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 emerging role of long non-coding RNAs, microRNAs, and an accelerated epigenetic age in Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease with variable clinical manifestations. Recent studies highlighted the contribution of epigenetic alterations to HD progress and onset. The potential crosstalk between different epigenetic layers and players such as aberrant expression of non-coding RNAs and methylation alterations has been found to affect the pathogenesis of HD or mediate the effects of trinucleotide expansion in its pathophysiology. Also, microRNAs have been assessed for their roles in the modulation of HD manifestations, among them are miR-124, miR-128a, hsa-miR-323b-3p, miR-432, miR-146a, miR-19a, miR-27a, miR-101, miR-9*, miR-22, miR-132, and miR-214. Moreover, long non-coding RNAs such as DNM3OS, NEAT1, Meg3, and Abhd11os are suggested to be involved in the pathogenesis of HD. An accelerated DNA methylation age is another epigenetic signature reported recently for HD. The current literature search collected recent findings of dysregulation of miRNAs or lncRNAs as well as methylation changes and epigenetic age in HD.

Emerging Role of Non-coding RNAs in Autism Spectrum Disorder.

Autism spectrum disorders (ASD) embrace a diverse set of neurodevelopmental diseases with a multifaceted genetic basis. Non-coding RNAs (ncRNAs) are among putative loci with critical participation in the development of ASD. Expression of some lncRNAs, namely RP11-466P24.2, SYP-AS1, STXBP5-AS1, and IFNG-AS1 has been decreased in ASD, while AK128569, CTD-2516F10.2, MSNP1AS, RPS10P2-AS1, LINC00693, LINC00689, NEAT1, TUG1, and Shank2-AS lncRNAs have been over-expressed in ASD. Expression of several miRNAs which are implicated in the immunological developmental, immune responses, and protein synthesis as well as those participating in the regulation of PI3K/Akt/mTOR and EGFR signaling pathways is dysregulated in the context of ASD. In the present article, we describe investigations which appraised the role of lncRNAs, miRNAs, and circRNAs in the pathobiology of ASD.

Discrimination of Patients with Prostate Cancer from Healthy Persons Using a Set of Single Nucleotide Polymorphisms.

PURPOSE: Prostate cancer is the second cancer diagnosed in males. It accounts for about 4% of cancer-related mortality in men. Several genetic polymorphisms in different genes have been identified that alter the risk of this kind of malignancy. MATERIALS AND METHODS: We used the random forest (RF) algorithm for prediction of prostate cancer risk in Iranian population using 13 different single nucleotide polymorphisms (SNPs) in four genes (ANRIL, HOTAIR, IL-6 and IL-8). The samples were divided into a training set (n=320) and a test set (n=80) to evaluate the generalization power for training algorithm. For hyper-parameters tuning, we used randomized search with 5-fold cross-validation for the following hyper-parameters: (1) Number of trees or estimators in the forest (set from 3 to 500); (2) The maximum number of leaf nodes (set from 2 to 32); (3) The maximum number of features used for the best split (set from 5 to 13); and (4) Using bootstrap samples in the trees building (True or False). Accuracy, sensitivity, specificity, and F1-score in both training and test sets were reported. RESULTS: The most important SNP was ANRIL-rs1333048: A/A (Gini index= 0.096) followed by ANRIL- rs10757278: G/G (Gini index= 0.059). Training Dataset Outcomes were as follow: Accuracy: 0.896, Sensitivity: 0.85, Specificity: 0.944 and F1 Score: 0.891. Test Dataset Outcomes were as follow: Accuracy: 0.787, Sensitivity: 0.775, Specificity: 0.800 and F1 Score: 0.784. The AUC Scores were 0.966 and 0.841 for training and test datasets, respectively. CONCLUSION: The proposed panels of SNPs can predict risk of prostate cancer in Iranian population with appropriate accuracy.

Emerging role of lncRNAs in the regulation of Rho GTPase pathway.

