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Docosahexaenoic acid (DHA; 22:6n-3), which is enriched in the neuronal membrane, plays a variety of roles in the brain. Vesicular glutamate transporters (VGLUTs) are responsible for incorporating glutamine into synaptic vesicles. We investigated the influence of DHA on the fatty acid profile and the levels of VGLUT1 and VGLUT2 proteins in differentiated NG108-15 cells, a neuroblastoma-glioma hybrid cell line. NG108-15 cells were plated and 24 h later the medium was replaced with Dulbecco's modified Eagle's medium supplemented with 1% fetal bovine serum, 0.2 mM dibutyryl cAMP, and 100 nM dexamethasone, which was added to induce differentiation. After 6 d, the amount of DHA in the cells was increased by addition of DHA to the medium. VGLUT2 levels were increased by the addition of DHA. These data indicate that DHA affected the levels of VGLUT2 in NG108-15 cells under differentiation-promoting conditions, suggesting that DHA affects brain functions involving VGLUT2.

Background/aim The aim of the present study was to investigate whether idarubicin (IDR) induces oxidative DNA damage in the presence of copper (II). Materials and methods DNA damage was evaluated by pBR322 plasmid DNA cleavage. The formation of oxidative stress markers [O2 •- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] was analysed. Results IDR induced DNA damage and O2 •- and 8-OHdG generation in the presence of copper (II). Conclusion IDR induced oxidative DNA damage in the presence of copper (II). Since it has been reported that the concentration of copper in the serum of cancer patients is higher than that in healthy groups, IDR-induced oxidative DNA damage in the presence of copper (II) may play an important role in anticancer therapeutic strategies.

Free radicals, such as metabolic intermediates, reactive oxygen species, and metal enzymes, are key substances in organisms, although they can also cause various oxidative diseases. Thus, in vivo free radical imaging should be considered as the ultimate form of metabolic imaging. Unfortunately, electron spin resonance (ESR) imaging has inherent disadvantages, such as free radicals with large linewidths generating blurred images and the presence of two or more free radicals resulting in a complicated imaging procedure. Dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) is a noninvasive imaging method to visualize in vivo free radicals, theoretically, with the same resolution as the MRI anatomical resolution, and fixed low-field DNP-MRI provides unique information on oxidative diseases and cancer. However, the large gyromagnetic ratio of the electron spin, which is 660-fold greater than that of a proton, requires field cycling, wherein the external magnetic field should be varied during DNP-MRI observations. This causes difficulties in developing a DNP-MRI system for clinical purposes. We developed a novel field-cycling DNP-MRI system for a preclinical study. In the said system, the magnetic field is switched by rotationally moving two magnets, with a magnetic flux density of 0.3 T for MRI and 5 mT for ESR. The image quality was examined using various pulse sequences and ESR irradiation using nitroxyl radical as the phantom, and the optimum conditions were established. Using the system, we performed a preclinical study involving free radical imaging by placing the free radicals under the palm of a human hand.

Objective To elucidate the clinical and genetic features of patients with TNFRSF1A variants in Japan using data obtained from a nationwide survey conducted by the Ministry of Health, Labor, and Welfare of Japan study group for tumor necrosis factor receptor–associated periodic syndrome (TRAPS). Methods Inquiries were sent to 2,900 departments of internal medicine and pediatrics in all hospitals with more than 200 beds in Japan, asking whether they had patients in whom TRAPS was suspected. Genetic tests for TNFRSF1A, MEFV, and MVK were performed on 169 patients. Cell surface expression of TNFRSF1A variants was assessed using 293T cells. Results Ten patients from 10 independent families were found to have TNFRSF1A variants. We collected clinical and genetic information on 41 additional patients with TNFRSF1A variants and symptoms of inflammation from 23 independent families; 17 of these patients had not been described in the literature. The common clinical features of Japanese patients were fever of >38°C (100% of patients), arthralgia (59%), and rash (55%). The prevalence of abdominal pain (36%), myalgia (43%), and amyloidosis (0%) was significantly lower in Japanese patients than in Caucasian patients. The most common variant was T61I (appearing in 49% of patients), and it was identified in 7 of 363 healthy controls. Defects in cysteine residues and the T50M variant were associated with decreased cell surface expression, while other variants, including T61I, were not. Conclusion Patients with TNFRSF1A variants are very rare in Japan, as in other countries, but there are a number of clinical and genetic differences between Japanese and Caucasian patients. The pathogenic significance of the T61I variant remains unclear.

Congenital retinal nonattachment (CRNA) is a hereditary ocular disorder characterized by bilateral retinal dysplasia with blindness.1,2 The signs of CRNA include the presence of a retrolental fibro...

DNA has been recognized as an ideal material for bottom-up construction of nanometer scale structures by self-assembly. The generation of sequences optimized for unique self-assembly (GENESUS) program reported here is a straightforward method for generating sets of strand sequences optimized for self-assembly of arbitrarily designed DNA nanostructures by a generate-candidates-and-choose-the-best strategy. A scalable procedure to prepare single-stranded DNA having arbitrary sequences is also presented. Strands for the assembly of various structures were designed and successfully constructed, validating both the program and the procedure.

