Analysis of epigenetic clocks links yoga, sleep, education, reduced meat intake, coffee, and a SOCS2 gene variant to slower epigenetic aging.

DNA methylation (DNAm) clocks hold promise for measuring biological age, useful for guiding clinical interventions and forensic identification. This study compared the commonly used DNAm clocks, using DNA methylation and SNP data generated from nearly 1000 human blood or buccal swab samples. We evaluated different preprocessing methods for age estimation, investigated the association of epigenetic age acceleration (EAA) with various lifestyle and sociodemographic factors, and undertook a series of novel genome-wide association analyses for different EAA measures to find associated genetic variants. Our results highlighted the Skin&Blood clock with ssNoob normalization as the most accurate predictor of chronological age. We provided novel evidence for an association between the practice of yoga and a reduction in the pace of aging (DunedinPACE). Increased sleep and physical activity were associated with lower mortality risk score (MRS) in our dataset. University degree, vegetable consumption, and coffee intake were associated with reduced levels of epigenetic aging, whereas smoking, higher BMI, meat consumption, and manual occupation correlated well with faster epigenetic aging, with FitAge, GrimAge, and DunedinPACE clocks showing the most robust associations. In addition, we found a novel association signal for SOCS2 rs73218878 (p = 2.87 × 10-8) and accelerated GrimAge. Our study emphasizes the importance of an optimized DNAm analysis workflow for accurate estimation of epigenetic age, which may influence downstream analyses. The results support the influence of genetic background on EAA. The associated SOCS2 is a member of the suppressor of cytokine signaling family known for its role in human longevity. The reported association between various risk factors and EAA has practical implications for the development of health programs to improve quality of life and reduce premature mortality associated with age-related diseases.

Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks.

BACKGROUND: DNA methylation analysis has proven to be a powerful tool for age assessment. However, the implementation of epigenetic age prediction in diagnostics or routine forensic casework requires appropriate laboratory methods. In this study, we aimed to compare the performance of large-scale DNA methylation analysis protocols that show promise in terms of accuracy, throughput, multiplexing capacity, and high sensitivity. RESULTS: The protocols were designed to target a predefined panel of 161 genomic CG/CA sites from four known estimators of epigenetic age-related parameters, optimized and validated using artificially methylated controls or blood samples. We successfully targeted 96% of these loci using two enrichment protocols: Ion AmpliSeq™, an amplicon-based method integrated with Ion Torrent S5, and SureSelectXT Methyl-Seq, a hybridization-based method followed by MiSeq FGx sequencing. Both protocols demonstrated high accuracy and robustness. Although hybridization assays have greater multiplexing capabilities, the best overall performance was observed for the amplicon-based protocol with the lowest variability in DNA methylation at 25 ng of starting DNA, mean observed marker coverage of ~ 6.7 k reads, and accuracy of methylation quantification with a mean absolute difference between observed and expected methylation beta value of 0.054. The Ion AmpliSeq method correlated strongly with genome-scale EPIC microarray data (R = 0.91) and showed superiority in terms of methylation measurement accuracy. Method-to-method bias was accounted for by the use of linear transformation, which provided a highly accurate prediction of calendar age with a mean absolute error of less than 5 years for the VISAGE and Hannum age clocks used. The pace of aging (PoAm) and the mortality risk score (MRS) estimators included in our panel represent next-generation clocks, were found to have low to moderate correlations with the VISAGE and Hannum models (R < 0.75), and thus may capture different aspects of epigenetic aging. CONCLUSIONS: We propose a laboratory tool that allows the quantification of DNA methylation in cytosines underlying four different clocks, thus providing broad information on epigenetic aging while maintaining a reasonable number of CpG markers, opening the way to a wide range of applications in forensics, medicine, and healthcare.

The role of CYP19A1 and ESR2 gene polymorphisms in female androgenetic alopecia in the Polish population.

