Genetic landscape of Rett syndrome-like phenotypes revealed by whole exome sequencing.

BACKGROUND: Rett syndrome (RTT) is a characteristic neurological disease presenting with regressive loss of neurodevelopmental milestones. Typical RTT is generally caused by abnormality of methyl-CpG binding protein 2 (MECP2). Our objective to investigate the genetic landscape of MECP2-negative typical/atypical RTT and RTT-like phenotypes using whole exome sequencing (WES). METHODS: We performed WES on 77 MECP2-negative patients either with typical RTT (n=11), atypical RTT (n=22) or RTT-like phenotypes (n=44) incompatible with the RTT criteria. RESULTS: Pathogenic or likely pathogenic single-nucleotide variants in 28 known genes were found in 39 of 77 (50.6%) patients. WES-based CNV analysis revealed pathogenic deletions involving six known genes (including MECP2) in 8 of 77 (10.4%) patients. Overall, diagnostic yield was 47 of 77 (61.0 %). Furthermore, strong candidate variants were found in four novel genes: a de novo variant in each of ATPase H+ transporting V0 subunit A1 (ATP6V0A1), ubiquitin-specific peptidase 8 (USP8) and microtubule-associated serine/threonine kinase 3 (MAST3), as well as biallelic variants in nuclear receptor corepressor 2 (NCOR2). CONCLUSIONS: Our study provides a new landscape including additional genetic variants contributing to RTT-like phenotypes, highlighting the importance of comprehensive genetic analysis.

No Relationships between Psychosis and the Diazepam Binding Inhibitor rs2276596 (C/A) Polymorphism in Japanese: An Exploratory Study.

OBJECTIVE: Our recent study for the first time reported genotyping method of the diazepam binding inhibitor (DBI) rs2276596 polymorphism using a Polymerase Chain Reaction-Restriction Fragment Length Polymor- phism (PCR-RFLP), and revealed a significant relationships between this polymorphism and alcohol depend- ence. In this study, to facilitate elucidation of the pathogeneses of psychoses including schizophrenia and mood (affective) disorders, we investigated the relationship between the DBI rs2276596 polymorphism (C/A) and psychoses. METHOD: We analyzed the DBI genotypes using the PCR-RFLP method in healthy controls, and psychotics including schizophrenia and mood (affective) disorders (including recurrent depressive disorder and bipolar affective disorder) (ICD-10: F31, F33). RESULT: There was no significant difference in the rs2276596 genotype and allele frequencies of the DBI gene between these psychoses and healthy controls. CONCLUSION: The present data suggested that a mutated allele of the DBI was not one of the risk factors for schizophrenia and mood (affective) disorders, as for the rs2276596 polymorphism. [Original].

Identification of two novel Shank3 transcripts in the developing mouse neocortex.

SHANK3 is a synaptic scaffolding protein enriched in the post-synaptic density of excitatory synapses. Since several SHANK3 mutations have been identified in a particular phenotypic group of patients with autism spectrum disorder (ASD), SHANK3 is strongly suspected of being involved in the pathogenesis and neuropathology of ASD. Several SHANK3 isoforms are known to be produced in the developing brain, but they have not been fully investigated. Here, we identified two different amino-terminus truncated Shank3 transcripts. One transcript, designated as Shank3c-3, produces an isoform that contains the entire carboxyl-terminus, but the other transcript, designated as Shank3c-4, produces a carboxyl-terminus truncated isoform. During development, expression of the novel Shank3 transcripts increased after birth, transiently decreased at P14 and then gradually increased again thereafter. We also determined that methyl CpG-binding protein 2 (MeCP2) is involved in regulating expression of the novel Shank3 transcripts. MeCP2 is a transcriptional regulator that has been identified as the causative molecule of Rett syndrome, a neurodevelopmental disorder that includes autistic behavior. We demonstrated a difference between the expression of the novel Shank3 transcripts in wild-type mice and Mecp2-deficient mice. These findings suggest that the SHANK3 isoforms may be implicated in the synaptic abnormality in Rett syndrome. SHANK3 is a synaptic scaffolding protein and is suspected of being implicated in the pathogenesis and neuropathology of ASD. We here identified two different amino-terminus truncated Shank3 transcripts, Shank3c-3 and Shank3c-4, expressed from the intron 10 of the Shank3 gene, and also suggested the epigenetic regulation of their expression via methyl CpG-binding protein 2 (MeCP2) that has been identified as the causative molecule of Rett syndrome.

Relationship between alcohol dependence and the DBI rs2276596 (C/A) polymorphism in Japanese.

