Kaoru Sagisaka’s research while affiliated with Central Forensic Laboratory of the Police and other places

Allele frequencies for the eight STR loci Hum-CSF1P0, F13A01, F13B, FES/FPS, LPL, TH01, TPOX and VWA were investigated in Japanese and Chinese populations. No significant deviations from Hardy-Weinberg equilibrium could be found for all loci. In the Japanese population VWA, CSF1PO, TH01, FES/FPS and TPOX were found to be useful for forensic applications and in the Chinese population, VWA, CSF1PO, TH01 and TPOX were found to be useful. Allele distributions were similar between both populations except for FES/FPS.

Simple and rapid detection of HLA-DRB polymorphism has been performed using AMPLICOR HLA-DRB Typing Kit. We tried to apply this kit to various forensic samples. When DNA was extracted from the forensic samples using conventional phenol-chloroform method, addition of 7.5 mM MgCl2 was required to PCR amplification. HLA-DRB types were detected from DNA more than 0.1 ng by PCR amplification. Typing of unrelated 50 Japanese showed 38 different patterns, of which 30 patterns occurred once in the group. A total of 16 serotypes were deduced from the HLA-DRB DNA types. Out of them, high frequency serotypes were DR4 (24%), DR9 (18%) and DR15/16 (14%). This kit was very useful in forensic cases such as rape and in paternity cases. When we tried to detect HLA-DRB types from a single hair shaft of 3 cm in length, we were successful in detection from only one of five persons.

Terminal deoxynucleotidyl transferase(TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay is useful to detect apoptotic cells in situ. We examined by hematoxylin-eosin (H-E) and TUNEL assay whether or not postmortem delay affects the development of apoptotic signals of cells in various organs. Wistar Imamichi rats were radiated by X-ray and sacrificed six hours after radiation. The spleen, thymus, adrenal and testis were excised and kept in a moist chamber at room temperature. Each tissue was fixed after different time intervals 0, 6, 12, 24 hours and paraffin-embedded sections were made. In the no-radiation group, a few of TUNEL positive cells were observed in the spleen, thymus and testis sections, but not in the adrenal. No increase in the number of apoptotic cells was observed with postmortem delay. In the radiation group, we observed in the spleen and thymus, much increase in the number of TUNEL positive cells, of which nuclei were clearly and deeply stained, corresponding to the area where shrinking nuclei were observed in H-E section. In testis sections, there was a little increase in the number of positively stained cells, and no change was observed in H-E section. With postmortem delay, the margin of the TUNEL positive cells changed from clear to indistinct, and the positive area was spread around. Our results show that it is difficult to distinguish apoptotic cells from postmortem change. It is possible, however, to detect TUNEL positive cell together with postmortem changes as the spread of the TUNEL positive area after 24 hours postmortem delay. It is important to consider the effect of the postmortem change when we adapt TUNEL assay to autopsy cases.

We report a study of polymorphism for seven short tandem repeat (STR) loci in Japanese and Chinese populations. Among 104 to 134 individuals in the both population samples, eight alleles were revealed for locus PLA2, thirteen for D3S1359, eleven for FGA, eight for D8S315 (kw38), ten for D8S1132, five for CYP19, and seven for D3S2459. They correspondingly constituted 10 to 39 genotypes therein. For most of the STRs, there was only a single allele active as the most frequent one among the others, except locus D3S1359 in Chinese samples (two alleles, 206 bp and 210 bp, frequency = 0.273 each). Also, the population genotype configurations were locus specific, varying in the patterns of commonest genotypes on each locus, e.g., one pattern for loci CYP19, D3S1359, and D8S315, one and two for loci PLA2 and D3S2459, two for locus D8S1132, and one and four for locus FGA. The distributions of observed genotypes were in Hardy-Weinberg Equilibrium. Furthermore, the seven STRs were exhibited highly polymorphic and informative for the both populations, and the alleles could be easily separated in electrophoresis and correctly interpreted with side-to-side allelic ladders. Together, the results suggest that the tri- and tetra-meric STRs are useful genetic markers for forensic practice.

