The MTHFR C677T polymorphism was also analyzed by PCR-RFLP using Hinf I enzyme (Fermentas). PCR primers were: forward primer (5'-TGA AGG AGA AGG TGT CTG CGG GA-3') and reverse primer (5'-AGG ACG GTG CGG TGA GAG TG-3'). Polymerase chain reaction and Hinf I digestion condition were similar to that described for SOD2 -9 SNP except for the annealing temperature, which was 60°C in this case. PCR fragments from MTHFR 677 C allele don't cut with Hinf I and remain as a 198 bp product while fragments from MTHFR 677 T allele cut with Hinf I to give 175 bp and 23 bp fragments.

Methods

One hundred thirty unrelated elderly subjects (> 85 years, mean age 90.01 year) volunteered from different parts of Jordan to take part in this study. Another 135 unrelated young control subjects (range from 20 to 50 years, mean age 33.34 years) were matched long-lived individuals for geographical origin. Subjects with cardiovascular diseases, diabetes, or cognitive impairments were excluded from the study. The experimental design and the sample size were similar to most longevity studies reviewed by Glatt et al., [23]. Subject's mean ages were selected based on the mortality rate in the Jordanian population, which is approximately constant from childhood to late forties, thereafter, it starts gradually inclining to reach maximum in late seventies [24]. Therefore, individuals who reach more than 85 years are rare in Jordan. An official identifi- cation document was required to participate in the study. Acceptable documents include civil ID card, birth certifi- cate, family book, passport and military card, otherwise enrolment in the study was denied. All subjects received written and verbal explanation of the study before giving consent. The study protocol was approved by the Institu- tional Review Boards of Jordan University of Science and Technology.

The genotype distributions of the examined polymor- phisms were analyzed in agreement with Hardy-Weinberg equilibrium. To test association between longevity and the polymorphic loci, distributions of allele and genotype frequencies were compared between young and elderly groups using the chi-square and Fisher's exact tests. The test power was calculated for alleles frequency using Power and Sample Size Calculation Program (PS version 3.0.1, Vanderbilt University Medical Center, Nashville, TN, USA) and for genotype frequencies using SAS macro [25]. For all analysis, the power was more than 75%. The SPSS 15.0 statistical software package (SPSS Inc., Chicago, IL) was used for statistical analysis. P values smaller than 0.05 were considered significant.

Blood samples (1-3 ml) in EDTA tubes were obtained from all subjects. DNA was extracted from all samples using Wizard DNA Extraction Kit (Promega, Madison, USA) according to the manufacturer instructions. DNA

Results

Jordan is a small country located in Southwest Asia and classified among the low income countries. The popula-

tion is predominantly Arab (98%) and most of it is urban (70%) [26]. According to the 2007 census, the total pop- ulation of Jordan was 5.7 million, percentage of individu- als of 65 years of age or older was 4.1% and life expectancy at birth in the total population was 73 year [26].

women might follow different pathways to reach longev- ity [27,28]. In our sample, genotypes and alleles frequen- cies for examined SNPs were not different when males were considered alone (Chi square test: for -9 T/C SOD2, P = 0.691 and for 677 C/T MTHFR, P = 0.795, Table 2), or when females were considered alone (Chi square test: for -9 T/C SOD2, P = 0.317 and for 677 C/T MTHFR, P = 0.792, Table 3).

The average age of the elderly group in the study was 90.01 years. In Jordan, the mortality rate starts inclining exponentially at fifty year-old getting maximum level in the late seventies indicating that reaching above 85 year- old is a rarity (Khoury et al., 1999). Therefore, oldest old people (> 85 year-old) are considered exceptional individ- uals in Jordan.

Discussion

Oxidative stress is a condition where the redox balance between oxidant and antioxidant is shifted toward an oxi- dized state. In animals, oxidative stress increase with age- ing due to high production of free radicals by aged mitochondria and decreased cellular antioxidant capacity. The mitochondrial magnesium superoxide dismutase (SOD2) is considered the first line of defense against reac- tive oxygen species [4]. The gene for SOD2 has a common T to C polymorphism, resulting in a valine to alanine change at the 16 position of its mitochondrial targeting sequence (Ala16Val), which affects the structure of the protein [6], and reduces its entrance into the mitochon- dria [29] leading to increased oxidative stress.

Males to females ratio was 1.3:1 in the elderly group and 1:1 in the control group (P = 0.346). Number of relatives who exceeded 85 year-old was higher in the elderly group compared to the young control group (70.3% versus 59.8%, respectively, P = 0.013). The higher number of rel- atives who exceeded 85 year-old (>25%) in the elderly group indicates the presence of genetic component to lon- gevity in the Jordanian population.

Table 1 shows the frequency of homozygous and hetero- zygous genotypes for SOD2 T-9C and MTHFR C677T SNPs in our sample of elderly and young controls. The genotype frequencies of the SOD2 T-9C SNP of elderly and control groups were not statistically different (Chi square test, P = 0.576). Accordingly, the frequency of SNP -9 T to C was not significantly different between elderly and controls (Chi square test, P = 0.355). Similar results were observed with the MTHFR C677T SNP (Chi square test: for genotype frequencies, P = 0.944 and for allele fre- quencies, P = 0.727).