Abstract
Adipokines are important for regulation body metabolism and immune response. Many studies have shown that variants in adipokines genes play a role in age-associated diseases. In this study, we investigated the contribution of rs266729 (−11377G/C), rs2241766 (+45T/G), and rs1501299 (+276 G/T) SNPs of adiponectin gene (ADIPQO) and rs7799039 (−2548C/A) SNP of leptin (LEP) gene to human longevity phenotype in Jordanian population. Polymorphisms were genotyped in 110 randomly selected elderly subjects (>85 years old) with mean age of 90.2 years, and 120 young control subjects (range from 20 to 50 years) with mean age of 32.0 years. No significant differences were detected in the genotype and allele frequencies of examined gene variants between the two groups (p > 0.05). However, when gender was considered, genotypes and alleles frequencies of rs1501299 SNP in ADIPOQ gene and rs7799039 in LEP gene were significantly associated with longevity in men (p < 0.02) but not in women (p > 0.05). Thus, ADIPOQ and LEP genes polymorphisms might play a gender-specific role in the pathway to men's longevity.
Keywords:
Introduction
Adiponectin and leptin are adipokines secreted from white adipose tissue and function in a hormone-like manner to regulate body metabolism and immune responses [Citation1]. Adiponectin activates multiple signalling pathways, which mediate its anti-diabetic, anti-atherogenic and anti-inflammatory functions, thus, adiponectin has a protective role against age-related diseases [Citation2,Citation3]. Low level of adiponectin has been implicated in several late-onset disease states such as insulin resistance and type-2 diabetes [Citation4], cardiovascular disease [Citation5], hypertension [Citation6] and dyslipidemia [Citation7]. Several single nucleotide polymorphisms (SNPs) in ADIPOQ gene (adipocyte-, C1Q and collagen domain containing) that code for adiponectin, have been shown to affect either levels or activity of adiponectin and have been associated with age-related diseases. For example, the promoter rs266729 SNP lowers the circulating levels of adiponectin and has strong association with metabolic syndrome among Spanish and French populations [Citation8]. Furthermore, rs2241766 and rs1501299 SNPs in ADIPOQ gene, which alter adiponectin activity, are associated with metabolic phenotype [Citation9,Citation10].
Leptin, on the other hand, is increased significantly in individuals suffering from obesity, diabetes mellitus, hypertension or metabolic syndrome [Citation11]. A common promoter polymorphic site rs7799039 SNP in the leptin (LEP) gene is associated with transcriptional activity, leading to reduced circulating leptin concentrations in subjects with AA genotype [Citation11,Citation12]. This polymorphism is also associated with multiple age-related diseases in several populations such as breast cancer among Tunisians [Citation13], and non-small cell lung cancer and prostate cancer among Portuguese [Citation14,Citation15].
Since adiponectin and leptin play an important role in the body homeostasis and susceptibility to age-related diseases, allelic variants that change the levels and/or activity of adiponectin and leptin might be involved in the pathway of human longevity [Citation16]. Indeed, two allelic variants in ADIPOQ gene, rs17300539 (−11391G/A) and +2019A/−, are associated with longevity in Ashkenazi Jewish population [Citation17]. In the current study, the contribution of rs266729, rs2241766, and rs1501299 SNPs in ADIPOQ gene and rs7799039 SNP in LEP gene to longevity phenotype were investigated in Jordanian population. Results showed that rs1501299 SNP in ADIPOQ gene and rs7799039 SNP in promoter region of LEP gene play a gender-specific role in human longevity.
Material and methods
A sample of 110 unrelated elderly subjects (>85 years, mean age 90.2 years) was recruited from different parts of Jordan. Additionally, 120 unrelated control subjects (range from 20 to 50 years, mean age 32.0) were matched to long-lived individuals for geographical origin. Male to female ratio was 1.6:1 in the elderly group and 1.4:1 in the control group (p > 0.05). Subjects with cardiovascular diseases, diabetes or cognitive impairments were excluded from the study. The experimental design was similar to longevity studies reviewed by Glatt et al. [Citation18]. The age criterion was chosen based on the mortality rate in the Jordanian population which remains approximately constant from childhood until late 40s and then starts inclining exponentially at 50 years of age getting maximum level in the late 70s [Citation19]. According to the 2007 census, the total population of Jordan was 5.7 million, percentage of individuals of 65 years of age or older was 4.1%, and life expectancy at birth in the total population was 73 years [Citation20]. Therefore, reaching above 85 year-old is a rarity in Jordan and oldest old people are considered exceptional individuals in Jordan. To verify age, an official identification document was required from all participants in the study. Written informed consents were obtained from all participants in accordance with the requirements of the Institutional Review Board of Jordan University of Science and Technology.
