To study childhood trauma in patients with bipolar affective disorder and its association with leucocyte telomere length.

Background: Childhood traumatic (CT) events are more frequent in Bipolar Affective Disorder (BD) than in healthy individuals. As per existing studies, telomere shortening might be associated with psychiatric illnesses and aging-related disorders. One basis could be CT in BD aiding in telomere shortening. Methods: 100 BD patients and 100 healthy controls (HC) were matched for age and sex. All the participants were administered Childhood Trauma Questionnaire (CTQ). Subsequently, Quantitative Polymerase Chain Reaction (q-PCR) was performed in order to verify leukocyte telomere length (LTL) for both cases and controls. Results: Presence of subtypes of moderate to severe CT among cases revealed emotional abuse in 35%, physical abuse in 16%, and sexual abuse in 15%. BD patients had significantly shorter telomeres in comparison to HC. BD patients with CT had significantly shorter LTL as compared to healthy controls with CT. The association between CT and LTL was not statistically significant in cases as well as in controls. Conclusions: Our study revealed presence of CT (moderate to severe) in 46% of BD patients and 12% in age and sex-matched healthy controls. All CT subtypes except sexual abuse were significantly higher among cases than in healthy controls. Mean score of LTL among cases including that with CT was significantly lower than the healthy controls.

Alteration in serum miR126 expression in healthy adults observing Navratri fast.

Background: Fasting is practiced by various religions in the world. The previous studies show the effect of fasting on biochemical markers in healthy subjects; however, no study is available on its effect on gene expression or epigenetic markers. In the present study, miR126, a microRNA, was measured in serum samples of healthy adult subjects, and their correlation with biochemical profile was carried out during the short-term fasting of the Navratri festival. Methods: A total of 30 subjects who underwent fasting for 07 days during the Navratri festival were recruited for the study. The fasting blood samples were obtained at three different time points; day 1 of fasting, day 7 of fasting, and day 7 after completion of fasting period. The miR126 expression, fasting plasma glucose, and lipid profile were measured in all the three samples. Results: The miR126 levels showed a decreasing trend with a significant difference across the three time points (p-value = 0.006). Fasting plasma glucose increased continuously across three time points without showing any statistical significance. Serum total cholesterol (p = 0.001) and triglycerides (p = 0.001) levels were decreased initially and then increased after resuming normal diet. There was a medium-level negative correlation (-0.332) between baseline fasting glucose level and miR126 level (p = 0.068). Conclusion: The study revealed that serum levels of total cholesterol and triglyceride were more dynamic than the miR126 levels. A significant decrease in the miR126 expression across three time points is a promising outcome of this pilot study and indicates its role in short-term fasting. However, the fasting plasma glucose showed heterogeneous values without significant correlation with miR126 levels.

Correlation of CYP2R1 gene promoter methylation with circulating vitamin D levels among healthy adults.

Background & objectives: Despite being a tropical country, vitamin D deficiency is highly prevalent in India with studies indicating 40-99 per cent prevalence. Apart from calcium and phosphate metabolism, vitamin D is involved in cell cycle regulation, cardiovascular, hepatoprotection. The metabolism of vitamin D is regulated by vitamin D tool genes (CYP2R1/CYP27B1/CYP24A1/VDR). The promoter regions of some of these genes have CpG islands, making them prone to methylation induced gene silencing, which may cause a reduction in circulating vitamin D levels. Epigenetic basis of vitamin D deficiency is yet to be studied in India, and hence, this pilot study was aimed to analyze whether methylation levels of CYP2R1 gene were correlated with the levels of 25(OH)D in healthy, adult individuals in Indian population. Methods: In this cross-sectional study, healthy adults of 18-45 yr of age with no history of malabsorption, thyroidectomy, chronic illness or therapeutic vitamin D supplementation were recruited. DNA methylation analysis was carried out by methylation specific quantitative PCR. Serum calcium, phosphate and vitamin D levels were also quantified. Statistical analysis was done by R 4.0.5 software. Results: A total of 61 apparently healthy adults were analyzed. The serum vitamin D levels did not correlate with CYP2R1 methylation levels in our study population. Significant positive correlation was observed between age and serum vitamin D levels. Significant association of gender was found with CYP2R1 methylation levels. Interpretation & conclusions: This study found no significant correlation between levels of CYP2R1 methylation and circulating 25(OH)D deficiency. Further studies on the Indian population having a larger sample size including entire vitamin D tool genes, among different ethnic groups may be conducted to elucidate molecular etiology of circulating 25(OH)D deficiency. The high prevalence of normal serum calcium and phosphate levels among vitamin D deficient subjects in this study coupled with the strikingly high prevalence of the deficiency at the national level, may suggest the need to revise the cut-off criteria for vitamin D deficiency in the Indian population.

