Shohei Yamamura, Shouki Yatsushiro, Yuka Nagasaki, Kaori Abe, Yasuo Shinohara, Eiichi Tamiya, Yoshinobu Baba and Masatoshi Kataoka : Accurate detection of carcinoma cells by use of a cell microarray chip., PLoS ONE, Vol.7, No.3, e32370, 2012.
(要約)
Accurate detection and analysis of circulating tumor cells plays an important role in the diagnosis and treatment of metastatic cancer treatment. A cell microarray chip was used to detect spiked carcinoma cells among leukocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene; and the formation of monolayers of leukocytes in the microchambers was observed. Cultured human T lymphoblastoid leukemia (CCRF-CEM) cells were used to examine the potential of the cell microarray chip for the detection of spiked carcinoma cells. A T lymphoblastoid leukemia suspension was dispersed on the chip surface, followed by 15 min standing to allow the leukocytes to settle down into the microchambers. Approximately 29 leukocytes were found in each microchamber when about 600,000 leukocytes in total were dispersed onto a cell microarray chip. Similarly, when leukocytes isolated from human whole blood were used, approximately 89 leukocytes entered each microchamber when about 1,800,000 leukocytes in total were placed onto the cell microarray chip. After washing the chip surface, PE-labeled anti-cytokeratin monoclonal antibody and APC-labeled anti-CD326 (EpCAM) monoclonal antibody solution were dispersed onto the chip surface and allowed to react for 15 min; and then a microarray scanner was employed to detect any fluorescence-positive cells within 20 min. In the experiments using spiked carcinoma cells (NCI-H1650, 0.01 to 0.0001%), accurate detection of carcinoma cells was achieved with PE-labeled anti-cytokeratin monoclonal antibody. Furthermore, verification of carcinoma cells in the microchambers was performed by double staining with the above monoclonal antibodies. The potential application of the cell microarray chip for the detection of CTCs was shown, thus demonstrating accurate detection by double staining for cytokeratin and EpCAM at the single carcinoma cell level.
We describe the potential of microchip electrophoresis with a Hitachi SV1210, which can be used to evaluate the integrity of total RNA, for the analysis of mRNA expression. The ribonuclease (RNase) protection assay was performed by using microchip electrophoresis with cyanine 5 (Cy5) labeled 248-base antisense RNA probe (riboprobe) encoding adipose-type fatty acid binding protein (A-FABP) as the riboprobe. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary strand RNA. Results were obtained in 120 s, and the same amount of Cy5-labeled antisense riboprobe as used in the conventional method can be used. Furthermore, 8 times more sensitive detection of mRNA by microchip electrophoresis could be obtained. An obvious increase in the mRNA expression of A-FABP, which is known as a differentiation marker of adipocytes, occurred during the adipocyte differentiation of 3T3-L1 cells. These results clearly indicate the potential of microchip electrophoresis for the analysis of mRNA expression in cells.
Shouki Yatsushiro, Yuka Nagasaki, Shohei Yamamura, Yasuo Shinohara, Yoshinobu Baba and Masatoshi Kataoka : Highly sensitive DNA detection with a combination of 2 DNA-intercalating dyes for microchip electrophoresis., Journal of Pharmaceutical and Biomedical Analysis, Vol.55, No.1, 202-205, 2010.
(要約)
A highly sensitive DNA detection method using a combination of ethidium bromide (EtBr) and SYBR Green II (SG II) for microchip electrophoresis was developed. By use of the combination of these intercalating DNA-staining dyes for microchip electrophoresis with Hitachi SV1100 system, the fluorescence intensities corresponding to DNA fragments were obviously increased over those obtained with EtBr only, with accuracy of DNA sizing and quantification. The detection limit with EtBr and the combination of EtBr and SG II were 0.048 and 0.007ng/μl, respectively. This highly sensitive DNA detection just using the combination of these dyes offering high resolution in a short time will be useful for various biological analyses.