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous disorder characterized by abnormal vascularization of the peripheral retina, which can result in retinal detachment and severe visual impairment. In a large Dutch FEVR family, we performed linkage analysis, exome sequencing, and segregation analysis of DNA variants. We identified putative disease-causing DNA variants in proline-alanine-rich ste20-related kinase (c.791dup; p.Ser265ValfsX64) and zinc finger protein 408 ( ZNF408 ) (c.1363C>T; p.His455Tyr), the latter of which was also present in an additional Dutch FEVR family that subsequently appeared to share a common ancestor with the original family. Sequence analysis of ZNF408 in 132 additional individuals with FEVR revealed another potentially pathogenic missense variant, p.Ser126Asn, in a Japanese family. Immunolocalization studies in COS-1 cells transfected with constructs encoding the WT and mutant ZNF408 proteins, revealed that the WT and the p.Ser126Asn mutant protein show complete nuclear localization, whereas the p.His455Tyr mutant protein was localized almost exclusively in the cytoplasm. Moreover, in a cotransfection assay, the p.His455Tyr mutant protein retains the WT ZNF408 protein in the cytoplasm, suggesting that this mutation acts in a dominant-negative fashion. Finally, morpholino-induced knockdown of znf408 in zebrafish revealed defects in developing retinal and trunk vasculature, that could be rescued by coinjection of RNA encoding human WT ZNF408 but not p.His455Tyr mutant ZNF408. Together, our data strongly suggest that mutant ZNF408 results in abnormal retinal vasculogenesis in humans and is associated with FEVR.

The transcription factor c-MYC intron binding protein 1 (MIBP1) binds to various genomic regulatory regions, including intron 1 of c-MYC. This factor is highly expressed in postmitotic neurons in the fetal brain and may be involved in various biological steps, such as neurological and immunological processes. In this study, we globally characterized the transcriptional targets of MIBP1 and proteins that interact with MIBP1. Microarray hybridization followed by gene set enrichment analysis revealed that genes involved in the pathways downstream of MYC, NF-κB, and TGF-β were down-regulated when HEK293 cells stably overexpressed MIBP1. In silico transcription factor binding site analysis of the promoter regions of these down-regulated genes showed that the NF-κB binding site was the most overrepresented. The up-regulation of genes known to be in the NF-κB pathway after the knockdown of endogenous MIBP1 in HT1080 cells supports the view that MIBP1 is a down-regulator of the NF-κB pathway. We also confirmed the binding of the MIBP1 to the NF-κB site. By immunoprecipitation and mass spectrometry, we detected O-linked β-N-acetylglucosamine (O-GlcNAc) transferase as a prominent binding partner of MIBP1. Analyses using deletion mutants revealed that a 154-amino acid region of MIBP1 was necessary for its O-GlcNAc transferase binding and O-GlcNAcylation. A luciferase reporter assay showed that NF-κB-responsive expression was repressed by MIBP1, and stronger repression by MIBP1 lacking the 154-amino acid region was observed. Our results indicate that the primary effect of MIBP1 expression is the down-regulation of the NF-κB pathway and that this effect is attenuated by O-GlcNAc signaling.

The molecular bases and clinical features of hereditary angioedema (HAE) have not been systematically documented in Japan or in other Asian countries. Thus, the authors researched the genetic and clinical characteristics of Japanese patients with HAE.The authors analyzed the CIINH gene for mutations in 13 unrelated Japanese patients with HAE by means of the polymerase chain reaction and nucleotide sequencing. In addition, the authors searched the literature from January 1969 to October 2010 on Japanese patients with HAE.Seven of the mutations found were novel, including 4 missense mutations (8728TG, 8831CA, 16661TG and 16885CA), 2 frameshift mutations (2281_2350del70, 14158delT) and 1 large deletion (at least 1 kb-length deletion including exon 4), whereas 6 mutations had previously been reported in European populations.The genetic and clinical characteristics in Japanese patients with HAE may be similar to those in Western patients although our sample size is small and the authors identified 7 novel mutations.

Objective. Identification of the association of killer cell immunoglobulin-like receptor (KIR) genes withSLE and accompanying infections.Methods. Presence or absence of all 14 KIR genes was studied for association with SLE by case–controlstudies. A total of 417 SLE cases, 72 RA cases and 256 controls, all of Japanese descent, were enrolled.Results. The carrier frequency of KIR2DL5 was significantly decreased in SLE patients compared withhealthy controls [39.3 vs 50.4%; odds ratio (OR)=0.64; 95% CI 0.36, 0.92; P=0.005). When the preva-lence of severe infections was analysed in 184 SLE patients, whose medical records were available,KIR2DL5 carriers were at an increased risk of overall infection and viral infection (crude OR=2.66; 95%CI 1.43, 4.92; P=0.017 and crude OR=2.31; 95% CI 1.15, 4.62; P=0.017, respectively). After adjustingfor methylprednisolone pulse and/or cyclophosphamide pulse therapy, KIR2DL5 carriers were at signifi-cantly greater risk of infectious events overall (adjusted OR=2.45; 95% CI 1.24, 4.81; P=0.0095).However, KIR2DL5 carriers were marginally associated with an increased risk of viral infectious events(adjusted OR=2.03; 95% CI 0.94, 4.41; P=0.0718).Conclusion. KIR2DL5 was significantly associated with a decreased risk of SLE as well as an increasedrisk of infectious events overall in SLE patients. Our data suggest a further role of KIRs in the pathogen-esis of autoimmune diseases and infection.Key words: Systemic lupus erythematosus, Killer cell immunoglobulin-like receptor, Infection, Genetic analysis.

