DNA methylation plays an important role in the occurrence and development of age-related cataracts (ARC). This study aims to reveal potential epigenetic biomarkers of ARC by detecting modifications to the DNA methylation patterns of genes shown to be related to ARC by transcriptomics. The MethylationEPIC BeadChip (850 K) was used to analyze the DNA methylation levels in ARC patients and unaffected controls, and the Pearson correlation test was used to perform genome-wide integration analysis of DNA methylation and transcriptome data. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to perform functional analysis of the whole genome, promoter regions (TSS1500/TSS200), and the associated differentially methylated genes (DMG). Pyrosequencing was used to verify the methylation levels of the selected genes. The results showed that, compared with the control group, a total of 52,705 differentially methylated sites were detected in the ARC group, of which 13,858 were hypermethylated and 38,847 were hypomethylated. GO and KEGG analyses identified functions related to the cell membrane, the calcium signaling pathway, and their possible molecular mechanisms. Then, 57 DMGs with negative promoter methylation correlations were screened by association analysis. Pyrosequencing verified that the ARC group had higher methylation levels of C3 and CCKAR and lower methylation levels of NLRP3, LEFTY1, and GPR35 compared with the control group. In summary, our study reveals the whole-genome DNA methylation patterns and gene expression profiles in ARC, and the molecular markers of methylation identified herein may aid in the prevention, diagnosis, treatment, and prognosis of ARC.

BACKGROUND: Genetic and epigenetic factors are associated with the development of alcohol-associated liver disease (AALD). The single nucleotide polymorphisms (SNPs), rs738409 in Patatin-like phospholipase domain-containing protein (PNPLA3) and rs58542926 in Transmembrane 6 Superfamily Member 2 (TM6SF2) are strongly associated with AALD in different global populations, Hence, we analyzed the genetic risk score for these variants and deoxyribonucleic acid (DNA) methylation levels of the PNPLA3 and TM6SF2 genes among cases (alcohol liver cirrhosis) and controls (heavy drinkers without cirrhosis).
METHODS: We studied patients with alcohol use disorder (AUD) with cirrhosis (AUD-C + ve, n = 136) and without cirrhosis (AUD-C-ve, n = 107) drawn from the clinical services of St. John\'s Medical College Hospital (SJMCH) (Gastroenterology and Psychiatry) and Centre for Addiction Medicine (CAM), National Institute of Mental Health and Neurosciences, (NIMHANS). Genotype data was generated for rs738409 (PNPLA3) and rs58542926 (TM6SF2) and used to calculate unweighted genetic risk score (uGRS) and weighted genetic risk scores (wGRS). DNA methylation levels were estimated by pyrosequencing at PNPLA3 and TM6SF2 loci.
RESULTS: Overall we observed a significantly higher genetic risk score (weighted genetic risk score, wGRS) in individuals with alcohol use disorder compared to control population (p =  < 0.01). Further, uGRS and wGRS were associated with the diagnosis of cirrhosis, even after correcting for age of onset, quantity and frequency of drinking. We also found hypomethylation at CpG2 of TM6SF2 gene in AUD-C + ve compared to AUD-C-ve (P = 0.02).
CONCLUSIONS: We found that a genetic risk score based on SNPs in the PNPLA3 and TM6SF2 genes was significantly associated with cirrhosis in patients with AUD, suggesting a potential utility in identifying patients at risk and providing pre-emptive interventions. These may include interventions that aim to alter DNA methylation, which may be one of the mechanisms through which elevated genetic risk may influence the development of cirrhosis.

