Takahiro Umehara

Autopsy report for a caffeine intoxication case and review of the current literature

Caffeine (1,3,7-trimethylxanthine) is a popular mild central nervous system stimulant found in the leaves, seeds and fruits of various plants and in foodstuffs such as coffee, tea, and chocolate, among others. Caffeine is widely used and is not associated with severe side effects when consumed at relatively low doses. Although rarely observed, overdoses can occur. However, only a few fatal caffeine intoxication cases have been reported in the literature. Herein, we report the pathological examination results and information on caffeine concentrations in the blood, urine and main organs in a fatal caffeine intoxication case. Even though high caffeine concentrations were found in the systemic organs, no caffeine-related pathological changes were detected.

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.

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 next generation sequencing in sudden unexpected death in infancy: Postmortem diagnosis of fatty acid oxidation disorders

The recent introduction of metabolic autopsy in the field of forensic science has made it possible to detect hidden inherited metabolic diseases. Since the next generation sequencing (NGS) has recently become available for use in postmortem examinations, we used NGS to perform metabolic autopsy in 15 sudden unexpected death in infancy cases. Diagnostic results revealed a case of carnitine palmitoyltransferase II deficiency and some cases of fatty acid oxidation-related gene variants. Metabolic autopsy performed with NGS is a useful method, especially when postmortem biochemical testing is not available.

Immunohistochemical detection of CD14 and combined assessment with CD32B and CD68 for wound age estimation

Estimation of wound age is a major topic of study for forensic pathologists, but few markers exist that can indicate a specific period 1-5 days postinfliction, and a method to estimate wound age with high accuracy has not yet been established. This study examined CD14 as such a marker in mouse skin wounds of different ages (0min and 1, 2, 3, 5, 7, and 9 days) and in human subjects (group 1, 0-1 day; group 2, 1-5 days; group 3, >7 days) using Western blot analysis and/or immunohistochemical staining. In addition, we evaluated a combination of immunohistochemical markers in human skin wounds using transmembrane proteins, CD14, CD32B, and CD68, expressed on inflammatory cells. The expression of CD14 was detected only during 1-5 days postinfliction and, thus, the evaluation of CD14-expressing cells could specify wound age during 1-5 days postinfliction in mouse skin wounds. The ratio of samples assessed to be CD14(+) was significantly high in human skin wounds in group 2. Combined assessment using the three markers increased the specificity of diagnosis and shortened the range of wound age, compared with the assessment using a single marker. Our results indicate that CD14 may be a useful marker of wound age, 1-5 days postinfliction, and that combined assessment with CD14, CD32B, and CD68 may be a good method for the accurate estimation of wound age.

Pasteurella multocida Septicemia in a Patient with Cirrhosis: An Autopsy Report

More people are keeping pets in their homes but may not be sufficiently aware of the potential danger from infections. We report an autopsy case of a 57-year-old man affected by cirrhosis. Septic shock with Pasteurella multocida pneumonia was the cause of his death. P. multocida was the source of infection via the respiratory tract and caused pneumonia. Cirrhosis is one of the risk factors for P. multocida infection. A detailed patient history about animal exposure should be obtained and a differential diagnosis of P. multocida infection must be kept in mind.