We have employed a laser-capture microdissection technique and single-nucleotide polymorphism arrays to characterize genomic alterations associated with the development of glioblastoma multiforme (GBM). Combined analysis of loss of heterozygosity (LOH) and copy number revealed that more than half (56.3%) of the 254 identified LOH loci showed no copy-number alteration, indicating the presence of copy-number neutral LOH (cnLOH). Furthermore, we found a GBM case that showed cnLOH in 18 of the 22 autosomes. These results were confirmed by quantitative real-time PCR, microsatellite analysis, and fluorescence in situ hybridization. The high rate of cnLOH suggests that epigenetic abnormalities of many genes are involved in the development and progression of GBMs.

Mapping loss of heterozygosity (LOH) regions in the genomes of tumor tissues is a practical approach for identifying genes whose loss is related to tumorigenesis. Conventional LOH analyses using microsatellite or single nucleotide polymorphism (SNP) markers require the simultaneous examination of tumor- and matched normal-DNA. Here, we improved the previously developed SNP-based LOH assay using single strand conformation polymorphism (SSCP) analysis, so that LOH in tumor samples heavily contaminated with normal DNA can now be precisely estimated, even when matched normal DNA is not available. We demonstrate the reliability of the improved SSCP-based LOH detection method, called the LOH estimation by quantitative SSCP analysis using averaged control (LOQUS-AC), by comparing the results with those of the previous “LOH estimated by quantitative SSCP assay” (LOQUS) method. Using the LOQUS-AC assay, LOH was detected at a high consistency (98.1%) with the previous LOQUS method. We then applied this new method to characterize LOH profiles in 130 meningiomas, using 68 SNPs (i.e., a mean inter-SNP interval of 441 kbp) that are evenly distributed throughout chromosome 1p36. Benign, atypical and anaplastic meningiomas exhibited 1p36 LOH at frequencies of 48.39, 84.62 and 100.00%, respectively, using LOQUS-AC. Subsequently, we detected a candidate common LOH region on 1p36.11 that might harbor tumor suppressor genes related to malignant progression of meningioma. © 2007 Wiley-Liss, Inc.

In order to investigate the contribution of genetic variation in the human dopamine receptor D4 gene (DRD4) to the risk of developing schizophrenia, we carried out a genetic analysis of 27 polymorphisms in 216 schizophrenic patients and 243 healthy controls from the Kyushu region of Japan. Twenty-two single nucleotide polymorphisms (SNPs) and five insertion/deletion polymorphisms were analyzed in this study, including four novel SNPs and a novel mononucleotide repeat. Linkage disequilibrium (LD) and haplotype analyses reveal weak LD across the DRD4 gene. In univariate analysis female individuals with allele -521C had a higher risk for schizophrenia. However, this finding was not significant after correction for multiple hypothesis testing. No other polymorphisms or haplotypes differed between schizophrenic patients and controls. Likewise, multivariate analyses did not reveal any statistically significant associations.

Purpose: To report the clinical features of a patient with familial exudative vitreoretinopathy (FEVR) associated with homozygous R417Q mutation in the frizzled-4 gene (FZD4). Methods: Clinical examination and mutation analysis by direct sequencing. Results: A five-month-old girl was found to have leukocoria associated with retrolental fibroplasia in the right eye and a severe falciform retinal fold in the left eye. Mutational analysis revealed a homozygous R417Q mutation in the FZD4 gene. Her parents who carried the same mutation heterozygously exhibited milder ocular phenotype. Conclusions: Homozygous state for the FZD4 gene is possibly involved in the severity of the FEVR phenotype.

Background The functional single nucleotide polymorphism (SNP) in the MDM2 promoter region, SNP309, is known to be associated with various diseases, particularly cancer. Although many studies have been performed to demonstrate the mechanism of allele-specific expression (ASE) on SNP309, they have only utilized in vitro techniques. It is unknown whether ASE of MDM2 is ascribed solely to SNP309, in vivo. Methods We attempted to evaluate ASE of MDM2 in vivo using post-labeling followed by automated capillary electrophoresis under single-strand conformation polymorphism conditions. For measuring a quantitative difference, we utilized the SNPs on the exons of MDM2 as markers, the status of which was heterozygous in a large population. To address the cause of ASE beyond 20 %, we confirmed sequences of both MDM2-3’UTR and promoter regions. We assessed the SNP which might be the cause of ASE using biomolecular interaction analysis and luciferase assay. Results ASE beyond 20 % was detected in endometrial cancers, but not in cancer-free endometria samples only when an SNP rs1690916 was used as a marker. We suspected that this ASE in endometrial cancer was caused by the sequence heterogeneity in the MDM2-P2 promoter, and found a new functional polymorphism, which we labelled SNP55. There was no difference between cancer-free endometria and endometrial cancer samples neither for SNP55 genotype frequencies nor allele frequencies, and so, SNP55 alone does not affect endometrial cancer risk. The SNP55 status affected the DNA binding affinity of transcription factor Sp1 and nuclear factor kappa-B (NFκB). Transcriptional activity of the P2 promoter containing SNP55C was suppressed by NFκB p50 homodimers, but that of SNP55T was not. Only ASE-positive endometrial cancer samples displayed nuclear localization of NFκB p50. Conclusions Our findings suggest that both the SNP55 status and the NFκB p50 activity are important in the transcriptional regulation of MDM2 in endometrial cancers.

