Copy number variations in RNF216 and postsynaptic membrane-associated genes are associated with bipolar disorder: a case-control study in the Japanese population.

AIM: Bipolar disorder (BD) is a common psychiatric disorder characterized by alterations between manic/hypomanic and depressive states. Rare pathogenic copy number variations (CNVs) that overlap with exons of synaptic genes have been associated with BD. However, no study has comprehensively explored CNVs in synaptic genes associated with BD. Here, we evaluated the relationship between BD and rare CNVs that overlap with synaptic genes, not limited to exons, in the Japanese population. METHODS: Using array comparative genome hybridization, we detected CNVs in 1839 patients with BD and 2760 controls. We used the Synaptic Gene Ontology database to identify rare CNVs that overlap with synaptic genes. Using gene-based analysis, we compared their frequencies between the BD and control groups. We also searched for synaptic gene sets related to BD. The significance level was set to a false discovery rate of 10%. RESULTS: The RNF216 gene was significantly associated with BD (odds ratio, 4.51 [95% confidence interval, 1.66-14.89], false discovery rate < 10%). The BD-associated CNV that corresponded with RNF216 also partially overlapped with the minimal critical region of the 7p22.1 microduplication syndrome. The integral component of the postsynaptic membrane (Gene Ontology:0099055) was significantly associated with BD. The CNV overlapping with the intron region of GRM5 in this gene set showed a nominal significant association between cases and controls (P < 0.05). CONCLUSION: We provide evidence that CNVs in RNF216 and postsynaptic membrane-related genes confer a risk of BD, contributing to a better understanding of the pathogenesis of BD.

Study of the genetic association between selected 3q29 region genes and schizophrenia and autism spectrum disorder in the Japanese population.

Psychiatric disorders are highly inheritable, and most psychiatric disorders exhibit genetic overlap. Recent studies associated the 3q29 recurrent deletion with schizophrenia (SCZ) and autism spectrum disorder (ASD). In this study, we investigated the association of genes in the 3q29 region with SCZ and ASD. TM4SF19 and PAK2 were chosen as candidate genes for this study based on evidence from previous research. We sequenced TM4SF19 and PAK2 in 437 SCZ cases, 187 ASD cases and 524 controls in the Japanese population. Through targeted sequencing, we identified 6 missense variants among the cases (ASD & SCZ), 3 missense variants among controls, and 1 variant common to both cases and controls; however, no loss-of-function variants were identified. Fisher's exact test showed a significant association of variants in TM4SF19 among cases (p=0.0160). These results suggest TM4SF19 variants affect the etiology of SCZ and ASD in the Japanese population. Further research examining 3q29 region genes and their association with SCZ and ASD is thus needed.

Analysis of human neuronal cells carrying ASTN2 deletion associated with psychiatric disorders.

Recent genetic studies have found common genomic risk variants among psychiatric disorders, strongly suggesting the overlaps in their molecular and cellular mechanism. Our research group identified the variant in ASTN2 as one of the candidate risk factors across these psychiatric disorders by whole-genome copy number variation analysis. However, the alterations in the human neuronal cells resulting from ASTN2 variants identified in patients remain unknown. To address this, we used patient-derived and genome-edited iPS cells with ASTN2 deletion; cells were further differentiated into neuronal cells. A comprehensive gene expression analysis using genome-edited iPS cells with variants on both alleles revealed that the expression level of ZNF558, a gene specifically expressed in human forebrain neural progenitor cells, was greatly reduced in ASTN2-deleted neuronal cells. Furthermore, the expression of the mitophagy-related gene SPATA18, which is repressed by ZNF558, and mitophagy activity were increased in ASTN2-deleted neuronal cells. These phenotypes were also detected in neuronal cells differentiated from patient-derived iPS cells with heterozygous ASTN2 deletion. Our results suggest that ASTN2 deletion is related to the common pathogenic mechanism of psychiatric disorders by regulating mitophagy via ZNF558.

Phenotypes for general behavior, activity, and body temperature in 3q29 deletion model mice.

