) ) Association of longevity with IL-10 1082 G⁄A and TNF-a 308 G⁄A polymorphisms

to genetic factors (Karasik et al., 2005). People who survived beyond 85 years old (centenarians, octogenar- ians and nonagenarians) are characterized by marked delay or escape from age-related diseases, therefore analysis of genes that modulate susceptibility to age- related diseases in these populations may provide insights into the human longevity.

Summary

Cytokines are crucial for the regulation of inflammation development in humans. Many studies have shown that variations in cytokine genes might play a role in determining human longevity. This study examined the changes in the gene pool relevant to the ) 308 G⁄A polymorphism in the promoter region of the proinflam- matory cytokine tumour necrosis factor (TNF)-a gene and the ) 1082 G⁄A polymorphism in the promoter region of anti-inflammatory cytokine interleukin (IL)-10 gene with aging and survival selection occurs in the Jordanian population. IL-10 ) 1028 G⁄A and TNF-a ) 308 G⁄A were genotyped in 119 randomly selected elderly subjects (41 women and 78 men) with a mean age of 90.2 years and young control subjects of 118 (46 women and 72 men) with a mean age of 31.9 years. No significant differences were found in the genotype and allele frequencies of TNF-a gene variants between the two groups (P > 0.05) while the IL-10 genotype and allele frequencies were significantly associated with longevity in men (P < 0.05) but not in women (P < 0.05). Thus, IL-10 ) 1028 G⁄A polymorphism seems to play a role in the pathway to longevity in Jordanian men.

cesses that could intensify susceptibility to diseases (Krabbe et al., 2004; Griffin, 2006; et al., 2006; Sbarsi et al., 2007; Yamashina et 2007; Sansoni et al., 2008). As cytokines inflammatory status in humans, allelic variations inflammatory cytokine genes might affect human span. In fact, certain variations in cytokine genes shown to be associated with human longevity type (Cavallone et al., 2003; Scola et al., 2003, Carrieri et al., 2004; Caruso et al., 2004; et al., 2004; Franceschi et al., 2005; Candore et 2006). Tumour necrosis factor (TNF)-a and kin (IL)-10 are two cytokines with opposite in the inflammatory response. TNF-a acts nantly as a proinflammatory agent whereas IL-10 as an anti-inflammatory cytokine (Bidwell et 1999; Dinarello, 2000). Therefore, variations in levels of TNF-a and IL-10 might have an impact the inflammatory response with subsequent effect longevity. Two polymorphisms in the promoter of TNF-a (G⁄A transition at position ) 308) and ) (G⁄A transition at position 1082) are related to scriptional activity (Kroeger et al., 1997; et al., 1997; Crawley et al., 1999; Gibson et 2001; Cacev et al., 2008). Their functional is evident by the involvement in determining bility to immune-inflammatory diseases (Hajeer Hutchinson, 2000; Howell & Rose-Zerilli, 2006). addition, the IL-10 ) 1082 G⁄A and TNF-a ) 308 polymorphisms are associated with longevity in (Lio et al., 2002a, 2003; Cardelli et al., 2008)

Introduction

Longevity is a biological phenomenon means success- ful ageing resulting from a complex interaction between environmental and genetic factors (Kirkwood, 2005). Environmental factors include dietary habits, physical activity, smoking and health care, whereas genetic factors consist of the various loci playing key roles in body homeostasis (Dossey, 2002). Arguably, about one quarter of longevity trend can be attributed

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

Therefore, the purpose of the current study is to examine the association of TNF-a and IL-10 alleles with longevity in the Jordanian population. We found that the frequency of ) 308 A is low in Jordanian pop- ulation whereas the frequency of IL-10 1082 A allele

Received 24 January 2009; revised 6 May 2009; accepted 24 March 2010

Correspondence: Dr Omar Khabour, Department of Medical Labora- tory Sciences, Jordan University of Science and Technology, PO Box 3030, Irbid 22110, Jordan. Tel: +962 2 7201000 ext. 23784; Fax: +962 2 7201087; E-mail: khabour@just.edu.jo

) is very common. No association between 308 G⁄A TNF-a promoter SNP in our sample of Jordanian ) elderly with longevity was detected, whereas 1082 G⁄A IL-10 promoter SNP was associated with longev- ity in men but not in women.

with a modified sequence which creates a restriction site for the enzyme NcoI. The primer sequences used for this assay were: forward (5¢-agg caa tag gtt ttg agg gcc at-3¢); reverse (5¢-aca ctc ccc atc ctc cct gct-3¢). PCR-RFLP was performed as follows: 100 ng of DNA was amplified in a 25 lL final volume PCR reaction containing 1X Taq Buffer, 200 l dNTPs, 1 l of M M each primer and 1 unit Taq polymerase (all PCR reagents were from Promega). PCR was completed on Brinkmann Gradient Eppendorf Mastercycler. Cycling was performed at 95 (cid:3)C for 2 min and 35 cycles at 95 (cid:3)C for 30 s, 60 (cid:3)C for 15 s, and 72 (cid:3)C for 30 s, followed by a final extension of 10 min at 72 (cid:3)C. Fol- lowing amplification, PCR product was visualized on a 2% agarose gel stained with ethidium bromide. This confirmed the presence of a 116-bp product. NcoI enzyme digestion was carried out in 20 lL final vol- ume reaction using 3 unit NcoI (Fermentas, Glen Bur- nie, MD, USA) and 10 lL of PCR product. The reactions were incubated at 37 (cid:3)C for 4 h. The diges- tive products were visualized on 4% agarose gel stained with ethidium bromide. Materials from indi- viduals homozygous for TNF-a ) 308 A allele do not cut with NcoI and remains as a 116-bp product. The homozygous TNF-a ) 308 G allele cuts with NcoI to give 96- and 20-bp fragments.

Materials and methods

Study population