To elucidate the clinical and genetic features of patients with TNFRSF1A variants in Japan using data obtained from a nationwide survey conducted by the Ministry of Health, Labor, and Welfare of Japan study group for tumor necrosis factor receptor-associated periodic syndrome (TRAPS).Inquiries were sent to 2,900 departments of internal medicine and pediatrics in all hospitals with more than 200 beds in Japan, asking whether they had patients in whom TRAPS was suspected. Genetic tests for TNFRSF1A, MEFV, and MVK were performed on 169 patients. Cell surface expression of TNFRSF1A variants was assessed using 293T cells.Ten patients from 10 independent families were found to have TNFRSF1A variants. We collected clinical and genetic information on 41 additional patients with TNFRSF1A variants and symptoms of inflammation from 23 independent families; 17 of these patients had not been described in the literature. The common clinical features of Japanese patients were fever of38°C (100% of patients), arthralgia (59%), and rash (55%). The prevalence of abdominal pain (36%), myalgia (43%), and amyloidosis (0%) was significantly lower in Japanese patients than in Caucasian patients. The most common variant was T61I (appearing in 49% of patients), and it was identified in 7 of 363 healthy controls. Defects in cysteine residues and the T50M variant were associated with decreased cell surface expression, while other variants, including T61I, were not.Patients with TNFRSF1A variants are very rare in Japan, as in other countries, but there are a number of clinical and genetic differences between Japanese and Caucasian patients. The pathogenic significance of the T61I variant remains unclear.

We present genome-wide definitive haplotypes, determined using a collection of 74 Japanese complete hydatidiform moles, each carrying a genome derived from a single sperm. The haplotypes incorporate 281,439 common SNPs, genotyped with a high throughput array-based oligonucleotide hybridization technique. Comparison of haplotypes inferred from pseudoindividuals (constructed from randomized mole pairs) with those of moles showed some switch errors in resolution of phases by the computational inference method. The effects of these errors on local haplotype structure and selection of tag SNPs are discussed. We also show that definitive haplotypes of moles may be useful for elucidation of long-range haplotype structure, and should be more effective for detecting extended haplotype homozygosity indicative of positive selection.

Aims: To search for mutations in the frizzled 4 ( FZD4 ) gene in patients with familial exudative vitreoretinopathy (FEVR) and to delineate the defective gene associated clinical features. Methods: Direct sequencing following polymerase chain reaction of exons of FZD4 was performed for 24 probands with FEVR (18 familial and six sporadic), and some of their families. Clinical symptoms among individuals with mutations were assessed. Results: Four novel mutations were identified in four patients with familial and one with sporadic FEVR. Three of these mutations were missense (M105V, R417Q, and G488D) and one was a nonsense change (W319X). M105V, R417Q, and G488D co-segregated with the disease. None of these sequence changes was found among 300 chromosomes from 150 healthy volunteers. The severity of vitreoretinopathy in the individuals involved in this study varied, but no patient with mutations in FZD4 exhibited rhegmatogenous retinal detachment although this pathology is thought to be the most common type of retinal detachment in FEVR. Conclusion: FZD4 gene mutations were found in some cases of autosomal dominant and sporadic FEVR. FZD4 mutations were responsible for FEVR with variable clinical manifestations.

We describe a new environment of a single-stranded conformational polymorphism (SSCP) analysis using automated capillary array sequencers (e.g., ABI PRISM® 3100 and 3700). In this environment, electrophoretic conditions, settings for instrument management, and software for data analysis are adjusted for SSCP analysis. Highly reproducible results are obtained with this new system, and fragments with mutations and/or polymorphisms in different capillaries or different runs can be reliably detected. The relative peak heights between alleles are quantitative and reproducible between runs, and so allele frequencies of single nucleotide polymorphisms can be accurately estimated by a pooled DNA strategy. The method allows unattended, lowcost, and quantitative SSCP analysis using instruments that are widely accessible.

Detection of the loss of chromosomal regions in cancerous tissues has diagnostic and prognostic relevance, and the development of a reliable and cost-effective technique for this is clinically important. Here we present an efficient technique for quantitative detection of microsatellite alleles, using a post-PCR fluorescence-labeling procedure and multiplexed analysis. We also present a new statistical method for the interpretation of the data that permits reliable and sensitive evaluation of the allelic status of sampled DNA. A high-resolution analysis of allelic imbalance on chromosomes 1p, 10 and 19q in 28 glioma samples of various types using this method revealed that allelic imbalances are more frequent than have been reported, suggesting the diagnostic value of this method in examining the genetic profiles of gliomas.

