A Single Medical Marker for Diagnosis of Methamphetamine Addiction - DNA Methylation of SHATI/NAT8L Promoter Sites from Patient Blood.

BACKGROUND: Methamphetamine (METH) is one of the most widely distributed psychostimulants worldwide. Despite active counter measures taken by different countries, neither overall usage of METH nor the frequency of repeat users has reduced over the past decade. METH induces abuse and dependence as it acts on the central nervous system and temporarily stimulates the brain. The recidivism rate for abuse of stimulants in Japan is very high and therefore prevention of repeated usage is paramount. However, we lack information about the relationship between METH users and genomic changes in humans in Japan, which would provide important information to aid such efforts. OBJECTIVE: Shati/Nat8l is a METH-inducible molecule and its overexpression has protective effects on the brain upon METH usage. Here we investigated the effect of METH usage on DNA methylation rates at the promoter site of SHATI/NAT8L. We used DNA samples from human METH users, who are usually difficult to recruit in Japan. METHODS: We measured DNA methylation at SHATI/NAT8L promoter sites by pyrosequencing method using 193 samples of METH users and 60 samples of healthy subjects. In this method, DNA methylation is measured by utilizing the property that only non-methylated cytosine changes to urasil after bisulfite conversion. RESULTS: We found that the rate of DNA methylation at six CpG islands of SHATI/NAT8L promoter sites is significantly higher in METH users when compared to healthy subjects. CONCLUSION: These results suggest that the DNA methylation rate of SHATI/NAT8L promotor regions offers a new diagnostic method for METH usage.

Enrichment of deleterious variants of mitochondrial DNA polymerase gene (POLG1) in bipolar disorder.

AIM: Rare missense variants, which likely account for a substantial portion of the genetic 'dark matter' for a common complex disease, are challenging because the impacts of variants on disease development are difficult to substantiate. This study aimed to examine the impacts of amino acid substitution variants in the POLG1 found in bipolar disorder, as an example and proof of concept, in three different modalities of assessment: in silico predictions, in vitro biochemical assays, and clinical evaluation. We then tested whether deleterious variants in POLG1 contributed to the genetics of bipolar disorder. METHODS: We searched for variants in the POLG1 gene in 796 Japanese patients with bipolar disorder and 767 controls and comprehensively investigated all 23 identified variants in the three modalities of assessment. POLG1 encodes mitochondrial DNA polymerase and is one of the causative genes for a Mendelian-inheritance mitochondrial disease, which is occasionally accompanied by mood disorders. The healthy control data from the Tohoku Medical Megabank Organization were also employed. RESULTS: Although the frequency of carriers of deleterious variants varied from one method to another, every assessment achieved the same conclusion that deleterious POLG1 variants were significantly enriched in the variants identified in patients with bipolar disorder compared to those in controls. CONCLUSION: Together with mitochondrial dysfunction in bipolar disorder, the present results suggested deleterious POLG1 variants as a credible risk for the multifactorial disease.

Mutation screening of GRIN2B in schizophrenia and autism spectrum disorder in a Japanese population.

N-methyl-d-aspartate receptors (NMDARs) play a critical role in excitatory synaptic transmission and plasticity in the central nervous systems. Recent genetics studies in schizophrenia (SCZ) show that SCZ is susceptible to NMDARs and the NMDAR signaling complex. In autism spectrum disorder (ASD), several studies report dysregulation of NMDARs as a risk factor for ASD. To further examine the association between NMDARs and SCZ/ASD development, we conducted a mutation screening study of GRIN2B which encodes NR2B subunit of NMDARs, to identify rare mutations that potentially cause diseases, in SCZ and ASD patients (n = 574 and 152, respectively). This was followed by an association study in a large sample set of SCZ, ASD, and normal healthy controls (n = 4145, 381, and 4432, respectively). We identified five rare missense mutations through the mutation screening of GRIN2B. Although no statistically significant association between any single mutation and SCZ or ASD was found, one of its variant, K1292R, is found only in the patient group. To further examine the association between mutations in GRIN2B and SCZ/ASD development, a larger sample size and functional experiments are needed.

