Variable number of tandem repeat polymorphisms of DRD4: re-evaluation of selection hypothesis and analysis of association with schizophrenia.

Associations have been reported between the variable number of tandem repeat (VNTR) polymorphisms in the exon 3 of dopamine D4 receptor gene gene and multiple psychiatric illnesses/traits. We examined the distribution of VNTR alleles of different length in a Japanese cohort and found that, as reported earlier, the size of allele '7R' was much rarer (0.5%) in Japanese than in Caucasian populations (approximately 20%). This presents a challenge to an earlier proposed hypothesis that positive selection favoring the allele 7R has contributed to its high frequency. To further address the issue of selection, we carried out sequencing of the VNTR region not only from human but also from chimpanzee samples, and made inference on the ancestral repeat motif and haplotype by use of a phylogenetic analysis program. The most common 4R variant was considered to be the ancestral haplotype as earlier proposed. However, in a gene tree of VNTR constructed on the basis of this inferred ancestral haplotype, the allele 7R had five descendent haplotypes in relatively long lineage, where genetic drift can have major influence. We also tested this length polymorphism for association with schizophrenia, studying two Japanese sample sets (one with 570 cases and 570 controls, and the other with 124 pedigrees). No evidence of association between the allele 7R and schizophrenia was found in any of the two data sets. Collectively, this study suggests that the VNTR variation does not have an effect large enough to cause either selection or a detectable association with schizophrenia in a study of samples of moderate size.

Autistic-like phenotypes in Cadps2-knockout mice and aberrant CADPS2 splicing in autistic patients.

Autism, characterized by profound impairment in social interactions and communicative skills, is the most common neurodevelopmental disorder, and its underlying molecular mechanisms remain unknown. Ca(2+)-dependent activator protein for secretion 2 (CADPS2; also known as CAPS2) mediates the exocytosis of dense-core vesicles, and the human CADPS2 is located within the autism susceptibility locus 1 on chromosome 7q. Here we show that Cadps2-knockout mice not only have impaired brain-derived neurotrophic factor release but also show autistic-like cellular and behavioral phenotypes. Moreover, we found an aberrant alternatively spliced CADPS2 mRNA that lacks exon 3 in some autistic patients. Exon 3 was shown to encode the dynactin 1-binding domain and affect axonal CADPS2 protein distribution. Our results suggest that a disturbance in CADPS2-mediated neurotrophin release contributes to autism susceptibility.

Expression analysis of actin-related genes as an underlying mechanism for mood disorders.

In this study, we explored the newly postulated 'disturbed cytoskeletal' theory of mood disorders. Firstly, we identified Cap1, a gene for important mediator of actin turnover, as a cogent quantitative trait gene for depressive trait of mice by combining the results of our prior genetic and current genome-wide expression analyses. Then we rigorously examined 'core' actin-related gene expression in the frontal cortex of C57BL/6 (B6) (prone to depression) and C3H/He (C3) (resistant to depression) mice. We confirmed that Cap1 was down-regulated at both transcript and protein levels in B6. Other differentially regulated genes included cofilin1 and profilin1 (up-regulated in B6), and a Rho-family GTPase member (Pak1) (down-regulated in B6). Thirdly, we investigated the 'core' actin-pathway components in human postmortem prefrontal cortices, and observed trend for CAP1 reduction in the bipolar brains. These data suggest that the balance of actin dynamics might be altered towards actin depolymerization in mood disorders.

Association and synergistic interaction between promoter variants of the DRD4 gene in Japanese schizophrenics.

Recent association studies suggest that polymorphisms in the promoter and exon 1 upstream region of the dopamine D4 receptor (DRD4) gene play a functional role in the development of common psychiatric illnesses, although there are also conflicting results. In this study, we re-sequenced this region to identify all genomic variants, and tested them for association with schizophrenia. A total of 570 Japanese schizophrenic cases with matched controls were studied by genotyping all identified/validated common polymorphisms (-1106T>C, -906T>C, -809G>A, -616G>C, -521T>C, -376C>T, -291C>T and 12-bp repeat) and a known microsatellite (120-bp tandem duplication) in the upstream region. A single nucleotide polymorphism (SNP) -809G>A in the promoter region was found to be significantly associated with disease (P=0.018 and 0.032 for allelic and genotypic comparisons, respectively), although not surviving after Bonferroni correction. Logistic regression analysis showed that a combination of the four polymorphisms, -809G>A, -616G>C, -291C>T and the 12-bp repeat, conferred a susceptibility to schizophrenia. These results suggest that the upstream variants have a primary functional effect in the etiology of schizophrenia in the Japanese population.

Linkage disequilibrium analysis of the CHRNA7 gene and its partially duplicated region in schizophrenia.

