Etsuko Tsubota

Characterization and haplotype analysis of the polymorphic Y-STRs DYS443, DYS444 and DYS445 in a Japanese population.

Abstract From sequence database information we have newly identified three male-specific and polymorphic tetranucleotide STRs, DYS443 (GDB: 10807127), DYS444 (GDB: 10807128) and DYS445 (GDB: 10807129) on the Y chromosome. Analysis of 190 Japanese males revealed 6, 5 and 4 alleles in the DYS443, DYS444 and DYS445 systems, with calculated STR diversities of 0.68, 0.57 and 0.53, respectively. The cumulative haplotype diversity of the five Y-STRs DYS441, DYS442, DYS443, DYS444 and DYS445 was calculated to be 0.95 and therefore application of these STRs may yield very useful information for forensic individualization.

Identification and characterization of two novel human polymorphic STRs on the Y chromosome.

Abstract From sequence database information, we have identified two male-specific and polymorphic tetranucleotide STRs, DYS 441 (GDB:10013873) and DYS 442 (GDB: 10030304), on the Y chromosome. Analysis of 184 males allowed 7 and 5 alleles to be distinguished in the DYS 441 and DYS 442 systems, respectively, yielding 21 haplotypes. The gene diversities were 0.72 and 0.51, respectively and the haplotype diversity was 0.85.

A biochemical and genetic study on all non-synonymous single nucleotide polymorphisms of the gene encoding human deoxyribonuclease I potentially relevant to autoimmunity

A reduction of deoxyribonuclease I (DNase I) activity levels in the serum of patients with autoimmune diseases has been reported. The objectives of this study were to clarify genetic and biochemical aspects of 12 non-synonymous SNPs in the human gene (DNASE1), potentially giving rise to an alteration in the in vivo DNase I activity levels. Genotyping of all the non-synonymous SNPs was performed in healthy subjects of three ethnic groups including 15 populations using newly developed methods. Among them, only four SNPs, R-21S, Y95S, G105R, and Q222R were polymorphic in all or some populations; Asian group showed a relatively low genetic diversity of these SNPs. Furthermore, the distribution pattern of the common SNP Q222R was classified into three ethnic groups. The activity levels of the amino acid-substituted DNase I forms derived from SNPs R-21S, G105R, P132A, and P197S were significantly high compared with that of the wild-type; the polymorphic SNPs R-21S and G105R gave rise to a high activity-harboring DNase I isoform. On the other hand, activity levels from Q35H, R85G, V89M, C209Y, Q222R, and A224P were significantly low, but these SNPs, except Q222R, were not distributed in any of the populations. However, since these SNPs may produce potentially low levels of in vivo DNase I activity, a minor allele in each SNP will be served as a genetic risk factor for autoimmune diseases. These findings on non-synonymous SNPs in DNASE1 may provide a biochemical-genetic basis for the clarification of a possible relationship between DNase I and the diseases.

Allele frequencies and haplotypes for 28 Y-STRs in Ovambo population

Y-chromosomal 28 short tandem repeat (STR) loci were investigated in unrelated healthy individuals of the Ovambo population from Namibia (n=54). Sixteen Y-chromosome short tandem repeat (Y-STR) polymorphic loci (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS393, DYS391, DYS439, DYS635, DYS392, GATAH4, DYS437, DYS438, and DYS448) were analyzed using AmpFISTR Yfiler Polymerase Chain Reaction (PCR) Amplification Kit. DYS441-445 and DYS446, DYS447, DYS449, DYS450, DYS459a/b, DYS463 and DYS464a/b/c/d were investigated using a multiplex PCR system. Fifty-one haplotypes were identified in 54 Ovambos. The STR diversity values for Y-STRs loci ranged from 0.036 (DYS392) to 0.900 (DYS 385).

Multiplex single base extension method for simultaneous genotyping of non-synonymous SNP in the three human SOD genes

The superoxide dismutases (SOD) are a family of enzymes that function as the first line of antioxidant defense against highly reactive superoxide radicals. In SOD genes, a number of SNP have been identified and their associations with various diseases have been reported. In the present study, we applied a multiplex single base extension technique to genotype multiple non‐synonymous SNP in the SOD1, SOD2 and SOD3 genes simultaneously, and examined allele distributions in healthy Caucasian (German), Asian (Japanese) and African (Xhosa) populations. Of the ten SNP investigated, two (SOD2 Ala16Val, SOD3 Ala58Thr) were polymorphic in all three ethnic groups and the genotype distributions showed significant inter‐group differences. On the other hand, a small number of heterozygotes were observed for three SNP (SOD2 Ser10Ile, SOD3 Ala91Thr, SOD3 Arg231Gly) and no heterogeneity was observed for the remaining five (SOD1 Thr40Ile, SOD1 Asn87Ser, SOD2 Arg156Trp, SOD2 Gly76Arg, SOD2 Glu66Val). Analyses of associations between SOD genotypes and levels of plasma SOD activity demonstrated that SOD2 Ala16Val, a dimorphism leading to substitution in the mitochondrial targeting sequence of SOD2, significantly influences plasma SOD2 activity, and that SOD3 Arg231Gly, leading to substitution in the heparin‐binding domain of SOD3, significantly influences plasma total SOD activity.