The Ras homolog (Rho) family of small GTPases comprise several proteins with prominent roles in regulation of cell cycle transition, cell migration, and remodeling of actin cytoskeleton. Expression of these proteins is regulated by several factors among them are long non-coding RNAs (lncRNAs). The impact of lncRNAs on Rho GTPases signaling can be exerted through direct modulation of expression of these proteins or influencing expression of miRNAs that negatively regulate Rho GTPases. LINC00974/miR-122/RhoA, MALAT1/miR-429/RhoA, ZFAS1/miR-3924/RhoA/ROCK2, PCAT6/miR-326/RhoA/ROCK, SMILR/miR-141/RhoA/ROCK, DAPK1/miR-182/RhoA, GAS5/miR663a/RhoB, H19/miR-15b/CDC42/PAK1, TDRG1/miR-93/RhoC, TUG1/miR-498/CDC42, UCA1/miR-18a/Cdc42 and UCA1/miR-182/Cdc42 are examples of lncRNAs/miRNAs axes that regulate Rho GTPases. In the present manuscript, we describe the role of lncRNAs on Rho GTPases.

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.

Association between methylene tetrahydrofolate reductase polymorphisms and risk of ischemic stroke.

Background: The methylene tetrahydrofolate reductase (MTHFR) is a folate-dependent enzyme which catalyzes the conversion of homocysteine to methionine. Two single nucleotide polymorphisms (SNPs) within this gene namely rs1801133 (C677T) and rs1801131 (A1298C) have been associated with elevated risk of ischemic stroke and total serum homocysteine in some populations.Aim: To assess associations between MTHFR SNPs and risk of ischemic stroke in Iranian population.Methods: In the current case-control study, we genotyped rs1801133 and rs1801131 SNPs in 318 Iranian patients with history of ischemic stroke and 400 age- and sex-matched controls using tetra-primer amplification refractory mutation system-polymerase chain reaction method.Results: The rs1801133 was significantly associated with risk of stroke in recessive model (OR (95% CI) = 1.89 (1.12-3.20), p = 0.03). The CT haplotype (rs1801131 and rs1801133, respectively) was significantly over-represented in patients compared with controls (OR (95% CI) = 1.71 (0.25-2.32), p = 0.002).Conclusion: Consequently, our data demonstrate contribution of MTHFR variants in risk of ischemic stroke in Iranian population.

Identification of miRNA-mRNA Network in Autism Spectrum Disorder Using a Bioinformatics Method.

Autism spectrum disorder (ASD) includes a heterogeneous group of disorders with different contributing genetics and epigenetics factors. Aberrant expression of miRNAs has been detected in ASD children compared with normally developed children. Due to the heterogeneity of this disorder, there is no consensus on ASD-associated miRNAs; thus, it is necessary to develop a model for comprehensive assessment of the role of miRNAs in ASD. We interrogated the PubMed, Google Scholar, and Web of Science databases until the end of 2019 to identify ASD-associated miRNAs. In addition, mRNA-coding genes that contribute to the pathogenesis of ASD were downloaded from the SFARI GENE ( https://gene.sfari.org/ ). The obtained 201 miRNAs and 478 target mRNAs were imported into the Cytoscape software suite to construct a miRNA-mRNA network. A protein-protein interaction network was constructed for target mRNAs using the CluPedia program in Cytoscape. Using this approach, we detected five modules that were associated with neurexins and neuroligins, glutamatergic synapse, cell adhesion molecules, NOTCH, MECP2 and circadian clock pathways, L1CAM interactions, and neurotransmitter release cycle. Taken together, functional analysis of these genes led to determination of critical pathways related to CNS disorders. Thus, the suggested approach in the current study resulted in the identification of the most relevant pathways in the pathogenesis of ASD that can be used as biomarkers or therapeutic targets.

Association Analysis of ANRIL Polymorphisms and Haplotypes with Autism Spectrum Disorders.

