5-hydroxymethylcytosine (5-hmC), a derivative of 5-methylcytosine (5-mC), is abundant in the brain for unknown reasons. Our goal was to characterize the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC using glucosylation coupled with restriction enzyme digestion, and interrogation on microarrays. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary, in both human and mouse. This boundary change was mainly due to 5-hmC in the brain, but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent datasets and with single molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC, relative to alternatively-spliced exons. Our study suggests a novel role for 5-hmC in RNA splicing and synaptic function in the brain.
Epigenetic studies of DNA and histone modifications represent a new and important activity in molecular investigations of human disease. Our previous epigenome-wide scan identified numerous DNA methylation differences in post-mortem brain samples from individuals affected with major psychosis. In this article, we present the results of fine mapping DNA methylation differences at the human leukocyte antigen (HLA) complex group 9 gene (HCG9) in bipolar disorder (BPD). Sodium bisulfite conversion coupled with pyrosequencing was used to interrogate 28 CpGs spanning ∼700 bp region of HCG9 in 1402 DNA samples from post-mortem brains, peripheral blood cells and germline (sperm) of bipolar disease patients and controls. The analysis of nearly 40 000 CpGs revealed complex relationships between DNA methylation and age, medication as well as DNA sequence variation (rs1128306). Two brain tissue cohorts exhibited lower DNA methylation in bipolar disease patients compared with controls at an extended HCG9 region (P=0.026). Logistic regression modeling of BPD as a function of rs1128306 genotype, age and DNA methylation uncovered an independent effect of DNA methylation in white blood cells (odds ratio (OR)=1.08, P=0.0077) and the overall sample (OR=1.24, P=0.0011). Receiver operating characteristic curve A prime statistics estimated a 69-72% probability of correct BPD prediction from a case vs control pool. Finally, sperm DNA demonstrated a significant association (P=0.018) with BPD at one of the regions demonstrating epigenetic changes in the post-mortem brain and peripheral blood samples. The consistent multi-tissue epigenetic differences at HCG9 argue for a causal association with BPD.
Background Major depressive disorder (MDD) exhibits numerous clinical and molecular features that are consistent with putative epigenetic misregulation. Despite growing interest in epigenetic studies of psychiatric diseases, the methodologies guiding such studies have not been well defined. Methods We performed DNA modification analysis in white blood cells from monozygotic twins discordant for MDD, in brain prefrontal cortex, and germline (sperm) samples from affected individuals and control subjects (total N = 304) using 8.1K CpG island microarrays and fine mapping. In addition to the traditional locus-by-locus comparisons, we explored the potential of new analytical approaches in epigenomic studies. Results In the microarray experiment, we detected a number of nominally significant DNA modification differences in MDD and validated selected targets using bisulfite pyrosequencing. Some MDD epigenetic changes, however, overlapped across brain, blood, and sperm more often than expected by chance. We also demonstrated that stratification for disease severity and age may increase the statistical power of epimutation detection. Finally, a series of new analytical approaches, such as DNA modification networks and machine-learning algorithms using binary and quantitative depression phenotypes, provided additional insights on the epigenetic contributions to MDD. Conclusions Mapping epigenetic differences in MDD (and other psychiatric diseases) is a complex task. However, combining traditional and innovative analytical strategies may lead to identification of disease-specific etiopathogenic epimutations.
Objective To assess retrograde embolization for the treatment of varicocele and to examine the causes of surgical and radiological treatment failure. Patients and methods Of 154 patients with clinical varicocele associated with subfertility or symptoms who were treated, 100 underwent surgical high ligation, retrograde embolization under fluoroscopic control was attempted in 84 and 30 had both forms of treatment. Venographic findings were defined in those patients for whom embolization proved impossible and in those in whom prior high ligation had failed. Among subfertile patients, 64 had semen analyses before and at least 3 months after the procedure available for comparison. Those patients undergoing both radiological and surgical procedures were sent questionnaires to evaluate their experience. Results Retrograde embolization was technically successful in 68 (81%) of the 84 patients. Two early failures were associated with venous spasm provoked by technical inexperience, while difficulties in the remainder were caused by anomalous venous anatomy. In patients who had recurrent varicocele after previous ligation, venography showed incomplete ligation of collateral channels; 14 of 18 patients were successfully re‐treated by embolization. The sperm concentration improved significantly in 83% of patients undergoing embolization and in 63% of those surgically ligated. Patients who underwent both procedures expressed a strong preference for embolization. Conclusion In centres where there is a skilled interventional radiologist, embolization is an effective alternative to surgical ligation of varicocele. Carried out under local anaesthesia as an out‐patient procedure, it is cost‐effective, associated with minimal morbidity and most patients are able to return to normal daily activities immediately.