(キーワード)
DNA / DNA Fragmentation / Electrophoresis, Microchip / Ethidium / Fluorescent Dyes / Intercalating Agents / Limit of Detection / Microchemistry / Molecular Weight / Organic Chemicals / Plasmids / Reproducibility of Results / Spectrometry, Fluorescence
Shouki Yatsushiro, Shohei Yamamura, Yuka Nagasaki, Yasuo Shinohara, Eiichi Tamiya, Toshihiro Horii, Yoshinobu Baba and Masatoshi Kataoka : Rapid and highly sensitive detection of malaria-infected erythrocytes using a cell microarray chip., PLoS ONE, Vol.5, No.10, 2010.
(要約)
A cell microarray chip was used to detect for malaria-infected erythrocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene, and the formation of monolayers of erythrocytes in the microchambers was observed. Cultured Plasmodium falciparum strain 3D7 was used to examine the potential of the cell microarray chip for malaria diagnosis. An erythrocyte suspension in a nuclear staining dye, SYTO 59, was dispersed on the chip surface, followed by 10 min standing to allow the erythrocytes to settle down into the microchambers. About 130 erythrocytes were accommodated in each microchamber, there being over 2,700,000 erythrocytes in total on a chip. A microarray scanner was employed to detect any fluorescence-positive erythrocytes within 5 min, and 0.0001% parasitemia could be detected. To examine the contamination by leukocytes of purified erythrocytes from human blood, 20 µl of whole blood was mixed with 10 ml of RPMI 1640, and the mixture was passed through a leukocyte isolation filter. The eluted portion was centrifuged at 1,000×g for 2 min, and the pellet was dispersed in 1.0 ml of medium. SYTO 59 was added to the erythrocyte suspension, followed by analysis on a cell microarray chip. Similar accommodation of cells in the microchambers was observed. The number of contaminating leukocytes was less than 1 on a cell microarray chip.
Yuka Nagasaki, Maki Moritani, Toshihito Tanahashi, Dai Osabe, Kyoko Nomura, Yuka Fujita, Parvaneh Keshavarz, Kiyoshi Kunika, Naoto Nakamura, Toshikazu Yoshikawa, Eiichiro Ichiishi, Hiroshi Shiota, Natsuo Yasui, Hiroshi Inoue and Mitsuo Itakura : Lack of association of genetic variation in chromosome region 15q14-22.1 with type 2 diabetes in a Japanese population., BMC Medical Genetics, Vol.9, 22, 2008.
(要約)
BACKGROUND: Chromosome 15q14-22.1 has been linked to type 2 diabetes (T2D) and its related traits in Japanese and other populations. The presence of T2D disease susceptibility variant(s) was assessed in the 21.8 Mb region between D15S118 and D15S117 in a Japanese population using a region-wide case-control association test. METHODS: A two-stage association test was performed using Japanese subjects: The discovery panel (Stage 1) used 372 cases and 360 controls, while an independent replication panel (Stage 2) used 532 cases and 530 controls. A total of 1,317 evenly-spaced, common SNP markers with minor allele frequencies > 0.10 were typed for each stage. Captured genetic variation was examined in HapMap JPT SNPs, and a haplotype-based association test was performed. RESULTS: SNP2140 (rs2412747) (C/T) in intron 33 of the ubiquitin protein ligase E3 component n-recognin 1 (UBR1) gene was selected as a landmark SNP based on repeated significant associations in Stage 1 and Stage 2. However, the marginal p value (p = 0.0043 in the allelic test, OR = 1.26, 95% CI = 1.07-1.48 for combined samples) was weak in a single locus or haplotype-based association test. We failed to find any significant SNPs after correcting for multiple testing. CONCLUSION: The two-stage association test did not reveal a strong association between T2D and any common variants on chromosome 15q14-22.1 in 1,794 Japanese subjects. A further association test with a larger sample size and denser SNP markers is required to confirm these observations.
Toshihito Tanahashi, Keiko Shinohara, Parvaneh Keshavarz, Yuka Nagasaki, Katsuyuki Miyawaki, Kiyoshi Kunika, Maki Moritani, Naoto Nakamura, Toshikazu Yoshikawa, Hiroshi Shiota, Hiroshi Inoue and Mitsuo Itakura : The association of genetic variants in Krüppel-like factor 11 and Type 2 diabetes in the Japanese population., Diabetic Medicine, Vol.25, No.1, 19-26, 2008.