Autism spectrum disorder (ASD) has been shown to have a complex inheritance. Several single-nucleotide polymorphisms (SNPs) have been shown to be associated with risk of this neurodevelopmental disorder. In the current study, we genotyped four SNPs in a genomic hotspot for human disorders. The selected SNPs were located in adjacency of the antisense noncoding RNA in the INK4 locus (ANRIL) and have been shown to be associated with a number of human disorders. Genotyping was performed in 420 ASD cases and 420 normally developed children. After correction of P values for multiple comparisons, there was no significant difference in frequencies of rs1333045, rs1333048, rs4977574, and rs10757278 alleles, genotypes, or haplotypes between ASD children and children with normal development. However, one estimated haplotype (T A A A haplotype corresponding to rs1333045, rs1333048, rs4977574, and rs10757278 SNPs, respectively) tended to be more prevalent among cases compared with controls (OR (95% CI) = 1.77 (1.19-2.64), adjusted P value = 0.07). Besides, the T A G G tended to be less common among ASD cases compared with controls (OR (95% CI) = 0.64 (0.47-0.87), adjusted P value = 0.07). Although we could not detect significant difference in alleles, genotypes, or haplotypes frequencies between cases and controls, the trend toward association between two haplotypes and ASD risk implies that there might be a putative causative variant in the mentioned haplotypes whose association with ASD could be determined in larger cohorts of patients.

Origin of the Induced Pluripotent Stem Cells Affects Their Differentiation into Dopaminergic Neurons.

Neuronal differentiation of human induced pluripotent stem (iPS) cells, both in 2D models and 3D systems in vitro, allows for the study of disease pathomechanisms and the development of novel therapies. To verify if the origin of donor cells used for reprogramming to iPS cells can influence the differentiation abilities of iPS cells, peripheral blood mononuclear cells (PBMC) and keratinocytes were reprogrammed to iPS cells using the Sendai viral vector and were subsequently checked for pluripotency markers and the ability to form teratomas in vivo. Then, iPS cells were differentiated into dopaminergic neurons in 2D and 3D cultures. Both PBMC and keratinocyte-derived iPS cells were similarly reprogrammed to iPS cells, but they displayed differences in gene expression profiles and in teratoma compositions in vivo. During 3D organoid formation, the origin of iPS cells affected the levels of FOXA2 and LMX1A only in the first stages of neural differentiation, whereas in the 2D model, differences were detected at the levels of both early and late neural markers FOXA2, LMX1A, NURR1, TUBB and TH. To conclude, the origin of iPS cells may significantly affect iPS differentiation abilities in teratomas, as well as exerting effects on 2D differentiation into dopaminergic neurons and the early stages of 3D midbrain organoid formation.

Effects of host genetic variations on response to, susceptibility and severity of respiratory infections.

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global crisis, necessitating the identification of genetic factors that modulate the risk of disorder or its severity. The current data about the role of genetic risk factors in determination of rate of SARS-CoV-2 infection in each ethnic group and the severity of disorder is limited. Moreover, several confounding parameters such as the number of tests performed in each country, the structure of the population especially the age distribution, the presence of risk factors for respiratory disorders such as smoking and other environmental factors might be involved in the variability in disease course or prevalence of infection among different ethnic groups. However, assessment of the role of genetic variants in determination of the course of other respiratory infections might help in recognition of possible candidate for further analysis in patients affected with SARS-CoV-2. In the current review, we summarize the data showing the association between genomic variants and risk of acute respiratory distress syndrome, respiratory infections or severity of these conditions with an especial focus on the SARS-CoV-2.

In silico identification of MAPK14-related lncRNAs and assessment of their expression in breast cancer samples.