The regulation of soluble receptor for AGEs contributes to carbonyl stress in schizophrenia.

Our previous study showed that enhanced carbonyl stress is closely related to schizophrenia. The endogenous secretory receptor for advanced glycation end-products (esRAGE) is a splice variant of the AGER gene and is one of the soluble forms of RAGE. esRAGE is considered to be a key molecule for alleviating the burden of carbonyl stress by entrapping advanced glycation end-products (AGEs). In the current study, we conducted genetic association analyses focusing on AGER, in which we compared 212 schizophrenic patients to 214 control subjects. We also compared esRAGE levels among a subgroup of 104 patients and 89 controls and further carried out measurements of total circulating soluble RAGE (sRAGE) in 25 patients and 49 healthy subjects. Although the genetic association study yielded inconclusive results, multiple regression analysis indicated that a specific haplotype composed of rs17846798, rs2071288, and a 63 bp deletion, which were in perfect linkage disequilibrium (r2 = 1), and rs2070600 (Gly82Ser) were significantly associated with a marked decrease in serum esRAGE levels. Furthermore, compared to healthy subjects, schizophrenia showed significantly lower esRAGE (p = 0.007) and sRAGE (p = 0.03) levels, respectively. This is the first study to show that serum esRAGE levels are regulated by a newly identified specific haplotype in AGER and that a subpopulation of schizophrenic patients are more vulnerable to carbonyl stress.

Individual risk alleles of susceptibility to schizophrenia are associated with poor clinical and social outcomes.

Many patients with schizophrenia have poor clinical and social outcomes. Some risk alleles closely related to the onset of schizophrenia have been reported to be associated with their clinical phenotypes, but the direct relationship between genetic vulnerability to schizophrenia and clinical/social outcomes of schizophrenia, as evaluated by both practical clinical scales and 'real-world' function, has not been investigated. We evaluated the clinical and social outcomes of 455 Japanese patients with schizophrenia by severity of illness according to the Clinical Global Impression-Severity Scale (CGI-S) and social outcomes by social adjustment/maladjustment at 5 years after the first visit. We examined whether 46 single nucleotide polymorphisms (SNPs) selected from a Japanese genome-wide association study of susceptibility to schizophrenia were associated with clinical and social outcomes. We also investigated the polygenic risk scores of 46 SNPs. Allele-wise association analysis detected three SNPs, including rs2623659 in the CUB and Sushi multiple domains-1 (CSMD1) gene, associated with severity of illness at end point. The severity of illness at end point was associated with treatment response, but not with the severity of illness at baseline. Three SNPs, including rs2294424 in the C6orf105 gene, were associated with social outcomes. Point estimates of odds ratios showed positive relationships between polygenic risk scores and clinical/social outcomes; however, the results were not statistically significant. Because these results are exploratory, we need to replicate them with a larger sample in a future study.

Associations between the orexin (hypocretin) receptor 2 gene polymorphism Val308Ile and nicotine dependence in genome-wide and subsequent association studies.

