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Human arsenic (+3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite, and intronic single-nucleotide polymorphisms (SNPs: G7395A, G12390C, T14215C, T35587C, and G35991A) in the AS3MT gene were shown to be related to inter-individual variation in the arsenic metabolism. In the present study, the genotyping for these SNPs was developed using the polymerase chain reaction and restriction fragment length polymorphism technique. Applying this method, the genotype distribution among the Ovambo, Turkish, Mongolian, Korean, and Japanese populations was investigated, and our results were compared with those from other studies. G7395, G12390, T35587, and A35991 were predominant among the five populations in our study. However, a previous study in Argentina, C12390 and G35991 showed the highest allele frequency among the eight populations studied in other studies. The dominant allele of T14215C differed among populations: the T14215 allele was predominant in Argentina, the allele frequency of C14215 was higher than that of T14215 among Turks, Mongolians, Europeans, and American ancestry. In Korea and Japan, similar allele frequencies were observed in T14215 and C14215. Higher allele frequencies were observed in haplotype G7395/G12390/C14215/T35587 with frequencies of 0.40 (Turks), 0.28 (Mongolians), and 0.23 (Koreans). On the other hand, the allele frequency for G7395/G14215/T35587/A35991 was the highest among the Ovambos (0.32), and the frequency for G7395/G12390/C35587/G35991 was the highest among the Japanese (0.27). It is noteworthy that the Japanese haplotype differs from that of the Koreans and Mongolians, which indicates the importance of investigating other intronic polymorphisms in AS3MT, especially in Asians.

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.

Arsenic poisoning from drinking groundwater is a serious problem, particularly in developing Asian countries. Human arsenic (+3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite. Recently, a single nucleotide polymorphism (SNPs; rs17885947, M287T (T860C)) in the AS3MT gene was shown to be related to enzyme activity and considered to be related to genetic susceptibility to arsenic. In the present study, a useful genotyping method for M287T was developed using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique. Applying this method, the genotype distribution of M287T in Ovambo (n=185), Turkish (n=191), Mongolian (n=233), Korean (n=200), and Japanese (n=370) populations were investigated. The mutation frequencies in Asian populations were relatively lower than those of African and Caucasian populations, including those from previous studies: the frequencies of mutation in the Mongolian, Korean, and Japanese populations were 0.040, 0.010, and 0.010, respectively. In the course of this study, a PCR-based genotyping method that is inexpensive and does not require specialized equipment was developed. This method could be applied to a large number of residents at risk for arsenic poisoning.

A multiplex PCR system for five Y-STRs (DYS441, DYS442, DYS443, DYS444 and DYS445) has been improved to increase the probability of obtaining a DNA typing result from aged samples. Newly designed PCR primers for amplification of the DYS441 and DYS442 loci and optimization of PCR conditions enabled successful typing from blood and semen stains that had been stored for more than seven years at room temperature. Analysis of 340 Japanese males revealed 7, 5, 6, 5 and 4 alleles at the DYS441, DYS442, DYS443, DYS444 and DYS445 loci, respectively, yielding 122 haplotypes with a cumulative haplotype diversity of 0.97.

We have developed a new sensitive multiplex PCR system consisting of five male-specific and polymorphic tetranucleotide STRs--DYS441 (GDB: 10013873), DYS442 (GDB: 10030304), DYS443 (GDB: 10807127), DYS444 (GDB: 10807128), and DYS445 (GDB: 10807129) on the Y chromosome. Fifty pg DNA per 10 microL reaction volume was required for the correct typing of five STRs. Using this system, the five Y-STRs were correctly typed from blood and semen stains that had been stored for several years at room temperature.

Highlights • Associations of SNPs in iron homeostasis genes and iron concentration were examined. • TF genotypes rs12769 were significantly associated with blood iron concentration. • The rs12769 might be related to diseases and mortality risk. Abstract Iron is bound to mobile transferrin (TF) and ferritin in blood. TF receptors (TFRC and TFR2) regulate intracellular iron by delivering iron from TF into the cytoplasm. In this study, we examined the effects of 10 single nucleotide polymorphisms (SNPs) in each of the genes for TF and TF receptors on blood iron concentrations in Japanese subjects. Blood iron levels were determined by microwave plasma-atomic emission spectrometry and the SNPs were analyzed by polymerase chain reaction followed by restriction fragment length polymorphism analysis. Blood iron levels in males were significantly higher than those in females. Therefore, the analysis was performed only in males. Blood iron concentrations did not correlate with age and postmortem intervals in males. Among the 10 SNPs in TF , TFRC , and TFR2 genes, significant associations were observed between TF genotypes (rs12769) and male iron concentrations. Individuals with genotype GG in rs12769 had significantly higher blood iron concentrations than those with GA. Previous studies have shown the association between high tissue iron concentrations and disease, liver iron levels are higher in infants dying from sudden infant death syndrome and decreased blood iron concentrations were observed in critically ill children. Therefore, rs12769 in TF might be related to diseases and mortality risk. [ABSTRACT FROM AUTHOR]

