Fig. 1. A representative agarose gel image of digested and undigested PCR products withNlaIIIforIL-6polymorphism:(M:100 bpladder,lanes1and2:undigestedPCR product, lanes 3, 4 and 5: homozygote typical genotype (GG), lanes 6 and 7: heterozygote genotype (GC), lane 8: homozygote atypical genotype (CC).

It is very important to demonstrate the relationship between MT and IL-6 productions. The IL-6 (cid:2)174 G/C promoter region polymorphism affects cytokine production and pro-inflammatory cytokines induce the gene expression of MT. Increased levels of MT, IL-6 and low zinc ion availability are observed in diseases characterised by chronic inflammation and in syndromes of accelerated aging (Kwon et al., 2007).

reverse 50-GCC TCA GAC ATC TCC AGT CC-30. Then the PCR product (303 bp) was digested with NlaIII restriction enzyme (New England Biolabs, Hertfordshire, UK) and incubated at 37 8C for overnight. The undigested PCR product and digested products were separated on a 2% agarose gel electrophoresis, visualized by ethidium bromide staining under an ultraviolet illuminator, scanned and photographed using Syngene Monitoring System. Digestion of PCR product by NlaIII yields 233, 57 and 13 bp fragments for the G allele; 122, 111, 57 and 13 bp fragments for the C allele (Fig. 1).

The aim of the present study was to determine IL-6 (cid:2)174 G/C promoter region polymorphism (SNP rs1800795; Gene access number: NM_000600; Gene ID: 3569) and MT2A (cid:2)5 A/G core promoter region polymorphism (SNP rs 28366003; Gene access number: NM_005953; Gene ID: 4502) and to find out whether there is an association between the polymorphic characteristics and longevity in Turkish population.

For assay of the (cid:2)5 A/G single-nucleotide polymorphism in the core promoter region of MT2A gene, PCR was carried out using the forward primer 50-CGC CTG GAG CCG CAA GTG AC-30 and the reverse primer 50-TGG GCA TCC CCA GCC TCT TA-30 resulting in an amplified fragment of 241 bp in size. Then the PCR product (241 bp) was digested with 3 U of BsgI restriction enzyme (New England Biolabs, Hertfordshire, UK) and incubated at 37 8C for 2 h. The undigested PCR product and digested products were separated on a 3% agarose gel electrophoresis, visualized by ethidium bromide staining under an ultraviolet illuminator, scanned and photographed using Syngene Monitoring System. Digestion of PCR product by BsgI yields 144, 56 and 41 bp fragments for the A allele; 185 and 56 bp for the G allele (Fig. 2).

2. Materials and methods

2.1. Study subjects

In this study, blood samples were obtained from 354 genetically unrelated and healthy individuals without a known history of any chronic disease and cancer (mean age 55.35 yb 19.16 years; range 18–95 years), including 175 females (mean age 55.44  19.15 years; range 20–95 years) and 179 males (mean age 55.27 y+  19.22 years; range 18–92 years). Blood samples were acquired from volunteers referred to Ankara Oncology Education and Research Hospital for routine control. Informed consent was obtained from each individual who was selected randomly control group sample among from Turkish population. The geographical distributions of the subjects were from the Central Anatolia region and other regions representing the Turkish population from all regions of the country. A small questionnaire for gathering the demographic and ethnic information was also given to the individuals and the individuals stating themselves as Turkish were included in the study. The study design was approved by the institutionalethics committee (approval No: 92; 25.12.2008). Samplings were performed in accordance with the principles of The Declaration of Helsinki. Blood samples were kept at 4 8C while they were in active use.

2.3. Statistical analysis

Statistical analyzes were performed by using statistical package the social sciences (SPSS) 16.0 package program. The

 

Genomic DNAs were extracted from 100 ml whole blood samples with ‘‘Qiagen QIAamp DNA Mini Kit’’ according to the method recommended by the manufacturer.

Polymorphism analyze of the IL-6 (cid:2)174 G/C was determined according to method (Mazzatti et al., 2008) with slight modifica- tion and MT2A (cid:2)5 A/G were carried out according to those previously described (Kayaalti and So¨ylemezog˘lu, 2010) as below. In order to screen for (cid:2)174 G/C SNP in the promoter region of IL-6 gene, 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 and

In order to screen for (cid:2)174 G/C SNP in the promoter region of IL-6 gene, 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 and

Fig. 2. A representative agarose gel image of digested and undigested PCR products with BsgI for MT2A polymorphism: (M: 100 bp ladder, lane 1: undigested PCR product, lane 2: homozygote typical genotype (AA), lane 3: heterozygote genotype (AG) and lane 4: homozygote atypical genotype (GG).

Table 1

Distribution of IL-6 and MT2A genotypes and ages.

Polymorphisms n (%) Age (years) Mean(cid:3)S.D. Median Range IL-6 genotypes GG (C(cid:2)) 158 (44.6) 49.82(cid:3)20.45 50 18–92 GC (C+) 166 (46.9) 58.57(cid:3)17.02 55 22–95 CC (C+) 30 (8.5) 66.73(cid:3)14.00 64 47–92 GG (C(cid:2)) 158 (44.6) 49.82(cid:3)20.45 50 18–92 GC+CC (C+) 196 (55.4) 59.82(cid:3)16.82 56 22–95 MT2A genotypes AA (G(cid:2)) 320 (90.4) 56.18(cid:3)19.50 55 18–95 AG+GG (G+) 34 (9.6) 47.59(cid:3)13.45 50 22–81 * p<0.05.

Table 2

Distribution of the IL-6 and MT2A genotypes according to age groups, n (%).

Polymorphisms Age groups p< 20–40 41–60 61–80 >80 IL-6 C(cid:2) (GG) 61 (17.2) 50 (14.1) 23 (6.5) 24 (6.8) 0.0001** C+ (GC+CC) 17 (4.8) 100 (28.2) 41 (11.6) 38 (10.7) MT2A G(cid:2) (AA) 70 (19.8) 128 (36.2) 61 (17.2) 61 (17.2) 0.013* G+ (GC+GG) 8 (2.3) 22 (6.2) 3 (0.8) 1 (0.3)