Hidehiro Umehara

Blood diagnostic biomarkers for major depressive disorder using multiplex DNA methylation profiles: discovery and validation.

Aberrant DNA methylation in the blood of patients with major depressive disorder (MDD) has been reported in several previous studies. However, no comprehensive studies using medication-free subjects with MDD have been conducted. Furthermore, the majority of these previous studies has been limited to the analysis of the CpG sites in CpG islands (CGIs) in the gene promoter regions. The main aim of the present study is to identify DNA methylation markers that distinguish patients with MDD from non-psychiatric controls. Genome-wide DNA methylation profiling of peripheral leukocytes was conducted in two set of samples, a discovery set (20 medication-free patients with MDD and 19 controls) and a replication set (12 medication-free patients with MDD and 12 controls), using Infinium HumanMethylation450 BeadChips. Significant diagnostic differences in DNA methylation were observed at 363 CpG sites in the discovery set. All of these loci demonstrated lower DNA methylation in patients with MDD than in the controls, and most of them (85.7%) were located in the CGIs in the gene promoter regions. We were able to distinguish patients with MDD from the control subjects with high accuracy in the discriminant analysis using the top DNA methylation markers. We also validated these selected DNA methylation markers in the replication set. Our results indicate that multiplex DNA methylation markers may be useful for distinguishing patients with MDD from non-psychiatric controls.

A significant causal association between C-reactive protein levels and schizophrenia

Many observational studies have shown elevated blood CRP levels in schizophrenia compared with controls, and one population-based prospective study has reported that elevated plasma CRP levels were associated with late- and very-late-onset schizophrenia. Furthermore, several clinical studies have reported the efficacy of anti-inflammatory drugs on the symptoms in patients with schizophrenia. However, whether elevated CRP levels are causally related to schizophrenia is not still established because of confounding factors and reverse causality. In the present study, we demonstrated that serum CRP levels were significantly higher in patients with schizophrenia than in the controls by conducting a case-control study and a meta-analysis of case-control studies between schizophrenia and serum CRP levels. Furthermore, we provided evidence for a causal association between elevated CRP levels and increased schizophrenia risk by conducting a Mendelian randomization analysis. Our findings suggest that elevated CRP itself may be a causal risk factor for schizophrenia.

Sex differences of leukocytes DNA methylation adjusted for estimated cellular proportions

BackgroundDNA methylation, which is most frequently the transference of a methyl group to the 5-carbon position of the cytosine in a CpG dinucleotide, plays an important role in both normal development and diseases. To date, several genome-wide methylome studies have revealed sex-biased DNA methylation, yet no studies have investigated sex differences in DNA methylation by taking into account cellular heterogeneity. The aim of the present study was to investigate sex-biased DNA methylation on the autosomes in human blood by adjusting for estimated cellular proportions because cell-type proportions may vary by sex.MethodsWe performed a genome-wide DNA methylation profiling of the peripheral leukocytes in two sets of samples, a discovery set (49 males and 44 females) and a replication set (14 males and 10 females) using Infinium HumanMethylation450 BeadChips for 485,764 CpG dinucleotides and then examined the effect of sex on DNA methylation with a multiple linear regression analysis after adjusting for age, the estimated 6 cell-type proportions, and the covariates identified in a surrogate variable analysis.ResultsWe identified differential DNA methylation between males and females at 292 autosomal CpG site loci in the discovery set (Bonferroni-adjusted p < 0.05). Of these 292 CpG sites, significant sex differences were also observed at 98 sites in the replication set (p < 0.05).ConclusionsThese findings provided further evidence that DNA methylation may play a role in the differentiation or maintenance of sexual dimorphisms. Our methylome mapping of the effects of sex may be useful to understanding the molecular mechanism involved in both normal development and diseases.