Tardive dyskinesia (TD) develops in 20-30% of schizophrenia patients and up to 50% in patients > 50 years old. DNA methylation may play an important role in the development of TD.
DNA methylation analyses in schizophrenia with TD.
We conducted a genome-wide DNA methylation analysis in schizophrenia with TD using methylated DNA immunoprecipitation coupled with next-generation sequencing (MeDIP-Seq) in a Chinese sample including five schizophrenia patients with TD and five without TD (NTD), and five healthy controls. The results were expressed as the log2FC, fold change of normalized tags between two groups within the differentially methylated region (DMR). For validation, the pyrosequencing was used to quantify DNA methylation levels of several methylated genes in an independent sample (n = 30).
Through genome-wide MeDIP-Seq analysis, we identified 116 genes that were significantly differentially methylated in promotor regions in comparison of TD group with NTD group including 66 hypermethylated genes (top 4 genes are GABRR1, VANGL2, ZNF534, and ZNF746) and 50 hypomethylated genes (top 4 genes are DERL3, GSTA4, KNCN, and LRRK1). Part of these genes (such as DERL3, DLGAP2, GABRR1, KLRG2, LRRK1, VANGL2, and ZP3) were previously reported to be associated with methylation in schizophrenia. Gene Ontology enrichment and KEGG pathway analyses identified several pathways. So far, we have confirmed the methylation of 3 genes (ARMC6, WDR75, and ZP3) in schizophrenia with TD using pyrosequencing.
This study identified number of methylated genes and pathways for TD and will provide potential biomarkers for TD and serve as a resource for replication in other populations.

Idiopathic scoliosis (IS) is a multifactorial disease with a genetic background. The association of Ladybird Homeobox 1 (LBX1) polymorphisms with IS has been proven in multiple studies. However, the epigenetic mechanisms have not been evaluated. This study aimed to evaluate the LBX1 methylation level in deep paravertebral muscles in order to analyze its association with IS occurrence and/or IS severity. Fifty-seven IS patients and twenty non-IS patients were examined for the paravertebral muscles’ methylation level of the LBX1 promoter region. There was no significant difference in methylation level within paravertebral muscles between patients vs. controls, except for one CpG site. The comparison of the paravertebral muscles’ LBX1 promoter region methylation level between patients with a major curve angle of ≤70° vs. >70° revealed significantly higher methylation levels in 17 of 23 analyzed CpG sequences at the convex side of the curvature in patients with a major curve angle of >70° for the reverse strand promoter region. The association between LBX1 promoter methylation and IS severity was demonstrated. In patients with severe IS, the deep paravertebral muscles show an asymmetric LBX1 promoter region methylation level, higher at the convex scoliosis side, which reveals the role of locally acting factors in IS progression.

UNASSIGNED: Thalassemia is a monogenic, autosomal recessive, inherited disorder of the red blood cells caused by mutations or deletions in the globin gene. Approximately 6-10% of the Indonesian population carries the β-globin gene mutation; however, premarital screening is rarely conducted, and antenatal screening is optional. We explored the use of cell-free fetal DNA (cffDNA) as a potential non-invasive method of detecting the fetal β-globin gene mutation prenatally in pregnant women.
UNASSIGNED: Pregnant mothers (n = 10), who were known carriers of thalassemia and who had a history of having borne a baby with thalassemia major, and their carrier husbands (n = 4) were recruited after providing consent. EDTA blood was drawn, and maternal DNA, including cffDNA, and paternal DNA were isolated. Maternal contamination tests were conducted using the variable number tandem repeat test for ApoB and D1S80 loci. Allele quantification was performed by pyrosequencing. Known mutations from the bio-archived DNA of patients with thalassemia major (n = 16) were run alongside as a control.
UNASSIGNED: In total, 7 out of 10 cffDNA successfully passed the maternal contamination test. The results of the allele quantification showed that six fetuses were predictive carriers of IVS1nt5 and one was predictive normal, in line with the allele quantification for the bio-archived DNA from patients with thalassemia major. The minimum threshold percentage for mutant A allele at cd26 was 32%, mutant T allele at IVS1nt1 was 23%, and mutant C allele at IVS1nt5 was 39%.
UNASSIGNED: Taking cffDNA from the mother\'s blood proved useful as a non-invasive means of detecting the β-globin gene mutation using pyrosequencing allele quantification. This non-invasive method is of great interest for prenatal diagnosis in settings with limited facilities, as it minimizes the risk of abortion. Further study of other mutations of the β-globin gene is needed.

