Age prediction in living: Forensic epigenetic age estimation based on blood samples.

DNA methylation analysis in a variety of genes has brought promising results in age estimation. The main aim of this study was to evaluate DNA methylation levels from four age-correlated genes, ELOVL2, FHL2, EDARADD and PDE4C, in blood samples of healthy Portuguese individuals. Fifty-three samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for CpG dinucleotide methylation status. Linear regression models were used to analyze relationships between methylation levels and chronological age. The highest age-associated CpG in each locus was chosen to build a multi-locus age prediction model (APM), allowing to obtain a Mean Absolute Deviation (MAD) between chronological and predicted ages of 5.35 years, explaining 94.1% of age variation. Validation approaches demonstrated the accuracy and reproducibility of the proposed multi-locus APM. Testing the APM in 51 blood samples from deceased individuals a MAD of 9.72 years was obtained. Potential differences in methylation status between samples from living and deceased individuals could exist since the highest age-correlated CpGs were different in some genes between both groups. In conclusion, our study using the bisulfite PCR sequencing method is in accordance with the high age prediction accuracy of DNA methylation levels in four previously reported age-associated genes. DNA methylation pattern differences between blood samples from living and deceased individuals should be taken into account in forensic contexts.

DNA methylation age estimation in blood samples of living and deceased individuals using a multiplex SNaPshot assay.

Many studies in the forensic field have reported that analysis of DNA methylation is the most reliable method of predicting age. In a previous study, 5 CpG sites located in ELOVL2, FHL2, KLF14, C1orf132 and TRIM59 genes were tested for age prediction purposes in blood, saliva and buccal swab samples from Korean individuals using a multiplex methylation SNaPshot assay. The main goals of the present study were i) to replicate the same multiplex SNaPshot assay in blood samples from Portuguese individuals, ii) to compare DNA methylation status between two different populations and iii) to address putative differences in the methylation status between blood from living and deceased individuals. Blood samples from 59 living individuals (37 females, 22 males; aged 1-94 years-old) and from 62 deceased individuals (13 females, 49 males; aged 28-86 years-old) were evaluated. The specific primers were those previously described. Linear regression models were used to analyse relationships between methylation levels and chronological age using IBM SPSS software v.24. Our results allowed to build a final age prediction model (APM) for blood samples of living individuals with 3 CpG sites, at ELOVL2, FHL2 and C1orf132 genes, explaining 96.3% of age variation, with a mean absolute deviation (MAD) from chronological age of 4.25 years. Some differences were found in the extent of the age association in the targeted loci comparing Portuguese with Korean individuals. The final APM built for deceased individuals included 4 CpG sites, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explaining 79.3% of age variation, with a MAD of 5.36 years. Combining both sets of samples from living and deceased individuals, the most accurate APM with 4 CpGs, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explained 92.5% of variation in age, with a MAD of 4.97 years. In conclusion, our study replicated in blood samples of Portuguese living individuals a previous SNaPshot assay for age estimation. The possibility that age markers might be population specific and that postmortem changes can alter the methylation status among specific loci was suggested by our data. Our study showed the usefulness of the multiplex methylation SNaPshot assay for forensic analysis in blood samples of living and deceased individuals.

The diagnostic performance of serum LIM homeobox transcription factor 1 alpha in patients with gastric cancer.

The study was conducted to investigate the diagnostic performance of serum LIM homeobox transcription factor 1 alpha (LMX1A) in patients with gastric cancer (GC).The serum level of LMX1A in GC, benign, and healthy groups was measured using quantitative real time PCR (qRT-PCR) and compared with the student t test. The associations of serum LMX1A levels with clinical parameters were analyzed with chi-square test. The diagnostic value of serum LMX1A in GC was evaluated by receiver operating characteristic (ROC) curve.The level of serum LMX1A in GC group (1.309 ±â€Š0.553) was significantly lower than that in the benign group (2.174 ±â€Š0.676) and healthy group (2.598 ±â€Š0.826) (P < .01 for both). The decreased level of LMX1A was associated with large tumor size (P = .009), positive lymph node metastasis (P = .027), and advanced TNM stages (P = .002). Receiver operating characteristic (ROC) analysis demonstrated that serum LMX1A could discriminate GC patients from the healthy individuals, with the area under the curve (AUC) of 0.889 (95% confidence interval [CI] = 0.838-0.938) combining with the sensitivity and specificity of 82.68% and 82.61%. Additionally, serum LMX1A also exhibited high accuracy in discriminating between GC patients and benign gastric disease cases (AUC = 0.842, 95% CI = 0.782-0.901), with the sensitivity of 81.89% and specificity of 72.41%.Serum LMX1A may be an effective biomarker for early detection of GC.

Methylation of ELOVL2 gene as a new epigenetic marker of age.

The discovery of biomarkers able to predict biological age of individuals is a crucial goal in aging research. Recently, researchers' attention has turn toward epigenetic markers of aging. Using the Illumina Infinium HumanMethylation450 BeadChip on whole blood DNA from a small cohort of 64 subjects of different ages, we identified 3 regions, the CpG islands of ELOVL2, FHL2, and PENK genes, whose methylation level strongly correlates with age. These results were confirmed by the Sequenom's EpiTYPER assay on a larger cohort of 501 subjects from 9 to 99 years, including 7 cord blood samples. Among the 3 genes, ELOVL2 shows a progressive increase in methylation that begins since the very first stage of life (Spearman's correlation coefficient = 0.92) and appears to be a very promising biomarker of aging.