Probe-free imaging of redox intermediate radicals must become a crucial method and diagnosis. DNP (dynamic nuclear polarization)-MRI, a new imaging method for free radical (Lurie, et al. 1988), utilizes Overhauser effect that nuclear spin close to free radical is 300 times higher polarized than that of natural state by inducing the electron spin resonance of free radical. The advantage of DNP-MRI is the high sensitivity and resolution, theoretically similar to that in MRI. We installed the custom-made DNP-MRI from Philips and succeeded in the imaging of redox status in disease models and the spectroscopic imaging of redox intermediate radicals from FMN, FAD, CoQ10, and vitamin E. It has, however, the large disadvantage of poor sensitivity and heating by ESR irradiation, because of its low magnetic field (15mT MRI). Here, we developed the novel field-cycling DNP-MRI system for clinical trial, in which free radicals are visualized with 0.3T MRI as the polarized proton by irradiating the free radicals at 5mT, and demonstrated the clear images of the phantoms containing FAD radical with less than 0.5 mm of the spatial resolution. The image of FAD radical placed under healthy volunteer hand was superimposed on the anatomical MRI image. The intrinsic radical, melanin, could be also visualized. Overhauser effect is known to be occurred in the various kind of free radicals, metal-complex, etc., and also in lipid membranes. The newly developed DNP-MRI could demonstrate the functional/metabolic imaging by visualizing intrinsic radials, redox intermediates and/or metal enzymes in disease models and also in patients.

The most recent definition of glioblastoma with oligodendroglioma component (GBMO) assigned clinical significance to the observation of oligodendroglial foci within glioblastomas. However, the pathological mechanism of its histogenesis has not yet been determined. We report the genetic analysis of a GBMO case that evolved from an astrocyte lineage. A 37-year-old male underwent a third craniotomy for the removal of recurrent lesions of a secondary glioblastoma originating from a previous diffuse astrocytoma. The lesion in the right frontal lobe contained oligodendroglial foci within a glioblastoma background, while the remaining lesions showed only classic glioblastoma histology. Genetic analyses revealed distal 10q loss of heterozygosity (LOH) occurring de novo in the oligodendroglial tissue, as well as 10p, 17p LOH, and isocitrate dehydrogenase-1 gene (IDH1) mutations inherited from the previous lesions. The final recurrent glioblastoma underwent LOH on almost the entire of chromosome 10. Based on these results, the importance of an oligodendroglial component in glioblastomas may be limited.

Instability of short tandem repeats (STRs) in DNA during replication is observed in all organisms examined, and is causatively involved in various human diseases. We explore the mechanisms involved in instability by examining length changes occurring during the replication of [(CA) 20 TA] n and [(CAG) 20 TAG] n , in human cells. We show that the majority of alterations consist of an insertion or deletion of one repeat unit, and base substitutions or length changes involving many repeat units are rare. We also show that length changes of two-tract STRs are biased toward the 3′-end of the repeat tract, in reference to lagging strand synthesis. There are some differences between our observations and previous observations in microbes, e.g. the orientation effect was not observed in this study. The results of this study are discussed in terms of the molecular mechanisms leading to alterations in repeat tracts.

The membrane TNF-α is known to serve as a precursor of the soluble form of TNF-α. Although it has been reported the biological functions of the membrane TNF-α as a ligand, the outside-to-inside (reverse) signal transmitted through membrane TNF-α is poorly understood. Here we report a novel function mediated by outside-to-inside signal via membrane TNF-α into the cells expressing membrane TNF-α. Activation by anti-TNF-α Ab against membrane TNF-α on human T cell leukemia virus (HTLV) I-infected T cell line, MT-2, or PHA-activated normal human CD4+ T cells resulted in the induction of an adhesion molecule, E-selectin (CD62E), on the cells with the peak of 12–24 h, which completely disappeared by 48 h. When wild-type or mutant membrane TNF-α (R78T/S79T) resistant to proteolytic cleavage was introduced into Jurkat or HeLa cells, E-selectin was induced by the treatment with anti-TNF-α Ab with the similar kinetics. Membrane TNF-α-expressing Jurkat cells also up-regulated E-selectin when brought into cell-to-cell contact with TNF receptor-expressing HeLa cells. Northern blot analysis and RT-PCR analysis showed that the membrane TNF-α-mediated E-selectin expression was up-regulated at the level of transcription. These results not only confirmed our previous findings of reverse signaling through membrane TNF-α, but also presented evidence that E-selectin was inducible in cell types different from endothelial cells. It is strongly suggested that membrane TNF-α is a novel proinflammatory cell surface molecule that transmits bipolar signals in local inflammation.

