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 to be associated with 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 < 1x10^4) SNPs initially identified, we found rs10491334 (CAMKIV) (odds ratio [OR] 0.55; 95% confidence interval [CI] 0.42-0.73; p=2.88x10^5), a variant previously reported to be associated with diastolic blood pressure. It was also associated in a replication set of 116 LLIs and 160 controls (OR=0.54; 95% CI 0.32-0.90; p=9x10^3). Furthermore, in vitro analysis established that calcium/calmodulin-dependent protein kinase IV (CAMKIV) activates the survival proteins AKT, SIRT1, and FOXO3A. We found that homozygous carriers of rs10491334 have a significant reduction in CAMKIV expression. This, together 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 mediates a prosurvival activity by regulating the cellular response 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 response, cell cycle arrest, proliferation, and apoptosis.13,14 Protein kinase B (AKT)-dependent phosphorylation and sirtuin 1 (SIRT1)-dependent deacetylation of FOXO3A induce 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 

Long-living individuals (LLIs), that is, those that approach 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).13 The exceptional longevity of LLIs is to some extent genetically driven, as underlined by the familial clustering effect for extreme longevity and the reduced mortality of centenarian siblings compared to those of noncentenarians.4,5 To date, the most remarkable genetic findings for exceptional longevity observed in the genome of centenarians are the decreased frequency of detrimental alleles of apolipoprotein E (APOE) and the increase of protective alleles of forkhead box 1. 

Dipartimento 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 Immunologiche, Universita` di Napoli Federico II, Napoli, Italy. 4CEINGE-Biotecnologie Avanzate, Napoli, Italy. 5Istituto di Tecnologie BiomedicheConsiglio Nazionale delle R(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 for the study, which was approved by the Ethical Committee. The study was conducted following the ethical principles originating from the Declaration of Helsinki. The significance threshold was chosen by estimating the false-positive fraction for various significance thresholds P on simulated datasets. Data simulation involved generating 100 independent datasets comprising 317K SNPs, with MAF distribution ranging from 0.05 to 0.50, and 963 control samples to which 410 case and 553 control labels were randomly assigned. We then tested for association between each marker and the case/control status by assuming allelic, dominant, and recessive genetic models and selecting the most significant marker as the most informative for each SNP. For each significance threshold (from p-value 0.05 to p<1x10^-7), we estimated the mean fraction of false positive associations (a false positive association was defined as a SNP with p lower than the selected significance threshold P) over 100 simulated datasets for each P.

Determination of genotypes: Genotyping was performed with the Infinium II Assay-HumanHap BeadChip 317K duo system (Illumina, San Diego, California) using standard protocols of the Illumina HumanHap 317 Duo workflow (Illumina, San Diego, CA). All genotypes were evaluated using a quantitative quality score called GenCall (GC) score. The GC score ranges from 0 to 1 and reflects the proximity within a cluster plot of the intensities of that genotype to the centroid of the nearest cluster.