Therese M. Murphy

Methylation QTLs in the developing brain and their enrichment in schizophrenia risk loci

We characterized DNA methylation quantitative trait loci (mQTLs) in a large collection (n = 166) of human fetal brain samples spanning 56–166 d post-conception, identifying >16,000 fetal brain mQTLs. Fetal brain mQTLs were primarily cis-acting, enriched in regulatory chromatin domains and transcription factor binding sites, and showed substantial overlap with genetic variants that were also associated with gene expression in the brain. Using tissue from three distinct regions of the adult brain (prefrontal cortex, striatum and cerebellum), we found that most fetal brain mQTLs were developmentally stable, although a subset was characterized by fetal-specific effects. Fetal brain mQTLs were enriched amongst risk loci identified in a recent large-scale genome-wide association study (GWAS) of schizophrenia, a severe psychiatric disorder with a hypothesized neurodevelopmental component. Finally, we found that mQTLs can be used to refine GWAS loci through the identification of discrete sites of variable fetal brain methylation associated with schizophrenia risk variants.We characterized DNA methylation quantitative trait loci (mQTLs) in a large collection (n=166) of human fetal brain samples spanning 56–166 days post-conception, identifying >16,000 fetal brain mQTLs. Fetal brain mQTLs are primarily cis-acting, enriched in regulatory chromatin domains and transcription factor binding sites, and show significant overlap with genetic variants also associated with gene expression in the brain. Using tissue from three distinct regions of the adult brain (prefrontal cortex, striatum and cerebellum) we show that most fetal brain mQTLs are developmentally stable, although a subset is characterized by fetal-specific effects. We show that fetal brain mQTLs are enriched amongst risk loci identified in a recent large-scale genome-wide association study (GWAS) of schizophrenia, a severe psychiatric disorder with a hypothesized neurodevelopmental component. Finally, we demonstrate how mQTLs can be used to refine GWAS loci through the identification of discrete sites of variable fetal brain methylation associated with schizophrenia risk variants.

Hypermethylation in the ZBTB20 gene is associated with major depressive disorder

BackgroundAlthough genetic variation is believed to contribute to an individual’s susceptibility to major depressive disorder, genome-wide association studies have not yet identified associations that could explain the full etiology of the disease. Epigenetics is increasingly believed to play a major role in the development of common clinical phenotypes, including major depressive disorder.ResultsGenome-wide MeDIP-Sequencing was carried out on a total of 50 monozygotic twin pairs from the UK and Australia that are discordant for depression. We show that major depressive disorder is associated with significant hypermethylation within the coding region of ZBTB20, and is replicated in an independent cohort of 356 unrelated case-control individuals. The twins with major depressive disorder also show increased global variation in methylation in comparison with their unaffected co-twins. ZBTB20 plays an essential role in the specification of the Cornu Ammonis-1 field identity in the developing hippocampus, a region previously implicated in the development of major depressive disorder.ConclusionsOur results suggest that aberrant methylation profiles affecting the hippocampus are associated with major depressive disorder and show the potential of the epigenetic twin model in neuro-psychiatric disease.

The emergence of DNA methylation as a key modulator of aberrant cell death in prostate cancer

It is now well established that cancer cells exhibit a number of genetic defects in the machinery that governs programmed cell death and that sabotage of apoptosis is one of the principal factors aiding in the evolution of the carcinogenic phenotype. A number of studies have implicated aberrant DNA methylation as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many processes including apoptosis. To date, studies on the methylation profile of apoptotic genes have largely focused on cancers of the breast, colon and stomach, with only limited data available on prostate cancer. Here we discuss the major developments in the field of DNA methylation and its role in the regulation of aberrant apoptosis in prostate cancer. The most significant advances have involved the discovery of apoptotic gene targets of methylation, including XAF1, (fragile histidine triad (FHIT ), cellular retinol binding protein 1 (CRBP1), decoy receptor 1(DCR1), decoy receptor 2 (DCR2 ), target of methylation-induced silenceing 1 (TMS1), TNF receptor superfamily, member 6 (FAS), Reprimo (RPRM) and GLI pathogenesis-related 1 (GLIPR1). These genes are reported to be hypermethylated in prostate cancer and some offer potential as diagnostic and prognostic markers. We also introduce the concept of an apoptotic methylation signature for prostate cancer and evaluate its potential in a diagnostic, prognostic and therapeutic setting.

