To ensure external validity of genotyping methods, the results of a subset of polymorphisms/samples should be corroborated in a second laboratory using a different method (Chanock et al. 2007). As such, genotyping (polymorphisms K153R and E164K) of the samples of 134 centenarians (78 % of total) was also performed in the genomics laboratory of Vall ’ d Hebron Hospital (Barcelona, Spain) during the spring of 2012 using a different methodology, i.e. custom-designed Taqman® SNP genotyping assays (assay ID: C___282184_30 for the K153R variant and assay ID: C__57828961_10 for the E164K vari- ant) (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 pre-read and post-read steps 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.

Genotype assessment

Spanish cohort Genotyping was performed specifi- cally for research purposes. The researchers in charge of genotyping were totally blinded to the ’ participants identities, i.e. saliva samples were tracked solely with barcoding and personal identi- ties were only made available to the main study researcher who was not involved in actual genotyp- ing. In all the participants, we extracted genomic DNA from saliva samples. We used the classical – phenol chloroform DNA extraction protocol with alcoholic precipitation. Genomic DNA was resus- μ − pended in 50 l Milli-Q H O and stored at 20 °C. 2

cally for research purposes. The researchers in charge of genotyping were totally blinded to the ’ participants identities, i.e. saliva samples were tracked solely with barcoding and personal identi- ties were only made available to the main study researcher who was not involved in actual genotyp- ing. In all the participants, we extracted genomic DNA from saliva samples. We used the classical – phenol chloroform DNA extraction protocol with alcoholic precipitation. Genomic DNA was resus- μ − pended in 50 l Milli-Q H O and stored at 20 °C. 2 ’ In all samples (from both cases and controls groups), sequences corresponding to the E164K, I225T, K153R and P198A variants were amplified – during 2009 2012 by polymerase chain reaction (PCR) in the Genetics Laboratory of the European University of Madrid (Spain). The primers used were ′ ′ ′ 5 -GAAAACCCAAATGTTGCTTC-3 and 5 - ′ TGTCTAGCTTATGAGCTTAGGG-3 . The PCR con- ditions were as follows: initial denaturation at 95 °C for 10 min; 35 cycles at 95 °C for 1 min, 52 °C for 45 s and 72 °C for 1 min; and a final extension at 72 °C for 5 The resulting PCR products were genotyped by single-base extension (SBE) (Gonzalez-Freire et al. 2010). The primers used for E164K, I225T, K153R and ′ ′ P198A were 5 -CAAACACTGTTGTAGGAGTCT-3 , ′ ′ ′ 5 - C T G A AT C C A A C T TA G G C A - 3 , 5 - ′ ′ TTTAATACAATACAATAAAGTAGTAA-3 and 5 - ′ TTTTTTTTATCTCTGAAACTTGACATGAAC-3 , re- spectively. The PCR SBE conditions were 96 °C for 10 s, then 25 cycles at 50 °C for 5 s and 60 °C for 30 s. The resulting PCR products were detected in an ABI

groups), sequences corresponding to the E164K, I225T, K153R and P198A variants were amplified – during 2009 2012 by polymerase chain reaction (PCR) in the Genetics Laboratory of the European University of Madrid (Spain). The primers used were ′ ′ ′ 5 -GAAAACCCAAATGTTGCTTC-3 and 5 - ′ TGTCTAGCTTATGAGCTTAGGG-3 . The PCR con- ditions were as follows: initial denaturation at 95 °C for 10 min; 35 cycles at 95 °C for 1 min, 52 °C for 45 s and 72 °C for 1 min; and a final extension at 72 °C for 5 min.

Replication cohort In Italian participants, genomic DNA was purified from peripheral blood samples us- ing the QiaAmp DNA Mini kit (QIAGEN, Hilden, ’ Germany) according to the manufacturer s protocol. ’ In order to obtain unlimited quantities of centenarians DNA for different genotyping projects, an in vitro immortalization of genomic DNA was performed by means of whole-genome amplification using a com- mercially available kit (GenomiPhi, GE Healthcare, Piscataway, NJ, USA). Genotyping of the K153R polymorphism was performed in the Italian cohort during November 2012. The procedure for detecting the K153R polymorphism was based on PCR ampli- fication, restriction cleavage with BanII (New England Biolabs, Ipswich, MA, USA) and separation of the DNA fragments by electrophoresis, as previously de- scribed (Ferrell et al. 1999). For quality control, gen- otyping analyses were done blind with respect to centenarian/young control status, and a random 25 % of the samples were repeated. The concordance rate for duplicate genotyping was 100 %. Two investiga- tors independently reviewed all results.

The resulting PCR products were genotyped by single-base extension (SBE) (Gonzalez-Freire et al. 2010). The primers used for E164K, I225T, K153R and ′ ′ P198A were 5 -CAAACACTGTTGTAGGAGTCT-3 , ′ ′ ′ 5 - C T G A AT C C A A C T TA G G C A - 3 , 5 - ′ ′ TTTAATACAATACAATAAAGTAGTAA-3 and 5 - ′ TTTTTTTTATCTCTGAAACTTGACATGAAC-3 , re- spectively. The PCR SBE conditions were 96 °C for 10 s, then 25 cycles at 50 °C for 5 s and 60 °C for 30 s. The resulting PCR products were detected in an ABI PRISM (Applied Biosystems, Foster City, CA, USA).

To ensure proper internal control, for each geno- type analysis, we used positive and negative con- trols from different DNA aliquots which were previously genotyped by the same method accord- ing to recent recommendations for replicating ge- – notype phenotype association studies (Chanock et al. 2007). All the analyses were performed by two experienced independent investigators who were blinded to subject data.

Statistical analysis

All statistical analyses were performed using the PASW (v. 18.0 for Windows, Chicago), except for statistical power, which we calculated with the