We designed a spreadsheet package for the computation of plausibility of paternity, that can cope with highly polymorphic genetic markers and cases of deceased parties. The application program is Microsoft EXCEL, which is one of the best-selling spreadsheet software running on both Microsoft Windows and Macintosh OS. Komatsu's formula for paternity testing was mainly employed in the spreadsheet package. Probability of the mother-child-alleged father combination was calculated using "IF" function to compare the members' genotypes, whereas "VLOOKUP (or HLOOKUP)" function was employed to refer to a list of genes and their frequencies. In case of a phenotype consisting of several genotypes, the list of phenotypes versus genotypes was also given, to which the function referred. To extend these spreadsheets available for the test of deceased party, additional sheets were also created to estimate frequencies of alleged father's possible genotypes. These probabilities were calculated on the basis of types of his parents and siblings, those of his wife and their biological children, and those of both. This package would be cut out to compute the probability of paternity with extremely polymorphic loci with gentle user interface. Calculation time is satisfactorily short, although it requires considerably large disk space in some extremely complicated cases. Japanese version of this package is freely available at anonymous FTP site of the Department of Forensic Medicine, Tohoku University School of Medicine.

In a maternity test in which the putative mother was deceased, the cumulative probability of maternity (PM) was calculated at 0.822 from 24 genetic markers by the stochastical method. This PM may not be evaluated in the same way as that of usual paternity cases. We applied the same method to two families whose blood relationships were undoubted. We compared the PMs in the cases in which maternal genotypes were estimated and were defined. Also, we calculated the PMs in the case of real maternal relationship and false maternal one. The estimated PM from real maternity relationship was significantly higher than that from false maternal one.

The PCR-direct sequence method was applied to ABO genotyping. At the 261st nucleotide of the genes of A and B glycosyltrasferase, it was easily detected that the nucleotide was guanine in AA, AB and BB genotypes and that the nucleotide was ademine in only OO. In AO and BO, substitution of A to G was confirmed by the dye primer method, but it was difficult to detect correctly by the dye terminator method. At the 297th, nucleotide substitution between A and B alleles was confirmed by the both methods. As this position, O allele was subdivided into three types, OAOA, OGOG and OAOG. At the 703rd, nucleotide substitution between A and B alleles was easily detected by the both methods. The PCR-direct sequence method was suitable to confirm the nucleotide substitution or deletion directly and to prevent the mistyping by other methods.

A 3-year-old girl was found unresponsive in the bedroom and expired at a hospital. Autopsy revealed massive intra-abdominal bleeding due to laceration of the liver and mesenterium with multiple rib fractures and multiple fresh and old bruises. The time of the assault causing the liver trauma was questioned because the perpetrator, her mother's boyfriend, denied any outrages on that particular day although he confessed that he had physically abused her for several months. Microscopically, numerous polymorph leucocytes infiltrated exclusively surrounding the lacerated area of the liver. Many hepatocytes were necrotic and cord arrangement of the parenchymal cells was destroyed. There should be a certain time lag between the major assault and massive intra-abdominal hemorrhage, which was not inconsistent with the statement of the perpetrator.

Genomic imprinting is a gamete-specific modification resulting in the allele-specific expression of genes in somatic cells. A loss of imprinting (LOI) has been found in many embryonal and adult tumors, suggesting that it plays a role in tumor development. The incidence of LOI, however, does not seem to be ubiquitous among tumors because neuroblastoma and colorectal cancer revealed no LOI. We examined the involvement of LOI of IGF2 and H19 genes in human gliomas. The two genes were imprinted in normal brain subcortex tissues. In glioma, 8 of 14 informative cases (57%) revealed LOI in IGF2. The frequency did not depend on the tumor grade. For H19, in contrast, all 13 informative cases maintained imprinting. These results suggest that LOI of IGF2 but not H19 plays a role in the development of human glioma.

The state of DNA methylation in the c-fos gene was examined in human livers of different ages, cirrhosis and hepatocellular carcinoma. The degree of methylation in the intron 1 to exon 4 region increased with age, whereas all of the 10 cirrhosis samples revealed a decrease in methylation when compared to normal livers of similar ages. The 11 hepatocellular carcinomas showed varied alterations suggesting that the alteration of the c-fos gene methylation is related to aging as well as to early-step of hepatocarcinogenesis.