Carnitine palmitoyltransferase 2 gene polymorphism is a genetic risk factor for sudden unexpected death in infancy.

RATIONALE: Carnitine palmitoyltransferase (CPT) II is one of a pivotal enzyme in mitochondrial fatty acid oxidation, which is essential for energy production during simultaneous glucose sparing and a requirement for major energy supply, such as prolonged fasting or exercise. When infants require more energy than provided by the glycolytic system, they rely on the mitochondrial fatty acid oxidation pathway. Mutations of the CPT2 gene have been reported to cause sudden unexpected death in infancy (SUDI). A thermolabile phenotype of a CPT2 polymorphism (F352C) has been recently reported to reduce CPT II enzyme activity. The F352C variant results in energy crisis at high temperature and is suspected as a risk factor for acute encephalopathy. However, a relationship between CPT2 gene polymorphism and SUDI has not been described. METHODS: Single nucleotide polymorphisms of the CPT2 gene were investigated among 54 SUDI cases and 200 healthy volunteers. RESULTS: The frequency of the C allele was significantly higher in the SUDI group than in the control group [25.0% vs 16.0%, odds ratio (OR)=1.75, 95% confidence interval (CI)=1.05-2.92, p=0.030). The frequency of the F352C homozygote was significantly higher in the SUDI group than in control group (11.1% vs 3.5%, OR=3.45, 95% CI=1.11-10.73, p=0.036). CONCLUSION: The F352C CPT2 variant might be a genetic risk factor for SUDI.

Autopsy case of sudden maternal death from thrombotic thrombocytopenic purpura.

A 31-year-old pregnant woman was transferred to the emergency room at 27 weeks of gestation. She had one-day history of fever and upper abdominal pain. Soon after admission, she underwent cardiopulmonary arrest. Autopsy was performed and multiple microthrombi were seen within the small-caliber vessels of many organs, but not in the lungs. Immunohistochemical staining revealed that the thrombi were rich in von Willebrand factor. We also obtained results which showed severely deficient plasma a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS) 13 activity and positive ADAMTS13 inhibitor, confirming a diagnosis of thrombotic thrombocytopenic purpura. As far as we know, in Japan, this is the first autopsy report of sudden maternal death from thrombotic thrombocytopenic purpura. We expect that the routine laboratory application of ADAMTS13 assays for unknown thrombocytopenic patients during pregnancy may help in differential diagnosis at an earlier stage of the disease and facilitate tailor-made therapeutic intervention.

Latent adrenal Ewing sarcoma family of tumors: A case report.

Ewing sarcoma family of tumors (ESFT) is derived from the neural crest, which originates from basal embryo cells in the primitive neural tube. ESFT often arises at the bone, chest wall, and soft tissues of the thoracic region. However, ESFT that arises from the adrenal gland is much rarer and it is usually revealed by clinical symptoms. We report an autopsy case of suicidal hanging, in which adrenal ESFT was incidentally revealed. To our knowledge, this is the first case of latent ESFT arising from the adrenal gland. Autopsy can sometimes reveal latent disease. Some of these latent diseases are very rare and we would not be able to detect them without a complete autopsy. As forensic pathologists, we should attempt to perform a complete autopsy and report new discoveries for the development of medicine.

Metabolic autopsy with postmortem cultured fibroblasts in sudden unexpected death in infancy: diagnosis of mitochondrial respiratory chain disorders.