Whole genome analysis has identified rare copy number variations (CNV) that are strongly involved in the pathogenesis of psychiatric disorders, and 3q29 deletion has been found to have the largest effect size. The 3q29 deletion mice model (3q29-del mice) has been established as a good pathological model for schizophrenia based on phenotypic analysis; however, circadian rhythm and sleep, which are also closely related to neuropsychiatric disorders, have not been investigated. In this study, our aims were to reevaluate the pathogenesis of 3q29-del by recreating model mice and analyzing their behavior and to identify novel new insights into the temporal activity and temperature fluctuations of the mouse model using a recently developed small implantable accelerometer chip, Nano-tag. We generated 3q29-del mice using genome editing technology and reevaluated common behavioral phenotypes. We next implanted Nano-tag in the abdominal cavity of mice for continuous measurements of long-time activity and body temperature. Our model mice exhibited weight loss similar to that of other mice reported previously. A general behavioral battery test in the model mice revealed phenotypes similar to those observed in mouse models of schizophrenia, including increased rearing frequency. Intraperitoneal implantation of Nano-tag, a miniature acceleration sensor, resulted in hypersensitive and rapid increases in the activity and body temperature of 3q29-del mice upon switching to lights-off condition. Similar to the 3q29-del mice reported previously, these mice are a promising model animals for schizophrenia. Successive quantitative analysis may provide results that could help in treating sleep disorders closely associated with neuropsychiatric disorders.

Association between copy number variations in parkin (PRKN) and schizophrenia and autism spectrum disorder: A case-control study.

AIM: The present study aimed to examine the association between copy number variations (CNVs) in parkin (PRKN) and schizophrenia (SCZ) and autism spectrum disorder (ASD) in a large case-control sample. METHOD: Array comparative genomic hybridization was performed on 3111 cases with SCZ, 1236 cases with ASD, and 2713 controls. We systematically prioritized likely pathogenic CNVs (LP-CNVs) in PRKN and examined their association with SCZ and ASD. RESULTS: In total, 3014 SCZ cases (96.9%), 1205 ASD cases (97.5%), and 2671 controls (98.5%) passed quality control. We found that monoallelic carriers of LP-CNVs in PRKN were common (70/6890, 1.02%) and were not at higher risk of SCZ (p = 0.29) or ASD (p = 0.72). We observed that the distribution pattern of LP-CNVs in the Japanese population was consistent with those in other populations. We also identified a patient diagnosed with SCZ and early-onset Parkinson's disease carrying biallelic pathogenic CNVs in PRKN. The absence of Parkinson's symptoms in 10 other monoallelic carriers of the same pathogenic CNV further reflects the lack of effect of monoallelic pathogenic variants in PRKN in the absence of a second hit. CONCLUSION: The present findings suggest that monoallelic CNVs in PRKN do not confer a significant risk for SCZ or ASD. However, further studies to investigate the association between biallelic CNVs in PRKN and SCZ and ASD are warranted.

Case reports of two siblings with autism spectrum disorder and 15q13.3 deletions.

BACKGROUND: Copy number variations (CNVs) have been implicated in psychiatric and neurodevelopmental disorders. Especially, 15q13.3 deletions are strongly associated with autism spectrum disorder (ASD), intellectual disability (ID), schizophrenia (SCZ), attention deficithyperactivity disorder (ADHD), and mood disorder. CASE PRESENTATION: We present two siblings with ASD. They had a father with bipolar disorder (BD). Patient 1 is a 21-year-old female with ASD and mild ID, who had language delay and repetitive behavior in childhood, social difficulties, and refused to go to school because of bullying. She was hospitalized in a psychiatric hospital several times. Patient 2 is a 19-year-old male with ASD and ADHD. He did not have developmental delay, but had social difficulties and impulsiveness, then refused to go to school because of bullying. He was treated by a psychiatrist for anxiety and disrupted sleep rhythms. Array comparative genomic hybridization was performed for the siblings and parents. 15q13.3 deletions were detected in the siblings and their healthy mothers. No other pathogenic CNVs were detected. We performed whole-genome sequencing of the family and identified 13 rare missense variants in brain-expressed genes, which may be responsible for the phenotypic differences between the siblings and their mother. CONCLUSIONS: This study shows incomplete penetrance and variable expressivity in 15q13.3 deletions. We detected second-hit variants that may explain the phenotypic differences within this family. In addition, detecting 15q13.3 deletions may lead to early diagnosis and a better prognosis with careful follow-up.