Restless legs syndrome with chest and back restlessness as the initial symptom

RESTLESS LEGS SYNDROME (RLS) is characterized principally by leg paresthesia and motor restlessness. Some patients, however, occasionally have the same sensations in the arm, face and trunk as well as the leg. The initial symptom of RLS could be arm restlessness. We report a case of RLS with chest and back restlessness as the initial symptom. A 55-year-old healthy man with neither past nor family history of RLS or psychiatric diseases started to feel uncomfortable sensations in his back and chest. On that day he visited a physician and was hospitalized on suspicion of a thoracic disease. Physical examination was all within normal limits. He did not take any medicine. These symptoms deteriorated at night. He had severe insomnia. On day 2 or 3 the uncomfortable sensations extended to the arms and the legs. On day 7 he was referred to a psychiatrist. He impatiently complained of the creeping and itchy sensations of arms, legs, chest and back. He kept walking around in the waiting room, because these symptoms became relieved when he moved. Neurological examination showed no abnormality. We diagnosed the patient with RLS and prescribed clonazepam, which is known to relieve the symptoms of RLS.After he took a single dose of 0.5 mg that night, the next day all his symptoms disappeared. Brain magnetic resonance imaging (MRI) taken several days later showed no abnormality. He has been taking clonazepam (0.5 mg/day) and has remained symptom free for 1 year. The diagnosis of RLS is based primarily on the following four essential criteria of the International Restless Legs Syndrome Study Group: (i) desire to move the extremities, often associated with paresthesias/dysesthesias; (ii) motor restlessness; (iii) worsening of symptoms at rest with at least temporary relief by activity; and (iv) worsening of symptoms in the evening or night. This case fulfilled the diagnostic criteria, although the initial and predominating symptom occurred not in the legs but the chest and back. As far as we know, this is the first case in which initial symptoms occurred in the chest and the back. Even when uncomfortable sensations occur in any parts of the body, we might think about RLS as a differential diagnosis.

Altered KYN/TRP, Gln/Glu, and Met/methionine sulfoxide ratios in the blood plasma of medication-free patients with major depressive disorder

Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) is a comprehensive, quantitative, and high throughput tool used to analyze metabolite profiles. In the present study, we used CE-TOFMS to profile metabolites found in the blood plasma of 33 medication-free patients with major depressive disorder (MDD) and 33 non-psychiatric control subjects. We then investigated changes which occurred in the metabolite levels during an 8-week treatment period. The medication-free MDD patients and control subjects showed significant differences in their mean levels of 33 metabolites, including kynurenine (KYN), glutamate (Glu), glutamine (Gln), methionine sulfoxide, and methionine (Met). In particular, the ratios of KYN to tryptophan (TRP), Gln to Glu, and Met to methionine sulfoxide were all significantly different between the two groups. Among the 33 metabolites with altered levels in MDD patients, the levels of KYN and Gln, as well as the ratio of Gln to Glu, were significantly normalized after treatment. Our findings suggest that imbalances in specific metabolite levels may be involved in the pathogenesis of MDD, and provide insight into the mechanisms by which antidepressant agents work in MDD patients.

Association study of polymorphism in the serotonin transporter gene promoter, methylation profiles, and expression in patients with major depressive disorder

The serotonin transporter (5HTT) may be associated with the pathogenesis of major depressive disorder (MDD). The 5HTT‐linked polymorphic region (5HTTLPR) genotype may determine how levels of 5HTT mRNA are influenced by promoter methylation. We examined the association of 5HTT gene methylation, which influences gene expression, and the 5HTTLPR genotype before antidepressant treatment and expression before and after treatment. The aims of this study were (1) to investigate the association between 5HTT methylation or expression in leukocytes and depression and (2) to investigate a possible effect of 5HTT methylation, expression, and genotype on clinical symptoms in MDD. The 5HTTLPR genotype was significantly associated with mean methylation levels in patients only (patients: r = 0.40, p = 0.035, controls: p = 0.96). The mean methylation level was significantly increased in patients compared with controls (patients: 5.30 ± 0.24, controls: 4.70 ± 0.19, unpaired t‐test, p = 0.04). 5HTT expression using real‐time PCR and Taqman probes was increased in unmedicated patients compared with controls and then decreased 8 weeks after antidepressant treatment. The mean 5HTT expression level was not associated with the 5HTTLPR genotype in patients or controls. Increased depressive symptoms were related to decreased levels of methylation. Copyright

Polymorphism in the promoter of the gene for the serotonin transporter affects the age of onset of major depressive disorder in the Japanese population