Background. O6-methylguanine (O6-MeG)-DNA methyltransferase (MGMT) methylation status is a predictive factor for alkylating treatment efficacy in glioblastoma patients, but its prognostic role is still unclear. We performed a large, multicenter study to evaluate the association between MGMT methylation value and survival. Methods. We evaluated glioblastoma patients with an assessment of MGMT methylation status by pyrosequencing from nine Italian centers. The inclusion criteria were histological diagnosis of IDH wild-type glioblastoma, Eastern Cooperative Oncology Group Performance Status (ECOG-PS) ≤2, and radio-chemotherapy treatment with temozolomide. The relationship between OS and MGMT was investigated with a time-dependent Receiver Operating Characteristics (ROC) curve and Cox regression models. Results. In total, 591 newly diagnosed glioblastoma patients were analyzed. The median OS was 16.2 months. The ROC analysis suggested a cut-off of 15% for MGMT methylation. The 2-year Overall Survival (OS) was 18.3% and 51.8% for MGMT methylation <15% and ≥15% (p < 0.0001). In the multivariable analysis, MGMT methylation <15% was associated with impaired survival (p < 0.00001). However, we also found a non-linear association between MGMT methylation and OS (p = 0.002): median OS was 14.8 months for MGMT in 0−4%, 18.9 months for MGMT in 4−40%, and 29.9 months for MGMT in 40−100%. Conclusions. Our findings suggested a non-linear relationship between OS and MGMT promoter methylation, which implies a varying magnitude of prognostic effect across values of MGMT promoter methylation by pyrosequencing in newly diagnosed IDH wild-type glioblastoma patients treated with chemoradiotherapy.

Treatment strategies for alcohol use disorder (AUD) aim for abstinence or harm reduction. While deranged biochemical parameters reverse with alcohol abstinence, whether molecular changes at the epigenetic level reverse is not clearly understood. We investigated whether the reduction from high alcohol use reflects DNA methylation at the gene-specific and global level. In subjects seeking treatment for severe AUD, we assessed gene-specific (aldehyde dehydrogenase [ALDH2]/methylene tetrahydrofolate reductase [MTHFR]) and global (long interspersed elements [LINE-1]) methylation across three-time points (baseline, after detoxification and at an early remission period of 3 months), in peripheral blood leukocytes. We observed that both gene-specific and global DNA methylation did not change over time, irrespective of the drinking status at 3 months (52% abstained from alcohol). Further, we also compared DNA methylation in AUD subjects with healthy controls. At baseline, there was a significantly higher gene-specific DNA methylation (ALDH2: p < .001 and MTHFR: p = .001) and a significant lower global methylation (LINE-1: p = .014) in AUD as compared to controls. Our results suggest that epigenetic changes at the DNA methylation level associated with severe AUD persist for at least 3 months of treatment.