(要約)
Krüppel-like factor 11 (KLF11) is a transcriptional factor of the zinc finger domain family that regulates the expression of insulin. In North European populations, its common functional variant Q62R (rs35927125) is a strong genetic factor for Type 2 diabetes (P = 0.00033, odds ratio for G allele = 1.29, 95% CI 1.12-1.49). We examined the contribution of KLF11 variants to the susceptibility to Type 2 diabetes in a Japanese population. By re-sequencing Japanese individuals (n = 24, partly 96), we screened all four exons, exon/intron boundaries and flanking regions of KLF11. Verified single nucleotide polymorphisms (SNPs) were genotyped in 731 initial samples (369 control and 362 case subjects). Subsequently, we tested for association in 1087 samples (524 control and 563 case subjects), which were collected in different districts of Japan from the initial samples. We identified eight variants, including a novel A/C variant on intron 3, but no mis-sense mutations. In an association study, we failed to find any significant result of SNPs (minor allele frequency 8.2-46.2%) after correcting for multiple testing. Similarly, no haplotypes were associated with Type 2 diabetes. It is notable that the G allele in rs35927125 was completely absent in 1818 Japanese individuals. Genetic variants in KLF11 are unlikely to have a major effect of Type 2 diabetes in the Japanese population, although they were significantly associated in North European populations. These observations might help to determine the role of KLF11 variants in Type 2 diabetes in different populations.
(キーワード)
Adult / 分散分析 (analysis of variance) / Asian Continental Ancestry Group / Cell Cycle Proteins / Diabetes Mellitus, Type 2 / 女性 (female) / Gene Frequency / Genetic Predisposition to Disease / Genotype / Humans / インスリン (insulin) / 日本 (Japan) / Linkage Disequilibrium / 男性 (male) / Middle Aged / Repressor Proteins
Yukiko Yamashita, Hiroshi Inoue, Keshavarz Parvaneh, Katsuyuki Miyawaki, Yuka Nagasaki, Maki Moritani, Kiyoshi Kunika, Naoto Nakamura, Toshikazu Yoshikawa, Natsuo Yasui, Hiroshi Shiota, Toshihito Tanahashi and Mitsuo Itakura : SNPs in the KCNJ11-ABCC8 gene locus are associated with type 2 diabetes and blood pressure levels in the Japanese population., Journal of Human Genetics, Vol.52, No.10, 781-793, 2007.
(要約)
Many genetic association studies support a contribution of genetic variants in the KCNJ11-ABCC8 gene locus to type 2 diabetes (T2D) susceptibility in Caucasians. In non-Caucasian populations, however, there have been only a few association studies, and discordant results were obtained. Herein, we selected a total of 31 SNPs covering a 211.3-kb region of the KCNJ11-ABCC8 locus, characterized the patterns of linkage disequilibrium (LD) and haplotype structure, and performed a case-control association study in a Japanese population consisting of 909 T2D patients and 893 control subjects. We found significant associations between eight SNPs, including the KCNJ11 E23K and ABCC8 S1369A variants, and T2D. These disease-associated SNPs were genetically indistinguishable because of the presence of strong LD, as found previously in Caucasians. For the KCNJ11 E23K variant, the most significant association was obtained under a dominant genetic model (OR 1.32, 95% CI 1.09-1.60, P = 0.004). A meta-analysis of East Asian studies, comprising a total of 3,357 T2D patients (77.4% Japanese) and 2,836 control subjects (77.8% Japanese), confirmed the significant role of the KCNJ11 E23K variant in T2D susceptibility. Furthermore, we found evidence suggesting that the KCNJ11 E23K genotype is independently associated with higher blood-pressure levels.
(キーワード)
ATP-Binding Cassette Transporters / Aged / Asian Continental Ancestry Group / 血圧 (blood pressure) / Diabetes Mellitus, Type 2 / 女性 (female) / Humans / 日本 (Japan) / 男性 (male) / Middle Aged / Polymorphism, Single Nucleotide / Potassium Channels / Potassium Channels, Inwardly Rectifying / Receptors, Drug
Dai Osabe, Toshihito Tanahashi, Kyoko Nomura, Shuichi Shinohara, Naoto Nakamura, Toshikazu Yoshikawa, Hiroshi Shiota, Parvaneh Keshavarz, Yuka Nagasaki, Kiyoshi Kunika, Maki Moritani, Hiroshi Inoue and Mitsuo Itakura : Evaluation of sample size effect on the identification of haplotype blocks., BMC Bioinformatics, Vol.8, 200, 2007.