Several previous studies have reported a significant linkage between markers in the alpha 7 nicotinic cholinergic receptor subunit (CHRNA7) gene and either schizophrenia or the P50 sensory gating deficit, a schizophrenia endophenotype. However, CHRFAM7A, a partially duplicated gene 1.6Mb upstream of the CHRNA7 gene, has complicated further genetic analysis. We genotyped 14 polymorphic markers throughout the full-length CHRNA7 gene and the duplicated region in 188 unrelated Han Chinese patients with schizophrenia and 188 controls. The duplicated regions were assessed by genotyping up- and down-stream polymorphic markers in the vicinity of each region and analyzing the linkage disequilibrium (LD) between each pair of markers. No evidence of risk variants for schizophrenia in either the CHRNA7 gene or the partially duplicated region was found in the LD analysis. A significant deviation from the Hardy-Weinberg equilibrium (HWE) was found only in the genotypic distribution of SNP9 (IVS4-1912) in patients (p=0.00829), but not in controls. In conclusion, our LD analysis did not reveal any association between schizophrenia in our Han Chinese population and the CHRNA7 gene or its partially duplicated region. However, we could not exclude the possibility of a weak genetic effect due to the small sample size. Analyses of larger samples and higher-density markers, particularly around SNP9 (IVS4-1912), are still needed.

Genetic and expression analyses of the STOP (MAP6) gene in schizophrenia.

Accumulating evidence suggests that the pathologic lesions of schizophrenia may in part be due to the altered cytoskeletal architecture of neurons. Microtubule-associated proteins (MAPs) that bind to cytoskeletal microtubules to stabilize their assembly are prominently expressed in neurons. Of the MAPs, MAP6 (STOP) has a particular relevance to schizophrenia pathology, since mice deficient in the gene display neuroleptic-responsive behavioral defects. Here we examined the genetic contribution of MAP6 to schizophrenia in a case (n = 570) -control (n = 570) study, using dense single nucleotide polymorphism (SNP) markers. We detected nominal allelic (p = 0.0291) and haplotypic (global p = 0.0343 for 2 SNP-window, global p = 0.0138 for 3 SNP-window) associations between the 3' genomic interval of the gene and schizophrenia. MAP6 transcripts are expressed as two isoforms. A postmortem brain expression study showed up-regulation of mRNA isoform 2 in the prefrontal cortex (Brodmann's area 46) of patients with schizophrenia. These data suggest that the contribution of MAP6 to the processes that lead to schizophrenia should be further investigated.

Analysis of correlation between serum D-serine levels and functional promoter polymorphisms of GRIN2A and GRIN2B genes.

D-Serine is an endogenous coagonist that increases the opening of N-methyl-D-aspartate (NMDA)-type glutamate receptor channels. We previously reported a reduction of D-serine serum levels in schizophrenia, supporting the disease hypothesis of NMDA receptor-mediated hypo-neurotransmission. The serum levels of D-serine are thought to reflect brain d-serine content. It is important to understand whether there is a direct link between the altered D-serine levels and NMDA receptor expression in vivo or whether these are independent processes. Two polymorphisms are known to regulate the expression of NMDA receptor subunit genes: (GT)(n) (rs3219790) in the promoter region of the NR2A subunit gene (GRIN2A) and -200T > G (rs1019385) in the NR2B gene (GRIN2B). These polymorphisms are also reported to be associated with schizophrenia. Therefore, we examined the correlation between these two polymorphisms and d-serine serum levels in mentally healthy controls, schizophrenics and the combined group. We observed no significant genotype-phenotype correlations in any of the sample groups. However, analyses of larger sample numbers and the detection of additional polymorphisms that affect gene expression are needed before we can conclude that NMDA receptor expression and serum levels of d-serine, if involved in schizophrenia pathophysiology, are independent and additive events.

Enhanced antitumor effect of ultrasound in the presence of piroxicam in a mouse air pouch model.

The antitumor effects of piroxicam, a non-steroidal anti-inflammatory drug, on sarcoma 180 cells under ultrasonic irradiation were examined in a mouse air pouch model. When piroxicam was added to sarcoma 180 suspension under ultrasound irradiation (2 MHz, 10 W, 120 s), the mortality rate of tumor cells immediately after the irradiation and the survival rate of mice were significantly higher than those when ultrasound alone was applied, and these effects of piroxicam were dose-dependent. When D-mannitol was used with piroxicam, the mortality rate of the tumors cells after the irradiation was comparable with that when piroxicam alone was applied, but when L-histidine was used concurrently, the antitumor effect was significantly lower than that when piroxicam alone was applied. Histological examinations one week after the ultrasound irradiation in the presence of piroxicam showed sparse tumor tissue in the air pouch and normal appearance of the air pouch and surrounding tissue. The findings suggest that piroxicam enhances the anti-tumor effects of ultrasound in vivo by increasing the production of singlet oxygen without damage to tissue surrounding the tumor.

Effect of recombinant basic fibroblast growth factor (bFGF) on fibroblast-like cells from human rotator cuff tendon.

Rotator cuff tendon cells (RCC) derived from surgical samples showed fibroblast-like morphology. Histological staining demonstrated collagen secretion by RCC. Immunohistological findings revealed that RCC secreted type I and III collagen, but not type II collagen. In addition, the SDS-PAGE analysis suggested that RCC predominantly produced type I collagen. Basic fibroblast growth factor (bFGF) had a stimulatory effect on the proliferation of RCC dose-dependently up to 1 ng/ml. Administration of bFGF suppressed the secretion of collagens from RCC in a dose-dependent manner.