Mitogen-activated protein kinase (MAP kinase) pathways participate in regulation of several cellular processes involved in breast carcinogenesis. A number of non-coding RNAs including both microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) regulate or being regulated by MAPKs. We performed an in-silico method for identification of MAPKs with high number of interactions with miRNAs and lncRNAs. Bioinformatics approaches revealed that MAPK14 ranked first among MAPKs. Subsequently, we identified miRNAs and lncRNAs that were predicted to be associated with MAPK14. Finally, we selected four lncRNAs with higher predicted scores (NORAD, HCG11, ZNRD1ASP and TTN-AS1) and assessed their expression in 80 breast cancer tissues and their adjacent non-cancerous tissues (ANCTs). Expressions of HCG11 and ZNRD1ASP were lower in tumoral tissues compared with ANCTs (P values < 0.0001). However, expression levels of MAPK14 and NORAD were not significantly different between breast cancer tissues and ANCTs. A significant association was detected between expression of HCG11 and estrogen receptor (ER) status in a way that tumors with up-regulation of this lncRNA were mostly ER negative (P value = 0.04). Expressions of ZNRD1ASP and HCG11 were associated with menopause age and breast feeding duration respectively (P values = 0.02 and 0.04 respectively). There was a trend towards association between ZNRD1ASP expression and patients' age of cancer diagnosis. Finally, we detected a trend toward association between expression of NORAD and history of hormone replacement therapy (P value = 0.06). Expression of MAPK14 was significantly higher in grade 1 tumors compared with grade 2 tumors (P value = 0.02). Consequently, the current study provides evidences for association between lncRNA expressions and reproductive factors or tumor features.

Altered cytokine levels and immune responses in patients with SARS-CoV-2 infection and related conditions.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic in early 2020. The infection has been associated with a wide range of clinical symptoms. In the severely affected patients, it has caused dysregulation of immune responses including over-secretion of inflammatory cytokines and imbalances in the proportion of naïve helper T cells, memory helper T cells and regulatory T cells. Identification of the underlying mechanism of such aberrant function of immune system would help in the prediction of disease course and selection of susceptible patients for more intensive cares. In the current review, we summarize the results of studies which reported alterations in cytokine levels and immune cell functions in patients affected with SARS-CoV-2 and related viruses.

Angiotensin converting enzyme: A review on expression profile and its association with human disorders with special focus on SARS-CoV-2 infection.

Angiotensin-converting enzyme (ACE) and its homologue, ACE2, have been mostly associated with hypertensive disorder. However, recent pandemia of SARS-CoV-2 has put these proteins at the center of attention, as this virus has been shown to exploit ACE2 protein to enter cells. Clear difference in the response of affected patients to this virus has urged researchers to find the molecular basis and pathophysiology of the cell response to this virus. Different levels of expression and function of ACE proteins, underlying disorders, consumption of certain medications and the existence of certain genomic variants within ACE genes are possible explanations for the observed difference in the response of individuals to the SARS-CoV-2 infection. In the current review, we discuss the putative mechanisms for this observation.

The rs4759314 SNP within Hotair lncRNA is associated with risk of multiple sclerosis.

Recent studies have demonstrated the role of long non-coding RNAs (lncRNAs) in the pathophysiology of autoimmune disorders such as multiple sclerosis (MS). Among these transcripts is HOX transcript antisense intergenic RNA (HOTAIR) whose contribution in MS has been verified both in animal models and in human studies. In the current study, we genotyped three single nucleotide polymorphisms (SNPs) with this lncRNA (rs12826786, rs1899663 and rs4759314) in 403 Iranian MS patients and 420 healthy subjects. After correction of P values for multiple comparisons, the rs4759314 SNP was associated with risk of MS in allelic model (OR (95% CI)= 1.34 (1.08-1.67), adjusted P value=0.02). The other SNPs were not associated with risk of MS in any inheritance model. The C G A haplotype (rs12826786, rs1899663 and rs4759314, respectively) was less prevalent in cases compared with controls (OR (95% CI)= 0.73 (0.59-0.90), adjusted P value=0.03). The T G A haplotype was more common among cases compared with controls (OR (95% CI)= 1.58 (1.20-2.08), adjusted P value=0.01). Taken together, HOTAIR might be regarded as a risk locus for MS in Iranian population.

GRM7 polymorphisms are not associated with ischemic stroke in Iranian population.