Mitochondrial respiratory chain disorders are the most common disorders among inherited metabolic disorders. However, there are few published reports regarding the relationship between mitochondrial respiratory chain disorders and sudden unexpected death in infancy. In the present study, we performed metabolic autopsy in 13 Japanese cases of sudden unexpected death in infancy. We performed fat staining of liver and postmortem acylcarnitine analysis. In addition, we analyzed mitochondrial respiratory chain enzyme activity in frozen organs as well as in postmortem cultured fibroblasts. In heart, 11 cases of complex I activity met the major criteria and one case of complex I activity met the minor criteria. In liver, three cases of complex I activity met the major criteria and four cases of complex I activity met the minor criteria. However, these specimens are susceptible to postmortem changes and, therefore, correct enzyme analysis is hard to be performed. In cultured fibroblasts, only one case of complex I activity met the major criteria and one case of complex I activity met the minor criteria. Cultured fibroblasts are not affected by postmortem changes and, therefore, reflect premortem information more accurately. These cases might not have been identified without postmortem cultured fibroblasts. In conclusion, we detected one probable case and one possible case of mitochondrial respiratory chain disorders among 13 Japanese cases of sudden unexpected death in infancy. Mitochondrial respiratory chain disorders are one of the important inherited metabolic disorders causing sudden unexpected death in infancy. We advocate metabolic autopsy with postmortem cultured fibroblasts in sudden unexpected death in infancy cases.

Case report of death from falling: Did heart tumor cause syncope?

A healthy man in his 30s was working on the balustrade of stairs on the second floor. He suddenly fell downstairs without saying anything. On emergency hospitalization, chest echogram showed left hemothorax. Cardiac echogram showed a floating mass from the mitral valve in the left ventricle and severe mitral regurgitation. Surgery for hemothorax and pulmonary contusion was immediately undertaken. However, bleeding from pulmonary contusion could not be controlled and he underwent cardiopulmonary arrest. Autopsy showed a white, elastic, pendulous mass in the left atrium and a white mass in the lower lobe of the left lung. Tumor histology showed a reticular pattern, Schiller-Duval bodies, eosinophilic hyaline globules, and positive staining for α-fetoprotein. We diagnosed primary lung yolk sac tumor with metastatic intracardiac yolk sac tumor, a rare and highly malignant germ cell tumor. It usually arises in the ovaries and testes, and intracardiac yolk sac tumor is rare. Intracavitary tumors induce obstruction of inflow into and outflow from the ventricular cavity. The most common clinical presentation is dyspnea and syncope. In the present case, metastatic cardiac yolk sac tumor might have disturbed cardiac outflow and affected hemodynamics, probably causing syncope. Unfortunately, he was in a high place at that time and fell to receive pulmonary contusion that led to death. Autopsy may sometimes reveal latent diseases which might be related to the cause of death. We should perform autopsy thoroughly to diagnose not only the cause of death but also the factors leading to death.

Retrospective review of Japanese sudden unexpected death in infancy: the importance of metabolic autopsy and expanded newborn screening.

Sudden unexpected death in infancy is defined as sudden unexpected death occurring before 12 months of age. The common causes of sudden unexpected death in infancy are infection, cardiovascular anomaly, child abuse, and metabolic disorders. However, the many potential inherited metabolic disorders are difficult to diagnose at autopsy and may therefore be underdiagnosed as a cause of sudden unexpected death in infancy. In the present study we retrospectively reviewed 30 Japanese sudden unexpected death in infancy cases encountered between 2006 and 2009 at our institute. With postmortem blood acylcarnitine analysis and histological examination of the liver, we found two cases of long-chain fatty acid oxidation defects. Molecular analysis revealed that the one patient had a compound heterozygote for a novel mutation (p.L644S) and a disease-causing mutation (p.F383Y) in the carnitine palmitoyltransferase 2 gene. Furthermore, retrospective acylcarnitine analysis of the newborn screening card of this patient was consistent with carnitine palmitoyltransferase II deficiency. Metabolic autopsy and expanded newborn screening would be helpful for forensic scientists and pediatricians to diagnose fatty acid oxidation disorders and prevent sudden unexpected death in infancy.

Methamphetamine directly accelerates beating rate in cardiomyocytes by increasing Ca(2+) entry via L-type Ca(2+) channel.