De novo MCM6 variants in neurodevelopmental disorders: a recognizable phenotype related to zinc binding residues.

The minichromosome maintenance (MCM) complex acts as a DNA helicase during DNA replication, and thereby regulates cell cycle progression and proliferation. In addition, MCM-complex components localize to centrosomes and play an independent role in ciliogenesis. Pathogenic variants in genes coding for MCM components and other DNA replication factors have been linked to growth and developmental disorders as Meier-Gorlin syndrome and Seckel syndrome. Trio exome/genome sequencing identified the same de novo MCM6 missense variant p.(Cys158Tyr) in two unrelated individuals that presented with overlapping phenotypes consisting of intra-uterine growth retardation, short stature, congenital microcephaly, endocrine features, developmental delay and urogenital anomalies. The identified variant affects a zinc binding cysteine in the MCM6 zinc finger signature. This domain, and specifically cysteine residues, are essential for MCM-complex dimerization and the induction of helicase activity, suggesting a deleterious effect of this variant on DNA replication. Fibroblasts derived from the two affected individuals showed defects both in ciliogenesis and cell proliferation. We additionally traced three unrelated individuals with de novo MCM6 variants in the oligonucleotide binding (OB)-fold domain, presenting with variable (neuro)developmental features including autism spectrum disorder, developmental delay, and epilepsy. Taken together, our findings implicate de novo MCM6 variants in neurodevelopmental disorders. The clinical features and functional defects related to the zinc binding residue resemble those observed in syndromes related to other MCM components and DNA replication factors, while de novo OB-fold domain missense variants may be associated with more variable neurodevelopmental phenotypes. These data encourage consideration of MCM6 variants in the diagnostic arsenal of NDD.

Internationalization of higher medical education in the post-COVID-19 era.

COVID-19 pandemic has caused disruption in higher medical education and healthcare worldwide. To thrive in times of uncertainty, medical higher education institutions have to adapt to the post-COVID-19 era and innovate its international activities. To make a difference in societies locally, nationally and internationally, they will have to enhance their global presence. Internationalization is the best way to the exchanging of knowledge, enhancement of the medical curriculum, and mobilization of talent and resources for research and teaching. To remain competitive, universities will need to expand their international activities. This paper highlights several suggestions to enhance internationalization of medical higher education institutions in the post-COVID-19 era.

Slovenian Version of the Drug-Induced Extrapyramidal Symptoms Scale: Evaluation of Interrater and Test-Retest Reliability.

AIM: The Drug-Induced Extrapyramidal Symptom Scale (DIEPSS) is a multidimensional rating scale for the assessment of drug-induced extrapyramidal symptoms (EPS), developed in 1994. It is suitable for evaluating EPS considering the degree of influence EPS has on daily activities and the subjective distress that it causes. METHOD: This study to evaluate the interrater and test-retest reliability of the DIEPSS Slovenian version conducted at the University Medical Center Maribor in Slovenia in November 2018. RESULTS: Six raters performed the interrater assessment of 135 DIEPSS video clips with recordings of patients with EPS. A second assessment was then performed by 2 raters to evaluate the test-retest reliability, which was high (interclass correlation coefficients from 0.743 to 0.936). CONCLUSIONS: The results for the Slovenian language version of the DIEPSS show high interrater and test-retest reliability, with high concordance rates for all evaluated items (interclass correlation coefficient > 0.8).

Clinical characterization of patients with schizophrenia and 16p13.11 duplication: A case series.