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, Students 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.

Epigenetics in health and disease: heralding the EWAS era

Success in the identifi cation of genetic variants that aff ect complex human phenotypes, such as height, weight, and common diseases, is one of the major achievements in contemporary biomedical research. Insight into the functional complexity of the genome also draws attention to the probable role of nonsequence-based genomic variation in health and disease. Notably, substantial attention is focused on the role of epigenetic processes that might regulate gene expression via modifi cations to DNA, histone proteins, and chromatin in medical traits. Although the role of epigenetic mechanisms in some rare developmental syndromes and in cancer is well established, systematic examination of their contribution to common nonmalignant disease phenotypes is only just beginning. New microarray-based and sequencing-based technologies allow economical, high-throughput profi ling of epigenetic marks, with a primary focus on DNA methylation; the era of the epigenome-wide association study (EWAS) of large numbers of samples has begun. In The Lancet, Katherine Dick and colleagues describe the fi rst systematic analysis of the association between variation in DNA methylation and body-mass index (BMI). They report signifi cant associations between methylation at three probes targeting specifi c CpG sites within intron 1 of HIF3A and BMI in a discovery cohort, and subsequently confi rm them in two independent cohorts. For every 10% increase in methylation of the most signifi cant probe— cg22891070—BMI increased by 3·6% (95% CI 2·4–4·9), equating to about 0·98 kg/m2 for a person in the discovery cohort with a BMI of 27 kg/m2 on average. The increase in BMI was higher in individuals who had had a myocardial infarction (4·6%, 2·9–6·3) than in blood donors (2·3%, 0·4–4·1). To put the size of this epigenetic association into perspective, the minor allele of FTO—robustly associated with obesity-related traits—accounts for a more modest 0·39 kg/m2 increase in BMI. HIF3A encodes a component of the hypoxia inducible transcription factor that mediates the cellular response to hypoxia by regulating expression of many downstream genes. This transcription factor has been previously implicated in metabolism and obesity, providing a biologically plausible mechanism behind the reported association with BMI. Epigenetic epidemiology is an area of great research interest; in the past year, EWAS have been reported for several other human health phenotypes, such as multiple sclerosis, rheumatoid arthritis, pain sensitivity, and metabolic traits. Dick and colleagues used a powerful sequential-replication design to do one of the most systematic epigenetic studies of a human physiological phenotype yet reported. However, the results of any EWAS need to be interpreted carefully, with clear caveats when compared with genetic studies of complex traits. Unlike genetics, a range of potentially important confounding factors need to be considered, such as tissue or cell type, age, sex, drug exposure, and reverse causation. A primary concern in epigenetic epidemiology is the tissue-specifi c nature of the epigenome. In large wellphenotyped sample cohorts, such as the discovery cohort used in Dick and colleagues’ study, DNA from peripheral tissues (normally whole blood) is often the only source of biological material available. To circumvent this issue, Dick and colleagues subsequently examined the relation between DNA methylation at their top-ranked loci and BMI in adipose and skin tissue from an independent sample cohort, recording strong associations in adipose tissue but not skin. Furthermore, the investigators examined the correlation between DNA methylation and HIF3A expression in adipose tissue, reporting a signifi cant inverse correlation and drawing attention to the potential functional relevance of epigenetic variation at the identifi ed locus. This result is important, because it suggests that assessment of DNA methylation in whole blood can identify robust and biologically relevant epigenetic variation related to BMI. Of course, blood itself is a heterogeneous mix of epigenetically distinct cell types, with variation in cell counts between individuals a potentially huge confounder in EWAS analyses. Dick and colleagues included basic cell-count data that allowed some control for cellular heterogeneity; another approach is the use of robust algorithms to infer cellular composition from epigenomic data. However, if the cellular content of a tissue is strongly associated with the trait being studied— as is likely for infl ammatory or neurodegenerative disorders, for example—any apparent trait-associated epigenetic diff erences could partially refl ect diff erences in cellular composition, even after statistical correction. Published Online March 13, 2014 http://dx.doi.org/10.1016/ S0140-6736(14)60269-5

