Contents lists available at ScienceDirect Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev    

Mechanisms of Ageing and Development

journal homepage: www.elsevier.com/locate/mechagedev

functional single nucleotide polymorphism in promoter of ATM is associated longevity

Tie Chen a, Birong Dong a,*, Zhenchan Lu a, Baochun Tian a, Jian Zhang c, Jianlin Zhou c, Hongmei Wu a, Yanling Zhang a, Jinhui Wu a, Ping Lin b, Jie Zhang b, Hong Xu a, Xianming Mo a,**

aLaboratory of Stem Cell Biology and Department of Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University,

aLaboratory of Stem Cell Biology and Department of Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China bLaboratory of Experimental Oncology, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China cCollege of Life Science, Hunan Normal University, Changsha, Hunan 410081, China

Although the ‘ataxia telangiectasia mutated’ (ATM) gene plays an important role in physiological processes, such as sensing DNA damage, reducing oxidative stress and protecting telomeres length, little informationaboutATMandlongevityisavailable.Therefore,weaimtoexaminetheassociationbetween genetic variants in promoter of ATM and longevity in Chinese Nonagenarians/Centenarians. Genotyping was performed in 789 long-lived individuals (LLIs) and 886 ethnically matched control subjects. A single nucleotide polymorphism (SNP, rs189037) in the promoter region of ATM gene was identified, and significant association between CT genotype and longevity was observed. Meanwhile, the SNP was able to affect expression of ATM mRNA by differentially binding to AP-2a. The CC genotype strongly bound to AP-2a, and the TT genotype showed less binding affinity to AP-2a. The AP-2a strongly repressed the reporterexpressionintheCCgenotypeandshowedlessrepressionoftheTTgenotypedrivingexpression in vitro assay. Accordingly, TT genotype individuals had highest ATM mRNA expression, CT genotype individuals had moderate ATM mRNA expression, and the CC genotype individuals had the lowest ATM mRNA.

Keywords: Long-lived individuals Ataxia telangiectasia mutated Single nucleotide polymorphism AP-2a

 2010 Elsevier Ireland Ltd. All rights reserved.

and function are dramatically decreased. In humans, patients with mutations in the ATM gene are featured with immunodeficiency, insulin resistance, growth retardation, pigmentary abnormalities, progressive cerebellar degeneration, and increased susceptibility to cancer (McKinnon, 2004). These observations suggest that ATM is critical to lifespan.

1. Introduction

Reactive oxygen species (ROS) are now believed to be a major contributor to the pathogenesis of senescence through inducing DNA double-strand breaks (DSBs) and telomere dysfunction (Herbig et al., 2006). Once DNA damage happens, the ataxia telangiectasia mutated (ATM) gene detects it, and then ATM phosphorylates activates downstream kinases, such as ChK1, ChK2 and P53 (Shiloh, 2003). As a result, cells undergo transient cell- cycle arrest, apoptosis or senescence. Meanwhile, in our body, there exists an anti-oxidant system, which can reduce ROS levels. The ATM gene plays an important role in keeping balance in redox state. Mice with the ATM gene knocked out have progressive bone marrow failure resulting from a defect in hematopoietic stem cells function due to elevated ROS (Ito et al., 2004). The ATM gene is widely expressed in many tissues, such as brain, skin and endothelial cells (McKinnon, 2004). In aged mice, ATM expression

Recent studies in twins have indicated that longevity is moderately heritable and genetic variants account for (cid:2)25% (Puca et al., 2001). However, as of now, reports about polymorphisms of the ATM gene mainly focus on cancer; little information of the relationship between the variants of ATM promoter region and longevity is available. So we intend to determine whether the association between genetic variants in ATM promoter and longevity exists.

2. Materials and methods

2.1. Subjects

1761 blood samples were collected from 875 long-lived individuals (LLIs) and 886 unrelated subjects from July 2005 to December 2007. Informed consent was obtained from all participants (as well as their legal proxies), all of Han Chinese descent and from the same region. The breakdown of the participants are as follows: LLIs included 280 men and 595 women, from 90 to 108 years, mean age 94.6, SD  )+3 3.40, without major diseases; control group included 395 women and 491

* Corresponding author. Tel.: +

; fax: +

** Corresponding author. Tel.: +

; fax: +

E-mail addresses: birongdong@

com (B. Dong), xmingmo@yahoo.com (X. Mo).

0047-6374/$ – see front matter © 2 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2010.08.009

Table 1

Primers and product sizes for PCR-DHPLC and PCR-RFLP.

Forward primer Reverse primer PCR product size Melt temperature 50-acagcaggaaccacaataagg-30 50-ccaagtctgaggacggaagt-30 226bp 568C 50-ctgtcgtcacttccgtcctca-30 50-aagaagcaacgccaagcag-30 240bp 588C 50-gctgcttggcgttgctt-30 50-catgcgattggcggtctgg-30 287bp 568C

2.7. Luciferase reporter assay

men, from 18 to 85 years, mean age 49.06, SD D+2  20.95. Anybody has not lived up to 90 years old till now in the families of control individuals. Age was reported by participants themselves and was confirmed by calculating the dates of birth stated in their ‘‘household registration books’’, the certification of birth in China. Genomic DNA wasisolatedfromwholebloodanddrawnfromtheantecubitalvein,usingcommercial DNA isolation kits from QIAGEN (Chatsworth, CA) and according to standard procedures. The relevant institutional Ethics Committees approved this study.