Medical records as screening tools for child death review in Japan.

BACKGROUND: Although many child death review (CDR) systems have been developed in Japan, the optimal system is still being identified. The aim of this study is to identify the etiologies of child deaths and to propose a screening method for initiating the CDR process in Japan. METHODS: Clinical medical records (CMRs) in hospitals and autopsy records were surveyed for cases of deaths of children aged less than 15 years between 2014 and 2016 in Aichi Prefecture, Japan. The data were analyzed in three steps, and the findings were compared with the vital statistics. RESULTS: Of the 695 children whose death certificates were submitted to Aichi Prefecture, 590 could be traced to pediatric care hospitals. The distribution of causes of death was slightly different from the vital statistics, with 11.5% dying of extrinsic causes and 19.7% dying of unknown causes. Maltreatment was suspected in 64 cases, which was much higher than that in government statistics. Overall, 158 (26.8%) deaths were considered preventable. The number of unnatural deaths, which might be screened in, was calculated as 172 (29.2%) in the vital statistics, whereas the survey of CMRs revealed that 241 (40.8%) to 282 (47.8%) should be screened in. CONCLUSIONS: Surveying CMRs in hospitals may be a suitable method to detect and screen deaths to start the CDR process in Japan.

Examination of postmortem changes in the lungs, trachea, and bronchi in a rat model imaged with small-animal computed tomography.

Objectives: As less autopsies are performed, the need for postmortem computed tomography (PMCT) as an alternative is increasing. It is important to know how postmortem changes over time are reflected on CT, in order to improve the diagnostic capability of PMCT and replace forensic pathology evaluations such as time of death estimation. Methods: In this study, we examined temporal changes on postmortem chest CT images of a rat model. After acquiring antemortem images under isoflurane inhalation anesthesia, the rats were euthanized with a rapid intravenous injection of anesthetics. From immediately after death to 48 hours postmortem, chest images were acquired using small-animal CT. The 3D images were then evaluated on a workstation to measure the antemortem and postmortem air content in the lungs, trachea, and bronchi over time. Results: The air content in the lungs decreased, but the air content of the trachea and bronchi temporarily increased 1-12 hours postmortem, then decreased at 48 hours postmortem. Therefore, the measurement of trachea and bronchi volumes on PMCT could be an objective way to estimate the time of death. Conclusions: While the air content of the lungs decreased, the volume of the trachea and bronchi temporarily increased after death, indicating the potential to use such measurements to estimate time of death.

Fulminant group A streptococcal infection without gangrene in the extremities: Analysis of five autopsy cases.

Five autopsy cases of fulminant group A streptococcal infection without gangrene in the extremities are presented. Clinical course of the fulminant illness was short (2-4 days). One pathological autopsy case was aged (86-years-old), and hemorrhagic cystitis was observed. The other four forensic autopsy cases were young (24-38 years-old) with the mean age of 32, and the primary infective lesions were located in the postpartum endometrium, tonsil and bronchus (2 cases). Systemic coccal dissemination with poor neutrophilic reaction was seen in two of five cases. Bilateral renal cortical necrosis was noted in three cases (including two with bacterial embolism). Hemophagocytosis, probably resulting from hypercytokinemia, was characteristic in three cases without bacterial embolism. Gram-positive cocci colonizing the hemorrhagic and necrotizing lesions were consistently immunoreactive for streptococcal antigens and Strep A (a carbohydrate antigen on group A streptococci). Neutrophilic reaction was mild in the primary infected foci. Clinicians should note that fulminant streptococcal infection (streptococcal toxic shock syndrome) in young and immunocompetent patients may not be associated with gangrene in the extremities. Autopsy prosecutors (diagnostic and forensic pathologists) must recognize the difficulty in making an appropriate autopsy diagnosis, particularly when bacterial embolism is not associated.

Methylation analysis of circadian clock gene promoters in forensic autopsy specimens.

DNA methylation in gene promoter regions influences gene expression. Circadian clock genes play an important role in the formation of a biological clock and aberrant methylation of these genes contributes to several disorders. In this study, we examined forensic autopsy specimens to determine whether DNA methylation status in the promoter regions of nine circadian clock genes (Per1, Per2, Per3, Cry1, Cry2, Bmal1, Clock, Tim, and Ck1e) is related to a change in acquired diathesis and/or causes of death. Methylation-specific PCR and direct sequencing methods revealed that the promoters of Per1, Cry2, Bmal1, Clock, and Ck1e were unmethylated in all the forensic autopsy specimens, while the promoters of Per2, Per3, Cry1, and Tim were partially methylated. Methylation status varied between individuals and between tissues in the same patient. A detailed analysis of methylation patterns in the Cry1 promoter region revealed that the patterns also varied between individuals and the Cry1 promoter had highly methylated patterns in two cases that had been exposed to methamphetamine. These results suggest that the methylation status of clock gene promoters varies between individuals. Methamphetamine use may influence methylation in the Cry1 gene promoter region and disturb circadian rhythmicity.