Methamphetamine induces several cardiac dysfunctions, which leads to arrhythmia, cardiac failure and sudden cardiac death. Although these cardiac alterations elicited by methamphetamine were thought to be due to an indirect action of methamphetamine, namely, an excessive catecholamine release from synaptic terminals, while it seems likely that methamphetamine directly modulates the functioning of cardiomyocytes independent of neurotransmitters. However, the direct effects of methamphetamine on cardiomyocytes are still not clear. We show that methamphetamine directly accelerates the beating rate and alters Ca(2+) oscillation pattern in cultured neonatal rat cardiomyocytes. Adrenergic receptor antagonists did not block the methamphetamine-induced alterations in cardiomyocytes. Treatment with a ryanodine receptor type 2 inhibitor and a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor did not affect these responses, either. In contrast, the L-type Ca(2+) channel inhibitor nifedipine eradicated these responses. Furthermore, methamphetamine elevated the internal free Ca(2+) concentration in HEK-293T cells stably transfected with the L-type Ca(2+) channel alpha1C subunit. In neonatal rat cardiomyocytes, methamphetamine accelerates beating rate and alters Ca(2+) oscillation pattern by increasing Ca(2+) entry via the L-type Ca(2+) channels independent of any neurotransmitters.

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.

A case of diffuse axonal injury in violent death.

A 59-year-old man was carried to the hospital by three men. The deceased was unconscious at admission and his face was severely swollen with many subcutaneous hemorrhages and extensive edema. His death was confirmed 17 min after resuscitation. A judicial autopsy was performed the next day. Findings showed that the victim's face and head were reddish and swollen, and that subscalp bleeding was ubiquitous, but no skull fracture, epi- and subdural hematoma or subarachnoidal bleeding was observed. The brain itself was severely edematous but no bleeding was found. Although small hemorrhages were seen in the limbs and back, there were no marked wounds except to the head. To determine the cause of death, we performed a microscopic histochemical examination. Conventional H.E. staining disclosed eosinophilic change, concentration of nuclei, edema, gliosis, and oozing at the corpus callosum. To identify further details of the cause of death, we used Bodian staining, Kluver-Barrera staining, anti-beta amyloid immunostaining, and anti-neurofilament immunostaining. We found sinusoidal swelling of axons and waving axons, which are typical findings of Diffuse Axonal Injury (DAI), but no positive staining of beta amyloid. Focal lesions of the corpus callosum and of the dorsolateral quadrant of the rostral brain stem, and diffuse damage to axons are considered to constitute the DAI triad. We therefore diagnosed the cause of death as DAI. Our experience shows that it is important to use several staining methods for diagnosis of a variety of neuronal degenerative disorders. Several days later, we were informed by the police that several men had hit and kicked the victim in an attempt to lynch him. To compare with this case, we also report two other cases in which DAI was observed.

The present and future situation of "model project for investigation and analysis of medical practice associated deaths" in Osaka, Japan.

To enhance the quality and safety of medical care, the Ministry of Health, Labor and Welfare (MHLW) launched a model project in September 2005 for investigation and analysis of medical practice associated deaths in an attempt to move the existing system in a different direction. The project, initiated in Tokyo, Osaka, Nagoya, and Kobe, has now been implemented in nine prefectures. In the hope that the model project will lead to the nationwide development of medical safety investigating committees, the MHLW has submitted a provisional third plan. Based on our practical experience of the model project in Osaka, we present and discuss practical problems and legal issues involving surgeons' criminal punishment.

Variants of the melanocortin 1 receptor gene (MC1R) and P gene would be effective for estimating the population origin of an individual.

The population origin of an individual is often required to be determined from specimens left at a crime scene for estimating a suspect and individual identity. The melanocortin 1 receptor gene (MC1R) and P gene are associated with human pigmentation. Although there have been several reports that these genes are highly polymorphic in human populations, it is unclear if the allele variants can be used to estimate the population origin of an individual. We aimed to estimate the ethnic origin of a particular individual by using single nucleotide polymorphisms (SNPs). Four SNPs (MC1R gene: R163Q and P gene: IVS5+1001, IVS13+113 and H615R) were genotyped in 394 volunteers from 4 ethnically defined populations using a PCR-based assay. The results revealed that the allele variants were present with high frequency in Asian populations but were low in European and African populations. On the basis of these results, we defined a specific combination of a genotype (R163Q) and a diplotype group (IVS5+1001, IVS13+113 and H615R). This study indicates that the specific combination of a genotype and a diplotype group would be effective for estimating the population origin of an individual from a list of population groups.

