Kenshi Hayashi

Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction.

We report a rapid and sensitive method for the detection of base changes in given sequences of genomic DNA. This technique is based on the facts that specific regions of genomic sequences can be efficently labeled and amplified simultaneously by using labeled substrates in the polymerase chain reaction and that in nondenaturing polyacrylamide gels, the electrophoretic mobility of single-stranded nucleic acid depends not only on its size but also on its sequence. The process does not involve restriction enzyme digestion, blotting, or hybridization to probes. We found that most single base changes in up to 200-base fragments could be detected as mobility shifts. RAS oncogene activation was detected by this technique. We also show that the interspersed repetitive sequences of human, Alu repeats are highly polymorphic.

Detection of p53 Gene Mutations in Human Brain Tumors by Single-strand Conformation Polymorphism Analysis of Polymerase Chain Reaction Products

Single-strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP analysis) was used for detection of mutations of the p53 gene in surgical specimens of human brain tumors. Six of 45 brain tumors showed mobility shifts in the analyses. These six tumors also showed loss of a normal allele. The samples were examined further by direct sequencing. Results showed that four of them had single-base substitutions and the other two had deletions of one and eight base pairs. Five of the six mutations detected were clustered in highly conserved regions of the p53 gene. The frequency of p53 gene mutations in primary brain tumors examined was 9.8%. We also found two new polymorphic markers in the p53 gene, one in intron 7 and the other in an Alu repeat in exon 11. Both markers could be detected by SSCP analysis. Using these two markers, we found two cases of loss of heterozygosity in other brain tumor specimens. Results suggested that aberrations of the p53 gene were not correlated with the malignancy of some types of brain tumors such as anaplastic astrocytoma and glioblastoma, contrary to previous observations on colorectal cancers.

PCR-SSCP: a method for detection of mutations.

PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) analysis is one of the simplest and perhaps one of the most sensitive methods for detection of mutations based on PCR technology. The principles of PCR-SSCP, guidelines for experiments, and applications of this technique in various fields are reviewed.

Allele-specific polymerase chain reaction: a method for amplification and sequence determination of a single component among a mixture of sequence variants.

A new technique is described for amplifying individual alleles in a mixture of two or more alleles by the polymerase chain reaction (PCR) to determine their nucleotide sequence. This technique involves amplifying and separating target sequences by the PCR-mediated single-strand conformation polymorphism (PCR-SSCP) method, isolating each polymorphic DNA strand, and amplifying it by a second-stage PCR for its sequence determination. By this technique, the sequence of a minor constituent (approximately 3%) can be determined accurately.

MiRNA-196 Is Upregulated in Glioblastoma But Not in Anaplastic Astrocytoma and Has Prognostic Significance

Purpose: MicroRNAs (miRNA) are short noncoding RNAs that can play critical roles in diverse biological processes. They are implicated in tumorigenesis and function both as tumor suppressors and as oncogenes. The clinical significance of miRNA expression profiles in malignant gliomas remains unclear. Experimental Design: In this study, we examined the expression levels of 365 mature human miRNAs in 12 malignant gliomas, including 8 glioblastomas and 4 anaplastic astrocytomas, using TaqMan real-time quantitative PCR arrays. A validation study was done to corroborate a subset of the results, including expression levels of miR-196a, -196b, -21, and -15b, by analyzing 92 malignant gliomas by conventional real-time PCR. We modeled the relationship between the expression levels of these miRNAs and the survival rate of 39 glioblastoma patients by Kaplan-Meier method and multivariate analysis. Results: Expression profiles in glioblastomas and anaplastic astrocytomas suggested that 16 miRNAs were candidate markers associated with the malignant progression of gliomas. Among them, miR-196a showed the most significant difference (P = 0.0038), with miR-196b also having a high significance (P = 0.0371). Both miRNAs showed increased expression levels in glioblastomas relative to both anaplastic astrocytomas and normal brains in the validation study. Furthermore, patients with high miR-196 expression levels showed significantly poorer survival by the Kaplan-Meier method (P = 0.0073). Multivariate analysis showed that miR-196 expression levels were an independent predictor of overall survival in all 39 glioblastoma patients (P = 0.021; hazard ratio, 2.81). Conclusions: Our results suggest that miR-196 may play a role in the malignant progression of gliomas and may be a prognostic predictor in glioblastomas. Clin Cancer Res; 16(16); 4289–97. ©2010 AACR.