A pilot study on DNA hypermethylation status in promoter region of P16 gene in patients with sporadic breast cancer.

Background: Epigenetic modification of cancer-related genes plays a role over and above their genetic alterations and contributes to the tumor initiation and progression of breast cancer. Promoter methylation of tumor suppressor genes is one such epigenetic modification, which can be potential biomarker. In this study, promoter methylation status of p16 gene was studied in blood samples of patients with breast carcinoma. Methods: Seventy-five patients, freshly diagnosed with carcinoma of breast and 20 age and sex matched healthy control subjects were recruited for the study. DNA extracted from EDTA blood sample was bisulfite converted and subjected to methylation-specific PCR to amplify the p16 promoter region. Results: Out of 75 patients, 25 (33%) patients showed hypermethylation in promoter region of p16 gene, which was statistically significant in comparison with the control group (p < 0.05). In subgroup analysis, lymph node involvement, cancer grade, and histopathological finding did not show any difference with methylation status of p16 promoter. Conclusion: Significant hypermethylation of p16 promoter region in the blood of histopathologically proven cases of breast cancer was observed suggesting promoter hypermethylation of p16 may be a possible mechanism accounting for sporadic carcinoma of breast.

DNA methylation and gene expression pattern of ACE2 and TMPRSS2 genes in saliva samples of patients with SARS-CoV-2 infection

COVID-19 caused by SARS-CoV-2 became a pandemic affecting the health and economy of the world. Although it was known that this virus uses ACE2 protein along with TMPRSS2 to enter the host cell, the methylation pattern and gene expression of ACE2 and TMPRSS2 genes are not explored in saliva samples of patients infected with COVID-19. The study aimed to quantify promoter methylation of ACE2 and TMPRSS2 along with its mRNA expression in saliva samples of COVID-19 patients in order to understand the regulatory mechanism of these genes in SARS-CoV-2 infection. Saliva samples were collected from thirty male patients with SARS-CoV-2 infection and thirty age-matched healthy control male subjects. Q MS PCR and qRT PCR was performed to quantify the promoter DNA methylation and mRNA expression of ACE2 and TMPRSS2 respectively. Our study didnt find any significant difference between methylation and expression of these two genes in cases compared to control subjects. However there was significant positive correlation between DNA methylation of ACE2 and its gene expression. Among cases, the sample collected [≥]7 days after appearance of symptoms showed higher amount of methylation in both ACE2 and TMPRSS2 genes when compared to sample collected before 7 days. In conclusion, we found that ACE2 and TMPRSS2 methylation plays a role in COVID-19.

Prognostic significance of factor XIIIA promoter methylation status in aneurysmal subarachnoid haemorrhage (aSAH).

BACKGROUND: Aneurysmal subarachnoid hemorrhage is a life- threatening condition with high rate of disability and mortality. Apolipoprotein E (APOE) and Factor XIIIA (F13A) genes are involved in the pathogenetic mechanism of aneurysmal subarachnoid haemorrhage (aSAH). We evaluated the association of promoter methylation status of APOE and F13A gene and risk of aSAH. METHODS: For evaluating the effect of hypermethylation in the promoter region of these genes with risk of aSAH, we conducted a case -control study with 50 aSAH patients and 50 healthy control. The methylation pattern was analysed using methylation specific PCR. The risk factors associated with poor outcome after aSAH was also analysed in this study. The outcome was assessed using Glasgow outcome score (GOS) after 3 months from the initial bleed. RESULTS: The frequency of APOE and F13A methylation pattern showed insignificant association with risk of aSAH in this study. Gender stratification analysis suggests that F13A promoter methylation status was significantly associated with the risk of aSAH in male gender. Age, aneurysm located at the anterior communicating artery and diabetes mellitus showed significant association with poor outcome after aSAH. CONCLUSION: There was no significant association with APOE promoter methylation with the risk as well as outcome of patients after aSAH. F13A promoter methylation status was significantly associated with risk of aSAH in male gender, with no significant association with outcome after aSAH.

Role of APOE and IL18RAP gene polymorphisms in cervical spondylotic myelopathy in Indian population.

Cervical spondylotic myelopathy (CSM) is a progressive degenerative spine disease. It is not clear why certain patients develop symptomatic myelopathy whereas others do not, even in the presence of radiographic features of cervical stenosis. Genetic predisposition has been suggested, supported by familial occurrence of CSM. In this study we explored the demographic and radiographic features of CSM in Indian population and studied the association between polymorphisms in interleukin18RAP and apo-lipoprotein genes in CSM. A total of 100 CSM patients and 100 healthy control subjects were included in this study. Genotyping of APOE (rs7412 and rs429358) and IL18RAP (rs1420106 and rs917997) gene polymorphisms was performed by Taqman allelic discrimination assay. Comparison of allelic frequencies, ε2 versus ε3 (OR = 4.4, 95%CI = 1.23-15.73, P = 0.002) and ε2 versus ε4 (OR = 6.67, 95%CI = 1.58-28.04, P = 0.009) showed a statistically significant association for the risk of CSM. There was no significant association between different genotypes with sex, T2 signal intensity change and Nurick grade. Only patients having multiple level cervical prolapsed intervertebral disc (PIVD) on MRI, had a higher proportion of the ε2 allele as compared to controls (p = <0.0001). No significant association was found between IL18RAP gene polymorphisms (rs1420106 and rs917997) with the risk of CSM. ε2 allele was associated with the risk of CSM in Indian population. There was no significant association between the two single nucleotide polymorphisms (SNPs) of IL18RAP gene with risk of CSM.

