AKR1A1 Variant Associated With Schizophrenia Causes Exon Skipping, Leading to Loss of Enzymatic Activity.

Schizophrenia is a heterogeneous psychiatric disorder characterized by positive symptoms such as hallucinations and delusions, negative symptoms such as anhedonia and flat affect, and cognitive impairment. Recently, glucuronate (GlucA) levels were reported to be significantly higher in serum of patients with schizophrenia than those in healthy controls. The accumulation of GlucA is known to be related to treatment-resistant schizophrenia, since GlucA is known to promote drug excretion by forming conjugates with drugs. However, the cause of GlucA accumulation remains unclear. Aldo-keto reductase family one member A1 (AKR1A1) is an oxidoreductase that catalyzes the reduction of GlucA. Genetic loss of AKR1A1 function is known to result in the accumulation of GlucA in rodents. Here, we aimed to explore genetic defects in AKR1A1 in patients with schizophrenia, which may result in the accumulation of GlucA. We identified 28 variants of AKR1A1 in patients with schizophrenia and control subjects. In particular, we identified a silent c.753G > A (rs745484618, p. Arg251Arg) variant located at the first position of exon 8 to be associated with schizophrenia. Using a minigene assay, we found that the c.753G > A variant induced exon 8 skipping in AKR1A1, resulting in a frameshift mutation, which in turn led to truncation of the AKR1A1 protein. Using the recombinant protein, we demonstrated that the truncated AKR1A1 completely lost its activity. Furthermore, we showed that AKR1A1 mRNA expression in the whole blood cells of individuals with the c.753G > A variant tended to be lower than that in those without the variants, leading to lower AKR activity. Our findings suggest that AKR1A1 carrying the c.753G > A variant induces exon skipping, leading to a loss of gene expression and enzymatic activity. Thus, GlucA patients with schizophrenia with the c.753G > A variant may show higher GlucA levels, leading to drug-resistant schizophrenia, since drug excretion by GlucA is enhanced.

Identification and functional characterization of the extremely long allele of the serotonin transporter-linked polymorphic region.

SLC6A4, which encodes the serotonin transporter, has a functional polymorphism called the serotonin transporter-linked polymorphic region (5-HTTLPR). The 5-HTTLPR consists of short (S) and long (L) alleles, each of which has 14 or 16 tandem repeats. In addition, the extralong (XL) and other rare alleles have been reported in 5-HTTLPR. Although they are more frequent in Asian and African than in other populations, the extent of variations and allele frequencies (AFs) were not addressed in a large population. Here, we report the AFs of the rare alleles in a large number of Japanese subjects (N = 2894) consisting of two cohorts. The first cohort (case-control study set, CCSS) consisted of 1366 subjects, including 485 controls and 881 patients with psychosis (bipolar disorder or schizophrenia). The second cohort (the Arao cohort study set, ACSS) consisted of 1528 elderly subjects. During genotyping, we identified 11 novel 5-HTTLPR alleles, including 3 XL alleles. One novel allele had the longest subunit ever reported, consisting of 28 tandem repeats. We named this XL28-A. An in vitro luciferase assay revealed that XL28-A has no transcriptional activity. XL28-A was found in two unrelated patients with bipolar disorder in the CCSS and one healthy subject in the ACSS who did not show depressive symptoms or a decline in cognitive function. Therefore, it is unlikely that XL28-A is associated with psychiatric disorders, despite its apparent functional deficit. Our results suggest that unraveling the complex genetic variations of 5-HTTLPR will be important for further understanding its role in psychiatric disorders.

Promoter Activity-Based Case-Control Association Study on SLC6A4 Highlighting Hypermethylation and Altered Amygdala Volume in Male Patients With Schizophrenia.

