alejandro.lucia@uem.es

111 years) living mainly in nursing residencies of the Spanish central area (Meseta Castellana). This cohort included the oldest European individual (111 years old) alive in June 2012 (http://www.grg.org/Adams/E. ≥ HTM), and ~9 % of the cohort was aged 105 years. The most prevalent diseases were osteoarthritis (67 %), hypertension (57 %), dementia (49 %), and CAD (29 %). Two centenarians were free of any diagnosed disease. Approximately 13 % of the centenarians were virtually independent during daily living without assistance from other persons, i.e., their Barthel score ≥ was 80 and their score in the mini-mental state evaluation was >23 (Christensen et al. 2008).

young controls and thus provided preliminary evidence of association between this polymorphism and extreme longevity (Emanuele et al. 2010). Of note, the centenarians of the pioneer report by Emanuele et al. were free of CAD and dementia. It thus remains to be more clearly determined to what extent the potential effect of rs1333049 on extreme longevity depends mainly on its association with CAD/dementia risk or on other potential mechanisms. This question can be answered, at least partly, by studying centenarians who are not free of these two conditions and both healthy and diseased younger controls. In addition, it is important to – corroborate previous findings obtained in candidate gene association studies using replication, independent cohorts of different ethnic/geographic origins. It was therefore the purpose of our study to determine, using – a case control design, the association of the rs1333049 SNP and extreme longevity in a Spanish (Caucasian) cohort and an independent (Japanese) cohort. Cases “ ” were normal centenarians (i.e., not necessarily free of CAD or dementia), and we studied two control healthy groups of younger adults, i.e., without ( ) or with CAD. Based on the aforementioned studies and in recent research with centenarians from Southern Europe (Emanuele et al. 2010), we hypothesized that the risk C-allele rs1333049 would be less frequent in centenarians compared with ethnically matched younger controls, at least if these controls have CAD.

Healthy controls (n=362) Inclusion criteria for this ≤ group were (1) man or woman aged 50 years and (2) free of any diagnosed cardiometabolic disease. We extracted genomic DNA from saliva samples of – 143 men and 219 women during the years 2008 2010.

Controls with CAD (n=99) Inclusion criteria for this ≤ group were (1) man or woman aged 65 years and (2) with diagnosed CAD. We extracted genomic DNA ’ from patients (66 men, 33 women) saliva samples in the cardiology department of two Spanish hospitals ’ (Vall d Hebron, Barcelona and Gregorio Marañón, Madrid) during the summer of 2013.

— Participants Japanese cohort

Written consent was obtained from each participant. The study protocol was approved by the corresponding institutional ethics committee (National Institute of Health and Nutrition and Medical Research Institute, Tokyo Medical Dental University, Japan, for controls and Keio University, Japan, for centenarians) and was in accordance with the Declaration of Helsinki for Human Research of 1974 (last modified in 2008). All of the study participants were of the same Asian (Japanese) descent.

Methods

— Participants Spanish cohort

Written consent was obtained from each participant. The study protocol was approved by the institutional ethics committee (Universidad Europea de Madrid, Spain) and was in accordance with the Declaration of Helsinki for Human Research of 1974 (last modified in 2008). All the study participants were of the same Caucasian (Spanish) descent for three or more generations; the majority (~90 %) of them were born and lived most of their lives in the same areas of Spain (Meseta Castellana, ~600 m altitude).

The Japanese cohort of cases was gathered from two prospective cohorts: the Tokyo Centenarians Study (TCS) and the Semi- supercentenarians Study in Japan (SSC-J). A detailed description of population-based recruitments for the TCS has been previously reported (Gondo et al.

Cases (centenarians, n=175) – During 2009 2012, we obtained DNA from saliva samples in centenarians of

The SSC-J is a nationwide longitudinal survey mainly consisting of individuals aged 105 years or older, which started in 2002. In 2002, we recruited 135 SSC in whole Japan. After 2002, our recruitment strategy has relied on responses to local governments and nursing homes in the whole country and direct inquiries to our research team. Consequently, a total of 450 centenarians (58 men, 392 women) were enrolled into SSC-J by the end of November 2011.

Spanish cohort Genotyping was performed in the ’ genomics laboratory of Vall d Hebron Hospital (Barcelona, Spain) during the summer and autumn of 2012 (centenarians and healthy controls) and summer of 2013 (CAD controls). The two experienced researchers in charge of genotyping were totally ’ blinded to the participants identities, i.e., saliva samples were tracked solely with bar coding and personal identities were only made available to the main study researcher who was not involved in actual genotyping. We used the classical phenol-chloroform DNA extraction protocol with alcoholic precipitation. μ Genomic DNA was resuspended in 50 l milli-Q H O 2 − and stored at 20 °C. We used custom-designed Taqman® SNP genotyping assays (assay ID C_1754666_10) (Applied Biosystems, Foster City, CA, USA). All samples were run in a 7.500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). The analysis had a pre-read and post-read step of the plate consisting of 1 min at 60 °C before and after the PCR cycle. The cycle conditions were 10 min at 95 °C, followed by 40 cycles of 15 s at 92 °C, and 1 min at 60 °C. To ensure proper internal control, for each genotype analysis, we used positive and negative controls from different DNA aliquots which were previously genotyped by the same method.