To facilitate elucidation of the pathogenesis of alcohol dependence, we investigated the relationship between a genetic variant of diazepam biding inhibitor (DBI) C/A polymorphism (rs2276596) and alcohol dependence. We determined the DBI genotypes using a novel method involving PCR-RFLP in healthy controls and alcoholics with a diagnosis of alcohol dependence by ICD-10 (F10.20). There was a significant difference in the rs2276596 polymorphism C/A allele frequency of the DBI gene (P < 0.0001) between alcoholics and healthy controls. The present data suggested that a mutant allele of the DBI was one of the risk factors for alcohol dependence as for the rs2276596 polymorphism.

NMDA receptor regulates migration of newly generated neurons in the adult hippocampus via Disrupted-In-Schizophrenia 1 (DISC1).

In the mammalian brain, new neurons are continuously generated throughout life in the dentate gyrus (DG) of the hippocampus. Previous studies have established that newborn neurons migrate a short distance to be integrated into a pre-existing neuronal circuit in the hippocampus. How the migration of newborn neurons is governed by extracellular signals, however, has not been fully understood. Here, we report that NMDA receptor (NMDA-R)-mediated signaling is essential for the proper migration and positioning of newborn neurons in the DG. An intraperitoneal injection of the NMDA-R antagonists, memantine, or 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) into adult male mice caused the aberrant positioning of newborn neurons, resulting in the overextension of their migration in the DG. Interestingly, we revealed that the administration of NMDA-R antagonists leads to a decrease in the expression of Disrupted-In-Schizophrenia 1 (DISC1), a candidate susceptibility gene for major psychiatric disorders such as schizophrenia, which is also known as a critical regulator of neuronal migration in the DG. Furthermore, the overextended migration of newborn neurons induced by the NMDA-R antagonists was significantly rescued by exogenous expression of DISC1. Collectively, these results suggest that the NMDA-R signaling pathway governs the migration of newborn neurons via the regulation of DISC1 expression in the DG.

[Relationship between GSK-3 beta -50T/C and DBI +529A/T polymorphisms in Japanese alcoholics].

As a help of the relationship between bipolar disorder and alcoholism, we studied the relationship between GSK-3 beta -50T/C polymorphism, which is reported to the relationship for bipolar disorder, and Japanese alcoholics. And we investigated the relationship between GSK-3 beta -50T/C polymorphism and DBI +529A/T polymorphisms, which is reported to one of the risk factor for alcoholism. We analyzed the GSK-3 beta genotype using a polymerase chain reaction (PCR), and DBI genotype using PCR with confronting two-pair novel primers (PCR-CTPP) in 75 health controls and 64 alcoholics. In this study, there was no significant difference in the frequency of GSK-3 beta -50T/C polymorphism between alcoholics and controls (p = 0.883), and there was no significant difference in the frequency of DBI +529A/T polymorphism (p = 0.131). Also, there was no relationship between GSK-3 beta -50T/C polymorphism and DBI +529A/T allele in alcoholism (p = 0.907). We suggested that bipolar disorder may not be one of the pathogenesis of alcoholism, and that there was no relationship between GSK-3 beta -50T/C polymorphism and DBI +529A/T polymorphism.

Down syndrome cell adhesion molecule like-1 (DSCAML1) links the GABA system and seizure susceptibility.

The Ihara epileptic rat (IER) is a mutant model with limbic-like seizures whose pathology and causative gene remain elusive. In this report, via linkage analysis, we identified Down syndrome cell adhesion molecule-like 1(Dscaml1) as the responsible gene for IER. A single base mutation in Dscaml1 causes abnormal splicing, leading to lack of DSCAML1. IERs have enhanced seizure susceptibility and accelerated kindling establishment. Furthermore, GABAergic neurons are severely reduced in the entorhinal cortex (ECx) of these animals. Voltage-sensitive dye imaging that directly presents the excitation status of brain slices revealed abnormally persistent excitability in IER ECx. This suggests that reduced GABAergic neurons may cause weak sustained entorhinal cortex activations, leading to natural kindling via the perforant path that could cause dentate gyrus hypertrophy and epileptogenesis. Furthermore, we identified a single nucleotide substitution in a human epilepsy that would result in one amino acid change in DSCAML1 (A2105T mutation). The mutant DSCAML1A2105T protein is not presented on the cell surface, losing its homophilic cell adhesion ability. We generated knock-in mice (Dscaml1A2105T) carrying the corresponding mutation and observed reduced GABAergic neurons in the ECx as well as spike-and-wave electrocorticogram. We conclude that DSCAML1 is required for GABAergic neuron placement in the ECx and suppression of seizure susceptibility in rodents. Our findings suggest that mutations in DSCAML1 may affect seizure susceptibility in humans.

The relationship between alcoholism and DBI gene polymorphism in Japanese--genotyping of the +529A/T in DBI gene polymorphism based on PCR.

