Evaluation of six blood-based age prediction models using DNA methylation analysis by pyrosequencing

Abstract

DNA methylation has been identified as the most promising molecular biomarker for the prediction of age. Several DNA methylation-based models have been proposed for age prediction based on blood samples, using mainly pyrosequencing. These methods present different performances for age prediction and have rarely, if ever, been evaluated and intercompared in an independent validation study. Here, for the first time, we evaluate and compare six blood-based age prediction models (Bekaert 1 , Park 2 , Thong 3 , Weidner 4 , and the Zbiec-Piekarska 1 5 and Zbiec-Piekarska 2 6 ), using DNA methylation analysis by pyrosequencing on 100 blood samples from French individuals aged between 19–65 years. For each model, we perform correlation analysis and evaluate age-prediction performance (mean absolute deviation (MAD) and standard error of the estimate (SEE)). The best age-prediction performances were found with the Bekaert and Thong models (MAD of 4.5–5.2, SEE of 6.8–7.2), followed by the Zbiec-Piekarska 1 model (MAD of 6.8 and SEE of 9.2), while the Park, Weidner and Zbiec-Piekarska 2 models presented lower performances (MAD of 7.2–8.7 and SEE of 9.2–10.3). Given these results, we recommend performing systematic, independent evaluation of all age prediction models on a same cohort to validate the different models and compare their performance.

Improvements and inter-laboratory implementation and optimization of blood-based single-locus age prediction models using DNA methylation of the ELOVL2 promoter

“…To our knowledge, only a single-locus model based on two CpGs of the ELOVL2 promoter and multiple linear regression has been proposed to date, in which the MAD of age predictions were of 5.03 and 5.75 in the training (303 samples) and testing (124 samples) sets respectively 25 . Despite multi-locus age prediction models generally presented better age prediction accuracy than the ELOVL2 single-locus model in their original studies, our recent study evaluating six blood based age prediction models using DNA methylation analysis by pyrosequencing showed that the performances of multi-locus models could sometimes be poorer in independent validation studies 26 . This could be attributed to inter-laboratory variations and discrepancies resulting from slight experimental differences accumulated during the different stages of sample processing, which could potentially increase as the number of PCR and pyrosequencing assays increases 26 .…”

“…Several multi-locus age prediction models based on DNA methylation of blood or other body fluids relied on less than a dozen markers using mainly pyrosequencing to several tens or hundreds of loci using epigenotyping arrays 12,21,26 . To our knowledge, only a single-locus model based on two CpGs of the ELOVL2 promoter and multiple linear regression has been proposed to date, in which the MAD of age predictions were of 5.03 and 5.75 in the training (303 samples) and testing (124 samples) sets respectively 25 .…”

“…DNA quantification was performed using the Qubit dsDNA HS assay Kit on a Qubit 3 Fluorometer (Thermo Fischer Scientific) according to the manufacturer's instructions. These DNA samples were already analysed in our previous study 26 and were used to perform a new sodium bisulfite treatment in the present study.…”

“…PCR amplification. ELOVL2 promoter region was amplified as described in 26 DNA methylation analysis by pyrosequencing. 10 µL of PCR product was purified and prepared for pyrosequencing (sequencing oligo ELOVL2_Seq: ACA ACC AAT AAA TAT TCC TAA AAC T and pyrosequencing analysis sequence: CCR 1 TGA AAC R 2 TTG AAG ACCR 3 CCR 4 CR 5 CR 6 AAA CCR 7 AC) according to a previously described protocol 36,37 .…”

“…The models presenting the best age prediction accuracy were the multi-locus models of Bekaert and Thong (MAD of 4.5-5.2 years and SEE of 6.8-7.2 years) followed by the single-locus model of Zbiec-Piekarska 1 (MAD of 6.8 year and SEE of 8.6 years) while the models presenting the worst age prediction performances (MAD of 7.2-8.7 years and SEE of 9.2-10.3 years) were the three other multi-locus models of Weidner, Park and Zbiec-Piekarska 2 26 . The latter MAD were much higher than the ones described in their original studies, and we suggested that these differences could be principally attributed to inter-laboratory variations during the implementation of the different pyrosequencing assays 26 . Thus, the use of several loci and pyrosequencing assays might increase the variability in the predicted age estimates of the models when run in different laboratories.…”