BACKGROUND: Many genetic and environmental factors are involved in the etiology of nicotine dependence. Although several candidate gene variations have been reported by candidate gene studies or genome-wide association studies (GWASs) to be associated with smoking behavior and the vulnerability to nicotine dependence, such studies have been mostly conducted with subjects with European ancestry. However, genetic factors have rarely been investigated for the Japanese population as GWASs. To elucidate genetic factors involved in nicotine dependence in Japanese, the present study comprehensively explored genetic contributors to nicotine dependence by using whole-genome genotyping arrays with more than 200,000 markers in Japanese subjects. RESULTS: The subjects for the GWAS and replication study were 148 and 374 patients, respectively. A two-stage GWAS was conducted using the Fagerström Test for Nicotine Dependence (FTND), Tobacco Dependence Screener (TDS), and number of cigarettes smoked per day (CPD) as indices of nicotine dependence. For the additional association analyses, patients who underwent major abdominal surgery, patients with methamphetamine dependence/psychosis, and healthy subjects with schizotypal personality trait data were recruited. Autopsy specimens with various diseases were also evaluated. After the study of associations between more than 200,000 marker single-nucleotide polymorphisms (SNPs) and the FTND, TDS, and CPD, the nonsynonymous rs2653349 SNP (located on the gene that encodes orexin [hypocretin] receptor 2) was selected as the most notable SNP associated with FTND, with a p value of 0.0005921 in the two-stage GWAS. This possible association was replicated for the remaining 374 samples. This SNP was also associated with postoperative pain, the initiation of methamphetamine use, schizotypal personality traits, and susceptibility to goiter. CONCLUSIONS: Although the p value did not reach a conventional genome-wide level of significance in our two-stage GWAS, we obtained significant results in the subsequent analyses that suggest that the rs2653349 SNP (Val308Ile) could be a genetic factor that is related to nicotine dependence and possibly pain, schizotypal personality traits, and goiter in the Japanese population.

Identification of Rare, Single-Nucleotide Mutations in NDE1 and Their Contributions to Schizophrenia Susceptibility.

BACKGROUND: Nuclear distribution E homolog 1 (NDE1), located within chromosome 16p13.11, plays an essential role in microtubule organization, mitosis, and neuronal migration and has been suggested by several studies of rare copy number variants to be a promising schizophrenia (SCZ) candidate gene. Recently, increasing attention has been paid to rare single-nucleotide variants (SNVs) discovered by deep sequencing of candidate genes, because such SNVs may have large effect sizes and their functional analysis may clarify etiopathology. METHODS AND RESULTS: We conducted mutation screening of NDE1 coding exons using 433 SCZ and 145 pervasive developmental disorders samples in order to identify rare single nucleotide variants with a minor allele frequency ≤5%. We then performed genetic association analysis using a large number of unrelated individuals (3554 SCZ, 1041 bipolar disorder [BD], and 4746 controls). Among the discovered novel rare variants, we detected significant associations between SCZ and S214F (P = .039), and between BD and R234C (P = .032). Furthermore, functional assays showed that S214F affected axonal outgrowth and the interaction between NDE1 and YWHAE (14-3-3 epsilon; a neurodevelopmental regulator). CONCLUSIONS: This study strengthens the evidence for association between rare variants within NDE1 and SCZ, and may shed light into the molecular mechanisms underlying this severe psychiatric disorder.

Frontotemporal dementia with parkinsonism linked to chromosome 17 with the MAPT†R406W mutation presenting with a broad distribution of abundant senile plaques.

We report the autopsy results of a patient with familial dementia who was diagnosed as having frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) with an R406W mutation in the microtubule-associated protein tau (MAPT) gene. This patient showed Alzheimer's disease (AD)-like clinical manifestations from the age of 59, with reduced ß-amyloid1-42 (Aß42 ) and elevated total and phosphorylated tau levels in the cerebrospinal fluid. He did not present with any apparent parkinsonism throughout the disease course. His autopsy at age 73 showed atrophy and neurodegeneration in many brain regions, particularly in the antero-medial temporal cortex and hippocampus, followed by the frontal lobes, with abundant neurofibrillary tangles. In addition, a diffuse distribution of Aß-positive senile plaques, including many neuritic plaques, was observed and classified as stage C according to the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) criteria. These results suggest that analyzing of the MAPT gene is essential for diagnosing familial dementia, even if amyloid markers such as Aß42 in the cerebrospinal fluid and amyloid imaging are positive, or if neuropathological findings indicate a diagnosis of AD.