Misidentification of ethyl chloride in the routine GC-FID analysis for alcohol.

UNLABELLED: GC-FID is the method of choice for alcohol screening and quantitative analysis in modern forensic medical practice. Although specific enough for routine use, some results could be misleading. In the current article we present a case of sexual asphyxia with drug and volatile substance abuse. Toxicological analysis revealed the presence of methamphetamine at a concentration of 1.3 microg/mL in blood. An ethanol-like peak was detected during our routine GC-FID test for alcohol (methylethylketone IS). Subsequent GC-MS analysis identified the peak as ethyl chloride. Levels of 0.05 mg/mL in blood and 0.01 mg/mL in urine were measured. Two facts proved misleading in our case. First: very small difference of 0.027 between the ethyl chloride and ethanol peaks in relative retention times at the GC-FID chromatograms. Second: missing evidence for the use of ethyl chloride at the scene-neither cans of the substance were found, nor such information was available otherwise. CONCLUSION: there is a substantial risk for mistaking ethyl chloride for ethanol, when ethyl chloride abuse is unanticipated. In the case of slightest uncertainty a GC-MS analysis should be employed to reliably determine the actual substance.

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.

Variants of the melanocortin 1 receptor gene (MC1R) and P gene as indicators of the population origin of an individual.

The population origin of an individual is often requested to be determined from specimens left at a crime scene for identifying a suspect and individual identity. The melanocortin 1 receptor gene (MC1R) and P gene are associated with human pigmentation. Although several studies have reported that these genes are highly polymorphic in human populations, it is unclear if the allele variants can be used to determine the population origin of an individual. We aimed to determine the ethnic origin of an individual by using single nucleotide polymorphisms (SNPs). Eighteen SNPs in the MC1R gene and P genes were genotyped in 52 individuals by the direct sequencing method, and 4 SNPs (MC1R gene: R163Q and P gene: IVS5 + 1001, IVS13 + 113, and H615R) were selected on the basis of differences in frequencies. Subsequently, we genotyped these four SNPs in 422 volunteers from six ethnically defined populations using a polymerase chain reaction-based assay. The results revealed that the allele variants were present with high frequencies in Asian populations but were low in European and African populations. On the basis of these results, we defined a specific combination of a genotype (R163Q) and a diplotype group (IVS5 + 1001, IVS13 + 113, and H615R). This study indicates that the specific combination of a genotype and a diplotype group would be effective in estimating the population origin of an individual from a list of population groups.

Cardiac ion channel gene mutations in sudden infant death syndrome.

Sudden infant death syndrome (SIDS) is multifactorial and may result from the interaction of a number of environmental, genetic, and developmental factors. We studied three major genes causing long QT syndrome in 42 Japanese SIDS victims and found five mutations, KCNQ1-K598R, KCNH2-T895M, SCN5A-F532C, SCN5A-G1084S, and SCN5A-F1705S, in four cases; one case had both KCNH2-T895M and SCN5A-G1084S. All mutations were novel except for SCN5A-F532C, which was previously detected in an arrhythmic patient. Heterologous expression study revealed significant changes in channel properties of KCNH2-T895M, SCN5A-G1084S, and SCN5A-F1705S, but did not in KCNQ1-K598R and SCN5A-F532C. Our data suggests that nearly 10% of SIDS victims in Japan have mutations of the cardiac ion channel genes similar to in other countries.

Determination of a newly encountered designer drug "p-methoxyethylamphetamine" and its metabolites in human urine and blood.

