Association between SOD2 T-9C and MTHFR C677T polymorphisms and longevity: a study in Jordanian population Omar F Khabour*, Essa S Abdelhalim and Ahmad Abu-Wardeh

Address: Department of Medical Laboratory Sciences, Jordan University of Science and Technology, Irbid 22110, Jordan

Address: Department of Medical Laboratory Sciences, Jordan University of Science and Technology, Irbid 22110, Email: Omar F Khabour* - khabour@just.edu.jo; Essa S Abdelhalim - watan48@yahoo.com; Ahmad Abu- Wardeh - ams_just_scholarship@yahoo.com * Corresponding author

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Abstract

Background: Aging in animals is associated with high body oxidative stress, which might affect susceptibility and onset of age-related diseases, and the subsequent life span. Therefore, genes that modulate oxidative stress might play a role in determining longevity. In this study, we investigated whether the gene pool relevant to the SOD2-9T/C and MTHFR 677C/T polymorphisms changes as the Jordanian population ages.

Methods: Polymorphisms were genotyped in 130 elderly subjects (57 females and 73 males, mean age: 90.01 years) and 135 young control subjects (67 females and 68 males, mean age: 33.43 years).

Results: No significant differences were found in the genotype and allele frequencies of examined SOD2 and MTHFR gene variants between the elderly group and young controls (P > 0.05), nor when each gender was considered separately (P > 0.05).

SOD2-9T/C and MTHFR 677C/T gene polymorphisms do not seem to be important Jordanian population for longevity phenotype.

Background

superoxide anion into hydrogen peroxide that is detoxi- fied into water by glutathione peroxidases and catalase [5]. Presence of the C allele at -9 position of SOD2 gene results in substitution of alanine for valine (Ala16Val) in the mitochondrial targeting sequence [6]. This substitu- tion partially retains SOD2 enzyme within the narrow inner membrane import pore and lowers the enzyme activity [7,8]. The importance of SOD2 -9T/C polymor- phism is indicated by its association with several age related diseases such as cancer [9,10] and diabetic neph- ropathy [11].

Aging involves increases in oxidative stress status pre- sented by elevated levels of oxidized forms of biomole- cules in the body of the organism [1]. This leads to tissue damage and decreases in body functions, homeostasis, and tolerance to chronic diseases [2,3]. Thus, genes that modulate oxidative stress might play a role in human lon- gevity.

In this study, we investigated association of SOD2 -9T/C SNP and MTHFR 677 C/T SNP with longevity in Jorda- nian population. The SOD2 gene codes for the mitochon- drial manganese superoxide dismutase, a major cellular antioxidative stress enzyme [4]. SOD2 dismutates the

The MTHFR gene codes for methylenetetrahydrofolate reductase that catalyzes the conversion of 5,10-methyl-

samples were stored at -20°C until used. The concentra- tion of the extracted DNA was measured using Smart- Spect™ 3000 (Bio-Rad, Hertfordshire, UK).

enetetrahydrofolate to 5-methyltetrahydrofolate. The lat- ter serves as a methyl donor in the reaction converting homocysteine to methionine [12]. The T allele at 677 position of MTHFR gene causes substitution of alanine to valine and the resulting decreases in enzyme activity and increases in body homocysteine concentrations [13]. Excess homocysteine undergoes auto-oxidation in plasma, so that free oxygen radicals are produced thereby enhancing endothelial tissue damage and inflammation [14]. In addition, excess homocysteine can directly impair DNA methylation, resulting in altered gene expression [15]. The MTHFR 677 C/T polymorphism has been shown to be associated with ischemic stroke [16], cancer [17] and coronary artery disease [18].

SOD2 T-9C polymorphism was typed using RFLP-PCR protocol. Briefly, 20 μl reaction mixture containing 5 ng of template DNA, 0.75 unit GoTaq polymerase (Promega, Madison, USA), and a final concentration of 200 mM each deoxynucleotide and 1× reaction buffer, and 1 mM of forward (5'-ACC AGC AGG CAG CTG GCG CCG G-3') and reverse (5'-GCG TTG ATG TGA GGT TCC AG-3') primers. Cycling was performed at 95°C for 15 min and 35 cycles at 94°C for 30 s, 65°C for 30 s and 72°C for 30 s, followed by a final extension of 7 min at 72°C. PCR products were detected using electrophoresis on 4% agar- ose, confirming the presence of a 107 bp product. The NgoMIV enzyme (Fermentas. GmbH, St. Leon-Rot, Ger- many) digestion was carried out in 20 ul reaction mixture containing 3 units of enzyme and 10 μl of PCR product at 37°C for 4 hours. Materials from individuals homozygous for SOD2 -9 T allele don't cut with NgoMIV and remain as a 107 bp product. The homozygous SOD2 -9 C allele cuts with NgoMIV to give 89 bp and 18 bp frag- ments.

Previous reports are variable on the association between SOD2 -9 SNP or MTHFR 677 SNP with human longevity. For example, positive association has been reported between SOD2 -9 SNP and Ashkenazi males [19], but not Italian population [20]. Similarly, positive association has been reported between MTHFR SNP, and Swiss popula- tion or Ashkenazi women [19,21], but not Irish popula- tion [22]. In this study, we report absence of association between -9 T/C SOD2 SNP or 677 C/T MTHFR SNP with longevity in the examined sample of Jordanian elderly.