We evaluated the performance of pyrosequencing, a genotypic test which detects TB and XDR-defining mutations within 6 h, directly on CSF samples for diagnosing TB meningitis(TBM).
This retrospective, diagnostic accuracy study was conducted in Hinduja hospital, Mumbai from May-2017 to May-2019. 107 consecutive patients with physician-suspected TBM for whom CSF pyrosequencing was requested were screened. Seven patients with incomplete data were excluded. Diagnostic accuracy of pyrosequencing was compared with Xpert MTB/Rif and TBMGIT (TB Mycobacterial Growth Indicator Tube) culture against the uniform case definition of definite or probable TBM. Susceptibility concordance rate of pyrosequencing with TBMGIT culture and Xpert MTB/Rif was determined.
The study cohort comprised of 100 patients[Definite(n = 33), Probable(n = 20), Possible(n = 30), Alternative(n = 17)] with 50% males[median age(years):38(Range:2-87)]. Against the uniform case definition, pyrosequencing had 98·11%(95%CI 89·93-99·95; n = 52/53) sensitivity and 97·79%(86·31-99·67; n = 44/45) negative predictive value(NPV) compared with 43.39%(29·83-57·72; n = 23/53,p < 0.0001) sensitivity and 61.04%(55·31-66·48; n = 47/77) NPV for Xpert MTB/Rif and 45·28%(31·56-59·55; n = 24/53,p < 0.0001) sensitivity and 61·84%(55·92-67·43; n = 47/76) NPV for TBMGIT culture. Susceptibility concordance rate of pyrosequencing with phenotypic Drug Susceptibility Testing was 91.3%(n = 21/23) and with Xpert MTB/Rif was 95·45%(n = 21/22).
CSF pyrosequencing is significantly more sensitive than Xpert MTB/Rif and TBMGIT culture for diagnosing TBM. Additionally, it facilitates early therapeutic decision-making by providing information on XDR-defining mutations.

OBJECTIVE: To study the difference in age estimation based on quantitative analysis of DNA methylation by MassARRAY and pyrosequencing techniques.
METHODS: The methylation levels of 9 CpG sites from two independent whole blood sample sets (containing 65 and 62 samples) were detected using MassARRAY and pyrosequencing techniques. Z-score transformation was used to remove the batch effects of different techniques, and a linear regression model was used for age prediction.
RESULTS: For age prediction using the MassARRAY system, the 65 samples showed a mean absolute difference (MAD) of 2.49 years before Z-score transformation of the data and 2.44 years after the transformation, similar to the results in the 62 samples (MAD of 3.36 years before and 3.42 years after Z-score transformation). For data typed from pyrosequencing, the 65 samples showed a MAD of 4.20 years before and 2.76 years after data Z-score transformation, also similar to the results in the 62 samples (MAD of 3.92 years before and 3.63 years after data transformation).
CONCLUSIONS: Z-score transformation can effectively reduce the system batch effect between MassARRAY and pyrosequencing. Data from the MassARRAY system allows direct age estimation without further data processing, while the pyrosequencing data may increase the error in age estimation, which can be corrected by Z-score transformation of the data.

BACKGROUND: Epigenetics is one of the factors shaping natural variability observed among human populations. A small proportion of heritable inter-population differences are observed in the context of both the genome-wide methylation level and the methylation status of individual CpG sites. It has been demonstrated that a limited number of carefully selected differentially methylated sites may allow discrimination between main human populations. However, most of the few published results have been performed exclusively on B-lymphocyte cell lines.
RESULTS: The goal of our study was to identify a set of CpG sites sufficient to discriminate between populations of European and Chinese ancestry based on the difference in the DNA methylation profile not only in cell lines but also in primary cell samples. The preliminary selection of CpG sites differentially methylated in these two populations (pop-CpGs) was based on the analysis of two groups of commercially available ethnically-specific B-lymphocyte cell lines, performed using Illumina Infinium Human Methylation 450 BeadChip Array. A subset of 10 pop-CpGs characterized by the best differentiating criteria (|Mdiff| > 1, q < 0.05; lack of the confounding genomic features), and 10 additional CpGs in their immediate vicinity, were further tested using pyrosequencing technology in both B-lymphocyte cell lines and in the primary samples of the peripheral blood representing two analyzed populations. To assess the population-discriminating potential of the selected set of CpGs (further referred to as \"composite pop (CEU-CHB)-CpG marker\"), three classification methods were applied. The predictive ability of the composite 8-site pop (CEU-CHB)-CpG marker was assessed using 10-fold cross-validation method on two independent sets of samples.
CONCLUSIONS: Our results showed that less than 10 pop-CpG sites may distinguish populations of European and Chinese ancestry; importantly, this small composite pop-CpG marker performs well in both lymphoblastoid cell lines and in non-homogenous blood samples regardless of a gender.