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.

Allele frequencies of a SNP and a 27-bp deletion that are the determinant of earwax type in the ABCC11 gene.

Allele frequencies for a SNP (rs17822931) and a 27-bp deletion that are the determinant of earwax type in the ABCC11 gene were investigated in seven Japanese, one Korean, and one German populations. The SNP will be useful as one of ancestry information markers, because it showed marked difference in frequencies between Asian and European populations.

Analysis of age-related carbonylation of human vitreous humor proteins as a tool for forensic diagnosis.

Age-related changes of protein carbonylation due to oxidative damage in human vitreous humors were analyzed. The vitreous samples were collected from 51 autopsied bodies (male: 27, 13-87 years-old, female: 24, 18-80 years-old) whose postmortem interval was less than 4 days. Total protein carbonyl content was assessed by colorimetry using 2,4-dinitrophenylhydrazine and particular carbonylated proteins were identified by immunostaining of vitreous proteins resolved by SDS-PAGE and MALDI-TOF MS with peptide mass fingerprinting. No significant correlation was found between total protein carbonyl content and biological age. However, two major proteins, albumin and transferrin, were found to be heavily carbonylated in the samples from individuals age 40 or over. Furthermore, an immunostained cluster of proteins at 50-60 kDa was discernible in the samples of aged individuals, and it was revealed to contain alpha enolase, pigment epithelial differentiating factor, type 2 keratin subunit protein, and S-arrestin. The results of this study suggest that some retinal proteins detected in the vitreous humor sample would be markers of age-related oxidative stress and biological age.

A study on mRNA expressions of fibronectin in dermal and cerebral wound healing for wound age estimation.

We investigated mRNA expressions of fibronectin for wound age estimation during dermal and cerebral wound healing. Fibronectin mRNA expressions in the injured skin peaked at 8h post-injury. The expressions were detected in endothelial cells before and after injury, whereas they were detectable in the epidermal cells at 1-240 h, in fibroblasts at 1-72 h, in neutrophils and macrophages at 8-72 h, respectively. However, the expressions in epidermal cells became relatively weak in the subacute phase. Fibronectin mRNA expressions of the injured cerebrum increased after the intervention and peaked at 48 h, whereas there was a slight decrease during 24h post-injury. Although fibronectin mRNA was seen exclusively in the endothelial cells of the intact cerebrum, it was also detected in astrocytes during wound healing. From these findings, it was considered that fibronectin played an important role in dermal and cerebral wound healing. Expression of fibronectin mRNA was considered to indicate the acute phase of dermal wound healing, and the subacute phase of cerebral wound healing.

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.

A hypervariable STR polymorphism in the CFI gene: mutation rate and no linkage disequilibrium with FGA.

A hypervariable short tandem repeat (STR) polymorphism in intron 7 of the human complement factor I gene (CFI) was investigated to estimate the mutation rate in Japanese samples and to test linkage disequilibrium (LD) with an STR in the fibrinogen alpha chain gene (FGA). The expected heterozygosity and the mutation rate of CFI were estimated to be 0.917 and 0.002, respectively. No LD was observed between CFI and FGA. CFI is a useful supplementary marker for forensic science.

Hypothalamic transcript profiling in hypothermia using SuperSAGE.

Understanding of molecular mechanisms underlying hypothermia is of primary importance in devising strategies to diagnose hypothermia. We investigated the hypothalamic transriptome in hypothermia. For transcriptomic analyses, SuperSAGE, an improved method of serial analysis of gene expression, was used. Totally, 62,208 and 54,084 tags were collected from the hypothalami of normal and hypothermia, respectively. And 367 transcripts were differentially expressed at a statistically significant level. That is, 157 and 210 transcripts among them were expressed at a higher level in normal and hypothermic hypothalami. Results obtained by SuperSAGE and quantitative PCR were consistent in 6 selected genes, although levels of differences detected by the 2 methods were not exactly the same. mRNA expressions in the hypothalamus were considered to be useful for hypothermic diagnosis. Various methods have been applied for gene expression analyses and biomarker detections. However in forensic pathology, SuperSAGE would be a promising method, especially in gene discoveries and transcriptomic analyses.

Analysis of the methylation profiles in imprinted genes applicable to parental allele discrimination.

This study investigated the distribution of methylated CpGs around several SNPs in five imprinted genes, which are useful to discriminate the differentially methylated parental allele (DMPA), by bisulfite-sequencing method. The genomic DNAs from peripheral blood, saliva and relatively fresh postmortem tissues except for testis showed a differentially methylated status in each parental allele, however, DNAs from the other sources (nail, hair, semen, bloodstains and testis) revealed unexpected methylation patterns with no obvious tendency. Therefore, it would be better to have a cautiousness when the forensic evidences under various conditions are examined by the DMPA detection method.

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.

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).

Studies on mRNA expression of tissue-type plasminogen activator in bruises for wound age estimation.

We investigated mRNA expression of tissue-type plasminogen activator (tPA) and inflammatory cell dynamics for wound age estimation of bruises in mice. Neutrophils were detected from 1 h post-injury. Up to 8 h, they accumulated in subcutaneous tissue and the lower part of the dermis, and thereafter they extended to all the layers. Macrophages became detectable 3 h post-injury, and moderate infiltration of lymphocytes was seen from 144 h. In addition, epidermal thickening was also seen from 72 h. tPA mRNA expression peaked at 1 h, and increased slightly at 72 h post-injury. tPA mRNA was detected in epidermal cells, fibroblasts, and endothelial cells before and after injury, from 3 h in neutrophils and from 72 h in macrophages, respectively. This study presents the time-dependent expression of tPA mRNA in bruises in relation to temporal histologic characteristics during wound healing, which was considered to be useful for wound age estimation. Furthermore, it is suggested that tPA plays an important role in the first step of tissue remodeling.

Systematic classification of alleles of the glycophorin A (MN blood group) gene.

Ten alleles (five M and five N alleles) of the MN blood group system with normal antigenicity were found by sequencing the glycophorin A (GPA) gene. This study demonstrates the systematic classification of these alleles to major or minor variations of the standard alleles. GPA-specific fragments ranging from 150 to 3.8 kb in length were amplified from the templates, and exons 1-7 and introns 1-6 were sequenced. The data were analyzed phylogenetically to classify these alleles into major groups or clusters. The ten alleles were grouped into four major clusters M10X (M101-M103), M20X (M201 and M202), N10X (N101-N104) and N20X (N201), where 'X' represents a digit indicating minor variations. This grouping was supported by phylogenetic analysis. The cluster system of GPA alleles is highly informative for genetic screening.

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.

Studies on mRNA expression of basic fibroblast growth factor in wound healing for wound age determination.

We investigated the mRNA expression of basic fibroblast growth factor (bFGF) for wound age determination during dermal, cerebral, hepatic and renal wound healing in mice. The bFGF mRNA expression in the injured skin peaked at 1 h and was detected in epidermal cells, fibroblasts, endothelial cells and neutrophils. In the injured cerebrum the expression increased from 1 h and peaked at 48 h. In the intact cerebrum, bFGF was detected exclusively in the endothelial cells, whereas it was also detected in astrocytes during wound healing. Time-dependent expression of bFGF mRNA in skin and cerebrum was considered to be useful for wound age determination. On the other hand it was suggested that bFGF mRNA in astrocytes could be a vital sign of the acute phase. In hepatic and renal injuries, however, bFGF mRNA expression increased slightly in endothelial cells at 24 h, in neutrophils of the liver and in the glomeruli of the kidneys.