Blood samples (1–3 ml) in ethylenediaminetetraacetic acid (EDTA) tubes were obtained from all subjects. Deoxyribonucleic acid (DNA) was extracted from all samples using Wizard DNA Extraction Kit (Promega, Madison, USA) according to manufacturer instructions. DNA samples were stored at −20°C until used. The concentration of the extracted DNA was measured using SmartSpect™3000 (Bio-Rad, Hertfordshire, UK). Three polymorphic sites of ADIPOQ were genotyped according to Li et al. [Citation21] using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). summarises the primers sequence, annealing temperature and restriction enzymes (Fermentas. GmbH, St. Leon-Rot, Germany) that were used. For each polymorphic site; 20 μl reaction mixtures containing 5 ng of template DNA, 0.75 unit GoTaq polymerase (Promega), and a final concentration of 200 μM of each deoxynucleotide and 1X reaction buffer, 1 μM of each forward and reverse primers. PCR products were detected using electrophoresis on 3% agarose, confirming the presence of a product. The restriction enzyme digestion was carried out in 20 μl containing 3 units of enzyme and 10 μl of PCR product and incubated at optimum temperature (see ) for 4 h. The LEP rs7799039 SNP was also analysed using PCR-RFLP as described by Stunff et al. [Citation11]. PCR primers, annealing temperature and restriction enzymes are shown in .
Table I. Primers sequences, PCR conditions and restriction enzymes used for genotyping of SNPs.
The genotype distributions of the examined polymorphisms were analysed in agreement with Hardy–Weinberg equilibrium. Statistical evaluation of results was done by comparing allele, genotype and predicted phenotype distributions using the χ2 test. If n < 5, exact Fisher statistic was used. The SPSS 15.0 statistical software package (SPSS Inc., Chicago, IL) was used for all statistical evaluations. Because several significant associations were observed among the 24 associations examined in the study, which exceeds the number that might be observed by chance (one significant association/24 tests), a p-values of < 0.05 was considered significant without adjustments for multiple comparisons.
Result
shows the frequency of homozygous and heterozygous genotypes for rs2241766, rs1501299 and rs266729 ADIPOQ gene SNPs, and for rs7799039 LEP gene promoter SNP in the elderly versus the young group. No differences in the genotype frequencies of the examined ADIPOQ SNPs (p = 0.231 for rs2241766 SNP, p = 0.172 for rs1501299 and p = 0.809 for rs266729 SNP) and the frequency of various alleles (p = 0.164 for rs2241766 T/G alleles, p = 0.078 for rs1501299 G/T alleles and p = 0.635 for rs266729 C/G alleles) were detected between the elderly and the young group. Similarly, rs7799039 LEP gene promoter SNP was not different between the two groups (for genotype frequencies: p = 0.082 and for allele frequencies: p = 0.051).
Table II. Frequencies of ADIPOQ and LEP alleles and genotypes in control and elderly groups.
Several studies indicated that gender was a main variable in the genetics of longevity and suggested that men and women might follow different pathways to reach longevity [Citation22–25]. Therefore, the effect of ADIPOQ and LEP genes SNPs on longevity were re-evaluated separately in each gender. Genotype frequency and allele frequency for rs1501299 ADIPOQ SNP and rs7799039 LEP gene promoter SNP were significantly different between the elderly and the young groups in men (genotype frequencies: p = 0.011 for rs1501299 SNP and p = 0.021 for rs7799039 SNP; alleles frequencies: p = 0.011 for rs1501299 SNP and p = 0.011 for rs7799039 SNP, ) but not in women (p = 0.788 for rs1501299 SNP and p = 0.962 for rs7799039 SNP, alleles frequencies: p = 0.621 for rs1501299 SNP and p = 0.990 for rs7799039 SNP, ). On the other hand, no significant difference was detected between the elderly and young control groups when gender was considered in the rs2241766 and rs266729 SNPs of ADIPOQ gene (for both SNPs, men: p > 0.05, , and women: p > 0.05, ).
Table III. Frequencies of ADIPQO and LEP alleles and genotypes in control and elderly men subjects.
Table IV. Frequencies of ADIPOQ and LEP alleles and genotypes in control and elderly women subjects.
Discussion
Adiponectin and leptin are adipocyte-derived protein hormones that play a pivotal role in regulation of immune response and body metabolism [Citation1]. Changes in plasma level/activity of adiponectin and leptin in adipocytes contribute to several age-associated diseases in human such as metabolic syndrome, coronary artery disease, hypertension, type-2 diabetes and cancer [Citation4–7,Citation13,Citation16,Citation26,Citation27].