As a help of elucidate of the pathogenesis of alcohol dependence, we investigated the relationship between the genetic variants of the diazepam biding inhibitor (DBI) gene polymorphism and alcoholism. We analyzed the DBI genotypes using a polymerase chain reaction with confronting two-pair novel primers (PCR-CTPP) method in 180 healthy controls and 43 alcoholic. There was a significant difference in the +529A/T (rs8192503) polymorphism allele frequency of the DBI gene (P = 0.0421) between the current alcoholics and healthy controls. The present data using the novel PCR method suggested that mutation allele of the DBI gene polymorphism was one of the risk factors for alcoholism.

[The relationship between smoking behavior in young people and CYP2A6 gene polymorphisms, between them and personality traits assessed by NEO-FFI].

We performed a survey on nicotine dependence among young students. We examined the CYP2A6 gene polymorphism using PCR-RFLP. We examined the personality traits using NEO-FFI. Samples were taken from young students of which 87 were smokers and 142 were non-smokers and we tried to clarify the relationship between the nicotine metabolizing ability (CYP2A6), personality, and smoking behavior. As a result, the frequency of the CYP2A6*4C gene was significantly higher among non-smokers than smokers, and the Openness score by NEO-FFI was higher among smokers than non-smokers. We concluded that the CYP2A6*4C gene and the Openness personality trait may be a cause of smoking among the young smokers. Therefore, there is a possibility that smoking behavior in youths may be affected not only by the CYP2A6 gene but also by the Openness personality trait.

Cholecystokinin (CCK) and the CCKA receptor gene polymorphism, and smoking behavior.

We analyzed genetic variants of the promoter region of the cholecystokinin (CCK; which modulates the release of dopamine) gene, and intron 1 and exon 5 of the CCKA receptor gene, and performed association analyses of nicotine dependence using an allele-specific amplification (ASA) method and PCR-RFLP methods. There was a significant difference between the current smoking and nonsmoking groups in the allele frequency of the CCK-45C/T polymorphism. However, there was no significant difference in the CCKA PstI polymorphism, and the HincII polymorphism was not detected in our study. Our data suggest that polymorphisms of the CCK gene may be one of the risk factors for smoking behavior.

[Relationship between of carotid ultrasonography in Japanese hypertensive subjects for intima-media thickness and plaque, and candidate gene polymorphism--possibility of early detection of arteriosclerotic disease].

The purpose of this study was to determine the relationship between organic changes in carotid artery walls and candidate gene polymorphism in Japanese sufferers of essential hypertension. Carotid Ultrasonography was used to measure intima-media thickness (IMT) and presence of plaque formation. Patients were divided into two groups; a hypertension (HT) group and a healthy control (C). Group HT consisted of 273 subjects (56.5 +/- 11.3y) and group C consisted of 500 subjects (55.6 +/- 11.4y). 5HTR2A (T102C, A-1438G) and beta3-AR (Trp64Arg) genetic polymorphism for genetic analysis were performed on 43 subjects (63.5 +/- 8.5y) in group HT and 24 subjects (60.3 +/- 6.8y) in group C. IMT showed 0.81 +/- 0.28mm in Group C and 1.05 +/- 0.22mm in Group HT in patients aged 50 or above, respectively. The thickening was significantly greater in Group HT than in Group C (p < 0.01). Plaque was present in 45.1% in Group C and 70.2% in Group HT in patients aged 50 or above, respectively. It was significantly present greater in Group HT than in Group C (p < 0.01). Results of genetic analysis showed no difference between Group HT and Group C for either genotype of allele frequency of T102C and A-1438G, or of beta3-AR. It is suggested that increase of IMT and formation of plaque are risk factors in patients aged 50 or above with hypertension, and that careful observation of the carotid artery will be effective for early detection of arteriosclerosis, and to predict the symptoms thereof.

Novel Therapeutic Approach for Autism Spectrum Disorder: Focus on SHANK3.

SHANK3 is a synaptic scaffolding protein and plays an important role in neuronal development. SHANK3 interacts with various synaptic molecules, including post-synaptic density-95 (PSD-95), homer and GluR1 AMPA receptor. SHANK3 gene is a causable gene of the Phelan- McDermid syndrome (also known as the 22q13.3 deletion syndrome), whose manifestation is global developmental delay and autistic behavior, especially shows severe speech and language deficit. Additionally since cumulative gene analysis in autistic subjects identified several mutations in SHANK3 gene, including deletion and duplication in a particular region, abnormality of SHANK3 gene is thought the be related with the neuropathology of autism spectrum disorder (ASD). We here review the recent findings in regard to the roles of SHANK3 in higher brain functions, molecular-biologic studies of the complex expression of Shank3 transcripts and production of SHANK3 isoforms, and behavioral studies of Shank3-mutant mice, including our recent findings, and discuss a novel therapeutic approach for ASD.