Objective. In order to study the role of the p53 tumour suppressor gene in the proliferation of rheumatoid arthritis (RA) synovium, we analysed the mutation of p53 in the synovial fibroblast-like type B synoviocyte from RA patients. Methods. Synovial fibroblast-like type B synoviocytes were prepared from the synovial tissues from nine Japanese patients with RA. The p53 cDNA region from exons 4-11 was screened for mutations by the streamlined mutation detection method in which polymerase chain reaction (PCR) products are post-labelled and are analysed by automated capillary electrophoresis using single-strand conformation polymorphism conditions, followed by direct sequencing of the subclones of the PCR products. Results. p53 mutation with possible functional alteration was detected in four of the nine RA patients (44.4%). Of a total of 262 p53 cDNA subclones, 10 subclones were carrying 10 p53 mutations, eight of which were associated with amino acid alterations or protein truncation. Of the p53 functional mutations, a substitution of Gly at amino acid residue 245 to Asp (G245D) was identified in two patients in three subclones. G245D was the first mutation that was recurrently identified in different RA individuals. G245D is also one of the relatively common mutations in human cancers. Conclusions. In some patients with RA, dysfunction of p53 might play a role in the proliferation of the synovial tissue. G245D mutation might especially need further study as it is the first recurrently identified p53 mutation in RA and is also one of the frequently identified mutations in human cancers.

Novel mutation in PAX3 gene in Waardenburg syndrome accompanied by unilateral macular degeneration

Effective use of knowledge of human genome sequences in studies of hereditary diseases or cancer heavily depends on efficient methods for detection of mutations in individual samples. We describe here a simple and efficient mutation scanning system in which PCR products arepost-labeled with two different fluorescent dyes in one tube, and analyzed by an automatedcapillary electrophoresis system using single-strand conformation polymorphism (SSCP) conditions (PLACE–SSCP). With the appropriate use of an internal control DNA, differences in electrophoretic mobilities between a reference and samples are precisely evaluated, then the presence of mutations is statistically judged. Thirty-three of 34 known mutations in fragments of three unrelated sequence contexts up to 741 bp were detected using one electrophoresis condition at the confidence level of

Sensitivity of single-strand conformation polymorphism analysis of PCR products (PCR-SSCP analysis) is known to be decreased when the DNA fragments are longer than 300 bp. We examined effects of buffer ions in an attempt to extend the length limit of the analysis. It has been noted that addition of glycerol to the gel containing Tris-borate buffer enhances the sensitivity, but the effects of glycerol have been left unexplained. We found that the effects of glycerol are caused by the reduction of pH of the buffer by the reaction of glycerol and borate ion. We further extended these observations and found that sensitivity of SSCP can be greatly improved by running the electrophoresis in low pH buffer systems. Using a new buffer system and running the electrophoresis at a fixed temperature, we detected 27 of 31 known mutations of factor IX gene in six different sequence contexts ranging in length from 300 to 800 bp.

Purpose To describe the clinical features of members of a family with acorea, microphthalmia and cataract syndrome. In addition, to perform linkage analysis on family members to determine possible candidate genes. Methods Comprehensive ophthalmic examinations were performed on five affected members of a family consisting of a paternal grandmother, father and three children. In addition, DNA was extracted from nine family members (the five affected and four normal members) and used for genome-wide single nucleotide polymorphism genotyping and linkage analysis. Results All of the affected patients had acorea or fibrous occlusion of the pupil, microphthalmia and cataracts in both eyes. They also had microcornea and iridocorneal dysgenesis. Examination of the crystalline lens was hindered by the abnormal iris surface, but cataracts were detected by ultrasound biomicroscopy. Surgical reconstruction of the pupil allowed a better view of the posterior pole of the eye, and ophthalmoscopy showed a normal retina and optic disc. No systemic abnormalities were observed. Linkage analysis did not reach significance but narrowed the location of possible candidate genes to chromosomes 1, 5, 8, 11 and 17. Conclusions This acorea, microphthalmia and cataract syndrome has not previously been reported. Genetic analyses indicate that this syndrome is probably due to an autosomal dominant mutation.

We show that RNA can serve as a primer in PCR. Use of rTth DNA polymerase is essential because it has strong reverse transcriptase activity. RNA primers can be obtained by in vitro transcription and are less costly than DNA primers, which are chemically synthesized. RNA-primed PCR also opens the possibility that a specific amplification reaction can be achieved in the absence of knowledge of the target nucleotide sequence.

Purpose To search for mutations in the TSPAN12 gene in 90 Japanese probands with familial exudative vitreoretinopathy (FEVR) and their family members and to determine the types and frequencies of the mutations. Design Laboratory investigation and clinical case analyses. Methods Direct sequencing after polymerase chain reaction of the coding exons of TSPAN12 was performed for 90 probands with FEVR and some of their family members. The clinical signs and symptoms that were characteristic of individuals with TSPAN12 mutations were determined. Results Three families were found to carry 2 mutations in TSPAN12 . One of these mutations was a new missense change, L245P, and the other was an already reported nonsense mutation, L140X, in 2 families. Mutations in TSPAN12 accounted for 3% of Japanese FEVR patients and 8% of the FEVR families who did not have mutations in any of the known FEVR genes, FZD4 , LRP5 , and NDP . The clinical signs and symptoms varied among the patients, but the retinal findings with TSPAN12 mutations were not different from those with mutations in the known FEVR-causing genes. Conclusions Mutant TSPAN12 is responsible for approximately 3% of FEVR patients in Japan. The results provide further evidence that mutations in TSPAN12 are FEVR causing and that the gene products most likely play a role in the development of retinal vessels.

