism is located within the caspase recruitment domain (CARD) of caspase-5, which mediates interaction with other proteins and likely influences the activity of this enzyme. If alanine- and threonine-containing variants of caspase-5 have a somewhat distinct repertoire of interacting proteins, a combination of these alleles may render inflammatory responsiveness to a wider spectrum of stimuli. However, the actual functional significance of CASP5 Ala90Thr polymorphism remains unknown.