Miyaishi, Satoru

Allele and genotype frequencies for 15 short tandem repeat (STR) polymorphisms--D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA--in a Japanese population were estimated. No deviations of the observed allele frequency from Hardy-Weinberg equilibrium expectations were found for any of the systems studied. Between 2 new pentanucleotide STR loci, Penta E and Penta D, for which there is only limited data regarding the allelic distribution in Japanese, the Penta E locus was found to be highly polymorphic and exhibited a tri- or tetra-modal distribution pattern having allelic peaks with 5, 11, 15 and 20 repeats. The distribution was significantly different from that of the other ethnic groups. Statistical parameters of forensic importance, the power of discrimination (PD), observed and expected heterozygosity values (H), polymorphism information content (PIC), power of discrimination (PD), matching probability (pM), power of exclusion (PE), and typical paternity index (PI), were calculated for the loci. These parameters indicated the usefulness of the loci in forensic personal identification and paternity testing among Japanese. The systems Penta E, FGA, D18S51 and D8S1179 were the most informative. This method was successfully applied to forensic personal identification and paternity testing among Japanese, thereby confirming its efficacy for forensic practice.

Healthy subjects 40 years old were used as controls in a study of stellate cells (S-100 protein-containing cells, or S-100 cells) in subjects with chronic alcoholism and fatty liver or fatty cirrhosis. S-100 cells were sparsely found in the adenohypophysis of control subjects, and these cells sometimes formed small clusters. However, in chronic alcoholics with fatty liver or fatty cirrhosis, the number of stellate cells in the anterior pituitary tended to be 17 times higher than it was in the control group. No increase in the number of S-100 positive cells that constitute the large and small follicles in the intermediate pituitary. The physiological function of the S-100 protein has not yet been identified. The fact that an increase in prolactin-secreting and growth hormone-secreting cells, as well as a decrease in gonadotrophs were observed in the hypophysis of alcoholics suggests that the function of stellate cells may be closely related to these phenomena. Our results also imply that the stellate cells found in the anterior and intermediate pituitary differ in function although they both produce S-100 proteins.

In this study we used paraffin-embedded human pituitary obtained from 248 autopsy cases and identified mixed cell follicles by the immunohistochemical method. We examined the number and size of the mixed cell follicles, and the ratio of each component cell of these follicles, in the anterior pituitary at various age groups. The number of follicles increased with age, and the size of the follicles also tended to enlarge with age. Statistical analysis showed that a high correlation existed between age and the number or the size of the mixed cell-follicles formed by various adenohypophyseal cells. In addition, when the proportions of the different cell types that formed the follicles were examined, sex differences were observed with aging for the GH cells, the PRL cells, and the gonadotroph (GTH) cells, while no changes were observed with aging in both men and women for the ACTH cells and TSH cells. These results indicate that the number, size, and ratio of each component cell of follicles in the anterior pituitary are adequately applicable for the purpose of age estimation in routine forensic medicine.

In this study, sex determination using polymerase chain reaction (PCR) on tooth material was evaluated from the viewpoint of forensic medicine. The sensitivity of PCR for detection of the Y chromosome-specific alphoid repeat sequence and the X chromosome-specific alphoid repeat sequence was 0.5 pg of genomic DNA. Sex could be determined by PCR of DNA extracted from the pulp of 16 freshly extracted permanent teeth and dentine including the surface of the pulp cavity of 6 freshly extracted milk teeth. Sex could be determined using the pulp in all 20 teeth (10 male and 10 female) preserved at room temperature for 22 years. For the pulp of teeth stored in sea water, the sex could be determined in all 8 teeth immersed for 1 week and in 5 of 6 teeth immersed for 4 weeks. In the remaining 1 tooth, in which sex determination based on the pulp failed, the sex could be determined correctly when DNA extracted from the tooth hard tissue was examined. For teeth stored in soil, the sex could be determined accurately in all 8 teeth buried for 1 week, 7 of 8 teeth buried for 4 weeks, and in all 6 teeth buried for 8 weeks. When teeth were heated for 30 min, sex determination from the pulp was possible in all teeth heated to 100, 150, and 200 degrees C, and even in some teeth heated to 250 degrees C. When this method was applied to actual forensic cases, the sex of a mummified body estimated to have been discovered half a year to 1 year after death could be determined readily by examination of the dental pulp. In the skeletons of 2 bodies placed under water for approximately 1 year and approximately 11 years and 7 months, pulp tissues had been dissolved and lost, but sex determination was possible using DNA extracted from hard dental tissues. These results indicate that this method is useful in forensic practices for sex determination based on teeth samples.

We developed a method for human identification of forensic biological materials by PCR-based detection of a human-specific sequence in exon 3 of the myoglobin gene. This human-specific DNA sequence was deduced from differences in the amino acid sequences of myoglobins between humans and other animal species. The new method enabled amplification of the target DNA fragment from 30 samples of human DNA, and the amplified sequences were identical with that already reported. Using this method, we were able to distinguish human samples from those of 21 kinds of animals: the crab-eating monkey, horse, cow, sheep, goat, pig, wild boar, dog, raccoon dog, cat, rabbit, guinea pig, hamster, rat, mouse, whale, chicken, pigeon, turtle, frog, and tuna. However, we were unable to distinguish between human and gorilla samples. This method enabled us to detect the target sequence from 25 pg of human DNA, and the target DNA fragment from blood stored at 37 degrees C for 6 months, and from bloodstains heated at 150 degrees C for 4 h or stored at room temperature for 26 years. Herein we also report a practical application of the method for human identification of a bone fragment.</P>

We have improved on conventional methods for HLA-DRB1 genotyping and devised a new method that is simple, cost-effective, and adequately applicable to routine forensic practice. This method consists of group-specific polymerase chain reaction (PCR) of the exon 2 region of the HLA-DRB1 gene and simultaneous detection of single nucleotide polymorphisms (SNPs) at multiple sites using multiplex primer extension reactions. With this method, we successfully detected HLA-DRB1 genotypes from the following materials: the peripheral blood of 142 donors, 6 aged saliva stains of known DRB1 genotype stored for 5-10 years at room temperature, 10 aged bloodstains of unknown DRB1 genotype stored for 29 years at room temperature, and minimal bloodstains and saliva stains from 3 donors of known DRB1 genotypes. Furthermore, we were able to type DRB1 alleles of the minor component in mixed samples at a proportion of 1/1,000 or 1/10,000. In a criminal case, DRB1 alleles detected from mixed bloodstains on a sword found at the scene enabled us to explain the case. This method is expected to be useful for forensic medicine.