The majority of complete hydatidiform moles (CHMs) harbor duplicated haploid genomes that originate from sperm. This makes CHMs more advantageous than conventional diploid cells for determining haplotypes of single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) as all of the genetic variants in a CHM genome are homozygous. Here we report SNP/CNV haplotype structures determined by analyzing 100 CHMs from Japanese subjects using high-density DNA arrays. The obtained haplotype map should be useful as a reference for the haplotype structure of Asian populations. We resolved common CNV regions (merged CNV segments across the examined samples) into CNV events (clusters of CNV segments) on the basis of mutual overlap and found that the haplotype backgrounds of different CNV events within the same CNV region were predominantly similar, perhaps due to inherent structural instability.

The single strand conformation polymorphism (SSCP) method is a sensitive technique used to detect subtle sequence differences in PCR-amplified DNA fragments as separated peaks in electrophoretic analysis. In this chapter, we focus on SSCP analysis for quantifying polymorphic alleles rather than scanning for mutations. Short fragments carrying single nucleotide polymorphisms are amplified from individual and pooled DNA samples, then the products are labeled with fluorescent dyes and analyzed by automated capillary electrophoresis under nondenaturing conditions. Dedicated software, QSNPlite, interprets trace data of the electrophoresis to identify alleles of individuals and quantify these alleles in the pool. The software can also incorporate sequencing data to assign alleles at the nucleotide level. The procedures described here are being used in association studies that compare allele frequencies between cases and controls to identify genes responsible for common diseases.

Group IVA cytosolic phospholipase A2 (cPLA2α) plays a key role in tumorigenesis via generating arachidonic acids as the substrate of cyclooxygenase. The aim of this study was to elucidate the possible associations between cPLA 2 α gene polymorphisms and phenotypic features of patients with familial adenomatous polyposis (FAP). A tag single nucleotide polymorphisms (SNPs)-based genotype–phenotype association study of the cPLA 2 α gene was conducted in 73 Japanese patients from 59 families with FAP. Based on the HapMap database, seven tag SNPs of the cPLA 2 α gene were selected and genotyped by direct sequencing analysis. The genotype–phenotype association in relation to the adenomatous polyposis coli (APC) gene mutation was also assessed. The single SNP analysis showed that rs3820185 C allele [odds ratio (OR), 2.5; 95% confidence interval (CI), 1.2–4.9] and rs127446200 GG genotype (OR, 10.9; 95%CI, 1.6–69.8), were more frequent in patients with gastric fundic gland polyposis (FGP) than in those without. Rs12749354 C allele was more frequently found in patients with small intestinal adenoma (OR, 7.0; 95% CI, 1.5–30.4; p = 0.008). This association was also significant when adjusted for covariates (age, sex, and APC mutation) in a logistic regression analysis (adjusted OR, 7.4; 95% CI, 1.2–64.2; p = 0.027). The cPLA 2 α gene may be a possible disease modifier gene in FAP.

The haplotype map constructed by the HapMap Project is a valuable resource in the genetic studies of disease genes, population structure, and evolution. In the Project, Caucasian and African haplotypes are fairly accurately inferred, based mainly on the rules of Mendelian inheritance using the genotypes of trios. However, the Asian haplotypes are inferred from the genotypes of unrelated individuals based on population genetics, and are less accurate. Thus, the effects of this inaccuracy on downstream analyses needs to be assessed. We determined true Japanese haplotypes by genotyping 100 complete hydatidiform moles (CHM), each carrying a genome derived from a single sperm, using Affymetrix 500 K Arrays. We then assessed how inferred haplotypes can differ from true haplotypes, by phasing pseudo-individualized true haplotypes using the programs PHASE, fastPHASE, and Beagle. We found that, at various genomic regions, especially the MHC locus, the expansion of extended haplotype homozygosity (EHH), which is a measure of positive selection, is obscured when inferred Asian haplotype data is used to detect the expansion. We then mapped the genome using a new statistic, XDiHH, which directly detects the difference between the true and inferred haplotypes, in the determination of EHH expansion. We also show that the true haplotype data presented here is useful to assess and improve the accuracy of phasing of Asian genotypes., Author Summary Precise haplotype maps are preferred for the performance of a variety of genetic studies including identification of disease-associated loci and dissection of evolutionary mechanisms such as selection and recombination. For diploid organisms, the haplotype information appears as the genotypes when we obtain the information using widely used high-throughput techniques. The process of extracting haplotype information from genotypes is called phasing, which can be accurately done if the genotypes are from related individuals, such as parent–child trios, by considering the constraints imposed by the rules of Mendelian inheritance. For the genotype data without family information, phasing is done by one of the methods that are based on haplotype clustering, and the inferred haplotypes are known to be less accurate. Here, we experimentally determined genome-wide definitive haplotypes using a collection of Japanese complete hydatidiform moles (CHM), each of which carries a genome derived from a single sperm. Using these resources, we asked if the definitive haplotype data can detect long-distance information that has been obscured when we rely solely on the haplotypes inferred by clustering. We also show that by introducing definitive haplotypes as references, inference of haplotypes of unrelated individuals is significantly improved.