SHANK3 as an autism spectrum disorder-associated gene.

SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses, and plays important roles in the formation, maturation, and maintenance of synapses. Haploinsufficiency of the SHANK3 gene causes a developmental disorder, 22q13.3 deletion syndrome (known as Phelan-McDermid syndrome), that is characterized by severe expressive language and speech delay, hypotonia, global developmental delay, and autistic behavior. Since several SHANK3 mutations have been identified in a particular phenotypic group in patients with autism spectrum disorder (ASD), the SHANK3 is strongly suspected of being involved in the pathogenesis and neuropathology of ASD. Five CpG-islands have been identified in the SHANK3 gene, and tissue-specific expression of SHANK3 is regulated by DNA methylation in an epigenetic manner. Cumulative evidence has shown that several SHANK3 variants are expressed in the developing rodent brain and that their expression is regulated by DNA methylation of intragenic promoters. We identified novel SHANK3 transcripts whose transcription started at the vicinity of the CpG-island 2 in the mouse brain. Shank3 mutant mice exhibit autistic-like behaviors, including impaired social interaction and repetitive behaviors. In this article we review recent findings in regard to higher brain functions of SHANK3, epigenetic regulation of SHANK3 expression, and SHANK3-related ASD that were obtained from genetic analyses in ASD patients, molecular biological studies using developing mouse brains, and studies of Shank3 mutant mice.

Novel variants of the SHANK3 gene in Japanese autistic patients with severe delayed speech development.

The 22q13.3 deletion syndrome is characterized by a significant delay in language development, mental retardation, hypotonia, and autistic features. Cumulative evidence has shown that haploinsufficiency of the SHANK3 gene is a major cause of the neurological symptoms of the 22q13.3 deletion syndrome. Shank3, a multidomain protein containing the SH3 and PDZ domains, is thought to play an important role in the formation and function of synapses in the developing brain. In this study, we analyzed the SHANK3 gene in 128 autistic patients with manifestations similar to those seen in the 22q13.3 deletion syndrome. The results showed a 6-amino acid deletion upstream of the SH3 domain, a missense variant (arginine to histidine at amino acid position 656) in the PDZ domain, and the insertion or deletion of a repeated 10-bp GC sequence located 9-bp downstream from the 3' end of exon 11. None of these variants was found in 228 controls.

CYP2A6 gene polymorphism and personality traits for NEO-FFI on the smoking behavior of youths.

We performed a survey on the relationship between nicotine dependence and psychological (the personality traits using neuroticism extroversion openess-five factor inventory (NEO-FFI)/nicotine metabolism (the CYP2A6 gene polymorphism) factors among Japanese young students to elucidate the mechanism of the development of nicotine dependence. As a result, the frequency of the CYP2A6*4C gene (enzyme activity deficit) was significantly (p<0.05) higher among nonsmokers than smokers, and the Openness score by NEO-FFI was higher among smokers than nonsmokers. However, there were no significant differences in the Neuroticism, Extraversion, Agreeableness, and Conscientiousness scores by NEO-FFI between smokers and nonsmokers. We conclude that the CYP2A6*4C gene and the Openness personality trait may affect the development of the smoking behavior of youth.

Questionnaire on animal-assisted therapy (AAT): The expectation for AAT as a day-care program for Japanese schizophrenic patients.

Animal-assisted therapy (AAT) was developed to promote human social and emotional functioning as a day-care program for psychiatric patients. In this study, we determined which animals 481 schizophrenic patients liked and what they thought about AAT, using an original questionnaire. It was found that more than 80% of the present patients liked animals and that they thought contact with animals was useful as a novel therapy. They had much interest in, and many hopes, for AAT.

Whole deletion of CYP2A6 gene (CYP2A6AST;4C) and smoking behavior.

The relationship between nicotine metabolism of CYP2A6 and the smoking behavior in a Japanese population was investigated. The CYP2A6 genotypes were determined by the PCR method. There was a significant difference in the frequency of the CYP2A6AST;4C allele, which is a whole deleted allele for the human CYP2A6 gene, between smokers and nonsmokers. The frequency of the CYP2A6AST;4C allele was higher in nonsmokers than in smokers, whereas the frequency of CYP2A6AST;1A/AST;1B heterozygotes with a higher activity of nicotine metabolism was lower. In this study it was suggested that the CYP2A6AST;4C allele may prevent the carrier from smoking and the CYP2A6AST;1A/AST;1B heterozygote may be at risk for developing smoking behavior.