Risk and protective genetic variants in suicidal behaviour: association with SLC1A2, SLC1A3, 5-HTR1B & NTRK2 polymorphisms

BackgroundSuicidal behaviour is known to aggregate in families. Patients with psychiatric disorders are at higher risk for suicide attempts (SA), however protective and risk genetic variants for suicide appear to be independent of underlying psychiatric disorders. Here we investigate genetic variants in genes important for neurobiological pathways linked to suicidal behaviour and/or associated endophenotypes, for association with SA among patients with co-existing psychiatric illness. Selected gene-gene and gene-environment interactions were also tested.MethodsDNA was obtained from bloods of 159 patients (76 suicide attempters and 83 non-attempters), who were profiled for DSM-IV Axis I psychiatric diagnosis. Twenty-eight single nucleotide polymorphisms (SNPs) from 18 candidate genes (COMT, 5-HT2A, 5-HT1A, 5-HTR1B, TPH1, MAO-A, TPH2, DBH, CNR1, BDNF, ABCG1, GABRA5, GABRG2, GABRB2, SLC1A2, SLC1A3, NTRK2, CRHR1) were genotyped. Genotyping was performed by KBioscience. Tests of association between genetic variants and SA were conducted using Chi squared and Armitage Trend tests. Binary logistical regression analyses were performed to evaluate the contribution of individual genetic variants to the prediction of SA, and to examine SNPs for potential gene-gene and gene-environment interactions.ResultsOur analysis identified 4 SNPs (rs4755404, rs2269272, rs6296 and rs1659400), which showed evidence of association with SA compared to a non-attempter control group. We provide evidence of a 3-locus gene-gene interaction, and a putative gene-environment interaction, whereby genetic variation at the NTRK2 locus may moderate the risk associated with history of childhood abuse.ConclusionPreliminary findings suggest that allelic variability in SLC1A2/3, 5-HTR1B and NTRK2 may be relevant to the underlying diathesis for suicidal acts.

Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer.

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled‐Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC‐3 cells with 5‐Aza‐CdR reactivated 39 genes (≥ 2‐fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high‐grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

DNA methylation profiling in inflammatory bowel disease provides new insights into disease pathogenesis

BACKGROUND AND AIMSnInflammatory bowel diseases (IBDs) are heterogeneous disorders with complex aetiology. Quantitative genetic studies suggest that only a small proportion of the disease variance observed in IBD is accounted for by genetic variation, indicating a potential role for differential epigenetic regulation in disease aetiology. The aim of this study was to assess genome-wide DNA methylation changes specifically associated with ulcerative colitis (UC), Crohns disease (CD) and IBD activity.nnnMETHODSnDNA methylation was quantified in peripheral blood mononuclear cells (PBMCs) from 149 IBD cases (61 UC, 88 CD) and 39 controls using the Infinium HumanMethylation450 BeadChip. Technical and functional validation was performed using pyrosequencing and the real-time polymerase chain reaction. Cross-tissue replication of the top differentially methylated positions (DMPs) was tested in colonic mucosa tissue samples obtained from paediatric IBD cases and controls.nnnRESULTSnA total of 3196 probes were differentially methylated between CD cases and controls, while 1481 probes were differentially methylated between UC cases and controls. There was considerable (45%) overlap between UC and CD DMPs. The top-ranked IBD-associated PBMC differentially methylated region (promoter region of TRIM39-RPP2) was also significantly hypomethylated in colonic mucosa from paediatric UC patients. In addition, we confirmed TRAF6 hypermethylation using pyrosequencing and found reduced TRAF6 gene expression in PBMCs of IBD patients.nnnCONCLUSIONSnOur data provide new insights into differential epigenetic regulation of genes and molecular pathways, which may contribute to the pathogenesis and activity of IBD.