BACKGROUND: Chromosome 16p13.11 duplication is a well-known genetic risk factor for schizophrenia (SCZ) (odds ratio = 1.84). However, no case reports focusing on patients with SCZ and 16p13.11 duplication have been published. Therefore, here, we report the detailed clinical cases of four patients with SCZ and 16p13.11 duplication who were identified in our previous whole-genome copy number variant (CNV) study. CASE PRESENTATION: In the four patients with SCZ and 16p13.11 duplication detected by array comparative genomic hybridization, one patient was found to have treatment-resistant SCZ and an additional pathogenic rare CNV. Two of the four patients in this study had environmental risk factors that may have been involved in the development of SCZ. CONCLUSIONS: The results of this case series suggest that a genetic cohort study would be useful for evaluating which genetic and environmental risk factors could better explain the variable expressivity of 16p13.11 duplication. Furthermore, this work could be useful for elucidating the pathophysiology of SCZ.

The genetic architecture of schizophrenia: review of large-scale genetic studies.

Schizophrenia is a complex and often chronic psychiatric disorder with high heritability. Diagnosis of schizophrenia is still made clinically based on psychiatric symptoms; no diagnostic tests or biomarkers are available. Pathophysiology-based diagnostic scheme and treatments are also not available. Elucidation of the pathogenesis is needed for development of pathology-based diagnostics and treatments. In the past few decades, genetic research has made substantial advances in our understanding of the genetic architecture of schizophrenia. Rare copy number variations (CNVs) and rare single-nucleotide variants (SNVs) detected by whole-genome CNV analysis and whole-genome/-exome sequencing analysis have provided the great advances. Common single-nucleotide polymorphisms (SNPs) detected by large-scale genome-wide association studies have also provided important information. Large-scale genetic studies have been revealed that both rare and common genetic variants play crucial roles in this disorder. In this review, we focused on CNVs, SNVs, and SNPs, and discuss the latest research findings on the pathogenesis of schizophrenia based on these genetic variants. Rare variants with large effect sizes can provide mechanistic hypotheses. CRISPR-based genetics approaches and induced pluripotent stem cell technology can facilitate the functional analysis of these variants detected in patients with schizophrenia. Recent advances in long-read sequence technology are expected to detect variants that cannot be detected by short-read sequence technology. Various studies that bring together data from common variant and transcriptomic datasets provide biological insight. These new approaches will provide additional insight into the pathophysiology of schizophrenia and facilitate the development of pathology-based therapeutics.

X chromosome aneuploidies and schizophrenia: association analysis and phenotypic characterization.

AIM: The aims of the present study were: (i) to examine the association between schizophrenia (SCZ) and 47, XXY or 47, XXX in a large case-control sample; and (ii) to characterize the clinical features of patients with SCZ with these X chromosome aneuploidies. METHODS: To identify 47, XXY and 47, XXX, array comparative genomic hybridization (aCGH) was performed in 3188 patients with SCZ and 3586 controls. We examined the association between 47, XXY and 47, XXX and SCZ in males and females separately using exact conditional tests to control for platform effects. Clinical data were retrospectively examined for patients with SCZ with X chromosome aneuploidies. RESULTS: Of the analyzed samples, 3117 patients (97.8%) and 3519 controls (98.1%) passed our quality control. X chromosome aneuploidies were exclusively identified in patients: 47, XXY in seven patients (0.56%), 47, XXX in six patients (0.42%). Statistical analysis revealed a significant association between SCZ and 47, XXY (P = 0.028) and 47, XXX (P = 0.011). Phenotypic data were available from 12 patients. Treatment-resistance to antipsychotics and manic symptoms were observed in six patients each (four with 47, XXY and two with 47, XXX for both), respectively. Statistical analysis revealed that treatment-resistance to antipsychotics, mood stabilizer use, and manic symptoms were significantly more common in patients with 47, XXY than in male patients without pathogenic copy number variations. CONCLUSION: These findings indicate that both 47, XXY and 47, XXX are significantly associated with risk for SCZ. Patients with SCZ with 47, XXY may be characterized by treatment-resistance and manic symptoms.

Investigation of OLIG2 as a candidate gene for schizophrenia and autism spectrum disorder.