OBJECTIVE Recent research has suggested that a functional polymorphism in the promoter region of the serotonin transporter gene (serotonin transporter-linked polymorphic region, 5HTTLPR) may be implicated in gene-environment interactions leading to major depressive disorder (MDD). METHODS Our study examined the association between 5HTTLPR and clinical variables of MDD in the Japanese population. We genotyped 5HTTLPR in 216 patients with MDD and 213 age- and sex-matched controls. RESULTS The genotype distributions and allele frequencies were similar in the patients and controls. When the relationships between the polymorphism and several clinical variables (i.e., age of onset, number of episodes, presence of psychotic features, suicidal behavior, and family history) were examined, the dose of the long (l) allele had significant effects on the age of onset. CONCLUSION These results suggest that 5HTTLPR may not be entirely related to the development of MDD but may be related to the age of onset of MDD, which may be due to gene-environment interactions in the Japanese population. LIMITATIONS Because of the low frequencies of psychotic features and suicidal behavior, our results must be treated with caution until they are replicated in larger numbers of Japanese samples. MDD patients did not undergo a structured interview. Clinical information from the medical records may have not been complete.

1H-magnetic resonance spectroscopy study of glutamate-related abnormality in bipolar disorder

BACKGROUND Previous studies of patients with bipolar disorder (BD) using magnetic resonance spectroscopy (MRS) have shown neurophysiological abnormalities related to the glutamate (Glu)-glutamine (Gln) cycle, membrane turnover, and neuronal integrity, although the results were neither consistent nor conclusive. Recently it has been reported the Gln/Glu ratio is the most useful index, quantifying neuronal-glial interactions and the balance of glutamatergic metabolites In this MRS study, we elucidated the abnormalities of metabolites in a larger sample of patients with BD with a high-field MRI system. METHODS Sixty-two subjects (31 patients with BD and 31 healthy controls [HC]) underwent 3T proton MRS (1H-MRS) of the anterior cingulate cortex (ACC) and left basal ganglia (ltBG) using a stimulated echo acquisition mode (STEAM) sequence. RESULTS After verifying the data quality, 20 patients with BD and 23 age- and gender-matched HCs were compared using repeated-measures analysis of covariance (ANCOVA). Compared to the HC group, the BD group showed increased levels of Gln, creatine (Cr), N-acetyl aspartate (NAA), choline (Cho), and an increased ratio of Gln to Glu in the ACC, and increased Gln and Cho in the ltBG. These findings remained after the participants with BD were limited to only euthymic patients. After removing the influence of lithium (Li) and sodium valproate (VPA), we observed activated glutamatergic neurotransmission in the ACC but not in the ltBG. LIMITATIONS The present findings are cross-sectional and metabolites were measured in only two regions. CONCLUSIONS Our results support a wide range of metabolite changes in patients with BD involved in glutamatergic neurotransmission, membrane turnover, and neuronal integrity. Moreover, the elevation of Gln/Glu ratio suggested that hyperactivity of glutamatergic neurotransmission in the ACC is a disease marker for BD.

Effect of Clozapine on DNA Methylation in Peripheral Leukocytes from Patients with Treatment-Resistant Schizophrenia

Clozapine is an atypical antipsychotic, that is established as the treatment of choice for treatment-resistant schizophrenia (SCZ). To date, no study investigating comprehensive DNA methylation changes in SCZ patients treated with chronic clozapine has been reported. The purpose of the present study is to reveal the effects of clozapine on DNA methylation in treatment-resistant SCZ. We conducted a genome-wide DNA methylation profiling in peripheral leukocytes (485,764 CpG dinucleotides) from treatment-resistant SCZ patients treated with clozapine (n = 21) in a longitudinal study. Significant changes in DNA methylation were observed at 29,134 sites after one year of treatment with clozapine, and these genes were enriched for “cell substrate adhesion” and “cell matrix adhesion” gene ontology (GO) terms. Furthermore, DNA methylation changes in the CREBBP (CREB binding protein) gene were significantly correlated with the clinical improvements. Our findings provide insights into the action of clozapine in treatment-resistant SCZ.

No association between the COMT Val158Met polymorphism and the long-term clinical response in obsessive–compulsive disorder in the Japanese population

Catechol‐O‐methyltransferase (COMT) is an enzyme that participates in the metabolic inactivation of dopamine and norepinephrine, and the Met allele of the COMT Val158Met polymorphism is associated with lower enzymatic activity. The purpose of the present study was to investigate whether this functional variant is associated with obsessive–compulsive disorder (OCD) and the clinical responses in OCD.