Anxiety is associated with higher levels of global DNA methylation and altered expression of epigenetic and interleukin-6 genes.

Objectives Anxiety is associated with elevated levels of the inflammatory cytokine interleukin-6 (IL-6) and an increased risk for diseases with an inflammatory aetiology. In cancer, higher levels of IL-6 have been associated with increased expression of the epigenetic enzymes DNMT1 and Enhancer of Zeste Homolog 2 (EZH2). However, the relationship between IL-6 and DNA methyltransferases (DNMTs) and EZH2 expression has not previously been examined in anxious individuals. Methods Global DNA methylation levels were measured using the Methylflash Methylated DNA Quantification Kit and gene expression levels of the DNMT and EZH2 genes in anxious (n=25) and nonanxious individuals (n=22) were compared using quantitative real-time PCR. Specifically, we investigated whether global DNA methylation or aberrant expression of these genes was correlated with IL-6 mRNA and protein serum levels in anxious individuals. Results Anxious participants had significantly higher levels of global DNA methylation compared with controls (P=0.001). There were no differences in the mean mRNA expression levels of the DNMT1/3A/3B, EZH2 and IL-6 genes in anxious individuals compared with controls. However, the expression of DNMT1/3A, EZH2 and IL-6 genes increases with increasing Hospital Anxiety and Depression Scale-Anxiety scores in the anxious cohort only. Interestingly, IL-6 gene expression was correlated strongly with DNMT1/3A/3B and EZH2 expression, highlighting a potential relationship between IL-6 and important epigenetic regulatory enzymes. Conclusion This study provides novel insight into the relationship between anxiety, epigenetics and IL-6. Moreover, our findings support the hypothesis that changes in DNA methylation profiles may contribute to the biology of anxiety.

IGFBP7 promoter methylation and gene expression analysis in prostate cancer.

PURPOSEnIGFBP7 belongs to a family of insulin-like growth factor-1 regulatory binding proteins. IGFBP7 hypermethylation is associated with its down-regulation in various carcinomas. In prostate cancer IGFBP7 down-regulation has been widely reported but to our knowledge the mechanisms behind this event are unknown. We performed a denaturing high performance liquid chromatography screening and validation strategy to profile the methylation status of IGFBP7 in prostate cancer.nnnMATERIALS AND METHODSnWe combined denaturing high performance liquid chromatography and bisulfite sequencing to examine IGFBP7 methylation in a panel of prostate cancer cell lines. Quantitative methylation specific polymerase chain reaction was used to determine methylation levels in prostate tissue specimens of primary prostate cancer, histologically benign prostate adjacent to tumor, high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. IGFBP7 gene expression was measured by quantitative methylation specific polymerase chain reaction in cell lines and tissue specimens.nnnRESULTSnIGFBP7 was methylated in the 4 prostate cancer cell lines DU145, LNCaP, PC-3 and 22RV1. Quantitative methylation specific polymerase chain reaction analysis revealed that promoter methylation was associated with decreased IGFBP7 expression. Quantitative methylation specific polymerase chain reaction showed that IGFBP7 methylation was more frequently detected in prostate cancer (60% (31/52)) and high grade prostatic intraepithelial neoplasia (40% (6/15)) samples compared to histologically benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia (0%) samples.nnnCONCLUSIONSnTo our knowledge this is the first report of aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in prostate cancer cell lines. Results demonstrate that CpG methylation of IGFBP7 may represent a novel biomarker of prostate cancer and pre-invasive neoplasms. Thus, future examination of IGFBP7 methylation and expression in a larger patient cohort, including bodily fluids, is justified to further evaluate its role in a diagnostic and prognostic setting.