Diplotype analysis of the human cardiac sodium channel regulatory region in Japanese cases of sudden death by unknown causes.

Inherited mutations in the human cardiac sodium channel (SCN5A) gene cause arrhythmogenic diseases such as tachyarrhythmia and bradyarrhythmia. Moreover, mutation subsets in the coding region impair SCN5A function, potentially leading to sudden cardiac death (SCD). In the present study, we performed diplotype analysis of the regulatory region of the SCN5A gene in Japanese people who died suddenly because of an unknown cause (sudden death group; n=70) and controls (n=112). There were no significant differences at six polymorphic loci between the groups. However, 38 diplotypes of 6-nucleotide polymorphism variants were identified. One of these diplotypes-Dip.D (CTG-TC/CCG-TC)-occurred significantly more frequently in the sudden death group than in the controls (p<0.01, OR=5.18, 95% CI: 1.38-19.45). Dip.D has two variants (T-1062C and T-847G), and while it is unclear whether these directly affect mRNA expression, a common polymorphism in this region modulates SCN5A expression in vitro. Our results thus suggest that the transcription of the SCN5A Dip.D variant may be associated with arrhythmogenic diseases that can induce sudden death.

Association between the GST genetic polymorphisms and methamphetamine abusers in the Japanese population.

Glutathione S-transferase (GST) plays a major role in the detoxification of many compounds by conjugation with glutathione. GSTM1 and T1, which are important members of the GST multigene family, are polymorphic in humans. Complete deletion of the gene results in the null genotype and loss of function. However, it is not clear whether deletion of this gene is associated with a vulnerability to methamphetamine (MAP) abuse. To clarify the potential role and mechanisms of genetic polymorphisms of GSTM1 and T1 in susceptibility to MAP abuse in the Japanese population, we investigated GSTM1 and T1 polymorphisms in subjects with diagnosed MAP-related disorders and in control groups. The risk of MAP abuse associated with GSTM1 null genotype was significantly higher only in females than in subjects with the GSTM1 genotype. GSTM1 and GSTT1 null genotype combined conferred increased risk for MAP abuse compared with GSTM1 and GSTT1 genotype combined. In conclusion, we found that GSTM1 gene deletion may contribute to a vulnerability to MAP abuse in female subjects. Moreover, we identified an association between GSTM1 and GSTT1 null genotype combined and risk of MAP abuse in the Japanese population.

Cytoplasmic vacuolation in cultured rat astrocytes induced by an organophosphorus agent requires extracellular signal-regulated kinase activation.

There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders.

Y-chromosomal short tandem repeats haplotyping from vaginal swabs using a chelating resin-based DNA extraction method and a dual-round polymerase chain reaction.

Reported are 2 autopsy cases in which Y-chromosomal microsatellite short tandem repeats DYS19, DYS389I and II, DYS390, and DYS393 could be haplotyped with vaginal swabs by using a Chelex 100-based DNA extraction method and dual-round polymerase chain reaction. The extraction of DNA from vaginal swabs by using this method was as efficient or more efficient than using proteinase K and phenol-chloroform extraction or the alkaline lysis methods. Y-chromosomal microsatellite short tandem repeats haplotyping based on the dual-round polymerase chain reaction method provided genotypes from all the loci determined. Although amplification of Y-chromosomal microsatellite short tandem repeats loci is not directly involved in the existence of spermatozoa, it is considerably advantageous for male individualization from body fluid mixture stains in criminal cases.

Development of forensic diagnosis of acute sarin poisoning.

On March 20, 1995, the Tokyo subway system was subjected to a horrifying terrorist attack with sarin gas (isopropyl methylphosphonofluoridate) that left 12 persons dead and over 5000 injured. In order to diagnose the definite cause of death of the victims, a new method was developed to detect sarin hydrolysis products in the erythrocytes and formalin-fixed cerebella from four victims of sarin poisoning. Sarin-bound acetylcholinesterase (AChE) was solubilized from the specimens of sarin victims and digested with trypsin. The sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion. The digested sarin hydrolysis products were subjected to trimethylsilyl derivatization and detected by gas chromatography-mass spectrometry. Sarin hydrolysis products were detected in all sarin poisoning victims.

Chelating resin-based extraction of DNA from dental pulp and sex determination from incinerated teeth with Y-chromosomal alphoid repeat and short tandem repeats.

