Association Analyses of Insulin Signaling Pathway Gene Polymorphisms With Healthy Aging and Longevity in Americans of Japanese Ancestry

Brian J. Morris,1–3 Timothy A. Donlon,1 Qimei He,1 John S. Grove,1,4 Kamal H. Masaki,1,2 Ayako Elliott,1 D. Craig Willcox,1,5 and Bradley J. Willcox1,2

1Honolulu Heart Program (HHP)/Honolulu-Asia Aging Study (HAAS), Kuakini Medical Center, Hawaii. 2Department of Geriatric Medicine, John A. Burns School of Medicine, University of Hawaii, Honolulu. & Clinical Genomics Laboratory, School of Medical Sciences, University of Sydney, New South Wales, 4Public Health Sciences, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu. 5Department of Human Welfare, Okinawa International University, Ginowan, Japan.

Address correspondence to Brian J. Morris, DSc PhD, Honolulu Heart Program (HHP)/Honolulu-Asia Aging Study (HAAS), Kuakini Medical Center, 347 North Kuakini Street, HPM-9, Honolulu, Hawaii 96817. Email: brian.morris@sydney.edu.au

Evidence from model organisms suggests that the insulin/IGF-1 signaling pathway has an important, evolutionarily con- served influence over rate of aging and thus longevity. In humans, the FOXO3 gene is the only widely replicated insulin/ IGF-1 signaling pathway gene associated with longevity across multiple populations. Therefore, we conducted a nested case–control study of other insulin/IGF-1 signaling genes and longevity, utilizing a large, homogeneous, long-lived popu- lation of American men of Japanese ancestry, well characterized for aging phenotypes. Genotyping was performed of single nucleotide polymorphisms, tagging most of the genetic variation across several genes in the insulin/IGF-1 signal- ing pathway or related gene networks that may be influenced by FOXO3, namely, ATF4, CBL, CDKN2, EXO1, and JUN. Two initial, marginal associations with longevity did not remain significant after correction for multiple comparisons, nor were they correlated with aging-related phenotypes.

Key Words: Longevity—Molecular genetics—Insulin signaling genes—Human.

Received February 11, 2013; Accepted May 11, 2013

H ErITABILITy of extreme longevity is estimated as 0.33 and 0.48 in male and female centenarians (1). Although centenarians are enriched for longevity single nucleotide polymorphism (SNPs) (2), few have been identi- fied (3,4), possibly because of small effect sizes (5).

and uncoupling protein 3 and decreases phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase (12), and its neuronal expression affects Drosophila life span (13); (iii) cyclin-dependent kinase inhibitor 2B , which encodes INK4A and ArF, and whose genetic locus is asso- ciated with type 2 diabetes (14–16); (iv) the DNA repair enzyme exonuclease 1 (17); (v) V-Jun avian sarcoma virus 17 oncogene homolog (Jun), a transcription factor which, in a complex with Fos (to form the activator protein-1 com- plex), affects cell growth and tumorigenesis (18).

The insulin/IGF-1 signaling pathway is an example of an evolutionarily conserved biological pathway that affects aging (6,7). In humans a finding of association of genetic variation in the insulin/IGF-1 signaling pathway gene FOXO3A with extreme old age in our cohort of American men of Japanese ancestry (8) has now been replicated con- sistently in other populations for both sexes.

Methods

Here we report our findings from an exploratory study of SNPs in other evolutionarily conserved insulin/IGF-1 sign- aling pathway genes chosen on the basis of model organism data and human studies, namely, those encoding (i) activat- ing transcription factor 4, which activates stress-resistance genes (9), transcription of the asparagine synthase gene (ASNS) in response to nutrient stress (10), and suppresses the cell senescence/life span determining components INK4A/ ArF (locus CDKN2A) (11); (ii) Casitas B-cell lymphoma protein, which regulates whole-body energy expenditure and insulin action by suppression of the insulin receptor

Study Population

The study used a nested case–control design and participants drawn from the HHP/HAAS population described previously (8). These were American men of Japanese ancestry recruited in Oahu in 1965–1968 at ages 45–68 years and followed with regular examinations and blood work until the present, or death. “Cases” (longevity phenotype) were defined as HHP/HAAS participants who had survived to beyond the upper 1% of the 1910 U.S. birth cohort–specific survival (minimum 95 years

of age [19]; n = 213 as of August 2007, 176 died [age of death = 97.5 ± 2.1 SD; range 95–106 years] and 37 were still alive [mean age = 98.7 ± 2.1 SD; range 97–106 years]). Controls consisted of 402 individuals from the HHP/HAAS cohort who had died near the mean for the 1910 U.S. birth cohort–specific survival for middle-aged men (77 years). To achieve a case:control ratio of approximately 1:2, we sampled the HHP/HAAS study population for controls who died up to the age of 81 years (mean age at death = 78.5 ± 1.8 SD years; range 73–81 years), consistent with the 3.5 years longer life expectancy of American men of Japanese ancestry in Hawaii (20).

Statistical Analyses

Hardy–Weinberg equilibrium. Differences between cases and controls were evaluated by the Pearson χ2 test. The minimal detectable difference in allele frequency between cases and controls depends on the MAF among controls. For a nominal significance level of p = .05, a MAF for con- trols of 0.10 or 0.20 would require a MAF among cases of 0.155 and 0.271, respectively. Bonferroni adjustment for 15 tests requires p to be .05/15 (ie, .0033) for significance. For control MAF of 0.10 and 0.20, the minimal detectable dif- ference in MAF for cases would then be 0.176 and 0.296, respectively.