Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease

Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.     

Uniparental isodisomy of chromosome 2 causing MRPL44-related multisystem mitochondrial disease

Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T?>?G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T?>?G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.

     

Genetic variation in DNMT3B and increased global DNA methylation is associated with suicide attempts in psychiatric patients

Recently, a significant epigenetic component in the pathology of suicide has been realized. Here we investigate candidate functional SNPs in epigenetic‐regulatory genes, DNMT1 and DNMT3B, for association with suicide attempt (SA) among patients with co‐existing psychiatric illness. In addition, global DNA methylation levels [5‐methyl cytosine (5‐mC%)] between SA and psychiatric controls were quantified using the Methylflash Methylated DNA Quantification Kit. DNA was obtained from blood of 79 suicide attempters and 80 non‐attempters, assessed for DSM‐IV Axis I disorders. Functional SNPs were selected for each gene (DNMT1; n = 7, DNMT3B; n = 10), and genotyped. A SNP (rs2424932) residing in the 3′ UTR of the DNMT3B gene was associated with SA compared with a non‐attempter control group (P = 0.001; Chi‐squared test, Bonferroni adjusted P value = 0.02). Moreover, haplotype analysis identified a DNMT3B haplotype which differed between cases and controls, however this association did not hold after Bonferroni correction (P = 0.01, Bonferroni adjusted P value = 0.56). Global methylation analysis showed that psychiatric patients with a history of SA had significantly higher levels of global DNA methylation compared with controls (P = 0.018, Student's t‐test). In conclusion, this is the first report investigating polymorphisms in DNMT genes and global DNA methylation quantification in SA risk. Preliminary findings suggest that allelic variability in DNMT3B may be relevant to the underlying diathesis for suicidal acts and our findings support the hypothesis that aberrant DNA methylation profiles may contribute to the biology of suicidal acts. Thus, analysis of global DNA hypermethylation in blood may represent a biomarker for increased SA risk in psychiatric patients.     

Early stress predicts age at menarche and first birth,adult attachment,and expected lifespan

Life history theory suggests that in risky and uncertain environments the optimal reproductive strategy is to reproduce early in order to maximize the probability of leaving any descendants at all. The fact that early menarche facilitates early reproduction provides an adaptationist rationale for our first two hypotheses: that women who experience more risky and uncertain environments early in life would have (1) earlier menarche and (2) earlier first births than women who experience less stress at an early age. Attachment theory and research provide the rationale for our second two hypotheses: that the subjective early experience of risky and uncertain environments (insecurity) is (3) part of an evolved mechanism for entraining alternative reproductive strategies contingent on environmental risk and uncertainty and (4) reflected in expected lifespan. Evidence from our pilot study of 100 women attending antenatal clinics at a large metropolitan hospital is consistent with all four hypotheses: Women reporting more troubled family relations early in life had earlier menarche, earlier first birth, were more likely to identify with insecure adult attachment styles, and expected shorter lifespans. Multivariate analyses show that early stress directly affected age at menarche and first birth, affected adult attachment in interaction with expected lifespan, but had no effect on expected lifespan, where its original effect was taken over by interactions between age at menarche and adult attachment as well as age at first birth and adult attachment. We discuss our results in terms of the need to combine evolutionary and developmental perspectives and the relation between early stress in general and father absence in particular. This work was supported by The University of Melbourne Department of Obstetrics and Gynaecology. James S. Chisholm is Professor in the School of Anatomy and Human Biology at the University of Western Australia. He is an anthropologist whose interests lie in the fields of human behavioral biology, evolutionary ecology, life history theory, and parental investment theory, where he focuses on infant social-emotional development, the development of reproductive strategies, and the integration of evolutionary, developmental, and cultural psychology and public health. Julie A. Quinlivan is Associate Professor in Obstetrics and Gynaecology at the University of Melbourne and Head of the Maternity Care Program at the Royal Women’s Hospital, Melbourne. Her interests are teenage pregnancy, domestic violence, child abuse prevention, and high-risk pregnancy. Rodney W. Petersen is Senior Lecturer in Obstetrics and Gynaecology at the University of Melbourne and Senior Consultant in Obstetrics and Gynaecology at the Royal Women’s Hospital and Sunshine Hospital in Melbourne. His interests are in psychosocial aspects of women’s health and cancer. David A. Coall is a Ph.D. student in the School of Anatomy and Human Biology at the University of Western Australia. His main interest lies in the application of evolutionary theory within an epidemiological framework. He is currently working on the synthesis of life history theory, parental investment theory, and parent-offspring conflict theory in exploring factors that influence variation in human birth weight and placental weight.     

Portal protein diversity and phage ecology

Oceanic phages are critical components of the global ecosystem, where they play a role in microbial mortality and evolution. Our understanding of phage diversity is greatly limited by the lack of useful genetic diversity measures. Previous studies, focusing on myophages that infect the marine cyanobacterium Synechococcus, have used the coliphage T4 portal-protein-encoding homologue, gene 20 (g20), as a diversity marker. These studies revealed 10 sequence clusters, 9 oceanic and 1 freshwater, where only 3 contained cultured representatives. We sequenced g20 from 38 marine myophages isolated using a diversity of Synechococcus and Prochlorococcus hosts to see if any would fall into the clusters that lacked cultured representatives. On the contrary, all fell into the three clusters that already contained sequences from cultured phages. Further, there was no obvious relationship between host of isolation, or host range, and g20 sequence similarity. We next expanded our analyses to all available g20 sequences (769 sequences), which include PCR amplicons from wild uncultured phages, non-PCR amplified sequences identified in the Global Ocean Survey (GOS) metagenomic database, as well as sequences from cultured phages, to evaluate the relationship between g20 sequence clusters and habitat features from which the phage sequences were isolated. Even in this meta-data set, very few sequences fell into the sequence clusters without cultured representatives, suggesting that the latter are very rare, or sequencing artefacts. In contrast, sequences most similar to the culture-containing clusters, the freshwater cluster and two novel clusters, were more highly represented, with one particular culture-containing cluster representing the dominant g20 genotype in the unamplified GOS sequence data. Finally, while some g20 sequences were non-randomly distributed with respect to habitat, there were always numerous exceptions to general patterns, indicating that phage portal proteins are not good predictors of a phage's host or the habitat in which a particular phage may thrive.     

Genomic DNA methylation changes in response to folic acid supplementation in a population-based intervention study among women of reproductive age

Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 μg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 μg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (-14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation.     

The Epstein-Barr virus BRLF1 immediate-early gene product transactivates the human immunodeficiency virus type 1 long terminal repeat by a mechanism which is enhancer independent.

The Epstein-Barr virus (EBV) immediate-early gene product, BRLF1, transactivates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. BRLF1-induced transactivation of HIV-1 promoter constructs is accompanied by an increase in plasmid mRNA and is reporter gene independent. Previously, BRLF1 transactivation of EBV promoters has been mapped to regions which function as enhancer elements. Deletional analysis demonstrates that BRLF1 transactivation of the HIV-1 promoter does not require the HIV-1 enhancer. Thus, the EBV BRLF1 gene product may transactivate by at least two different mechanisms, one mechanism involving certain enhancer elements and another mechanism which is enhancer independent.