The annual number of suicides in Japan increased sharply in 1998, and since that time it has consistently exceeded 30,000 per year. In this study, we analyze a database of personal and background characteristics of 824 cases (605 men, 219 women) who completed suicide in Okayama Prefecture in 2002 and 2003. The data were obtained with cooperation from the police. Using the methodologies in a previous European study as a model, we classified the suicide methods into 8 categories. To examine the generational and regional differences in the choice of methods, we stratified the sample into 4 age groups (<-24, 2544, 4564, and >-65) and 2 regional groups (Okayama/Kurashiki vs. other areas). Our results on gender differences in 7 of the suicide methods were mostly similar to the European data. However, our data showed a remarkably higher proportionate male-to-female mortality ratio for poisoning by other substances (ICD-10, X65-X69 codes) (1.83, 1.15-2.92). In terms of generational differences in the choice of suicide methods, the Mantel-Haenszel test of homogeneity was significant for most of the categories in our study, suggesting an impact of age on how people commit suicide. There were no remarkable regional differences in our sample. An epidemic curve for suicides via carbon monoxide poisoning using charcoal briquets revealed a trend of time clustering not observed in the other 6 means. The database constructed and used in this study contains richer information than conventional death statistics and is expected to provide helpful knowledge and insights for future epidemiological studies.

We encountered a rare case of suffocation by an advertising balloon filled with pure helium gas. Suffocation caused by inhalation of atmosphere lacking in oxygen is not exceptional, but reports of death by suffocation due to a pure inert gas such as helium are very rare. In this case, the balloon mooring on the ground was enclosed, warning signs were displayed, and it was clear that entering the balloon filled with an atmosphere lacking in oxygen was extremely dangerous and should not be done; the accident did, however, occur. Accidents of this kind may occur in the future unless appropriate education and countermeasures are taken.

We describe a modified method for typing a polymorphic microsatellite D12S391 locus by PCR using a newly designed primer pair. This primer pair produces shorter D12S391 amplified fragments (104-156 bp) than the primer pair originally described by Lareu et al. (209-261 bp). The detection system for the D12S391 locus using the new primer pair and capillary electrophoresis (CE) analysis was evaluated using various forensic samples. The typing results from 70 DNA samples using the new primer pair and the original primer pair were completely identical. One hundred twenty-five amplified fragments from D12S391 alleles were sized correctly within +/- 0.25 bp of the D12S391 allelic ladder. A rare allele, 19.3, previously found only in Caucasians, was found for the first time in a Japanese subject, and it was clearly distinguished from allele 20 by the CE analysis. This detection system was sensitive and could detect D12S391 types from 16 pg of genomic DNA, and from a minor component at a ratio of 1:10 in mixed samples. This system was more useful for the analysis of degraded DNA than was the method using the original primer pair, and could detect D12S391 types from bloodstains that had been stored for 26 years. In addition, the specificity of the method was demonstrated using nonhuman DNA.

A sensitive method of HLA-DRB1 typing was devised using a semi-nested polymerase chain reaction (PCR) followed by a restriction fragment length polymorphism (RFLP) analysis (semi-nested PCR-RFLP method). The first-round amplification (30 cycles) of the semi-nested PCR was performed using DRB generic primer pairs and the second round of PCRs (20 cycles) were performed using DRB1 group-specific primers. The products of the second round PCRs were digested with restriction endonucleases for the typing of HLA-DRB1 alleles. By this method, HLA-DRB1 typing was possible from 10 pg of genomic DNA extracted from lymphocytes and from 0.5 microliter of 1,000 times diluted blood without DNA extraction. HLA-DRB1 alleles could be typed from a 2-mm long bloodstained cotton thread prepared from 10 times diluted blood and from a 2-mm thread of whole blood bloodstains stored at room temperature for 2 years. From the mixture of blood of two individuals with different genotypes, DRB1 alleles of the minor component were detected down to 1/1,000 of the major component. This semi-nested PCR-RFLP method is useful for HLA-DRB1 typing from extremely small amounts of DNA and from mixed samples.

We performed haptoglobin (Hp) genotyping by polymerase chain reaction using allele-specific primer-pairs. The major six genotypes of Hp were identified using this method. Among Japanese individuals living in Ehime and Okayama Prefectures, the allele frequencies were estimated to be Hp2 = 0.723 and Hp1s = 0.277. Genotyping of Hp was possible with 0.3 ng of DNA and with 0.125 microliter of blood. It was also possible with whole blood left at room temperature for a month and also with the bloodstains left at room temperature for three years. In the heated blood samples, both alleles, Hp2 and Hp1s, were detected in those heated at 100 degrees C for 2 h. In bloodstains, Hp2 and Hp1s were detected in samples heated at 100 degrees C for 2 h and 120 degrees C for 30 min. In addition, the genotype could be detected in samples other than blood such as saliva, hair roots, tissue sections and dental pulps. The present method for Hp genotyping is expected to become a useful method in forensic analysis.