Association Study on Long-Living Individuals from Southern Italy Identifies rs10491334 in the Gene That Regulates Survival Proteins

Alberto Malovini,1,2 Maddalena Illario,3 Guido Iaccarino,3 Francesco Villa,2 Anna Ferrario,2 Roberta Roncarati,2 Chiara Viviani Anselmi,2 Valeria Novelli,2 Ersilia Cipolletta,3 Eleonora Leggiero,4 Alessandro Orro,5 Maria Rosaria Rusciano,3 Luciano Milanesi,5 Angela Serena Maione,3 Gianluigi Condorelli,2,5 Riccardo Bellazzi,1 and Annibale A. Puca2,5

Abstract

Long-living individuals (LLIs) are used to study exceptional longevity. A number of genetic variants have been found associated in LLIs to date, but further identification of variants would improve knowledge on the mechanisms regulating the rate of aging. Therefore, we performed a genome-wide association study on 410 LLIs and 553 young control individuals with a 317K single-nucleotide polymorphism (SNP) chip to identify novel traits associated with aging. Among the top ( p < 1(cid:2)10(cid:3)4 ) SNPs initially identified, we found rs10491334 (CAMKIV) (odds ratio [OR] ¼ 0.55; 95% confidence interval [CI] 0.42–0.73; p ¼ 2.88(cid:2)10(cid:3)5 ), a variant previously reported associated with diastolic blood pressure, associated also in a replication set of 116 LLIs and 160 controls (OR ¼ 0.54; 95% CI 0.32–0.90; p ¼ 9(cid:2)10(cid:3)3 ). Furthermore, in vitro analysis established that calcium/calmodulin- dependent protein kinase IV (CAMKIV) activates the survival proteins AKT, SIRT1, and FOXO3A, and we found that homozygous carriers of rs10491334 have a significant reduction in CAMKIV expression. This, to- gether with the observed reduction in minor-allele carriers among centenarians, points to a detrimental role for the SNP. In conclusion, prolongevity genes are activated by CAMKIV, the levels of which are influenced by rs10491334, a SNP associated with human longevity.

Introduction

APOE is involved in lipid transport to the lymphatic system; its gene was the first to be successfully replicated as one important in exceptional longevity.12 FOXO3A medi- ates a prosurvival activity by regulating the cellular re- sponse to stimuli ranging from radical stress to growth factors (insulin-like growth factor-1 [IGF-1]) and by acting on the transcription of genes involved in radical stress re- sponse, cell cycle arrest, proliferation, and apoptosis.13,14 Protein kinase B (AKT)-dependent phosphorylation and sirtuin 1 (SIRT1)-dependent deacetylation of FOXO3A in- duce prosurvival activity by activating genes linked to the stress response and to cell cycle arrest and by inhibiting proapoptotic genes.15 In humans, genetic alterations of the IGF-1 receptor that alter the IGF signaling pathway confer an increase in propensity for longevity.16 Single-nucleotide polymorphisms (SNPs) of AKT have also been associated with human longevity.11

(LLIs), that is, those that ap- proach 100 years of age, are a model of successful aging. Most of them have a compression of disability and morbidity toward the end of their life and tend not to show changes normally observed during aging, such as decreases in insulin sensitivity and in heart rate variability (HRV).1–3 The ex- ceptional longevity of LLIs is to some extent genetically driven, as underlined by the familial clustering effect for extreme longevity and the reduced mortality of centenarians’ siblings compared to those of noncentenarians.4,5 To date, the most remarkable genetic findings for exceptional lon- gevity observed in the genome of centenarians are the de- creased frequency of detrimental alleles of apolipoprotein E (APOE) and the increase of protective alleles of forkhead box

1Dipartimento di Informatica e Sistemistica, Universita` degli Studi di Pavia, Pavia, Italy. 2IRCCS Multimedica, Milano, Italy. 3Dipartimento di Biologia e Patologia Cellulare e Molecolare, and Dipartimento di Medicina Clinica e Scienze Cardiovascolari ed Im- munologiche, Universita` di Napoli Federico II, Napoli, Italy. 4CEINGE-Biotecnologie Avanzate, Napoli, Italy. 5Istituto di Tecnologie Biomediche–Consiglio Nazionale delle Ricerche, Segrate (MI), Italy.

estimated using the confidence intervals rule. Haplotype phases were inferred using the default expectation maximi- zation (EM) algorithm.

The identification and the study of genetic variants that influence exceptional longevity in humans are important because novel targets for prevention and therapy of a large spectrum of age-related diseases could be discovered.17 Genome-wide association studies (GWAS) are hypothesis- generating studies that can be used to this end. We have performed a GWAS to identify SNPs that are highly associ- ated with exceptional longevity in LLIs enrolled in the Southern Italian Centenarian Study (SICS).9

Power calculation

We performed power calculations using QUANTO soft- ware23 applying the following settings: (1) Longevity prev- alence in the general population &0.0001; (2) allele frequency ranging from 0.05 to 0.95; (3) significance threshold for the screening analysis set to p<1(cid:2)10(cid:3)4, evaluating allelic, dominant, and recessive genetic models.

Methods

SICS collection

Choice of the significance threshold

We recruited and phenotyped 582 LLIs (age range 90–109 years) and 784 young control individuals (age range 18–45 years) as part of the SICS. SICS LLIs were thoroughly in- vestigated for demographic characteristics, medical history (past and present diseases), level of independence (Barthel Score),18 and cognitive status (Cognitive Score) (Supple- mentary Table 1)(Supplementary Data can be accessed at) SICS subjects were enrolled by Associazione Longevita`.9,19 All subjects donated blood samples for DNA study and gave written informed consent to the study, which was approved by Ethical Committee. The study was conducted in accor- dance with the ethical principles that have their origins in the Declaration of Helsinki.