Masafumi Ohtsubo

Antisense oligonucleotide of WAF1 gene prevents EGF-induced cell-cycle arrest in A431 cells

A431 cells hyperproduce EGF receptors and possess inactive p53 proteins. It has been suggested that a cyclin-dependent kinase (CDK) inhibitor p21/WAF1 plays a crucial role in the EGF-induced cell-cycle arrest of A431 cells. Here, we investigated the role of WAF1 gene transcription in the EGF-induced cell-cycle arrest by transfecting the 18-mer antisense oligonucleotide which corresponds to the 5′ region of WAF1 gene (AS/WAF1). When A431 cells were treated with EGF, a cascade of responses were observed, including immediate hyperphosphorylation of EGF receptor on tyrosine residues, accumulation of WAF1 mRNA and p21/WAF1 protein, dephosphorylation of RB protein which is a substrate of CDK-cyclin, and cell-cycle arrest. In the presence of AS/WAF1, EGF induced the tyrosine-phosphorylation of EGF receptor, but WAF1 mRNA was reduced to a half; accumulation of p21/WAF1 protein and its downstream responses were no longer observed; A431 cells grew continuously. Thus, the transfection of antisense efficiently prevented A431 cells from the EGF-induced arrest. These observations suggest that p21/WAF1 protein is a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells.

Elevated Transcription Factor Specificity Protein 1 in Autistic Brains Alters the Expression of Autism Candidate Genes

BACKGROUND Profound changes in gene expression can result from abnormalities in the concentrations of sequence-specific transcription factors like specificity protein 1 (Sp1). Specificity protein 1 binding sites have been reported in the promoter regions of several genes implicated in autism. We hypothesize that dysfunction of Sp1 could affect the expression of multiple autism candidate genes, contributing to the heterogeneity of autism. METHODS We assessed any alterations in the expression of Sp1 and that of autism candidate genes in the postmortem brain (anterior cingulate gyrus [ACG], motor cortex, and thalamus) of autism patients (n = 8) compared with healthy control subjects (n = 13). Alterations in the expression of candidate genes upon Sp1/DNA binding inhibition with mithramycin and Sp1 silencing by RNAi were studied in SK-N-SH neuronal cells. RESULTS We observed elevated expression of Sp1 in ACG of autism patients (p = .010). We also observed altered expression of several autism candidate genes. GABRB3, RELN, and HTR2A showed reduced expression, whereas CD38, ITGB3, MAOA, MECP2, OXTR, and PTEN showed elevated expression in autism. In SK-N-SH cells, OXTR, PTEN, and RELN showed reduced expression upon Sp1/DNA binding inhibition and Sp1 silencing. The RNA integrity number was not available for any of the samples. CONCLUSIONS Transcription factor Sp1 is dysfunctional in the ACG of autistic brain. Consequently, the expression of potential autism candidate genes regulated by Sp1, especially OXTR and PTEN, could be affected. The diverse downstream pathways mediated by the Sp1-regulated genes, along with the environmental and intracellular signal-related regulation of Sp1, could explain the complex phenotypes associated with autism.

Identification of 11 novel mutations in USH2A among Japanese patients with Usher syndrome type 2

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A, which accounts for 74–90% of USH2 cases. This is the first study reporting the results of scanning for USH2A mutations in Japanese patients with USH2. In 8 of 10 unrelated patients, we identified 14 different mutations. Of these mutations, 11 were novel. Although the mutation spectrum that we identified differed from that for Caucasians, the incidence of mutations in USH2A was 80% for all patients tested, which is consistent with previous findings. Further, c.8559‐2A>G was identified in four patients and accounted for 26.7% of mutated alleles; it is thus a frequent mutation in Japanese patients. Hence, mutation screening for c.8559‐2A>G in USH2A may prove very effective for the early diagnosis of USH2.

YPEL5 protein of the YPEL gene family is involved in the cell cycle progression by interacting with two distinct proteins RanBPM and RanBP10

YPEL5 is a member of the YPEL gene family that is highly conserved in the eukaryotic species and apparently involved in a certain cell division-related function. In this study, we examined the functional and phylogenetic aspects of YPEL5 protein in more detail. During cell cycle, YPEL5 protein was detected at different subcellular localizations; at interphase, it was located in the nucleus and centrosome, then it changed location sequentially to spindle poles, mitotic spindle, and spindle midzone during mitosis, and finally transferred to midbody at cytokinesis. Knockdown of YPEL5 function by siRNA or anti-sense morpholino oligonucleotide inhibited the growth of cultured COS-7 cells and early development of medaka fish embryos, indicating its involvement in cell cycle progression. Interestingly, RanBPM (Ran Binding Protein in the Microtubule organizing center, encoded by RANBP9) was identified as a YPEL5-binding protein by yeast two-hybrid method. A paralog of RanBPM, namely RanBP10 (encoded by RANBP10), was found to be another YPEL5-binding protein, and these two protein genes are highly conserved each other. Comparative genomic analysis allowed us to define a new gene family consisting of RanBPM and RanBP10, named Scorpin, providing a basis to better understand how they interact with YPEL5.

Mutation analysis of the MYO7A and CDH23 genes in Japanese patients with Usher syndrome type 1

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 1 (USH1), the second common type of USH, is frequently caused by MYO7A and CDH23 mutations, accounting for 70–80% of the cases among various ethnicities, including Caucasians, Africans and Asians. However, there have been no reports of mutation analysis for any responsible genes for USH1 in Japanese patients. This study describes the first mutation analysis of MYO7A and CDH23 in Japanese USH1 patients. Five mutations (three in MYO7A and two in CDH23) were identified in four of five unrelated patients. Of these mutations, two were novel. One of them, p.Tyr1942SerfsX23 in CDH23, was a large deletion causing the loss of 3 exons. This is the first large deletion to be found in CDH23. The incidence of the MYO7A and CDH23 mutations in the study population was 80%, which is consistent with previous findings. Therefore, mutation screening for these genes is expected to be a highly sensitive method for diagnosing USH1 among the Japanese.