The single strand conformation polymorphism (SSCP) method is a sensitive technique used to detect subtle sequence differences in PCR-amplified DNA fragments as separated peaks in electrophoretic analysis. In this chapter, we focus on SSCP analysis for quantifying polymorphic alleles rather than scanning for mutations. Short fragments carrying single nucleotide polymorphisms are amplified from individual and pooled DNA samples, then the products are labeled with fluorescent dyes and analyzed by automated capillary electrophoresis under nondenaturing conditions. Dedicated software, QSNPlite, interprets trace data of the electrophoresis to identify alleles of individuals and quantify these alleles in the pool. The software can also incorporate sequencing data to assign alleles at the nucleotide level. The procedures described here are being used in association studies that compare allele frequencies between cases and controls to identify genes responsible for common diseases.

LEC (Long-Evans with a cinnamon-like coat color) rats develop hepatocellular carcinomas (HCCs) spontaneously. We examined mutations of codons 12, 13, and 61 of the Ha-ras, Ki-ras, and N-ras genes in four HCCs by the polymerase chain reaction (PCR)-single-stranded DNA direct sequencing method. No ras gene mutations were observed, suggesting that ras activation is not involved in spontaneous hepatocarcinogenesis in LEC rats. The expression of mRNAs for c-myc, Ha-ras, c-raf, and the protein phosphatase 2A alpha gene (PP-2A alpha) was also examined in the four HCCs by northern blot analysis. Three of the four HCCs had c-myc expression levels approximately 30-fold higher than that in the liver of control Long-Evans rats with an agouti coat color (LEA), a sibling line of LEC rats, while the remaining HCC had an expression level sevenfold higher than that of control. In contrast, the expression levels of the Ha-ras, c-raf, and PP-2A alpha genes were the same as those in the livers of control rats. Studies of c-myc expression and mitotic index in five other HCCs, two hyperplastic nodules, and two nontumorous portions of livers of HCC-bearing LEC rats that had chronic-phase hepatitis suggested that the high level of c-myc gene expression was not due only to increased cell proliferation but might possibly be more integrally involved in hepatocarcinogenesis.

Identification of the genes responsible for systemic lupus erythematosus (SLE).All the exons and putative promoter regions of 53 candidate genes (TNFRSF6/Fas, TNFSF6/FasL, Fli1, TNFSF10/TRAIL, TNFSF12/TWEAK, Bcl-2, PTEN, FADD, TRADD, CDKN1A, TNFRSF1A/TNFR1, TNFRSF4/OX40, TNFSF4/OX40L, TNFSF5/CD40L, TNFSF13B/BAFF, ICOS, CTLA4, CD28, FYN, G2A, CR2, PTPRC/CD45, CD22, CD19, Lyn, PDCD1, PTPN6, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3, CD3Z, DNASE1, APCS, MERTK, C3, C1QA, C1QB, C1QG, C2, MBL2, IGHM, IL-2, IL-4, IL-10, IFNG, TNFA, MAN2A1, TNFRSF11A/RANK, TNFRSF11B/OPG, TNFSF11/OPGL) were screened for single nucleotide polymorphisms (SNPs) and their association with SLE was assessed by case-control studies. A total of 509 cases and 964 controls of Japanese descent were enrolled.A total of 316 SNPs was identified. When analysed in the Japanese population, the allele frequencies of T at rs7951 and G at rs2230201 of the C3 gene were 0.110 and 0.626, respectively, in SLE patients; significantly higher than the frequencies of 0.081 and 0.584, respectively, in controls [odds ratio (OR) = 1.40, 95% confidence interval (CI) = 1.05-1.86, P = 0.016 and OR=1.19, 95% CI = 1.01-1.41, P = 0.038, respectively]. The mean serum C3 level of carriers of the rs7951 T allele was significantly lower than that of non-carriers of the T allele in 87 SLE patients whose medical records were available (P = 0.0018).rs7951 T allele of the C3 gene was significantly associated with SLE, and decreased serum level of C3 seems to be correlated with this allele.

We examined the expression of ret proto-oncogene (proto-ret) in surgically resected human neuroblastomas. Slot blot RNA hybridization revealed that all 29 neuroblastomas examined expressed the proto-ret, the relative intensity of the hybridization ranging from 1 to 48. No correlation was found between the level of expression of proto-ret and the clinical stage. The level of expression was also not correlated with N-myc amplification, the patient's age or the histological type of the tumor. Based on the previous finding that proto-ret expression is very rarely detected in tumor cell lines other than those of neuroblastoma, proto-ret expression was suggested to be a characteristic of neuroblastomas, and possibly to be involved in the genesis of neuroblastomas.

We present a newly developed software called "QSNPlite" that comprehensively interprets the data of SSCP and sequencing analyses obtained from capillary array electrophoresis systems used in the quantitative characterization of SNPs. QSNPlite assists in the genotyping of individuals with SNPs and in estimating the allele frequencies of SNPs using pooled DNA. We show that this estimation is accurate (mean absolute error, 1.4%) by comparing the results of the pooled analysis using QSNPlite with the true frequencies based on the allele counting after performing individual genotypings. The QSNPlite program runs on Windows XP and can be used to determine the allele frequencies of SNPs among a large number of individuals, such as in association studies of disease-responsible genes using the candidate gene approach.