Recently, a Japanese study showed that plasma adiponectin levels were significantly higher in elderly subjects from a longevity district compared to subjects from a non-longevity district [Citation28] suggesting a possible role of adipokines in the pathway to human longevity. In this study, the possible association of three polymorphisms in ADIPOQ gene and one polymorphism in LEP gene with longevity phenotype was examined. Results showed that rs1501299 ADIPOQ SNP and rs7799039 LEP gene promoter SNP are associated with longevity in men but not in women. Previous studies have shown that the rs1501299 SNP is associated with low serum adiponectin [Citation29,Citation30], and increased susceptibility to coronary artery disease [Citation26,Citation30,Citation31] and type-2 diabetes [Citation32]. Similarly, the rs7799039 LEP SNP is associated with transcriptional activity. It is located proximal to a binding site for the Sp1 transcription factor in the promoter region, and high expression occurs in the presence of CC genotype [Citation11,Citation12,Citation33]. The clinical significance of this polymorphism has been demonstrated by its association with age-related diseases including cancer [Citation13–15]. The finding that rs1501299 ADIPOQ and rs7799039 leptin SNPs play a role in human longevity provides more evidence of the clinical importance of these SNPs in age-related diseases.
It has been suggested that gender is a major variable in the genetics of longevity in different populations. For instance, Tyrosine Hydroxylase gene, TNF-α, and mtDNA haplogroup are associated with longevity in men [Citation22,Citation34,Citation35] while haemochromatosis, IL2 and interferon-gamma gene polymorphisms are associated with longevity in women [Citation25,Citation36,Citation37]. Recently, Atzmon et al. [Citation17] demonstrated that two SNPs, rs17300539 and +2019A/− in ADIPOQ gene are associated with longevity in men of the Ashkenazi Jewish population. The finding that rs1501299 ADIPOQ SNP and rs7799039 LEP gene promoter SNP are associated with men's longevity but not in women in Jordanian population supports the hypothesis of gender differences in the pathway to human longevity. Gender variations in hormonal signalling pathways, body metabolism, immunity response, oxidative damage tolerance might account for such differences [Citation38].
Results of this study showed absence of association between rs2241766 and rs266729 SNPs of ADIPOQ gene and longevity in both genders. Previous studies are variable concerning the association of rs2241766 or rs266729 SNP in ADIPOQ gene with age-associated diseases. Positive association of rs2241766 SNP has been reported in Korean population with coronary artery disease [Citation39], and in Iranian population with type-2 diabetes [Citation40]. In contrast, negative association with disease has been reported in Polish population with type-2 diabetes, and Italian population with coronary artery diseases [Citation31,Citation32]. For – rs266729 SNP, positive association was reported in Spanish subjects with metabolic syndrome [Citation8], while no association was detected between rs266729 SNP and survival among Ashkenazi Jewish population [Citation17]. Therefore, ADIPOQ polymorphisms/age-related diseases associations might have a population specific component. In addition, it is possible that Jordanians have different polymorphisms in the ADIPOQ gene that might buffer out or modulate the effect of the rs2241766 and rs266729 SNPs.
Jordan is a small country located in Southwest Asia. The population is predominantly Arab (98%) [Citation20] originally from Jordan and Palestine with approximately equal percentages. Since Jordanian and Palestinian share common ancestry, stratification due to migration of Palestinians to Jordan in 1948 is not expected to affect the results. In addition, the subjects (elderly and young) were recruited from different parts of Jordan without taking into consideration their origin and therefore, the sample is homogeneous.
One of the limitations of our study was the lack of data regarding plasma levels of adiponectin and leptin, however, the functional relevance of the studied polymorphisms is well documented [Citation11,Citation12,Citation29,Citation30,Citation33]. In addition, several other polymorphisms in the examined genes (rs17300539 and +2019A/−) were not investigated in this study. Examining plasma levels of adiponectin and leptin in subjects in addition to genotyping of other SNPs in examined loci and their relation with longevity in Jordanian population will be a matter of future studies.
In conclusion, results of this study indicate that rs1501299 ADIPOQ SNP and rs7799039 LEP gene promoter SNP play a gender-specific role in human longevity.
Acknowledgements
This work has been done with funds from the Deanship of Scientific Research in Jordan University of Science and Technology, grant number 182/2007 and 207/2007 to O. K. The authors thank Mr. Jameel M. Bernawi and Mr. Essa S. Abdelhalim for their help with recruitments of subjects.
Related Research Data
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