Purpose X-ray repair cross-complementing group 1 (XRCC1) repairs single-strand breaks in DNA. Several reports have shown the association of single nucleotide polymorphisms (SNPs) ( Arg194Trp, Pro206Pro, Arg280His, Arg399Gln) in XRCC1 to diseases. Limited population data are available regarding SNPs in XRCC1, especially in African populations. In this study, genotype distributions of four SNPs in worldwide populations were examined and compared with those reported previously. Materials and Methods Four SNPs ( Arg194Trp, Pro206Pro, Arg280His, Arg399Gln) in XRCC1 from genomic DNA samples of 10 populations were evaluated by using polymerase chain reaction followed by restriction fragment length polymorphism analysis. Results The frequency of the minor allele corresponding to the Trp allele of XRCC1Arg194Trp was higher in Asian populations than in African and Caucasian populations. As for XRCC1Pro206Pro, Africans showed higher minor allele frequencies than did Asian populations, except for Tamils and Sinhalese. XRCC1 Arg280His frequencies were similar among Africans and Caucasians but differed among Asian populations. Similarly, lower mutant XRCC1 Arg399Gln frequencies were observed in Africans. Conclusions This study is the first to show the existence of a certain genetic heterogeneity in the worldwide distribution of four SNPs in XRCC1. [ABSTRACT FROM AUTHOR]

Many nonsynonymous single-nucleotide polymorphisms (SNPs) in the human deoxyribonuclease I-like 1 (DNase 1L1) gene, possibly implicated in the blocking of endocytosis-mediated foreign gene transfer, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all 21 nonsynonymous human DNase 1L1 SNPs was performed in 16 different populations representing three ethnic groups using the PCR-restriction fragment length polymorphism technique. All of the nonsynonymous SNPs, except for SNP p.Val122Ile in Caucasian populations, exhibited a monoallelic distribution in all of the populations. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, two activity-abolishing and four activity-reducing SNPs were confirmed to be functional. Although all of the nonsynonymous SNPs that affected the catalytic activity showed extremely low genetic heterogeneity, it seems plausible that a minor allele of six SNPs producing a loss-of-function or extremely low-activity variant could serve directly as a genetic risk factor for diseases. Especially, the amino acid residues in activity-abolishing SNPs were conserved in animal DNases 1L1. Furthermore, results of phylogenetic analysis suggest that DNase 1L1 might have appeared latest among the DNase I family during the course of molecular evolution. [ABSTRACT FROM AUTHOR]

Abstract: We have focused on the 14 SNPs including all the non-synonymous and autoimmunity-related ones in the DNase II gene (DNASE2). The distribution of each allele and haplotype in these SNPs was examined in eight Asian, three African, three Mexican and two Caucasian populations using the newly developed PCR–RFLP methods. Eight SNPs among nine non-synonymous ones were monomorphic, indicating that a specific allele generating the intact activity-harboring DNase II in these SNPs is well conserved in worldwide populations. On the other hand, five other SNPs (−1951G>A, −1066G>C, −390A>C, +2630T>C, and +6235G>C) related to autoimmunity exhibited polymorphism common in worldwide populations, and especially their distributions were ethnic-dependent in the same manner as those of haplotypes. Furthermore, a strong linkage between SNPs −1951G>A and −1066G>C was confirmed in most populations. This study was the first to report any worldwide population analysis regarding all the non-synonymous and autoimmunity-related SNPs in the DNASE2, providing genetic information on the DNASE2 as a genetic marker for personal identification and/or genetic factor for susceptibility to autoimmunity. [Copyright &y& Elsevier]

In this study, a novel polymerase chain reaction (PCR)-based genotyping method to identify three Corbicula species growing in Japan (Corbicula japonica, Corbicula sandai, and Corbicula laena) was developed. Using an allele-specific PCR method, C. japonica could be identified and specifically distinguished from the species C. sandai and C. laena in both tissues and various processed foods. In addition, the discrimination of C. sandai and C. laena was possible using the PCR-restriction fragment length polymorphism (RFLP) method with Nla III as a restriction enzyme. Moreover, a preliminary experiment to determine the district in which C. japonica grows from among seven areas was performed. The use of the PCR-RFLP method made it possible to identify C. japonica from Lake Ogawara. By using electrophoretic genotyping methods in the present study, C. japonica species could be identified and the species of a particular growing district could be specified easily and reproducibly. This method is useful for protecting bioresources and maintaining ecosystems. The present study represents a contribution to the field because the strategy presented here can be applied to the forensic identification of other species, such as other bivalve and Mollusca. [ABSTRACT FROM AUTHOR]

