Nori Nakayashiki

Multiplex amplified product-length polymorphism analysis for rapid detection of human mitochondrial DNA variations

A number of mutations in coding and noncoding regions of mitochondrial DNA (mtDNA) have previously been studied. In the present study, we simultaneously typed six mutation sites in the coding region by use of amplified product‐length polymorphism (APLP) analysis. The mtDNA variations of 2471 individuals from 20 populations of Japanese, Korean, Chinese, and German were examined and classified into 18 haplotypes. Two of these haplotypes, B1 (estimated ancestral haplotype) and C1, were distributed among all populations tested. However, the haplotypes A1, A2, B2, B3, and C2 were mostly restricted to the Mongoloid populations, whereas haplotypes B5 and C5 appeared almost exclusively in the German population. Phylogenetic analysis by the neighbor‐joining method revealed that the Japanese populations were more closely related to each other than to the other East Asian populations surveyed. The multiplex APLP method is suitable for large‐scale screening studies of mtDNA variability because it is both rapid and economical.

A histological study on the mechanism of epidermal nuclear elongation in electrical and burn injuries.

Abstract Epidermal nuclear elongation is one of the most important signs for the diagnosis of electrical injury. In this study, we investigated the mechanism responsible for this phenomenon by comparing the findings from burn injuries and those from contusions. Electrical and burn injuries were made in the dorsal skin of rats using energy ranging from 100 to 790 joules for electrical injury, and 170–690 joules for burn injury. Contusions were also made by compressing the skin with a vice. In electrical and burn injuries, the dermis under the epidermal elongated nuclei was homogeneous and without empty spaces between collagen bundles and the number of dermal fibroblasts per 0.01 mm2 below the damaged epidermis decreased significantly (P < 0.05). The incidence of this change correlated with the depth of denatured dermal collagen fibres and in both types of injuries, dermal cells had no nuclear antigenicity for ubiquitin. The width of the injured epidermis with nuclear elongation decreased significantly (P < 0.05) and the elongated nuclei were parallel to the basal membrane. In electrical injury however, nuclear elongation occurred more frequently near the external root sheath. Nuclear elongation of fibroblasts and external root sheath cells was also found, but those of sebaceous gland cells were not detected. Epidermal elongated nuclei were also found in contusions. The evidence strongly suggests that epidermal nuclear elongation in electrical and burn injuries is due to dermal expansion by heat.

Wound age estimation by simultaneous detection of 9 cytokines in human dermal wounds with a multiplex bead-based immunoassay: An estimative method using outsourced examinations

Wound age estimation for human dermal wounds was performed based on quantification of interleukin 1beta (IL 1beta), IL 5, IL 7, IL 12 p70, IL 13, IL 17, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP 1), and macrophage inflammatory protein 1beta (MIP 1beta). IL 5, IL 12 p 70, IL 13, and IL 17 increased from the early phase, MCP 1 exclusively in the middle phase, and IL 1beta, G-CSF, and MIP 1beta from the middle phase to the late phase. IL 7 decreased from the early phase. Among the cytokines analyzed in the present study, MCP 1 was the most plentiful cytokine. In addition, an outsourced examination, which could be available to any forensic institute, was performed in two cases for confirmative purposes. Many factors have been proposed as markers for dermal wound age estimation, but the set of cytokines selected for the outsourced examination in the present study wound be useful in daily forensic practice.

A parent-of-origin detectable polymorphism in the hypermethylated region upstream of the human H19 gene

The H19 gene is a paternally imprinted gene located on chromosome 11p15.5. In this study the H19FR haplotype polymorphism including three SNPs upstream of the H19 gene was investigated. Six genotypes derived from three alleles were detected in the Japanese population by means of PCR and subsequent constant denaturing gel electrophoresis. Based on the methylation status of the genomic DNA from blood samples, selective detection of the parental allele for H19FR was examined by using two types of enzyme, the methylation-sensitive restriction enzymes HpaII or HhaI and McrBC. Genomic DNA digested by either HpaII or HhaI, revealed a single band derived from the paternal allele, as a result of cleavage of unmethylated recognition sites on the maternal allele. On the contrary, the use of McrBC, which can digest a methylated paternal sequence, resulted in exclusively amplifying the maternal allele. This method could be one of the useful techniques for discriminating the parental origin of alleles.