Factor XIII polymorphism and risk of aneurysmal subarachnoid haemorrhage in a south Indian population.

BACKGROUND: The rupture of a brain aneurysm causes bleeding in the subarachnoid space and is known as aneurysmal subarachnoid haemorrhage (aSAH). In our study, we evaluated the association of factor XIII polymorphism and the risk of Aneurysmal subarachnoid haemorrhage (aSAH) in South Indian population. METHODS: The study was performed in 200 subjects with aSAH and 205 healthy control subjects. Genotyping of rs5985(c.103G > T (p.Val35Leu)) and rs5982(c.1694C > T (p.Pro564Leu)) polymorphism was performed by Taqman® allelic discrimination assay. RESULTS: In our study, Val/Leu genotype frequency was higher in control subjects (18%) compared to aSAH patients (9%).The Val/Leu genotype was associated with lower risk of aSAH (OR = 0.48, 95%CI = 0.26-0.88, p = 0.02). When compared with Val allele, Leu allele was significantly associated with lower risk of aSAH (OR = 0.55, 95%CI = 0.32-0.95, p = 0.03). In subtyping, we found a significant association of Leu/Leu genotype with the Basilar top aneurysm (OR = 3.59, 95%CI = 1.11-11.64, p = 0.03). In c.1694C > T (p.Pro565Leu) variant, Pro/Pro Vs Pro/Leu genotype (OR = 2.06, 95%CI = 1.10-3.85, p = 0.02) was significantly associated with higher risk of aSAH. The 564Leu allelic frequency in aSAH patients (36%) was higher when compared with that in healthy controls (30%) in our study. When allele frequency (Pro Vs Leu) was compared, 564Leu allele was found to be significantly associated with higher aSAH risk (OR = 1.36, 95%CI = 1.01-1.83, p = 0.04). (OR = 1.36, 95%CI = 1.01-1.83, p = 0.04). Regarding rs5985 and rs5982, significant association was found in the log-additive model (OR = 0.57, 95%CI = 0.33-0.97, p = 0.034; OR = 1.32, 95%CI = 1.00-1.72, p = 0.043). CONCLUSION: These results suggest that 34Leu allele was a protective factor for lower risk of aSAH whereas 564Leu allele was associated with higher risk of aSAH in South Indian population.

Down regulation of human telomerase reverse transcriptase (hTERT) expression by BIBR1532 in human glioblastoma LN18 cells.

Increased telomerase activity can be blocked by targeting the hTERT activity at both RNA and catalytic subunits. Various inhibitors had been used to regulate hTERT activity in glioblastoma cell lines and showed promising results. The present study hypothesized that the telomerase specific inhibitor BIBR1532 can effectively down-regulate the telomerase activity in LN18 glioblastoma cell line. LN18 glioblastoma cell line was treated with various concentrations of BIBR1532 at different time intervals. MTT assay was performed to determine cell viability after BIBR1532 treatment. hTERT mRNA and protein expression were determined by qRT-PCR and western blotting, respectively. Flow cytometry and TRAP assay was performed to detect the rate of apoptosis and telomerase activity in treated and control samples. One-way ANOVA was performed to compare the mean values of variables in control and BIBR1532 treated groups. LN18 cells showed a significant dose dependent cytotoxic effect after treatment with BIBR1532. hTERT mRNA expression in cells treated with 25, 100 and 200 µM BIBR1532 treated groups was decreased ~ 21, ~ 61.2, and ~ 77%, respectively (p < 0.05). We also observed that, BIBR1532 treatment reduced the expression of hTERT protein in LN18 cells in a dose dependent manner. The Flow cytometry data showed that, the drug induced significant increase in the total percentage of apoptotic cells with 200 µM concentration of BIBR1532 at all time points. BIBR1532 exhibited potent inhibition of telomerase activity in a dose-dependent manner in LN18 cells. BIBR1532 could induce apoptosis in LN18 cells through the downregulation of telomerase activity at transcriptional and translational level. We conclude that BIBR1532 may be a therapeutic agent to suppress telomerase activity, however, further efforts are necessary in order to explore this therapeutic strategy.