(要約)
Genome-wide maps of linkage disequilibrium (LD) and haplotypes have been created for different populations. Substantial sharing of the boundaries and haplotypes among populations was observed, but haplotype variations have also been reported across populations. Conflicting observations on the extent and distribution of haplotypes require careful examination. The mechanisms that shape haplotypes have not been fully explored, although the effect of sample size has been implicated. We present a close examination of the effect of sample size on haplotype blocks using an original computational simulation. A region spanning 19.31 Mb on chromosome 20q was genotyped for 1,147 SNPs in 725 Japanese subjects. One region of 445 kb exhibiting a single strong LD value (average |D'|; 0.94) was selected for the analysis of sample size effect on haplotype structure. Three different block definitions (recombination-based, LD-based, and diversity-based) were exploited to create simulations for block identification with theta value from real genotyping data. As a result, it was quite difficult to estimate a haplotype block for data with less than 200 samples. Attainment of a reliable haplotype structure with 50 samples was not possible, although the simulation was repeated 10,000 times. These analyses underscored the difficulties of estimating haplotype blocks. To acquire a reliable result, it would be necessary to increase sample size more than 725 and to repeat the simulation 3,000 times. Even in one genomic region showing a high LD value, the haplotype block might be fragile. We emphasize the importance of applying careful confidence measures when using the estimated haplotype structure in biomedical research.
Maki Moritani, Kyoko Nomura, Toshihito Tanahashi, Dai Osabe, Yuka Fujita, Shinohara Syuichi, Yuka Nagasaki, Parvaneh Keshavarz, Eiji Kudo, Naoto Nakamura, Toshikazu Yoshikawa, Eiichiro Ichiichi, Yoichiro Takata, Natsuo Yasui, Hiroshi Shiota, Kiyoshi Kunika, Hiroshi Inoue and Mitsuo Itakura : Genetic association of single nucleotide polymorphisms in endonuclease G-like 1 gene with type 2 diabetes in a Japanese population., Diabetologia, Vol.50, No.6, 1218-1227, 2007.
(要約)
In order to identify type 2 diabetes disease susceptibility gene(s) in a Japanese population, we applied a region-wide case-control association test to the 20.4 Mb region between D3S1293 and D3S2319 on chromosome 3p24.3-22.1, supported by linkage to type 2 diabetes and its related traits in Japanese and multiple populations. We performed a two-stage association test using 1,762 Japanese persons with 485 gene-centric, evenly spaced, common single nucleotide polymorphism (SNP) markers with minor allele frequency >0.1. For mouse studies, total RNA was extracted from various organs of BKS.Cg-+Lepr(db)/+Lepr(db) and control mice, and from MIN6, NIH3T3 and C2C12 cell lines. We detected a landmark SNP375 (A/G) (rs2051211, p = 0.000046, odds ratio = 1.33, 95% CI 1.16-1.53) in intron 5 of the endonuclease G-like 1 (ENDOGL1) gene. Systematic dense SNPs approach identified a susceptibility linkage disequilibrium (LD) block of 116.5 kb by |D'|, an LD units map and a critical region of 2.1 kb by r (2) in ENDOGL1. A haplotype-based association test showed that an at-risk haplotype is associated with disease status (p = 0.00001). The expression of ENDOGL1 was rather ubiquitous with relatively abundant expression in the brain and also in a pancreatic islet beta cell line. Mouse Endogl1 expression increased in pancreatic islets of hyperglycaemic BKS.Cg-+Lepr(db)/+Lepr(db) mice compared with that in control mice. Based on the population genetics, fine mapping of LD block and haplotype analysis, we conclude that ENDOGL1 is a candidate disease-susceptibility gene for type 2 diabetes in a Japanese population. Further analysis in a larger sample size is required to substantiate this conclusion.