Investigation of the methylation status around parent-of-origin detectable SNPs in imprinted genes

Methylation of CpG dinucleotides was investigated in five regions by bisulphite treatment of gDNA, PCR and cloning/sequencing. The gDNA was prepared from peripheral blood, saliva, semen, nails and hair from the head. In gDNA from peripheral blood, three regions were investigated in 16, 23 and 24 individuals, respectively (Fig. 2). In gDNA from other sources, three or five regions were investigated in five individuals (Fig. 3). In many of the sequenced fragments, all the CpG dinucleotides were either methylated or not, which support the idea that the parental origin of an allele may be determined by the methylation status of the allele. However, the methylation of CpG dinucleotides varies across the fragment in some of the sequenced fragments, especially from semen samples, which indicate that it may be difficult to determine the parental origin from some gDNA sources by restriction-enzyme analysis (DMPA method).

An improved method for MN genotyping by the polymerase chain reaction

A novel method of human MN blood group genotyping is reported using the polymerase chain reaction. Genotyping is based on two base substitutions characteristic of M and N alleles in the 2nd exon of the glycophorin A gene. Using a newly designed primer trio, PCR products for M (255 bp) and N (270 bp) alleles are rapidly and simultaneously detected by a single PCR procedure and subsequent polyacrylamide gel electrophoresis. This method enables MN genotyping from not only minute but also degraded DNA samples.

A hypervariable STR polymorphism in the complement factor I (CFI) gene: Asian-specific alleles.

In this study, a short tandem repeat (STR) polymorphism in intron 7 of the human complement factor I (CFI) gene was studied in 637 DNA samples obtained from African, German, Thai, and Japanese populations and German and Japanese families. A total of 41 alleles were observed and classified into two groups, L and H, based on size differences. Group H, which consisted of 16 alleles, was observed only in Thai and Japanese populations at frequencies of 0.162 and 0.116, respectively, and was strongly associated with c.1217A in exon 11 (CFI*Ah). The heterozygosity values ranged from 0.89 in German to 0.93 in Thai populations. This STR would be a useful supplementary marker for forensic individualization.

An application of PCR-single strand conformation polymorphism to MN genotyping.

A PCR-based genotyping of MN blood group system was investigated for DNA samples taken from a population of 409 northern Japanese. DNA fragment (257bp) including exon 2 of glycophorin A (GPA) gene, in which encodes the determinants of MN antigens, was specifically amplified. On the analysis of PCR-single-strand conformation polymorphism (PCR-SSCP) for M alleles, band patterns of M(G) and M(T) were easily discriminated each other. For N alleles, three band patterns were observed, and we tentatively named these alleles as N(1), N(2) and N(V). The N(1) allele appeared predominantly and N(2) had two base substitutions at 1st (C-->A) and 56th (C-->T) in exon 2 of N(1). The other N(V), which was detected from a pair of a mother and her child, possessed a single base substitution at 23rd (A-->G) in intron 2. The allele frequencies of M(G), M(T), N(1) and N(2) were 0.4450, 0.0978, 0.4303 and 0.0269, respectively. The polymorphism information content and the probability of paternity exclusion by this MN genotyping were estimated to be 0.5252 and 0.3219, respectively.

The global distribution of the p.R1193Q polymorphism in the SCN5A gene

The SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. The p.R1193Q (c.3578G>A) polymorphism in SCN5A is known to accelerate inactivation of the sodium channel current, and has been identified in patients with Brugada and long QT syndromes. In the present study, we investigated the frequency of the p.R1193Q substitution in more than 4000 genomic DNA samples from 34 Asian, European, and African populations using TaqMan and/or APLP (amplified product length polymorphism) assays. Allele A (p.1193Q) was detected in most Asian populations, but was sporadically observed or absent in European and African populations. These results demonstrated that the p.R1193Q substitution is characteristic of Asian populations.

Investigation of Japanese-specific alleles: most are of Jomon lineage.

Japanese-specific alleles are expected to be powerful markers for the differentiation of the Japanese from other people. In this study, three single nucleotide polymorphisms (SNPs) in the GALNT11, H19, and PLA2G12A genes were analyzed in 2396 DNA samples from 25 global populations, and the derived alleles suggested that Japanese-specific alleles exist on autosomes. To identify new Japanese-specific alleles, candidate SNPs obtained from the HapMap database were investigated using 875 DNA samples from nine populations. A total of 67 (nearly) Japanese-specific derived alleles were observed. Of them, 57 showed higher frequencies in the Ryukyuans, living in the southernmost part of the Japanese Archipelago, than in the Wajins living in mainland Japan, and 43 were also present in Koreans at low frequencies. Jomon skeletons excavated from Hokkaido, the northernmost island of Japan, showed higher frequencies of the three derived alleles in the GALNT11, H19, and PLA2G12A genes than the Ryukyuans, suggesting that most of the 57 derived alleles observed at the high frequencies in the Ryukyuans originated from the Jomon lineage. These novel markers will be useful in the field of forensics.