No association between cardiac ion channel variants and sudden infant death

The cause of sudden unexpected death (SUD) in infancy remains unknown. Recent molecular autopsy studies have identified inherited metabolic disease and arrhythmia as a possible cause of SUD, but in Japan there have been few studies on sudden infant death (Table S1). In our previous metabolic autopsy and arrhythmia-related molecular autopsy studies, we noted that some cases of SUD were caused by these diseases, but only a small number of cases involved disease-causing gene variants, and most cases were not explained by mutations of these genes. Some polymorphisms have been identified as being related to SUD in infancy. Serotonin transporter polymorphism was first reported by Narita et al. We also reported that the carnitine palmitoyltransferase2 variant is a risk factor. Other than these, there are variants of arrhythmia channel genes, which are detected in healthy controls and which affect the cardiac ion channel currents. There are no studies, however, on the genetic frequencies of these arrhythmia channel gene variants, and it is controversial as to whether these variants are associated with SUD in infancy. In the 71 cases in the previous study, six involved arrhythmia-related and five involved metabolic disease-related pathogenic variants. After these 11 cases were excluded, the remaining cases were defined as molecular autopsy-negative SUD. To determine whether disease-related polymorphisms are associated with SUD, we compared the frequencies between SUD cases and healthy controls. A total of 60 cases were analyzed as previously described. Six single-nucleotide variants were retrieved (Table 1). Further details are available in Table S2. This study was approved by the ethics committees of Nagasaki University Graduate School of Medicine and Fukuoka University School of Medicine, and performed in accordance with the Declaration of Helsinki. The genotype distributions are listed in Table 1. The estimated haplotype distributions in H558R, P1090L, and R1193Q-sodium voltage-gated channel alpha subunit 5 (R1193Q-SCN5A) are listed in Table S3. There were no significant differences between the groups. The most important problem when studying SUD is that it is a heterogeneous disease. In this sense, these 60 molecular autopsy-negative SUD cases were a purer group of SUD, given the exclusion of 11 cases in the previous molecular autopsy study. This study is the first report of molecular autopsy-negative SUD. There are several disease-related polymorphisms that do not directly cause the disease, but which might be genetic risk factors. The G38S-potassium voltage-gated channel subfamily E member 1 (G38S-KCNE1), G643S-KCNQ1, K897T-KCNH2, and H558R or P1090L-SCN5A substitutions were benign or likely benign in the NCBI database (http://www.ncbi.nlm.nih. gov/clinvar/), although some studies reported an effect on the cardiac currents. The R1193Q-SCN5A substitution is controversial. In the 60 molecular autopsy-negative cases, there were no significant differences between SUD cases and the controls. Thus, these variants are not significantly associated with molecular autopsy-negative SUD, and may not be genetic risk factors. Miura reported that H558R or P1090L-SCN5A does not affect the cardiac currents, but these haplotypes also with R1193Q-SCN5A might be at risk for Brugada syndrome. In this study, there were also no significant differences in the haplotype estimation. One patient had H558R and R1193QSCN5A variants, and the cause of death in this particular case might have been related to these variants, but, as a whole, these haplotypes were not risk factors for SUD in the present study. There were limitations in this study. First, the number of SUD cases was only 60, which is still too small to enable discussion of associations, although this study included the highest number of SUD in Japan. Second, there are many more candidate genes for sudden death, most of which were not Correspondence: Takuma Yamamoto, MD PhD, Division of Forensic Pathology and Science, Unit of Social Medicine, Course of Medical and Dental Sciences, Graduate School of Biomedical Sciences, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki, Nagasaki 852-8523, Japan. Email: t-yamamoto@nagasaki-u.ac.jp *Present address: Department of Legal Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan. Received 28 September 2017; revised 20 December 2017; accepted 14 February 2018. doi: 10.1111/ped.13532 Genetic analysis in sudden infant death 483

Identification of potential markers of fatal hypothermia by a body temperature-dependent gene expression assay

Diagnosis of fatal hypothermia is considered to be difficult in forensic practice and even if findings due to cold exposure are evident, cold exposure is not necessarily a direct cause of death. Identification of useful molecular markers for the diagnosis of fatal hypothermia has not been successful. In this study, to identify novel molecular markers that inform the diagnosis of fatal hypothermia, we focused on skeletal muscle, which plays a role in cold-induced thermogenesis in mammals. We made rat models of mild, moderate, and severe hypothermia and performed body temperature-dependent gene expression analysis in the iliopsoas muscle using next-generation sequencing (NGS). NGS showed that after severe hypothermia, the expression levels of 91 mRNAs were more than double those in mild and moderate hypothermia and control animals. Gene ontology (GO) analysis indicated that these mRNAs are involved in a number of biological processes, including response to stress and lipids, and cellular response to hypoxia. The expression of four genes [connective tissue growth factor (Ctgf), JunB proto-oncogene, AP-1 transcription factor subunit (Junb), nuclear receptor subfamily 4, group A, member 1 (Nr4a1), and Syndecan 4 (Sdc4)] and the level of one protein (CTGF) were induced only by severe hypothermia. These genes and protein are involved in muscle regeneration, tissue repair, and lipid metabolism. These results indicate that heat production to maintain body temperature in a process leading to fatal hypothermia might be performed by the iliopsoas muscle, and that Ctgf, Junb, Nr4a1, and Sdc4 genes are potential diagnostic markers for fatal hypothermia.

Targeting miR‐223 in neutrophils enhances the clearance of Staphylococcus aureus in infected wounds

Argonaute 2 bound mature microRNA (Ago2‐miRNA) complexes are key regulators of the wound inflammatory response and function in the translational processing of target mRNAs. In this study, we identified four wound inflammation‐related Ago2‐miRNAs (miR‐139‐5p, miR‐142‐3p, miR‐142‐5p, and miR‐223) and show that miR‐223 is critical for infection control. miR‐223Y/− mice exhibited delayed sterile healing with prolonged neutrophil activation and interleukin‐6 expression, and markedly improved repair of Staphylococcus aureus‐infected wounds. We also showed that the expression of miR‐223 was regulated by CCAAT/enhancer binding protein alpha in human neutrophils after exposure to S. aureus peptides. Treatment with miR‐223Y/−‐derived neutrophils, or miR‐223 antisense oligodeoxynucleotides in S. aureus‐infected wild‐type wounds markedly improved the healing of these otherwise chronic, slow healing wounds. This study reveals how miR‐223 regulates the bactericidal capacity of neutrophils at wound sites and indicates that targeting miR‐223 might be of therapeutic benefit for infected wounds in the clinic.