A newly synthesized designer drug, para-methoxyethylamphetamine (PMEA) was unexpectedly detected in the postmortem specimens of fatality involving drug intoxication in 2005, Japan. For unequivocal identification, the isomeric discrimination of PMEA and its positional-isomers was performed by GC/MS with the trifluoroacetylation. In order to prove the intake of PMEA, the characteristic metabolites of PMEA were also identified by GC/MS analysis of the urine specimen with trifluoroacetylation. As a result, para-methoxyamphetamine, para-hydroxyethylamphetamine (POHEA) and para-hydroxyamphetamine were identified as the major metabolites of PMEA. For the quantitative analyses of PMEA and its three metabolites in body fluids, an automated column-switching LC/MS procedure was developed, and applied to the postmortem blood and urine specimens. In this fatal case, blood concentration of PMEA was estimated to be 12.2 microg/mL and this level seemed extremely high in comparison with lethal blood-levels of its analogues, representing acute-intoxication of the victim. Based on the quantitative results, PMEA was found to be extensively metabolized to POHEA via O-demethylation, partly followed by its conjugation.

Methamphetamine-related sudden death with a concentration which was of a 'toxic level'.

We reviewed 32 cases where a forensic autopsy detected methamphetamine in the blood, and all of these autopsies were performed at two institutes between 1991 and 2003. In accordance with several criteria, the blood concentration in 11 cases was classified as above the toxic level, and 10 of these cases were diagnosed as methamphetamine poisoning. In 20 cases (62.5% of total cases), the blood concentration was of a 'toxic level', and 10, 2 and 1 of these cases were diagnosed as methamphetamine poisoning, cardiomyopathy and intracerebral hemorrhage, respectively. Since it is unclear how the effects of methamphetamine may contribute to the death of an individual, a diagnosis of the exact cause of death is often difficult to make in cases where the blood concentration of methamphetamine was of a 'toxic level'. Therefore, a diagnosis has to be carefully made in consideration of the pathological findings, the pharmacological effects of methamphetamine and the process until death in such cases. Additionally, the mechanism of methamphetamine-related death needs to be more fully studied to enable an appropriate diagnosis to be made easily.

Sensitive detection of polyalanine expansions in PHOX2B by polymerase chain reaction using bisulfite-converted DNA.

Congenital central hypoventilation syndrome, also known as Ondine's curse, is characterized by idiopathic abnormal control of respiration during sleep. Recent studies indicate that a polyalanine expansion of PHOX2B is relevant to the pathogenesis of this disorder. However, it is difficult to detect the repeated tract because its high GC content inhibits conventional polymerase chain reaction (PCR) amplification. Here, we describe a bisulfite treatment for DNA in which uracil is obtained by deamination of unmethylated cytosine residues. Deamination of DNA permitted direct PCR amplification that yielded a product of 123 bp for the common 20-residue repetitive tract with replacement of C with T by sequencing. It settled allele dropouts accompanied by insufficient amplification of expanded alleles. The defined procedure dramatically improved detection of expansions to 9 of 10 congenital central hypoventilation syndrome patients examined in a previous study. The chemical conversion of DNA before PCR amplification facilitates effective detection of GC-rich polyalanine tracts.

Sudden infant death syndrome is not associated with the mutation of PHOX2B gene, a major causative gene of congenital central hypoventilation syndrome.

Sudden infant death syndrome (SIDS) is a major cause of infant death, but its etiology is unknown. There are several independent risk factors for SIDS, and prone sleeping is a major risk factor. SIDS is probably based on a compromise in arousal response to breathing or blood pressure during sleep. Congenital central hypoventilation syndrome (CCHS or Ondine's curse) is a disorder characterized by an idiopathic failure of the autonomic control of breathing and has been regarded as one of the compromised conditions in SIDS. Recently, mutations of the PHOX2B gene have been detected in half to two-thirds of CCHS patients. We therefore analyzed the PHOX2B gene in 23 cases of SIDS and did not find any mutations, except for three polymorphic nucleotidic substitutions. The mutation of PHOX2B is thus not likely associated with SIDS.