Human Rho guanine nucleotide exchange factor 11 gene is associated with schizophrenia in a Japanese population.

OBJECTIVE: The human Rho guanine nucleotide exchange factor 11 (ARHGEF11) gene is one of the candidate genes for type 2 diabetes mellitus (T2DM). ARHGEF11 is mapped to chromosome 1q21, which has susceptible risk loci for T2DM and schizophrenia. We hypothesized that ARHGEF11 contributes to the pathogenesis of schizophrenia. METHOD: We selected eight single nucleotide polymorphisms of ARHGEF11 that had significant associations with T2DM for a case-control association study of 490 patients with schizophrenia and 500 age-matched and sex-matched controls. RESULTS: We did not find any differences in allelic, genotypic associations, or minor allele frequencies with schizophrenia. Analysis of the rs6427340-rs6427339 haplotype and the rs822585-rs6427340-rs6427339 haplotype combination provided significant evidence of an association with schizophrenia (global permutations p = 0.00047 and 0.0032, respectively). C-C of the rs6427340-rs6427339 haplotype and A-C-C of the rs822585-rs6427340-rs6427339 haplotype carried higher risk factors for schizophrenia (permutation p = 0.0010 and 0.0018, respectively). A-C-T of the rs822585-rs6427340-rs6427339 haplotype had a possible protective effect (permutation p = 0.031). CONCLUSION: These results provide new evidence that ARHGEF11 may constitute a risk factor for schizophrenia.

Aripiprazole is effective for treatment of delayed sleep phase syndrome.

Delayed sleep phase syndrome (DSPS) comprises a persistent or recurrent pattern of sleep disturbances, sleep disruption that leads to insomnia and/or excessive daytime sleepiness, and impaired functioning in social, occupational, or other spheres. Aripiprazole (APZ), a second-generation antipsychotic, manifests a novel mechanism of action by serving as a partial agonist of both D2 and serotonergic 5-HT1A receptors and antagonist of 5-HT2A receptors. We have used APZ to treat DSPS. One reason it was effective may be that the insomnia induced by daytime APZ was effective in treating the patient's daytime sleepiness. Another reason may be APZ increases histamine release which controls sleep-wake cycles. Thus, APZ may be therapeutic for DSPS.

Replication and cross-phenotype study based upon schizophrenia GWASs data in the Japanese population: support for association of MHC region with psychosis.

Recent genome-wide association studies (GWASs) of schizophrenia (SCZ) identified several susceptibility genes and suggested shared genetic components between SCZ and bipolar disorder (BD). We conducted a genetic association study of single nucleotide polymorphisms (SNPs) selected according to previous SCZ GWAS targeting psychotic disorders (SCZ and BD) in the Japanese population. Fifty-one SNPs were analyzed in a two-stage design using first-set screening samples (all SNPs: 1,032 SCZ, 1,012 BD, and 993 controls) and second-set replication samples ("significant" SNPs in the first-set screening analysis: 1,808 SCZ, 821 BD, and 2,321 controls). We assessed allelic associations between the selected SNPs and the three phenotypes (SCZ, BD, and "psychosis" [SCZ + BD]). Nine SNPs revealed nominal association signals for all comparisons (P(uncorrected) < 0.05), of which two SNPs located in the major histocompatibility complex region (rs7759855 in zinc finger and SCAN domain containing 31 [ZSCAN31] and rs1736913 in HLA-F antisense RNA1 [HLA-F-AS1]) were further assessed in the second-set replication samples. The associations were confirmed for rs7759855 (P(corrected) = 0.026 for psychosis; P(corrected) = 0.032 for SCZ), although the direction of effect was opposite to that in the original GWAS of the Chinese population. Finally, a meta-analysis was conducted using our two samples and using our data and data from Psychiatric GWAS Consortium (PGC), which have shown the same direction of effect. SNP in ZSCAN31 (rs7759855) had the strongest association with the phenotypes (best P = 6.8 × 10(-5) for psychosis: present plus PGC results). These data support shared risk SNPs between SCZ and BD in the Japanese population and association between MHC and psychosis.