A procedure utilizing Chelex 100, chelating resin, was adapted to extract DNA from dental pulp. The procedure was simple and rapid, involved no organic solvents, and did not require multiple tube transfers. The extraction of DNA from dental pulp using this method was as efficient, or more so, than using proteinase K and phenol-chloroform extraction. In this study, the Chelex method was used with amplification and typing at Y-chromosomal loci to determine the effects of temperature on the sex determination of the teeth. The extracted teeth were incinerated in a dental furnace for 2 minutes at 100 degrees C, 200 degrees C, 300 degrees C, 400 degrees C, and 500 degrees C. After the isolation of DNA from the dental pulp by the Chelex method, alphoid repeats, and short tandem repeats, the human Y chromosome (DYZ3), DYS19, SYS389, DYS390, and DYS393 could be amplified and typed in all samples incinerated at up to 300 degrees C for 2 minutes. The DYS389 locus in some samples could not be amplified at 300 degrees C for 2 minutes. An autopsy case is described in which genotypings of DYS19, DYS390, and DYS393 from dental pulp obtained from a burned body were needed. The data presented in this report suggest that Chelex 100-based DNA extraction, amplification, and typing are possible in burned teeth in forensic autopsy cases.

Personal identification from human remains by mitochondrial DNA sequencing.

The authors report four cases in which severely damaged human remains were identified by mitochondrial DNA (mtDNA) sequencing. Degraded DNA was extracted from highly adipoceratous tissues using the phenol-chloroform method and polymerase chain reaction amplified for sequencing of two hypervariable regions, hypervariable region 1 and hypervariable region 2, of mitochondrial DNA. They also sequenced these regions of blood samples that were obtained from the presumptive mother or sister of the human remains. The sequencing results were compared with each other and with the Anderson's sequence. It was concluded from the sequence data that a lower part of a body in case 1 and some organs in case 2 were from the same woman, and a human head in case 3 and a female body in case 4 were from the relative of a presumptive mother and a sister, respectively.

Utility of Y-STR haplotype and mtDNA sequence in personal identification of human remains.

The utility of Y-STR haplotype and mtDNA sequence in personal identification from human remains is demonstrated. In five cases, severely damaged human remains were identified by Y-chromosomal short tandem repeat (STR) (Y-STR) haplotyping and mitochondrial DNA (mtDNA) sequencing. DNA was extracted from highly adipoceratous tissues using the phenol-chloroform method and polymerase chain reaction amplified for Y-STR haplotyping and sequencing of two hypervariable regions, HV1 and HV2, of the mtDNA. The authors also profiled the Y-STR haplotype and mtDNA sequence of a blood sample that was obtained from the presumptive brother of the decedent. Y-STR haplotyping and mtDNA sequencing results were compared with one another and with the Anderson sequence. It was concluded from these data that the lower part of the body (Case 1), the human head (Case 2), the upper part of the body (Case 3), and the right arm (Case 4) were from the same individual, whereas another lower part of a body (Case 5) was from a relative of the presumptive brother. A novel descriptive way of a statistical characteristic, gene or haplotype diversity, is shown.

An autopsy case of poisoning by massive absorption of cresol a short time before death.

A 65-year-old male patient who was hospitalized with schizophrenia died about 15 min later after ingestion of a large volume of saponated cresol solution in a mental hospital. Fatal levels of free p- and m-cresol in the heart blood were detected at 458.8 and 957.3 microg/ml, respectively, which far exceeded the fatal levels determined previously. The levels in the heart muscle, liver and spleen tissues were also extremely high, and there was 250 ml of cresol-odor-emitting fluid in the stomach. The levels of glucuronic-acid-conjugated p- and m-cresols in the heart blood were 38.2 and 85.6 microg/ml, respectively. Although the high levels of cresols in the heart blood may be due to diffusion from the stomach contents, it is surmised that the essential levels of free and conjugated forms in blood were at least 99 and 240 microg/ml, respectively, considering the results of postmortem examinations and some case reports. It was concluded that about 340 microg/ml of the total cresols was absorbed in a very short period following oral ingestion of saponated cresol solution in this case.

Y-STR haplotype data and allele frequency of the DXS10011 locus in a Japanese population sample.

The distribution of allele frequency of X-chromosomal STR, DXS10011, from 99 unrelated Japanese, 72 male and 27 female, were determined by PCR amplification and PAGE. At the same time, haplotype frequencies of five Y-chromosomal STR loci, DYS19, DYS389I, DYS389II, DYS390 and DYS393 from male samples were determined.

Mitochondrial sequence haplotype in the Japanese population.

Sequence polymorphysms of the mitochondrial DNA (mtDNA) control region, hypervariable regions I and II, from 50 unrelated Japanese were determined by PCR amplification and cycle sequencing.