Oligomerization of Optineurin and Its Oxidative Stress- or E50K Mutation-Driven Covalent Cross-Linking: Possible Relationship with Glaucoma Pathology

The optineurin gene, OPTN, is one of the causative genes of primary open-angle glaucoma. Although oligomerization of optineurin in cultured cells was previously observed by gel filtration analysis and blue native gel electrophoresis (BNE), little is known about the characteristics of optineurin oligomers. Here, we aimed to analyze the oligomeric state of optineurin and factors affecting oligomerization, such as environmental stimuli or mutations in OPTN. Using BNE or immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we demonstrated that both endogenous and transfected optineurin exist as oligomers, rather than monomers, in NIH3T3 cells. We also applied an in situ proximity ligation assay to visualize the self-interaction of optineurin in fixed HeLaS3 cells and found that the optineurin oligomers were localized diffusely in the cytoplasm. Optineurin oligomers were usually detected as a single band of a size equal to that of the optineurin monomer upon SDS-PAGE, while an additional protein band of a larger size was observed when cells were treated with H2O2. We showed that larger protein complex is optineurin oligomers by immunoprecipitation and termed it covalent optineurin oligomers. In cells expressing OPTN bearing the most common glaucoma-associated mutation, E50K, covalent oligomers were formed even without H2O2 stimulation. Antioxidants inhibited the formation of E50K-induced covalent oligomers to various degrees. A series of truncated constructs of OPTN was used to reveal that covalent oligomers may be optineurin trimers and that the ubiquitin-binding domain is essential for formation of these trimers. Our results indicated that optineurin trimers may be the basic unit of these oligomers. The oligomeric state can be affected by many factors that induce covalent bonds, such as H2O2 or E50K, as demonstrated here; this provides novel insights into the pathogenicity of E50K. Furthermore, regulation of the oligomeric state should be studied in the future.

The KMDB/MutationView: a mutation database for human disease genes

The KMDB/MutationView is a graphical database of mutations in human disease-causing genes and its current version consists of nine category-based sub-databases including diseases of eye, heart, ear, brain, cancer, syndrome, autoimmunity, muscle and blood. The KMDB/MutationView stores mutation data of 97 genes involved in 87 different disease and is accessible through http://mutview.dmb.med. keio.ac.jp.

Novel USH2A mutations in Japanese Usher syndrome type 2 patients: marked differences in the mutation spectrum between the Japanese and other populations

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A. In a recent mutation screening of USH2A in Japanese USH2 patients, we identified 11 novel mutations in 10 patients and found the possible frequent mutation c.8559-2A>G in 4 of 10 patients. To obtain a more precise mutation spectrum, we analyzed further nine Japanese patients in this study. We identified nine mutations, of which eight were novel. This result indicates that the mutation spectrum for USH2A among Japanese patients largely differs from Caucasian, Jewish and Palestinian patients. Meanwhile, we did not find the c.8559-2A>G in this study. Haplotype analysis of the c.8559-2G (mutated) alleles using 23 single nucleotide polymorphisms surrounding the mutation revealed an identical haplotype pattern of at least 635 kb in length, strongly suggesting that the mutation originated from a common ancestor. The fact that all patients carrying c.8559-2A>G came from western Japan suggests that the mutation is mainly distributed in that area; indeed, most of the patients involved in this study came from eastern Japan, which contributed to the absence of c.8559-2A>G.

MutationView/KMcancerDB: A database for cancer gene mutations

It is known that cancers are caused by accumulated mutations in various genes and consequent functional alterations of proteins that are important for maintenance of normal cellular functions. The changes in nucleotide sequences and expression patterns of cancer‐related genes are being extensively studied to better understand the mechanisms of tumorigenesis and to develop methods for DNA protein diagnosis and drug discovery. At present, a number of computer databases for molecular information on cancer‐related genes are available publicly through the internet. These databases deal with familial cancer and sporadic cancer at the levels of germline mutation or somatic mutation, genomic or chromosomal abnormalities, and changes in the expression levels of relevant genes. Previously, we constructed a human gene mutation database named MutationView (http://mutview.dmb.med.keio.ac.jp/) and have accumulated mutation data for ∼300 genes that are involved mainly in monogenic diseases. Forty‐two genes are cancer‐related and therefore a separate cancer database named KMcancerDB was constructed. MutationView/KMcancerDB utilizes a graphic display function for both queries and search results much more often than other existing databases, making the system quite user friendly. MutationView/KMcancerDB provides a highly sophisticated search function for all genes through a single internet URL. In the present paper, we briefly review various useful databases for cancer‐related genes, and describe MutationView/KMcancerDB in more detail. (Cancer Sci 2007; 98: 259–267)

Three novel mutations of the PAX6 gene in Japanese aniridia patients

AbstractMutations in the PAX6 gene of Japanese aniridia patients were analyzed. Four types of mutations including one known (474delC) and three novel (786_787ins10, 678_688del11 and 572_575delAATCins14) were found in six patients from four families. A patient with the mutation 572_575delAATCins14 also manifested VATER association. This is the first case of aniridia accompanied by VATER association. All of mutations found in this study are frameshift type, resulting in premature termination of translation. The database for PAX6 gene mutation has been made using a graphical data display system MutationView (http://mutview.dmb.med.keio.ac.jp/).