SSCP analysis of long DNA fragments in low pH gel

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. Hum Mutat 10:400–407, 1997.

Outside-to-inside signal through the membrane TNF-alpha induces E-selectin (CD62E) expression on activated human CD4+ T cells.

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.

Mutations in TSPAN12 cause autosomal-dominant familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Although mutations in three genes (LRP5, FZD4, and NDP) are known to cause FEVR, these account for only a fraction of FEVR cases. The proteins encoded by these FEVR genes form part of a signaling complex that activates the Norrin-beta-catenin signaling pathway. Recently, through a large-scale reverse genetic screen in mice, Junge and colleagues identified an additional member of this signaling complex, Tspan12. Here, we report that mutations in TSPAN12 also cause autosomal-dominant FEVR. We describe seven mutations identified in a cohort of 70 FEVR patients in whom we had already excluded the known FEVR genes. This study provides further evidence for the importance of the Norrin-beta-catenin signaling pathway in the development of the retinal vasculature and also indicates that more FEVR genes remain to be identified.

DNA sequence polymorphisms in Alu repeats

We have developed an efficient method for detection of sequence differences in genomic DNA based on a new principle (M. Orita et al., 1989, Genomics 5: 874-879). Using this method, we show here that approximately half the Alu repeats interspersed in the human genome are significantly polymorphic. Analysis of Alu repeat polymorphism should be useful in construction of a high-resolution map and also in identifying genotypes of individuals for clinical and other purposes because the repeats are ubiquitous and the technique for their detection is simple.

Prognostic value of the preserved expression of the E‐cadherin and catenin families of adhesion molecules and of β‐catenin mutations in synovial sarcoma

This study addresses the immunohistochemical expression of the E‐cadherin and catenin families and mutations of the β‐catenin gene detected by PCR–SSCP in synovial sarcoma. Immunohistochemical analysis was performed for 72 cases, with follow‐up data available on 62. The prognostic value of the expression of these proteins was evaluated. Reduced immunoreactivity for E‐cadherin and α‐catenin was significantly correlated with a poor survival rate (p=0.0040 and 0.0053, respectively). According to multivariate analysis, low AJC stage (stages I and II: p<0.0001), the preservation of α‐catenin expression (p=0.0001), and a low necrotic rate (<50%: p=0.0139) were independent favourable prognostic factors. Widespread aberrant staining of β‐catenin protein within cytoplasm and/or nuclei was observed in 28 cases (38.9%) and was significantly correlated with poor survival (p=0.0122). In addition, there was a trend towards a correlation between widespread aberrant staining of β‐catenin and the MIB‐1 labelling index (p=0.0535). Mutational analysis of exon 3 of the β‐catenin gene was performed for 49 cases. Nucleotide sequencing analysis revealed that four (8.2%) contained point mutations (three in codon 32, GAC to TAC; one in codon 37, TCT to TTT). Survival data were available for three out of four cases with β‐catenin mutations; two of these patients died within 1 year (died of disease at 6 and 11 months, respectively). These results suggest that E‐cadherin and α‐catenin undertake important roles as intercellular adhesion molecules; their preserved expression is associated with a better overall survival rate in synovial sarcoma and may have prognostic value. Abnormal levels of β‐catenin, with or without mutation, could contribute to the development and progression of synovial sarcoma, through increasing the proliferative activity of the tumour cells. Copyright