Genetic association study between the detected risk variants based upon type II diabetes GWAS and psychotic disorders in the Japanese population.

Several epidemiological and genetic studies have suggested that the risk of type II diabetes (T2D) is likely to overlap with the susceptibility to psychotic disorders such as schizophrenia (SCZ) and bipolar disorder (BD). In this study, we aimed to examine the association of single-nucleotide polymorphisms (SNPs) detected in previous T2D genome-wide association studies (GWAS) with SCZ, BD and psychosis (SCZ plus BD). A total of 37 SNPs were selected from the literature. A two-stage analysis was conducted using a first set of screening samples (total N=3037) and a second set of replication samples (N=4950). None of the SNPs showed a significant association to the screening samples after correction for multiple testing. To avoid type II error, we genotyped the top three SNPs in BCL11A, HMG20A and HNF4A showing associations with any of the phenotypes (Puncorrected <0.01) using independent samples to replicate the nominal associations. However, we were unable to find any significant associations based on the screening results (Puncorrected>0.05). Our findings did not support the shared genetic risk between T2D and psychotic disorders in the Japanese population. However, further replication using a larger sample size is required.

Genetic variants on 3q21 and in the Sp8 transcription factor gene (SP8) as susceptibility loci for psychotic disorders: a genetic association study.

BACKGROUND: Recent genome-wide association studies (GWASs) investigating bipolar disorder (BD) have detected a number of susceptibility genes. These studies have also provided novel insight into shared genetic components between BD and schizophrenia (SCZ), two major psychotic disorders. To examine the replication of the risk variants for BD and the pleiotropic effect of the variants associated with BD, we conducted a genetic association study of single nucleotide polymorphisms (SNPs) that were selected based upon previous BD GWASs, which targeted psychotic disorders (BD and SCZ) in the Japanese population. METHODS: Forty-eight SNPs were selected based upon previous GWASs. A two-stage analysis was conducted using first-set screening (for all SNPs: BD = 1,012, SCZ = 1,032 and control = 993) and second-set replication samples (for significant SNPs in the screening analysis: BD = 821, SCZ = 1,808 and control = 2,149). We assessed allelic association between BD, SCZ, psychosis (BD+SCZ) and the SNPs selected for the analysis. RESULTS: Eight SNPs revealed nominal association signals for all comparisons (Puncorrected<0.05). Among these SNPs, the top two SNPs (associated with psychosis: Pcorrected = 0.048 and 0.037 for rs2251219 and rs2709722, respectively) were further assessed in the second-set samples, and we replicated the signals from the initial screening analysis (associated with psychosis: Pcorrected = 0.0070 and 0.033 for rs2251219 and rs2709722, respectively). The meta-analysis between the current and previous GWAS results showed that rs2251219 in Polybromo1 (PBRM1) was significant on genome-wide association level (P = 5×10(-8)) only for BD (P = 9.4×10(-9)) and psychosis (P = 2.0×10(-10)). Although the association of rs2709722 in Sp8 transcription factor (SP8) was suggestive in the Asian population (P = 2.1×10(-7) for psychosis), this signal weakened when the samples size was increased by including data from a Caucasian population (P = 4.3×10(-3)). CONCLUSIONS: We found 3p21.1 (including PBRM1, strong linkage disequilibrium made it difficult to pinpoint the risk genes) and SP8 as risk loci for BD, SCZ and psychosis. Further replication studies will be required for conclusive results.

Lack of association of EGR2 variants with bipolar disorder in Japanese population.