A number of genomic mutations that are thought to be strongly involved in the development of schizophrenia (SCZ) and autism spectrum disorder (ASD) have been identified. Abnormalities involving oligodendrocytes have been reported in SCZ, and as a related gene, oligodendrocyte lineage transcription factor 2 (OLIG2) has been reported to be strongly associated with SCZ. In this study, based on the common disease-rare variant hypothesis, target sequencing of candidate genes was performed to identify rare mutations with a high effect size and the possibility that the identified mutations may increase the risks of SCZ and ASD in the Japanese population. In this study, the exon region of OLIG2 was targeted; 370 patients with SCZ and 192 with ASD were subjected to next-generation sequencing. As a result, one rare missense mutation (A33T) was detected. We used the Sanger method to validate this missense mutation with a low frequency (<1%), and then carried out a genetic association analysis involving 3299 unrelated individuals (1447 with SCZ, 380 with ASD, and 1472 healthy controls) to clarify whether A33T was associated with SCZ or ASD. A33T was not found in either case group, and in only one control. We did not find evidence that p.A33T is involved in the onset of ASD or SCZ; however, associations with this variant need to be evaluated in larger samples to confirm our results.

Exome sequencing analysis of Japanese autism spectrum disorder case-control sample supports an increased burden of synaptic function-related genes.

Autism spectrum disorder (ASD) is a highly heritable, complex disorder in which rare variants contribute significantly to disease risk. Although many genes have been associated with ASD, there have been few genetic studies of ASD in the Japanese population. In whole exomes from a Japanese ASD sample of 309 cases and 299 controls, rare variants were associated with ASD within specific neurodevelopmental gene sets, including highly constrained genes, fragile X mental retardation protein target genes, and genes involved in synaptic function, with the strongest enrichment in trans-synaptic signaling (p = 4.4 × 10-4, Q-value = 0.06). In particular, we strengthen the evidence regarding the role of ABCA13, a synaptic function-related gene, in Japanese ASD. The overall results of this case-control exome study showed that rare variants related to synaptic function are associated with ASD susceptibility in the Japanese population.

Cross-Disorder Analysis of Genic and Regulatory Copy Number Variations in Bipolar Disorder, Schizophrenia, and Autism Spectrum Disorder.

BACKGROUND: We aimed to determine the similarities and differences in the roles of genic and regulatory copy number variations (CNVs) in bipolar disorder (BD), schizophrenia (SCZ), and autism spectrum disorder (ASD). METHODS: Based on high-resolution CNV data from 8708 Japanese samples, we performed to our knowledge the largest cross-disorder analysis of genic and regulatory CNVs in BD, SCZ, and ASD. RESULTS: In genic CNVs, we found an increased burden of smaller (<100 kb) exonic deletions in BD, which contrasted with the highest burden of larger (>500 kb) exonic CNVs in SCZ/ASD. Pathogenic CNVs linked to neurodevelopmental disorders were significantly associated with the risk for each disorder, but BD and SCZ/ASD differed in terms of the effect size (smaller in BD) and subtype distribution of CNVs linked to neurodevelopmental disorders. We identified 3 synaptic genes (DLG2, PCDH15, and ASTN2) as risk factors for BD. Whereas gene set analysis showed that BD-associated pathways were restricted to chromatin biology, SCZ and ASD involved more extensive and similar pathways. Nevertheless, a correlation analysis of gene set results indicated weak but significant pathway similarities between BD and SCZ or ASD (r = 0.25-0.31). In SCZ and ASD, but not BD, CNVs were significantly enriched in enhancers and promoters in brain tissue. CONCLUSIONS: BD and SCZ/ASD differ in terms of CNV burden, characteristics of CNVs linked to neurodevelopmental disorders, and regulatory CNVs. On the other hand, they have shared molecular mechanisms, including chromatin biology. The BD risk genes identified here could provide insight into the pathogenesis of BD.

Sequencing of selected chromatin remodelling genes reveals increased burden of rare missense variants in ASD patients from the Japanese population.

