Contents lists are available at the 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. 

A functional single nucleotide polymorphism (SNP) in the promoter of ATM is associated with longevity. Tie Chen, Birong Dong, Zhenchan Lu, Baochun Tian, Jian Zhang, Jianlin Zhou, Hongmei Wu, Yanling Zhang, Jinhui Wu, Ping Lin, Jie Zhang, Hong Xu, Xianming Mo.

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 and reducing oxidative stress while protecting telomere length; little information about ATM and longevity is available. Therefore we aim to examine the association between genetic variants in the 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 the ATM gene was identified and a 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 reporter expression in the CC genotype and showed less repression of the TT genotype driving expression in vitro assay. Accordingly, TT genotype individuals had the 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. 

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; 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 antioxidant 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 and function are dramatically decreased. 

Recent studies in twins have indicated that longevity is moderately heritable and genetic variants account for 25% (Puca et al., 2001). However, as of now, reports about polymorphisms of the ATM gene mainly focus on cancer; little information about 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 is as follows: LLIs included 280 men and 595 women, from 90 to 108 years, mean age 94.6, SD ±3.40, withoutDilution of templates and the analysis of amplified products was performed by an image analyzer (ChemiDocTM XRS, BIO-RAD, USA). B-Actin was used as an internal control (Giacconi et al., 2004). To validate genetic variations, we randomly selected 41 DNA samples from 29 variant and 12 invariant subjects for sequencing. One single nucleotide polymorphism (SNP, rs189037) in the third amplicon of the ATM promoter (Fig. 1C) was identified. The SNP contains CC, CT, and TT genotypes in the tested human individuals. 

2.6. Electrophoretic mobility shift assay (EMSA) Synthetic double-stranded and 50 biotin-labeled and unlabeled probes were obtained from Takara. In brief, purified human recombinant GST-AP-2a protein and labeled probes were incubated in a 30 ml binding reaction mixture for 30 min at 4°C. To prevent nonspecific binding, poly(dI-dC) and salmon sperm DNA were added to the mixture. Then, the samples were separated on a 6% polyacrylamide gel at 4°C for 90 min at 100 V in 0.5 M Tris-glycine buffer when the gels were pre-run for 50 min at 100 V. After electrophoresis, transfer was performed at 4°C and 250 mA for 90 min. The products were detected by Stabilized Streptavidin-Horseradish Peroxidase Conjugate (Roche). Unlabeled probes at a 100-fold molar excess were added to the reaction for competition.