Objectives: Ischemic stroke is the main neurological cause of acquired incapability in adults and a prominent cause of mortality. Several association studies have been conducted to explore the role of candidate genes in this neurological condition.Methods: In the present study, we aimed at identification of association between Glutamate Metabotropic Receptor 7 (GRM7) and risk of ischemic stroke in Iranian population. Two intronic variants within this gene (rs6782011 and rs779867) were genotyped in 318 sporadic ischemic stroke cases and 300 unrelated, healthy controls individuals.Results: No significant difference was found in allele, genotype or haplotype frequencies of these SNPs between cases and controls after correction for multiple comparisons.Conclusion: Consequently, the assessed GRM7 variants are not implicated in risk of ischemic stroke in Iranian population.

The rs594445 in MOCOS gene is associated with risk of autism spectrum disorder.

Molybdenum cofactor sulfurase (MOCOS) gene encodes an enzyme which is involved in purine metabolism. Recent experiments have shown down-regulation of MOCOS in adult nasal olfactory stem cells of individuals with autism spectrum disorder (ASD). In the current study, we genotyped two single nucleotide polymorphisms (SNPs) within coding regions of MOCOS gene (rs594445 and rs1057251) in 406 ASD patients and 411 age and sex-matched controls. The A allele of the rs594445 SNP was more prevalent among ASD cases compared with controls (OR (95% CI) = 1.33 (1.07-1.64), adjusted P value = 0.02). This SNP was associated with risk of ASD in co-dominant (AA vs. CC: OR (95% CI) = 2.00 (1.22-3.23), adjusted P value = 0.04) and recessive (AA vs. CC + AC: OR (95% CI) = 1.86 (1.16-2.98), adjusted P value = 0.02) models. The other SNP was not associated with risk of ASD in any inheritance model. There was no LD between rs594445 and rs1057251 SNPs (D' = 0.03, r2 = 0.14). The C T haplotype (rs594445 and rs1057251, respectively) had a protective role against ASD (OR (95% CI) = 0.76 (0.62-0.92), adjusted P value = 0.02). Other estimated haplotypes distributed equally between cases and controls. Based on the results of current study, the rs594445 SNP might be regarded as a risk locus for ASD in Iranian population.

A Single Nucleotide Polymorphism in GAS5 lncRNA is Associated with Risk of Bladder Cancer in Iranian Population.

Down-regulation of the long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) has a pathogenic role in bladder cancer. Moreover, genomic variants of this lncRNA have been associated with risk of diverse cancers. In the present project, we genotyped two putative functional SNPs (rs2067079 and rs6790) in 122 bladder cancer patients and 150 age- and sex-matched healthy subjects. The rs2067079 was associated risk of bladder cancer in recessive inheritance model (TT vs.CC + CT: OR (95% Confidence interval (CI)) = 2.67 (1.27-5.62), adjusted P value = 0.02). The T G haplotype (rs2067079 and rs6790) increased the risk of bladder cancer in the assessed population (OR (95% CI) = 1.73 (1.18-2.56), adjusted P value = 0.02). Consequently, in the current project we introduced a novel risk locus for bladder cancer in Iranian population.

The rs12826786 in HOTAIR lncRNA Is Associated with Risk of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) encompasses a group of neurodevelopmental disorders with complex pattern of inheritance. Several single nucleotide polymorphisms (SNPs) within coding or non-coding regions of genome have been associated with risk of this disorder. In the current study, we genotyped rs12826786, rs1899663, and rs4759314 SNPs within HOX transcript antisense RNA (HOTAIR) in 427 ASD cases and 430 normally developed children. The rs12826786 was associated with ASD in allelic (T vs. C: OR (95% CI) = 1.29 (1.07-1.57), adjusted P value = 0.03) and recessive (TT vs. TC + CC: OR (95% CI) = 1.60 (1.10-2.32), adjusted P value = 0.04) models. However, the other SNPs were not associated with ASD in any inheritance model. No estimated haplotype within HOTAIR was associated with risk of ASD in the assessed population. Based on the results of the current investigation, the rs12826786 can be regarded as a risk locus for ASD in Iranian population.