(キーワード)
Adult / Aged / Body Mass Index / Case-Control Studies / Diabetes Mellitus, Type 2 / Endodeoxyribonucleases / Endonucleases / Female / Genetic Predisposition to Disease / Hemoglobin A, Glycosylated / Humans / Japan / Male / Middle Aged / Polymorphism, Single Nucleotide / Reference Values
Toshihito Tanahashi, Dai Osabe, Kyoko Nomura, Syuichi Shinohara, Hitoshi Kato, Eiichiro Ichiishi, Naoto Nakamura, Toshikazu Yoshikawa, Yoichiro Takata, Tatsuro Miyamoto, Hiroshi Shiota, Parvaneh Keshavarz, Yuka Nagasaki, Kiyoshi Kunika, Maki Moritani, Hiroshi Inoue and Mitsuo Itakura : Association study on chromosome 20q11.21-13.13 locus and its contribution to type 2 diabetes susceptibility in Japanese., Human Genetics, Vol.120, No.4, 527-542, 2006.
(要約)
Several linkage studies have predicted that human chromosome 20q is closely related to type 2 diabetes, but there is no clear evidence that certain variant(s) or gene(s) have strong effects on the disease within this region. To examine disease susceptibility variant in Japanese, verified SNPs from the databases, with a minor allele frequency larger than 0.15, were selected at 10-kb intervals across a 19.31-Mb region (20q11.21-13.13), which contained 291 genes, including hepatocyte nuclear factor 4alpha (HNF4alpha). As a result, a total of 1,147 SNPs were genotyped with TaqMan assay using 1,818 Japanese samples. By searching for HNF4alpha as a representative disease-susceptible gene, no variants of HNF4alpha were strongly associated with disease. To identify other genetic variant related with disease, we designed an extensive two-stage association study (725 first and 1,093 second test samples). Although SNP1146 (rs220076) was selected as a landmark within the 19.31 Mb region, the magnitude of the nominal P value (P = 0.0023) was rather weak. Subsequently, a haplotype-based association study showed that two common haplotypes were weakly associated with disease. All of these tests resulted in non-significance after adjusting for Bonferroni's correction and the false discovery rate to control for the impact of multiple testing. Contrary to the initial expectations, we could not conclude that certain SNPs had a major effect on this promising locus within the framework presented here. As a way to extend our observations, we emphasize the importance of a subsequent association study including replication and/or meta-analysis in multiple populations.
(キーワード)
Aged / Asian Continental Ancestry Group / Chromosome Mapping / Chromosomes, Human, Pair 20 / Diabetes Mellitus, Type 2 / Female / Gene Frequency / Genetic Predisposition to Disease / Genotype / Haplotypes / Hepatocyte Nuclear Factor 4 / Humans / 日本 (Japan) / Linkage Disequilibrium / Lod Score / Male / Middle Aged / Polymorphism, Single Nucleotide
Maki Moritani, Katsuhiko Togawa, Hiroshi Yaguchi, Yuka Fujita, Yuka Nagasaki, Hiroshi Inoue, Naoyuki Kamatani and Mitsuo Itakura : Identification of diabetes susceptibility loci in db mice by combined quantitative trait loci analysis and haplotype mapping., Genomics, Vol.88, No.6, 719-730, 2006.
(要約)
To identify the disease-susceptibility genes of type 2 diabetes, we performed quantitative trait loci (QTL) analysis in F(2) populations generated from a BKS.Cg-m+/+Lepr(db) and C3H/HeJ intercross, taking advantage of genetically determined obesity and diabetes traits associated with the db gene. A genome-wide scan in the F(2) populations divided by sex and db genotypes identified 14 QTLs in total and 3 major QTLs on chromosome (Chr) 3 (LOD 5.78) for fat pad weight, Chr 15 (LOD 6.64) for body weight, and Chr 16 (LOD 8.15) for blood glucose concentrations. A linear-model-based genome scan using interactive covariates allowed us to consider sex- or sex-by db-specific effects of each locus. For the most significant QTL on Chr 16, the high-resolution haplotype comparison between BKS and C3H strains reduced the critical QTL interval from 20 to 4.6 Mb by excluding shared haplotype regions and identified 11 nonsynonymous single-nucleotide polymorphisms in six candidate genes.
Yuka Nagasaki, S Yatsushiro, S. Yamamura, Kaori Abe, Yasuo Shinohara and Masatoshi Kataoka : High sensitive DNA detection with the combination of two kinds of fluorescent dyes by microchip electrophoresis, Pacifichem 2010, Honolulu, Dec. 2010.