Associations between altered DNA methylation of the serotonin transporter (5-HTT)-encoding gene SLC6A4 and early life adversity, mood and anxiety disorders, and amygdala reactivity have been reported. However, few studies have examined epigenetic alterations of SLC6A4 in schizophrenia (SZ). We examined CpG sites of SLC6A4, whose DNA methylation levels have been reported to be altered in bipolar disorder, using 3 independent cohorts of patients with SZ and age-matched controls. We found significant hypermethylation of a CpG site in SLC6A4 in male patients with SZ in all 3 cohorts. We showed that chronic administration of risperidone did not affect the DNA methylation status at this CpG site using common marmosets, and that in vitro DNA methylation at this CpG site diminished the promoter activity of SLC6A4. We then genotyped the 5-HTT-linked polymorphic region (5-HTTLPR) and investigated the relationship among 5-HTTLPR, DNA methylation, and amygdala volume using brain imaging data. We found that patients harboring low-activity 5-HTTLPR alleles showed hypermethylation and they showed a negative correlation between DNA methylation levels and left amygdala volumes. These results suggest that hypermethylation of the CpG site in SLC6A4 is involved in the pathophysiology of SZ, especially in male patients harboring low-activity 5-HTTLPR alleles.

Global DNA hypomethylation and its correlation to the betaine level in peripheral blood of patients with schizophrenia.

Accumulating evidence suggests that aberrant epigenetic regulation is involved in the pathophysiology of major psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BD). We previously showed that the plasma level of betaine (N,N,N-trimethylglycine), a methyl-group donor, was significantly decreased in patients with first episode schizophrenia (FESZ). In this study, we identified decrease of global DNA methylation level in FESZ (N = 24 patients vs N = 42 controls), and found that global DNA methylation level was inversely correlated with scores on the global assessment of functioning (GAF) scale, and positively correlated with plasma betaine level. Notably, correlations between levels of betaine and its metabolites (N,N-dimethylglycine and sarcosine, N-methylglycine) were lower or lost in FESZ plasma, but remained high in controls. We further examined global DNA methylation levels in patients with chronic SZ (N = 388) and BD (N = 414) as well as controls (N = 430), and confirmed significant hypomethylation and decreased betaine level in SZ. We also found that patients with BD type I, but not those with BD type II, showed significant global hypomethylation. These results suggest that global hypomethylation associated with decreased betaine level in blood cells is common to SZ and BD, and may reflect common pathophysiology such as psychotic symptoms.

Evaluation of the usefulness of saliva for DNA methylation analysis in cohort studies.

INTRODUCTION: Epigenetic information such as DNA methylation is a useful biomarker that reflects complex gene-environmental interaction. Peripheral tissues such as blood and saliva are commonly collected as the source of genomic DNA in cohort studies. Epigenetic studies mainly use blood, while a few studies have addressed the epigenetic characteristics of saliva. METHODS: The effects of methods for DNA extraction and purification from saliva on DNA methylation were surveyed using Illumina Infinium HumanMethylation450 BeadChip. Using 386 661 probes, DNA methylation differences between blood and saliva from 22 healthy volunteers, and their functional and structural characteristics were examined. CpG sites with DNA methylation levels showing large interindividual variations in blood were evaluated using saliva DNA methylation profiles. RESULTS: Genomic DNA prepared by simplified protocol from saliva showed a similar quality DNA methylation profile to that derived from the manufacturer provided protocol. Consistent with previous studies, the DNA methylation profiles of blood and saliva showed high correlations. Blood showed 1,514 hypomethylated and 2099 hypermethylated probes, suggesting source-dependent DNA methylation patterns. CpG sites with large methylation difference between the two sources were underrepresented in the promoter regions and enriched within gene bodies. CpG sites with large interindividual methylation variations in blood also showed considerable variations in saliva. CONCLUSION: In addition to high correlation in DNA methylation profiles, CpG sites showing large interindividual DNA methylation differences were similar between blood and saliva, ensuring saliva could be a suitable alternative source for genomic DNA in cohort studies. Consideration of source-dependent DNA methylation differences will, however, be necessary.