Introduction: Androgenetic alopecia is the most common type of non-cicatricial alopecia both in male and female patients. The mechanism that leads to hair loss is similar in both sexes, but the underlying cause, and especially the role of genes and sex hormones in the pathogenesis of the disease in women has not fully been explained as of yet. So far, a few attempts have been made to assess selected SNPs for CYP19A1 and ESR2 genes, but their results are not unequivocal and fully reproducible. Aim: To investigate the association of 13 CYP19A1 and 11 ESR2 gene SNPs with female androgenetic alopecia (FAGA) in a population of Polish patients, including some already genotyped SNPs of possible importance for FAGA pathophysiology in other populations. Material and methods: Twenty-four genetic polymorphisms were analysed for the ESR2 and CYP19A1 genes in 117 patients with FAGA and 128 healthy subjects treated at the Department of Dermatology in Krakow. Results: In the studied Polish population, none of the selected SNPs, frequently detected in the Caucasian population and linked with the transformation pathway of sex hormones, showed a significant association with FAGA. Conclusions: Further studies into the genetic background of androgenetic alopecia are needed. Ethnic differences as well as the size of the studied population may be of great significance for the obtained results.

A collaborative exercise on DNA methylation-based age prediction and body fluid typing.

DNA methylation has become one of the most useful biomarkers for age prediction and body fluid identification in the forensic field. Therefore, several assays have been developed to detect age-associated and body fluid-specific DNA methylation changes. Among the many methods developed, SNaPshot-based assays should be particularly useful in forensic laboratories, as they permit multiplex analysis and use the same capillary electrophoresis instrumentation as STR analysis. However, technical validation of any developed assays is crucial for their proper integration into routine forensic workflow. In the present collaborative exercise, two SNaPshot multiplex assays for age prediction and a SNaPshot multiplex for body fluid identification were tested in twelve laboratories. The experimental set-up of the exercise was designed to reflect the entire workflow of SNaPshot-based methylation analysis and involved four increasingly complex tasks designed to detect potential factors influencing methylation measurements. The results of body fluid identification from each laboratory provided sufficient information to determine appropriate age prediction methods in subsequent analysis. In age prediction, systematic measurement differences resulting from the type of genetic analyzer used were identified as the biggest cause of DNA methylation variation between laboratories. Also, the use of a buffer that ensures a high ratio of specific to non-specific primer binding resulted in changes in DNA methylation measurement, especially when using degenerate primers in the PCR reaction. In addition, high input volumes of bisulfite-converted DNA often caused PCR failure, presumably due to carry-over of PCR inhibitors from the bisulfite conversion reaction. The proficiency of the analysts and experimental conditions for efficient SNaPshot reactions were also important for consistent DNA methylation measurement. Several bisulfite conversion kits were used for this study, but differences resulting from the use of any specific kit were not clearly discerned. Even when different experimental settings were used in each laboratory, a positive outcome of the study was a mean absolute age prediction error amongst participant's data of only 2.7 years for semen, 5.0 years for blood and 3.8 years for saliva.

Impact of excessive alcohol abuse on age prediction using the VISAGE enhanced tool for epigenetic age estimation in blood.

DNA methylation-based clocks provide the most accurate age estimates with practical implications for clinical and forensic genetics. However, the effects of external factors that may influence the estimates are poorly studied. Here, we evaluated the effect of alcohol consumption on epigenetic age prediction in a cohort of extreme alcohol abusers. Blood samples from deceased alcohol abusers and age- and sex-matched controls were analyzed using the VISAGE enhanced tool for age prediction from somatic tissues that enables examination of 44 CpGs within eight age markers. Significantly altered DNA methylation was recorded for alcohol abusers in MIR29B2CHG. This resulted in a mean predicted age of 1.4 years higher compared to the controls and this trend increased in older individuals. The association of alcohol abuse with epigenetic age acceleration, as determined by the prediction analysis performed based on MIR29B2CHG, was small but significant (ß = 0.190; P-value = 0.007). However, the observed alteration in DNA methylation of MIR29B2CHG had a non-significant effect on age estimation with the VISAGE age prediction model. The mean absolute error in the alcohol-abusing cohort was 3.1 years, compared to 3.3 years in the control group. At the same time, upregulation of MIR29B2CHG expression may have a biological function, which merits further studies.

Epigenetic age prediction in semen - marker selection and model development.