Identification of the nucleotide substitution that generates the fourth polymorphic site in human deoxyribonuclease I (DNase I)

Abstract In addition to the three polymorphic sites responsible for protein polymorphism, a new polymorphic site has been identified in intron 7 of the human deoxyribonuclease I (DNase I) gene. Three phenotypes were observed on single-strand conformational polymorphism analysis of a 266-bp polymerase chain reaction-amplified fragment containing exon 7 and part of intron 7 of the human DNase I gene. DNA sequencing analysis demonstrated that a C-G substitution occurred at position 1978 in intron 7. This substitution was confirmed by restriction fragment length polymorphism analysis, since a new Msp1 site is created by the substitution. Population and family studies showed that the inheritance of the genotypes for DNase I C1978G polymorphism is controlled by two codominant alleles, tentatively designated DNASE1*1978C and *1978G. The gene frequencies in a Japanese population were significantly different from those in a Caucasian (German) population. The C1978G polymorphism is in linkage disequilibrium with the common DNase I protein phenotypes 1, 1–2, and 2.

Five age-dependently expressed genes in mouse brain revealed by the fluorescence differential display-PCR technique

We used a fluorescence differential display-PCR (FDD-PCR) technique to analyze the genes expressed in mouse brains collected at nine different developmental stages ranging from 3 days to 15 months after birth, and 5 age-dependently expressed genes were found. Age-dependent expression of each of these 5 genes was confirmed by quantitative real-time PCR analysis. Of the 5 genes, 4 (B1-B4) had high homology with the nucleotide sequences of cDNA clones of known mouse genes (myelin proteolipid protein, transferrin, embryo cDNA from the RIKEN full-length enriched library, and protein tyrosine phosphatase), and the rest (B5) with expressed sequence tags of an unknown gene. Sequencing analysis of the full-length cDNA constructed based on the B5 sequence demonstrated that the gene product of B5 was identical to G-substrate, a specific substrate for cGMP-dependent protein kinase. The expression patterns of known genes obtained in our study may provide a further opportunity to investigate the biological and physiological roles of the proteins they encode.

Cytochrome P450 2J2*7 polymorphisms in Japanese, Mongolians and Ovambos.

Human cytochrome P450 2J2 (CYP2J2) is abundant in cardiovascular tissue and active in the metabolism of arachidonic acid to eicosanoids that have potent vasodilatory properties. Variability of the CYP2J2 gene is highly constrained except for its proximal promoter: there is a relatively common and functionally relevant single nucleotide polymorphism, indicated by −50G > T polymorphism (CYP2J2*7). Although genetic variation is known among ethnic groups, data for allele frequency are limited to a few Caucasian, Asian, and one African populations. In the present study, genotype distribution of CYP2J2*7 polymorphisms was investigated using polymerase chain reaction and restriction fragment length polymorphism assay in Japanese (n = 338), Mongolian (n = 118), and Ovambo (n = 186) populations and the findings compared with other populations. The mutant (CYP2J2*7) frequencies in the Japanese, Mongolians, and Ovambos were 0.0621, 0.0339, and 0.0672, respectively. Except for the Taiwanese, a general uniformity in the polymorphism in the Asian populations was observed. The mutation frequency of Ovambos was relatively lower than that of the African‐American population. This study is the first to investigate the distribution of the CYP2J2*7 gene polymorphisms in Japanese, Mongolians, and Ovambos. These data will be informative and facilitate genetic association studies, in Asian and African populations for CYP2J2‐related diseases such as cardiovascular disorders. Copyright

Deoxyribonuclease I Phenotyping from Saliva Stains

Good typing results were obtained using a newly developed method for extraction and purification of deoxyribonuclease I (DNase I) from saliva stains. Previously, DNase I phenotyping from saliva stains has been unsuccessful because of low enzyme activity and heavy contamination. Salivary DNase I was extracted from stains using phosphate buffer containing Nonidet P-40. Extracts were purified using Phenyl Sepharose CL-4B gel. Electrophoresis was performed, and DNase I was successfully phenotyped. All of the DNase I phenotypes, which were obtained from saliva stains using this new method, were identical to the phenotypes determined from urine samples. Moreover, DNase I was correctly phenotyped from saliva stains that had been stored for over three months at room temperature or at 37 degrees C. These results suggest that DNase I polymorphisms provide valuable information for forensic characterization of saliva stains.

Time course of housekeeping gene expression changes in diffuse alveolar damage induced by hyperoxia exposure in mice

We have found diffuse alveolar damage (DAD) has taken place in some patients under mechanical ventilation with high-inspired oxygen concentrations. To clarify the molecular pathophysiology of this, the time course of gene expression changes induced by hyperoxia exposure in mouse lungs was examined using real-time quantitative polymerase chain reaction (real-time qPCR). Our raw data and those normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed that: (1) there is a decrease in levels of mRNAs for surfactant-associated protein C (SFTPC), cytochrome P450, 2F2 (CYP2F2), Claudin 1 (CLDN1), membrane-associated zonula occludens protein-1 (ZO-1), lysozyme (LYZS), and this suggests alveolar dysfunction and a disruption of the immune system, (2) we confirmed apoptotic conditions, such as significant up-regulations of mRNA levels in Myc and Galectin-3, and (3) hyperoxic conditions probably yielded reactive oxygen species (ROS), which resulted in a malignant cycle of ROS production by Myc overexpression [Shimada I, Matsui K, Brinkmann B, Hohoff C, Hiraga K, Tabuchi Y, et al. Novel transcript profiling of diffuse alveolar damage induced by hyperoxia exposure in mice: normalization by glyceraldehyde 3-phosphate dehydrogenase. Int J Legal Med 2008;122:373-83]. In this experiment, GAPDH was up-regulated when hyperoxia exposure was continued. Therefore, we reexamined our data and found that: (1) mRNA levels of other housekeeping genes, including beta(2)-microglobulin (beta2M), ribosomal protein: large P2 (RPLP2), and importin 8 (IPO8) altered to a lesser extent, (2) mRNA levels of beta2M and IPO8 were down-regulated when hyperoxia exposure was continued, and (3) our previous work was validated by normalization with these three housekeeping genes.