Population-neuroscience study of the Tokyo TEEN Cohort (pn-TTC): Cohort longitudinal study to explore the neurobiological substrates of adolescent psychological and behavioral development.

AIM: Adolescence is a crucial stage of psychological development and is critically vulnerable to the onset of psychopathology. Our understanding of how the maturation of endocrine, epigenetics, and brain circuit may underlie psychological development in adolescence, however, has not been integrated. Here, we introduce our research project, the population-neuroscience study of the Tokyo TEEN Cohort (pn-TTC), a longitudinal study to explore the neurobiological substrates of development during adolescence. METHODS: Participants in the first wave of the pn-TTC (pn-TTC-1) study were recruited from those of the TTC study, a large-scale epidemiological survey in which 3171 parent-adolescent pairs were recruited from the general population. Participants underwent psychological, cognitive, sociological, and physical assessment. Moreover, adolescents and their parents underwent magnetic resonance imaging (MRI; structural MRI, resting-state functional MRI, and magnetic resonance spectroscopy), and adolescents provided saliva samples for hormone analysis and for DNA analysis including epigenetics. Furthermore, the second wave (pn-TTC-2) followed similar methods as in the first wave. RESULTS: A total of 301 parent-adolescent pairs participated in the pn-TTC-1 study. Moreover, 281 adolescents participated in the pn-TTC-2 study, 238 of whom were recruited from the pn-TTC-1 sample. The instruction for data request is available at: http://value.umin.jp/data-resource.html. CONCLUSION: The pn-TTC project is a large-scale and population-neuroscience-based survey with a plan of longitudinal biennial follow up. Through this approach we seek to elucidate adolescent developmental mechanisms according to biopsychosocial models. This current biomarker research project, using minimally biased samples recruited from the general population, has the potential to expand the new research field of population neuroscience.

DNA methylation analyses of the candidate genes identified by a methylome-wide association study revealed common epigenetic alterations in schizophrenia and bipolar disorder.

AIM: Schizophrenia (SZ) and bipolar disorder (BD) have been known to share genetic and environmental risk factors, and complex gene-environmental interactions may contribute to their pathophysiology. In contrast to high genetic overlap between SZ and BD, as revealed by genome-wide association studies, the extent of epigenetic overlap remains largely unknown. In the present study, we explored whether SZ and BD share epigenetic risk factors in the same manner as they share genetic components. METHODS: We performed DNA methylation analyses of the CpG sites in the top five candidate regions (FAM63B, ARHGAP26, CTAGE11P, TBC1D22A, and intergenic region [IR] on chromosome 16) reported in a previous methylome-wide association study (MWAS) of SZ, using whole blood samples from subjects with BD and controls. RESULTS: Among the five candidate regions, the CpG sites in FAM63B and IR on chromosome 16 were significantly hypomethylated in the samples from subjects with BD as well as those from subjects with SZ. On the other hand, the CpG sites in TBC1D22A were hypermethylated in the samples from subjects with BD, in contrast to hypomethylation in the samples from subjects with SZ. CONCLUSION: Hypomethylation of FAM63B and IR on chromosome 16 could be common epigenetic risk factors for SZ and BD. Further comprehensive epigenetic studies for BD, such as MWAS, will uncover the extent of similarity and uniqueness of epigenetic alterations.

An epigenome-wide methylation study of healthy individuals with or without depressive symptoms.

Major depressive disorder is a common psychiatric disorder that is thought to be triggered by both genetic and environmental factors. Depressive symptoms are an important public health problem and contribute to vulnerability to major depression. Although a substantial number of genetic and epigenetic studies have been performed to date, the detailed etiology of depression remains unclear and there are no validated biomarkers. DNA methylation is one of the major epigenetic modifications that play diverse roles in the etiology of complex diseases. In this study, we performed an epigenome-wide association study (EWAS) of DNA methylation on subjects with (N = 20) or without (N = 27) depressive symptoms in order to examine whether different levels of DNA methylation were associated with depressive tendencies. Employing methylation-array technology, a total of 363,887 methylation sites across the genomes were investigated and several candidate CpG sites associated with depressive symptoms were identified, especially annotated to genes linked to a G-protein coupled receptor protein signaling pathway. These data provide a strong impetus for validation studies using a larger cohort and support the possibility that G-protein coupled receptor protein signaling pathways are involved in the pathogenesis of depression.