The early growth response gene 2 (EGR2) has been recently reported to be associated with bipolar disorder in the Korean population. However replication studies in independent cohorts of same and different ethnicities are essential for establishing the credibility of a genotype-phenotype association. With this notion, in the present study we have performed a replication study of the reported association of SNPs in EGR2 in a case-control study comprising of 867 unrelated Japanese bipolar disorder patients and 895 age-, sex- and ethnicity-matched controls. Results showed no significant differences in allele and genotype frequencies of EGR2 SNPs between bipolar disorder patients and controls and also in a sex-stratified genetic analysis. The haplotype and meta-analyses also showed no significant association with bipolar disorder. In conclusion, this is the first replication study of the previously reported association of EGR2 with bipolar disorder in a larger sample set and the results showed that the EGR2 gene is unlikely to contribute to the susceptibility of bipolar disorder in a Japanese cohort.

Evidence for shared genetic risk between methamphetamine-induced psychosis and schizophrenia.

Methamphetamine (METH) use can provoke psychotic reactions requiring immediate treatment, namely METH-induced psychosis. Although the distinction between METH-induced and primary psychosis is important for understanding their clinical courses, we do not have clear diagnostic procedure by their symptoms. Not only are there similarities between the clinical features of METH-induced psychosis and schizophrenia (SCZ), but there is also epidemiological evidence of a shared genetic risk between 'METH-related' disorders and SCZ, which makes the differentiation of these two conditions difficult. In this study, we conducted a genome-wide association study (GWAS) targeting METH-dependent patients. The METH sample group, used in the METH-dependence GWAS, included 236 METH-dependent patients and 864 healthy controls. We also included a 'within-case' comparison between 194 METH-induced psychosis patients and 42 METH-dependent patients without psychosis in a METH-induced psychosis GWAS. To investigate the shared genetic components between METH dependence, METH-induced psychosis, and SCZ, data from our previous SCZ GWAS (total N=1108) were re-analyzed. In the SNP-based analysis, none of the SNPs showed genome-wide significance in either data set. By performing a polygenic component analysis, however, we found that a large number of 'risk' alleles for METH-induced psychosis are over-represented in individuals with SCZ (Pbest=0.0090). Conversely, we did not detect enrichment either between METH dependence and METH-induced psychosis or between METH dependence and SCZ. The results support previous epidemiological and neurobiological evidence for a relationship between METH-induced psychosis and SCZ. These also suggest that the overlap between genes scored as positive in these data sets can have higher probability as susceptibility genes for psychosis.

Genome-wide association study of schizophrenia using microsatellite markers in the Japanese population.

OBJECTIVES: To search for schizophrenia susceptibility loci, we carried out a case-control study using 28601 microsatellite markers distributed across the entire genome. MATERIALS AND METHODS: To control the highly multiple testing, we designed three sequential steps of screening using three independent sets of pooled samples, followed by the confirmatory step using an independent sample set (>2200 case-control pairs). RESULTS: The first screening using pooled samples of 157 case-control pairs showed 2966 markers to be significantly associated with the disorder (P<0.05). After the second and the third screening steps using pooled samples of 150 pairs each, 374 markers remained significantly associated with the disorder. We individually genotyped all screening samples using a total of 1536 tag single nucleotide polymorphisms (SNPs) located in the vicinity of ~200 kb from the 59 positive microsatellite markers. Of the 167 SNPs that replicated the significance, we selected 31 SNPs on the basis of the levels of P values for the confirmatory association test using an independent-sample set. The best association signal was observed in rs13404754, located in the upstream region of SLC23A3. We genotyped six additional SNPs in the vicinity of rs13404754. Significant associations were observed in rs13404754, rs6436122, and rs1043160 in the cumulative samples (2617 cases and 2698 controls) (P=0.005, 0.035, and 0.011, respectively). These SNPs are located in the linkage disequilibrium block of 20 kb in size containing SLC23A3, CNPPD1, and FAM134A genes. CONCLUSION: Genome-wide association study using microsatellite markers suggested SLC23A3, CNPPD1, and FAM134A genes as candidates for schizophrenia susceptibility in the Japanese population.