aged 96–114 years and 596 females, aged 100–119 years. We selected cutoff ages of 96 years for males and 100 years for females of the NECS to focus on the extreme top 1% survival based on the U.S. Social Security Administration cohort life table (http://www. ssa.gov/OACT/NOTES/as116/as116LOT.html). NECS refer- ent cohort subjects consisted of 270 spouses of centenarian offspring and children of parents who died at the mean age of 73 years (average life expectancy for the parents’ birth cohort). Additional referent subjects were selected from the Illumina iControlDB database using genome-wide genetic matching as detailed in the methods (n= 1635). Note that approximately 100 male centenarians included in the pooling-based genome screen- ing overlap with this second set. Given that the overlap is relatively small (,10%) and that the subsequent analysis uses a different analytic approach (genotype data from individual subjects), we do not think the overlap is a significant concern.

In this study, we investigate two genes in the A(adenosine) to I (inosine) RNA editing pathway, a post-transcriptional process by which adenosine residues are converted to inosine resulting in a change in gene expression or protein function. Targets of RNA editing include a large number of genes as well as micro RNA. Thus, it is not surprising that such a non-specific cellular process would be involved in a general maintenance of cellular health and lifespan. However, such an implication has not been previously demonstrated.

Here, we first report significant association of these genes with EL in four centenarian studies that include the New England Centenarian Study (NECS), with more than 1,500 US individuals of primarily North European ancestry, aged between 90 and 119 years; the Southern Italian Centenarian Study (SICS) –a study of nonagenarians and centenarians from a closed population of Cilento, South Italy; the Ashkenazi Jewish Centenarian Study (AJCS), a study of approximately 300 nonagenarians and centenarians from a founder population of North Eastern European background, all resident in the US; and the Japanese Centenarian Study (JCS), a study of Japanese centenarians that has focused on ‘‘semi-supercentenarians’’ subjects living past 105 years [26]. The characteristics of the four populations allow us to assess the robustness of the associations to varying genetic background and environment.

From the SICS, we used genotype data from 271 males, ages 90–108 years and 188 females, aged 90–109 years (total= 459). Data from 200 male and 132 female SICS referent cohort subjects aged 18–48 years were used in this analysis. From the AJCS, genotype data were obtained from 299 oldest subjects (108 males aged 95 and older and 191 females aged 99 and older) and 269 younger referent cohort subjects (spouses of the offspring of centenarians, aged 85 and younger, without evidence of parental longevity). Four hundred and seventy oldest old subjects (82 males aged 100–110 years and 388 females, aged 100–116 years) and 538 referent cohort subjects (randomly selected Japanese subjects, aged 19–89 years) constituted the Japanese association study. Table 1 reports further summaries of subjects’ characteristics. Ages of the extreme old were validated with birth certificates (in the case

We further evaluate the functional significance of the RNA editing candidate genes in C. elegans lifespan studies and show that silencing orthologs of these genes reduces median survival by 50%. We also show that life span is fully restored by additional knockdown of an RNA interference gene, supporting the functional role of these genes in determining lifespan and implicating a novel axis for future aging studies.

Table 1. Study Subjects characteristics.

Results and Discussion

Selection of Candidate Genes

We selected the two genes to study for multiple reasons. First, in a preliminary genome wide screening using pooled DNA samples from approximately 130 male centenarians and 130 younger male controls from the NECS [27], we identified several single nucleotide polymorphisms (SNPs) in the RNA-editing genes ADARB1 (21q22.3), and ADARB2 (10p15.3) that were associated with extreme old age. ADARB1 exhibited the strongest evidence for genetic association with 5 SNPs that met genome-wide significance, with the posterior odds of allelic association .1,500 [27]. The probability of these 5 SNPs simultaneously associated under the null hypothesis of no association was 10213 based upon a hyper-geometric distribution. Second, this gene lies in chromo- some 21q21 and trisomy 21 (Down syndrome) resembles accelerated aging, with premature age-related changes including in the skin and hair, increased frequency of premature cataracts, hearing loss, menopause and Alzheimer’s disease [28] suggesting that genes in chromosome 21 could affect lifespan. Third, among the top genes identified from the preliminary genetic screen, RNA editing represents a general cellular process that might be expected to improve cellular health; and RNA editing activity has been associated with innate immune response [29,30] and age-related syndromes that include dementia and amyotrophic lateral sclerosis (ALS) [31].

Reported are summaries of the last contact ages. doi:10.1371/journal.pone.0008210.t001

of the JCS, the Basic Resident Registration Card). All subjects were enrolled by studies with Institutional Review Board approval and oversight.

errors thus suggesting that these are real associations and not artifacts.