Use of human methylation arrays for epigenome research in the common marmoset (Callithrix jacchus).

We examined the usefulness of commercially available DNA methylation arrays designed for the human genome (Illumina HumanMethylation450 and MethylationEPIC) for high-throughput epigenome analysis of the common marmoset, a nonhuman primate suitable for research on neuropsychiatric disorders. From among the probes on the methylation arrays, we selected those available for the common marmoset. DNA methylation data were obtained from genomic DNA extracted from the frontal cortex and blood samples of adult common marmosets as well as the frontal cortex of neonatal marmosets. About 10% of the probes on the arrays were estimated to be useful for DNA methylation assay in the common marmoset. Strong correlations existed between human and marmoset DNA methylation data. Illumina methylation arrays are useful for epigenome research using the common marmoset.

DNA methylation analysis of BDNF gene promoters in peripheral blood cells of schizophrenia patients.

Accumulating evidence suggests that epigenetic alterations in brain-derived neurotrophic factor (BDNF) promoters are associated with the pathophysiology of psychiatric disorders. Epigenetic changes in BDNF were reported not only in brain tissues but also in other tissues, including peripheral blood cells (PBC) and saliva. We examined DNA methylation levels of BDNF promoters I and IV using genomic DNA derived from PBC of healthy controls (n=100), and patients with schizophrenia (n=100), all from the Japanese population, by pyrosequencing. The examined CpG sites were chosen based on previous epigenetic studies that reported altered DNA methylation. We found a significantly higher level of methylation at BDNF promoter I in patients with schizophrenia compared to controls, although the difference was small. Subsequent analysis revealed that in controls, the methylation level of BDNF promoters was associated with sex, and the methylation difference observed in promoter I was more prominent in male patients with schizophrenia. Epigenetic alteration of BDNF in the PBC might reflect the pathophysiology of schizophrenia, and could be a potential biomarker.

DNA methylation of the BDNF gene and its relevance to psychiatric disorders.

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor, which is important for neuronal survival, development and synaptic plasticity. Accumulating evidence suggests that epigenetic modifications of BDNF are associated with the pathophysiology of psychiatric disorders, such as schizophrenia and mood disorders. Patients with psychiatric disorders generally show decreased neural BDNF levels, which are often associated with increased DNA methylation at the specific BDNF promoters. Importantly, observed DNA methylation changes are consistent across tissues including brain and peripheral blood, which suggests potential usefulness of these findings as a biomarker of psychiatric disorders. Here we review DNA methylation characteristics of BDNF promoters of cellular, animal and clinical samples and discuss future perspectives.

Bowline, a novel protein localized to the presomitic mesoderm, interacts with Groucho/TLE in Xenopus.

Cells in the prospective somite of Xenopus laevis embryos rotate in an orchestrated manner to form a segregated somite. The prospective somite boundaries are prepatterned by gene expressions in the unsegmented presomitic mesoderm (PSM). However, the roles of polarized gene expression in this boundary formation are not well elucidated. Here we identified a novel gene, bowline, which localizes to the anterior halves of S-II, III in the PSM of X. laevis. Bowline associated with corepressor XGrg-4, a Xenopus homolog of Groucho/TLE protein. A WRPW tetrapeptide motif in Bowline was prerequisite for coprecipitation with XGrg-4 and for downregulation of X-Delta-2 by bowline RNA injection. This study indicates that Bowline is a novel protein interacting with Groucho/TLE and may play a role in somitogenesis in X. laevis.