Chromatin remodelling is an important process in neural development and is related to autism spectrum disorder (ASD) and schizophrenia (SCZ) aetiology. To further elucidate the involvement of chromatin remodelling genes in the genetic aetiology of ASD and SCZ in the Japanese population, we performed a case-control study. Targeted sequencing was conducted on coding regions of four BAF chromatin remodelling complex genes: SMARCA2, SMARCA4, SMARCC2, and ARID1B in 185 ASD, 432 SCZ patients, and 517 controls. 27 rare non-synonymous variants were identified in ASD and SCZ patients, including 25 missense, one in-frame deletion in SMRACA4, and one frame-shift variant in SMARCC2. Association analysis was conducted to investigate the burden of rare variants in BAF genes in ASD and SCZ patients. Significant enrichment of rare missense variants in BAF genes, but not synonymous variants, was found in ASD compared to controls. Rare pathogenic variants indicated by in silico tools were significantly enriched in ASD, but not statistically significant in SCZ. Pathogenic-predicted variants were located in disordered binding regions and may confer risk for ASD and SCZ by disrupting protein-protein interactions. Our study supports the involvement of rare missense variants of BAF genes in ASD and SCZ susceptibility.

Contribution of copy number variations to the risk of severe eating disorders.

AIM: Eating disorders (EDs) are complex, multifactorial psychiatric conditions. Previous studies identified pathogenic copy number variations associated with NDDs (NDD-CNVs) in ED patients. However, no statistical evidence for an association between NDD-CNVs and EDs has been demonstrated. Therefore, we examined whether NDD-CNVs confer risk for EDs. METHODS: Using array comparative genomic hybridization (aCGH), we conducted a high-resolution CNV analysis of 71 severe female ED patients and 1045 female controls. According to the American College of Medical Genetics guidelines, we identified NDD-CNVs or pathogenic/likely pathogenic CNVs in NDD-linked loci. Gene set analysis was performed to examine the involvement of synaptic dysfunction in EDs. Clinical data were retrospectively examined for ED patients with NDD-CNVs. RESULTS: Of the samples analyzed with aCGH, 70 severe ED patients (98.6%) and 1036 controls (99.1%) passed our quality control filtering. We obtained 189 and 2539 rare CNVs from patients and controls, respectively. NDD-CNVs were identified in 10.0% (7/70) of patients and 2.3% (24/1036) of controls. Statistical analysis revealed a significant association between NDD-CNVs and EDs (odds ratio = 4.69, P = 0.0023). NDD-CNVs in ED patients included 45,X and deletions at KATNAL2, DIP2A, PTPRT, RBFOX1, CNTN4, MACROD2, and FAM92B. Four of these genes were related to synaptic function. In gene set analysis, we observed a nominally significant enrichment of rare exonic CNVs in synaptic signaling in ED patients (odds ratio = 2.55, P = 0.0254). CONCLUSION: Our study provides the first preliminary evidence that NDD-CNVs may confer risk for severe EDs. The pathophysiology may involve synaptic dysfunction.

Identification of rare mutations of the vasoactive intestinal peptide receptor 2 gene in schizophrenia.

OBJECTIVE: Studies showed that rare copy number variations (CNVs) encompassing the vasoactive intestinal peptide receptor 2 gene (VIPR2) were associated with schizophrenia, indicating VIPR2 is a risk gene for schizophrenia. We hypothesized that besides CNV, rare pathogenic single-nucleotide variant (SNV) or small insertion/deletion (Indel) of VIPR2 might be present in some patients and contribute to the pathogenesis of schizophrenia. METHODS: We performed genome-wide CNV analysis to screen CNV at the VIPR2 locus and targeted sequencing of all the exons of VIPR2 to search for SNV and indel in a sample of patients with chronic schizophrenia from Taiwan. RESULTS: We detected a 230-kb microduplication encompassing the VIPR2 in 1 out of 200 patients. Furthermore, we identified six ultrarare SNVs, including one splicing SNV and five missense SNVs, in 516 patients. In-silico analyses showed these SNVs had a damaging effect on the function of VIPR2. CONCLUSION: Our findings support the idea that besides CNV, rare pathogenic SNVs of VIPR2 might contribute to the pathogenesis of schizophrenia in some patients.