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.

Effect of rs11614913 Polymorphism on Mature miR196a2 Expression and its Target Gene HOXC8 Expression in Human Glioma.

miRNA polymorphisms are recently identified as a risk factor for various cancers, and it is associated with change in the expression of target genes in vitro. rs11614913 polymorphism in miR196a2 was associated with risk of glioma in Chinese population. In this study, we have evaluated the role of rs11614913 polymorphism and glioma risk in Indian population in 180 cases and controls. Seventy-two glioma tissue-blood pairs were also assessed for mutation in this SNP. Further, the effect of this polymorphism on mature miR196a2 expression and HOXC8 gene expression was analysed in 33 glioma tissue samples with different genotypes. Allelic discrimination assay was performed for genotyping and quantitative real time PCR for the expression of miR196a2 and HOXC8 gene. We could not find any association between rs11614913 polymorphism and glioma risk in Indian population. The rs11614913 genotyping of glioma tissue and blood pair revealed presence of mutations showing changes from C to T allele in majority of samples. The expression of the mature miR196a2 was significantly high in glioma samples, but there was no difference in expression with genotype. HOXC8 gene expression was not significantly different in glioma tissue when compared to non-glioma and interestingly there was a significant difference in expression with different genotypes, especially TT genotype was showing over expression when compared to other genotypes. Our study suggests that the rs11614913 polymorphism does not affect the mature miRNA expression, but shows its effect through target gene HOXC8 expression in glioma.

RNA interference mediated downregulation of human telomerase reverse transcriptase (hTERT) in LN18 cells.

Human telomerase reverse transcriptase (hTERT) gene is a biomarker for the targeted therapy in various cancers. Presence of increased telomerase activity is a common feature of all cancers including glioblastoma. Both RNA and catalytic subunits of hTERT are the target sites for blocking its activity. The current study focuses on the expression of hTERT in glioblastoma and its regulation using two different novel siRNAs (small interfering RNA). Our patient data demonstrated increased expression of hTERT, which could be correlated with carcinogenesis in glioma. In vitro studies in siRNA transfected LN18 cells confirmed significant cell death (p < 0.05) as evidenced by MTT and trypan blue exclusion assay. These results were further supported by flow cytometry data, which showed significant increase in early and late apoptosis. The hTERT mRNA expression was effectively downregulated by 45 and 39 % with siRNA1 and siRNA2, respectively. These results were further confirmed by immunoblotting analysis (p < 0.05). Our results suggest that both the siRNAs effectively down regulated the expression of hTERT at mRNA and protein levels, thereby decreasing cell viability and proliferation rate. Hence siRNA mediated downregulation of hTERT could be a potential therapeutic avenue in glioblastoma.

Two gene polymorphisms (rs4977756 and rs11515) in CDKN2A/B and glioma risk in South Indian population.

Gliomas are most common neoplasms in the CNS with unknown aetiology. Gene polymorphisms have been studied in glioma to check its risk in different population. CDKN2A, commonly altered tumor suppressor gene polymorphisms were recently shown to be associated with glioma in Caucasians. Present study evaluated potential association between two SNPs in CDKN2A/B gene with glioma risk in South Indian population with a total of 128 cases and 140 control subjects. Allelic discrimination assay was used for the genotyping and the association of each SNP with glioma risk were calculated using odds ratio and 95% CI. There was no association between rs4977756 polymorphism and glioma risk in south Indian population. GG genotype had a non-significant low risk in glioma (OR = 0.69). rs11515 polymorphism was not in Hardy Weinberg Equilibrium in our sample, so it was not considered for association studies. There was difference in genotype in tissue samples paired with blood samples for rs4977756 polymorphism, suggesting the importance of tissue SNP status in association studies. These results show that these two polymorphisms may not contribute to risk for glioma in South Indian population.

Polymorphisms of apolipoprotein E and aneurysmal subarachnoid haemorrhage: A meta-analysis.