DNA methylation analysis is becoming increasingly useful in biomedical research and forensic practice. The discovery of differentially methylated sites (DMSs) that continuously change over an individual's lifetime has led to breakthroughs in molecular age estimation. Although semen samples are often used in forensic DNA analysis, previous epigenetic age prediction studies mainly focused on somatic cell types. Here, Infinium MethylationEPIC BeadChip arrays were applied to semen-derived DNA samples, which identified numerous novel DMSs moderately correlated with age. Validation of the ten most age-correlated novel DMSs and three previously known sites in an independent set of semen-derived DNA samples using targeted bisulfite massively parallel sequencing, confirmed age-correlation for nine new and three previously known markers. Prediction modelling revealed the best model for semen, based on 6 CpGs from newly identified genes SH2B2, EXOC3, IFITM2, and GALR2 as well as the previously known FOLH1B gene, which predict age with a mean absolute error of 5.1 years in an independent test set. Further increases in the accuracy of age prediction from semen DNA will require technological progress to allow sensitive, simultaneous analysis of a much larger number of age correlated DMSs from the compromised DNA typical of forensic semen stains.

Development of the VISAGE enhanced tool and statistical models for epigenetic age estimation in blood, buccal cells and bones.

DNA methylation is known as a biomarker for age with applications in forensics. Here we describe the VISAGE (VISible Attributes through GEnomics) Consortium's enhanced tool for epigenetic age estimation in somatic tissues. The tool is based on eight DNA methylation markers (44 CpGs), bisulfite multiplex PCR followed by sequencing on the MiSeq FGx platform, and three statistical prediction models for blood, buccal cells and bones. The model for blood is based on six CpGs from ELOVL2, MIR29B2CHG, KLF14, FHL2, TRIM59 and PDE4C, and predicts age with a mean absolute error (MAE) of 3.2 years, while the model for buccal cells includes five CpGs from PDE4C, MIR29B2CHG, ELOVL2, KLF14 and EDARADD and predicts age with MAE of 3.7 years, and the model for bones has six CpGs from ELOVL2, KLF14, PDE4C and ASPA and predicts age with MAE of 3.4 years. The VISAGE enhanced tool for age estimation in somatic tissues enables reliable collection of DNA methylation data from small amounts of DNA using a sensitive multiplex MPS assay that provides accurate estimation of age in blood, buccal swabs, and bones using the statistical model tailored to each tissue.

DNA methylation-based age clocks: From age prediction to age reversion.

Aging as an irretrievable occurrence throughout the entire life is characterized by a progressive decline in physiological functionality and enhanced disease vulnerability. Numerous studies have demonstrated that epigenetic modifications, particularly DNA methylation (DNAm), correlate with aging and age-related diseases. Several investigations have attempted to predict chronological age using the age-related alterations in the DNAm of certain CpG sites. Here we categorize different studies that tracked the aging process in the DNAm landscape to show how epigenetic age clocks evolved from a chronological age estimator to an indicator of lifespan and healthspan. We also describe the health and disease predictive potential of estimated epigenetic age acceleration regarding different clinical conditions and lifestyle factors. Considering the revealed age-related epigenetic changes, the recent age-reprogramming strategies are discussed which are promising methods for resetting the aging clocks.

Murine cellular model of mucopolysaccharidosis, type IIIB (MPS IIIB) - A preliminary study with particular emphasis on the non-oxidative l-cysteine metabolism.

The lack of the N-alpha-glucosaminidase (Naglu) is responsible for the incidence of a rare disease - mucopolysaccharidosis, type IIIB (MPS IIIB). To date, studies have been conducted based on cells derived from patients suffering from MPS or using in vivo MPS mouse models. These limitations have allowed for defining our research goal - to create and characterize the first in vitro murine cellular MPS IIIB model. In the current work we present a new, stable cell line with confirmed accumulation of glycosaminoglycans. The line stability was achieved by immortalization using a lentivirus carrying the T-antigens of SV40. The Naglu-/- cells were confirmed to produce no Naglu enzyme. To confirm the proper functioning of the in vitro MPS IIIB model, we determined the activity and expression of cystathionine γ-lyase, rhodanese and 3-mercaptopyruvate sulfurtransferase, as well as the level of low molecular-weight thiols (reduced and oxidized glutathione, cysteine and cystine). The results were referred to our earlier findings originating from the studies on the tissues of the Naglu-/- mice that were used to create the lines. The results obtained in the Naglu-/- cells were in accordance with the results found in the mouse model of MPS IIIB. It suggests that the presented murine Naglu-/- cell lines might be a convenient in vitro model of MPS IIIB.