Abstract: Cytochrome P450 (CYP) 1A1, glutathione S-transferase (GST) M1, and GSTT1 gene polymorphisms have been shown to be associated with several diseases. In this study, CYP1A1 MspI, GSTM1 and GSTT1 gene polymorphisms were investigated in 134 Ovambo and 207 Mongolians, and the results were compared with those from previous studies. Using polymerase chain reaction/restriction fragment length polymorphism (PCR–RFLP) the frequency of CYP1A1 MspI mutation was determined. The multiplex PCR was used to determine the GSTM1 and GSTT1 polymorphism. The frequencies of wild-type, heterozygous variant and homozygous variant of the CYP1A1 MspI genotypes were 72.4%, 25.4% and 2.2%, and 22.7%, 55.6% and 21.7% in the Ovambos and Mongolians, respectively. The frequencies of GSTM1 (null) and GSTT1 (null) genotypes were 11.2% and 35.8%, and 46.4% and 25.6% in the Ovambos and Mongolians, respectively. The CYP1A1 MspI and GSTT1 (null) genotype distribution of the Ovambos was similar to that of African-Americans and some Caucasians. In contrast, the GSTM1 (null) genotype distribution was different from that of all other populations. Among Mongolians, the CYP1A1 MspI polymorphism showed the highest mutation frequencies, GSTM1 (null) was similar to that of East Asians, and GSTT1 (null) was different from that of almost all the Asians examined. [Copyright &y& Elsevier]

Recent studies of copy number variations (CNVs) associated with physical features, such as body mass index, body height or bone length, have suggested that such CNVs could serve as markers in forensic cases involving unidentified individuals. However, the process of cataloging CNVs has been slow because of the cumbersome nature and low reliability of the procedures involved. Here we describe a simple quantitative real-time PCR (Q-PCR) method for screening of medicolegally useful CNVs, which does not require reference DNA with known copy number. The first step is to prepare a chimeric plasmid vector including one copy each of the single-copy gene-specific sequence as the internal standard, and the target CNV-specific sequence. To assess the validity of this new method, we analyzed CNVs in the LTBP1 and ETV6 gene regions, both of which are candidate CNVs associated with body height. The PCR efficiencies for the single-copy (reference) gene and the target CNV were similar, indicating that quantitation was reliable. Furthermore, simulated analysis of the LTBP1 CNV using mock samples prepared by mixing vectors in varying proportions showed that this analytical method allowed correct determination of the LTBP1 copy number. These results demonstrated that our simple method has considerable potential for screening of trait-related CNVs that would be useful for forensic casework. [ABSTRACT FROM AUTHOR]

Highlights • A candidate CNV associated with body height was analyzed by qPCR method. • The accuracy of the results was confirmed with an Agilent 2100 Bioanalyzer. • Analysis of DNA from 232 Japanese revealed no statistically significant correlation. Abstract Recently it has been recognized that a considerable number of copy number variations (CNVs) are associated with diseases and other complex human traits. In our previous study, we developed a simple quantitative real-time PCR (Q-PCR) method for analysis of CNV copy number, which had the advantage of obviating the need for reference DNA with a known copy number. Using DNA samples obtained from 231 Japanese individuals, we applied this method for analyzing the copy number of a candidate CNV associated with body height, located in the neural precursor cell expressed, developmentally down-regulated 4-like, E3 ubiquitin protein ligase (NEDD4L) gene. In addition, the appropriateness of the results was evaluated and confirmed by quantification of amplicons with an Agilent 2100 Bioanalyzer. The NEDD4L gene encodes a member of the Nedd4 family of HECT domain E3 ubiquitin ligases. The target CNV located in the intron has been found to be significantly associated with height variation in Chinese. However, it remains unknown whether such an association exists in other populations, including Japanese. Analysis of the correlations between copy number and body height using ANOVA revealed no statistically significant correlations in Japanese. [ABSTRACT FROM AUTHOR]

Abstract: ABO grouping of biological specimens is informative for identifying victims and narrowing down suspects. In Japan and elsewhere, ABO grouping as well as DNA profiling plays an essential role in crime investigations. In the present study, we developed a new method for ABO genotyping using allele-specific primers and real-time PCR. The method allows for the detection of three single nucleotide polymorphisms (SNPs) at nucleotide positions 261, 796, and 803 in the ABO gene and the determination of six major ABO genotypes. This method required less than 2h for accurate ABO genotyping using 2.0ng of DNA. This method could be applicable for rapid and simple screening of forensic samples. [Copyright &y& Elsevier]

Abstract: Background: Several non-synonymous SNPs in the human DNase II gene, potentially relevant to autoimmunity, have been identified, but only limited population data are available. Also, the effects of these SNPs on the catalytic activity of the enzyme remain unknown. Methods: Genotyping of all the non-synonymous SNPs was performed in healthy subjects of 3 ethnic groups including 6 different populations using the PCR-RFLP technique. A series of mutants corresponding to each SNP was expressed in COS-7 cells and its activity was measured. Results: Five of the populations, including Japanese, Germans, Turks, Ghanaians and Ovambos, were typed as a single genotype at each SNP, but Koreans were not. Constructs derived from minor alleles at A58del, V284M, R298L and Q322Term exhibited drastically low or almost no activity. Conclusion: The DNase II gene shows relatively low genetic diversity with regard to these non-synonymous SNPs, suggesting that the enzyme has been well conserved. A minor allele at V284M is distributed with a frequency of 0.013 in the database, and it seems plausible that levels of DNase II activity for the heterozygote are lower than those in individuals with the predominant homozygote. Our results may have clinical implications in relation to the prevalence of autoimmune diseases. [Copyright &y& Elsevier]