Subarachnoid haemorrhage (SAH) is characterised by bleeding in the subarachnoid space in the brain. There are various polymorphisms in genes which are associated with this disease. We performed a systematic meta- analysis to investigate the relationship of APOE polymorphism on aSAH. A comprehensive literature search was done in the Pubmed database, Science Direct, Cochrane library and Google Scholar. The OR and 95% CI were evaluated for the gene and aSAH association using fixed and random effect models. Publication bias was assessed using Begg's funnel plot and Egger's regression test. All statistical evaluations were done using the software Review Manager 5.0 and Comprehensive Meta Analysis v2.2.023. A total of 9 studies were assessed on APOE polymorphism (1100 Cases, 2732 Control). Meta analysis results showed significant association in ε2/ ε2 versus ε3/ε3, ε2 versus ε3 genetic models and ε2 allele frequency. In subgroup analysis statistically significant association was observed in Asians in the genetic models ε2/ ε2 versus ε3/ε3, ε2/ε3 versus ε3/ε3, ε2 versus ε3 and also in ε2 allele frequency. However, in Caucasian population only ε2/ε2 versus ε3/ε3 genetic model showed significant association between APOE and risk of aSAH. In this meta-analysis study, the ε2/ε2 genotype is associated with increased risk of aSAH.

CD133 and BMI1 expressions and its prognostic role in primary glioblastoma.

Glioblastoma is the most common malignant brain tumour, generated by bulk of malignant cancer stem cells, which express various stem cell factors like CD133, BMI1 and nestin. There are several studies which show the importance of CD133 in cancer, but the function and interaction with other major oncogenes and tumour suppressor genes is still not understood. This study aimed to analyse the expression of CD133 mRNA and its correlations with BMI1 protein expression and TP53 mutations in newly diagnosed glioblastoma patients and its role in prognosis. Overexpression of CD133 mRNA and BMI1 protein was found in 47.6 and 76.2% patients respectively and TP53 mutations was seen in 57.1% of patients in our study.There was no correlation among TP53 mutations and expressions of CD133 and BMI1. We found that high level of BMI1 expression was favourable for the patient survival (P=0.0075) and high CD133 mRNA expression was unfavourable for the patient survival (P=0.0226).CD133 mRNA and BMI1 protein expression could independently predict the glioblastoma patient survival in multivariate analysis. In conclusion, the overexpression of these stem cell markers is a common event in glioblastoma progression and could be used as potential prognostic markers.

CDKN2A (p16) mRNA decreased expression is a marker of poor prognosis in malignant high-grade glioma.

Human high-grade glioma is heterogeneous in nature based on pathological and genetic profiling. Various tumour suppressor gene alterations are considered as prognostic markers in high-grade glioma. Gene expression of CDKN2A (p16) is used in various cancers as a prognostic biomarker along with methylation and deletion status of this gene. Expression levels of p16 mRNA were not studied as a biomarker in gliomas before. In this study, we have performed mRNA quantification analysis on 48 high-grade glioma tissues and checked for a possible prognostic role. The decreased expression of p16 mRNA in majority of the tumour tissues (57.1 %) was observed when compared to control tissues (P = 0.02). mRNA expression level was correlated with clinical variables also. p16 deletion status and BMI1 mRNA expression were also considered for comparison. p16 mRNA was negatively correlated with the BMI1 mRNA (P = <0.0001) but not with p16 deletion. p16 mRNA expression, midline shift in MRI and tumour type were able to predict patient survival in overall survival (OS) and progression-free survival (PFS). p16 mRNA could independently predict prognosis of OS (P = 0.0146) and PFS (P = 0.0305) in multivariate analysis. We have shown that p16 mRNA expression can act as an independent prognostic biomarker in high-grade glioma.

CDKN2A exon-wise deletion status and novel somatic mutations in Indian glioma patients.

Over the years, deletions of CDKN2A (p16) tumor suppressor gene has been studied using FISH and multiplex PCR, with major focus on exon 2 in various cancers, and the frequency of mutation is found to be varied in different studies. In this study, we analyzed the deletion status of all three exons of p16 and frequency of exon 2 somatic point mutations in glioma from the Indian population and its clinical implications. Multiplex PCR was carried out in order to check deletion of all 3 exons in 50 glioma samples. Nonconventional PCR-SSCP analysis and sequencing was done to identify mutations in 48 cases. Deletion of at least one of the three exons of p16 INK4A was observed in ten cases (20 %). The frequencies of exon-wise deletions were 10 % for exon 1, 4 % for exon 2, and 8 % for exon 3. Two out of 48 samples were positive for mutations in p16 exon 2. One sample had a transition of G to C on position 147 with a codon change TGG to TGC which does not contribute to the protein structure. Another sample had a transversion of A to G on the position 154 with a codon change ATG to GTG with change in amino acid methionine to valine in 52nd position. Deletion pattern was found to be varied in three exons. Frequency of p16 gene mutation was less in the Indian population (4.2 %), and this mutation does not contribute to any remarkable change in protein structure.