Contents lists are available at the ScienceDirect Archives of Gerontology and Geriatrics journal homepage: www.elsevier.com/locate/archger = ARCHIVES OF GERONTOLOGY        Archives of Gerontology and Geriatrics journal homepage: www.elsevier.com/locate/archger  Distributions of interleukin-6 (IL-6) promoter and metallothionein 2A (MT2A) core promoter region gene polymorphisms and their associations with aging in the Turkish population  Zeliha Kayaalt a,*, Levent Sahiner b, M. Emre Durakoglugil b, Tulin Soylemezoglu a  a Ankara University Institute of Forensic Sciences, Mamak Street, No:10, 06590, Dikimevi, Ankara, Turkey b Ankara Oncology Education and Research Hospital, Vatan Street, 06200, Yenimahalle, Ankara, Turkey  A B S T R A C T  Aging is determined by an interaction among genetic, environmental and lifestyle factors. As interleukin-6 (IL-6) and metallothioneins (MTs) are related to inflammation and oxidative stress response, their genes are appropriate candidates for aging, age-related diseases and infections. The aim of this study was to investigate the association between the IL-6 (174 G/C) promoter region and MT2A (5 A/G) core promoter region single nucleotide polymorphisms (SNPs) with longevity in the Turkish population. Blood samples were collected from 354 individuals between 18 and 95 years of age. Individuals were classified into four groups according to their ages as 20-40, 41-60, 61-80, >80. IL-6 and MT2A polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The mean ages of individuals with IL-6 174 C carriers and C+ carriers were 49.82 ± 20.45 years and 59.82 ± 16.82 years, respectively. For the MT2A polymorphism, mean ages were estimated as 56.18 ± 19.50 years for G carriers and 47.59 ± 13.45 years for G+ carriers. As a result, when the IL-6 and MT2A polymorphisms were compared with the mean ages and age groups, statistically significant associations were found (p < 0.001 and p < 0.05, respectively). In conclusion, these data support that the IL-6 174 C+ carriers and MT2A 5 G carriers may be more advantageous for longevity. 1. Introduction Polymorphisms (SNPs) have been identified (The National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/SNP/). IL-6 gene polymorphisms affect the rate of synthesis or degradation of IL-6. One of these polymorphisms in the IL-6 gene is the 174 G/C promoter region SNP and a significant association between the 174 G/C polymorphism and some age-related diseases and longevity has been shown (Caruso et al., 2004; Van Bodegom et al., 2007). Aging is determined by a complex interaction of genetic, epigenetic, and environmental factors, but a strong genetic component appears to have an impact on survival to extreme ages (Perls and Terry, 2003). Longevity is likely to be influenced by thousands of genes (Franceschi et al., 2000), and twin studies have indicated that approximately 20-30% of the overall variation in lifespan can be attributed to genetic factors (Hjelmborg et al., 2006). Inflammation and dysregulated production of inflammatory cytokines have an important role in the process of aging, as they affect an individual's capacity to counteract noxious events such as pathogen infection, neurodegenerative disorders, autoimmunity and cancer (Caruso et al., 2004; Van Bodegom et al., 2007). An acute inflammatory response is beneficial for survival, but a chronic inflammatory state contributes to age-associated diseases. A reliable marker of inflammation is interleukin-6 (IL-6), the circulating level of which is increased in chronic diseases and aging (Jylha et al., 2007). The IL-6 gene is highly polymorphic. The IL-6 gene, which is located on chromosome 7p21, is expressed in lymphocytes, fibroblasts, macrophages in response to different types of inflammatory stimuli (Berghe et al., 2000). IL-6 also controls induction and expression of metallothioneins (MTs) which maintain homeostasis of zinc and copper (Mazzatti et al., 2008). During stressStating themselves as Turkish, the participants of the study were included after approval from the institutional ethics committee (approval No: 92; 25.12.2008) according to the principles of The Declaration of Helsinki. Blood samples were stored at 4°C during active use. Statistical analysis was performed using the statistical package for social sciences (SPSS) 16.0 package program. Genomic DNAs were extracted from 100 ml whole blood samples with Qiagen QIAamp DNA Mini Kit, following the manufacturer's recommended method. Polymorphism analysis of IL-6 (cid:2)174 G/C was determined using a method based on Mazzatti et al. (2008) with slight modification, and MT2A (cid:2)5 A/G was carried out following the method described by Kayaalti and Soylemezoglu (2010). In order to screen for (cid:2)174 G/C SNP in the promoter region of IL-6 gene, a 303 bp fragment containing the promoter region was amplified by polymerase chain reaction (PCR) using the following primers: forward 50-TTG TCA AGA CAT GCC AAA GTG CT-30.

A representative agarose gel image of digested and undigested PCR products with BsgI for MT2A polymorphism is shown in Fig. 2. The distribution of IL-6 and MT2A genotypes and ages is presented in Table 1. The distribution of the IL-6 and MT2A genotypes according to age groups is shown in Table 2.

Frequencies of MT2A and IL-6 alleles and genotypes were obtained by direct count, and the departure from the Hardy-Weinberg equilibrium was evaluated using the Haldane exact test. Students t-test and one-way ANOVA analysis were used to compare metric variables, while categorical variables were compared using the x2 test. A p-value of less than 0.05 was considered statistically significant.