Background:Long-term therapy with metformin was shown to decrease the Vitamin B12 level and manifested as peripheral neuropathy.Aim:The aim of this study is to define the prevalence of Vitamin B12 deficiency in early Type 2 diabetic patients (duration ≤5 years or drug treatment ≤3 years) and the relationship among metformin exposure and levels of cobalamin (Cbl), folic acid, and homocysteine (Hcy) with severity of peripheral neuropathy.Methodology:This is a cross-sectional study involving randomly selected ninety patients (male 56, female 34) between age groups of 35 and 70 years, comparing those who had received >6 months of metformin (Group A) (n = 35) with those without metformin (Group B) (n = 35) and patients taking metformin with other oral hypoglycemic agent (Group C) (n = 20). Comparisons were made clinically, biochemically (serum Cbl, fasting Hcy, and folic acid), and with electrophysiological measures (nerve conduction studies of all four limbs). Comorbidities contributing to neuropathy were excluded from the study.Results:Group A patients (54.28%) were prone to develop peripheral neuropathy comparing Group B (28.57%) and Group C (35%). There was significantly low plasma level of Cbl in Group A (mean 306.314 pg/ml) than in Group B (mean 627.543 pg/ml) and Group C (mean 419.920 pg/ml). There was insignificant low-level plasma folic acid in Group A (16.47 ng/ml) than in Group B (16.81 ng/ml) and Group C (22.50 ng/ml). There was significantly high level of Hcy in Group A (mean 17.35 µmol/L) and Group C (mean 16.99 µmol/L) than in Group B (mean 13.22 µmol/L). Metformin users even for 2 years showed evidence of neuropathy on nerve conduction velocity though their body mass index and postprandial blood sugar were maintained. There was significant difference in between groups regarding plasma Cbl, folic acid, and Hcy level as significance level <0.05 in all three groups (F [2, 87] = 28.1, P = 0.000), (F [2, 87] = 7.43, P = 0.001), (F [2, 87] = 9.76, P = 0.000). Post hoc study shows significant (P < 0.05) lowering of Cbl and Hcy level in Group A (mean = 306.314, standard deviation [SD] = 176.7) than in Group C (mean = 419.92, SD = 208.23) and Group B (mean = 627.543, SD = 168.33).Discussion:Even short-term treatment with metformin causes a decrease in serum Cbl folic acid and increase in Hcy, which leads to peripheral neuropathy in Type 2 diabetes patients. A multicenter study with heterogeneous population would have increased the power of the study. We suggest prophylactic Vitamin B12 and folic acid supplementation or periodical assay in metformin user.
The purpose of this study was to assess the predictive values of central obesity and hyperandrogenemia in development of insulin resistance and dyslipidemia in the polycystic ovarian syndrome (PCOS) patients in our region. Differences of fasting blood glucose level, insulin resistance index HOMA-IR, lipid parameters, waist hip ratio (WHR), body mass index, LH/FSH ratio and testosterone levels between 45 PCOS cases and 35 age matched controls were obtained. Strength of association between different parameters in the case group was assayed by Pearson's correlation analysis. Dependence of insulin resistance and WHR on different predictors was assessed by multiple linear regression assay. Total cholesterol, LDL cholesterol, LH, FSH, LH/FSH ratio, WHR and insulin resistance were significantly higher in the case group (p \ 0.05). Serum testosterone showed strong correlation with insulin resistance and LH/FSH ratio (r = 0.432 and 0.747, p = 0.01 and 0.001 respectively) in the PCOS patients while WHR and serum testosterone level stood out to be most significant predictors for the insulin resistance (b = 0.361 and 0.498; p = 0.048 and 0.049 respectively). Hyperandrogenemia and central obesity were the major factors predicting development of insulin resistance and its related metabolic and cardiovascular complications in our PCOS patients. We suggest early monitoring for androgen level and WHR in these patients for predicting an ensuing insulin resistance and modulating the treatment procedure accordingly to minimise future cardiovascular risks.
BackgroundBranched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking.ResultsTo characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised.ConclusionsTaken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1022-6) contains supplementary material, which is available to authorized users.
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