Molecular autopsy for sudden death in Japan.

Japan has various death investigation systems; however, external examinations, postmortem computed tomography, macroscopic examinations, and microscopic examinations are performed regardless of the system used. These examinations can reveal morphological abnormalities, whereas the cause of death in cases with non-morphological abnormalities can be detected through additional examinations. Molecular autopsy and postmortem genetic analyses are important additional examinations. They are capable of detecting inherited arrhythmias or inherited metabolic diseases, which are representative non-morphological disorders that cause sudden death, especially in infants and young people. In this review, we introduce molecular autopsy reports from Japan and describe our experience with representative cases. The relationships between drug-related deaths and genetic variants are also reviewed. Based on the presented information, molecular autopsy is expected to be used as routine examinations in death investigations because they can provide information to save new lives.

Association of troponin I and macrophages in cardiac tamponade with Stanford type A aortic dissection.

Background: Acute aortic dissection has a high mortality rate, especially for Stanford type A with a dissected ascending aorta. Cardiac tamponade is one of the most common complications of acute type A aortic dissection (ATAAD) and can cause death. However, the pathogenesis is often unclear. We aimed to examine laboratory findings at the onset of disease and macrophage involvement. Methods: Hematological and biochemical parameters, and D-dimer, brain natriuretic peptide (BNP), and high-sensitivity troponin I (hs-cTnI) levels in 70 patients with ATAAD at our hospital were investigated. Additionally, the myocardium and aorta after autopsy of an ATAAD case with cardiac tamponade were pathologically examined. Results: Forty-four ATAAD cases were complicated by cardiac tamponade. The mean age of patients with cardiac tamponade and proportion of patients over 70 years of age were both significantly higher than for those without cardiac tamponade. Evaluable D-dimer values were higher than 0.5 µg/mL in all patients. Significantly elevated laboratory parameters in patients with cardiac tamponade included: lactate dehydrogenase, aspartate aminotransferase, C-reactive protein, lactate, BNP, and hs-cTnI. However, multivariate analysis showed only hs-cTnI was significantly associated with cardiac tamponade. Histological examination revealed numerous M2-like macrophages infiltrating the myocardium and dissecting aorta, expressing CC chemokine ligand (CCL)2 together with vascular endothelial growth factor-C and matrix metalloproteinase-9. The peripheral monocyte-to-neutrophil ratio (MNR) was also significantly higher in cardiac tamponade. Conclusions: In ATAAD patients with cardiac tamponade, hs-cTnI was significantly elevated and CCL2 expression was observed, which may be involved in the expression of M2-like macrophages via an increased MNR.

circRNAs May Be Involved in Dysfunction of Neutrophils of Type 2 Diabetic Mice through Regulation of Specific miRNAs.

Diabetes is known to delay wound healing, and this delay is attributed to prolonged inflammation. We found that microRNAs (miRNAs) might be involved in the dysfunction of diabetic-derived neutrophils, and dynamics of neutrophil and chronic inflammation might be initiated by miRNA-regulated genes. Moreover, studies of miRNA function in nephropathy have suggested that circular RNAs (circRNAs), which function as sponges of miRNA to regulate their expression, are potential biomarkers and new therapeutic targets for the diagnosis of diabetic nephropathy. Accordingly, to investigate the molecular mechanism of the regulation of inflammation in diabetic-derived neutrophils, we identified circRNAs in diabetic-derived neutrophils obtained from BKS.Cg-Dock7m +/+ Leprdb/J (Leprdb/db and Leprdb/+) mice using microarrays. Neutrophils from pooled bone marrow of three diabetic and three non-diabetic mice were isolated and total RNA was extracted. Microarray analysis was performed using the Arraystar Mouse Circular RNA Array. The results showed that three circRNAs were significantly increased and six circRNAs were significantly decreased in diabetic-derived neutrophils compared with non-diabetic-derived neutrophils. The expressions of some circRNAs in diabetic-derived neutrophils were more than double those in non-diabetic-derived neutrophils. The circRNAs contain binding sites of miRNAs, which were differentially expressed in diabetic-derived neutrophils. Our results suggest that circRNAs may be involved in the regulation of inflammation in diabetic-derived neutrophils.

rno-miR-203a-3p and Mex3B contribute to cell survival of iliopsoas muscle via the Socs3-Casp3 axis under severe hypothermia in rats.

Forensic diagnosis of fatal hypothermia is considered difficult because no specific findings, such as molecular markers, have been identified. Therefore, determining the molecular mechanism in hypothermia and identifying novel molecular markers to assist in diagnosing fatal hypothermia are important. This study aimed to investigate microRNA (miRNA) and mRNA expression in iliopsoas muscle, which plays a role in homeostasis in mammals, to resolve the molecular mechanism in hypothermia. We generated rat models of mild, moderate, and severe hypothermia, then performed body temperature-dependent miRNA and mRNA expression analysis of the iliopsoas muscle using microarray and next-generation sequencing. Analysis showed that rno-miR-203a-3p expression was lower with decreasing body temperature, while Socs3 expression was significantly increased only by severe hypothermia. Luciferase reporter assays suggested that Socs3 expression is regulated by rno-miR-203a-3p. Socs3 and Mex3B small interfering RNA-mediated knockdown showed that suppressing Mex3B could induce the activation of Socs3, followed by a change in caspase 3/7 activity and adenosine triphosphate levels in iliopsoas muscle cells. These findings indicate that rno-miR-203a-3p and Mex3B are deactivated by a decrease in body temperature, whereby it contributes to suppressing apoptosis by accelerating Socs3. Accordingly, the rno-miR-203a-3p-Socs3-Casp3 or Mex3B-Socs3-Casp3 axis may be the part of the biological defense response to maintain homeostasis under extreme hypothermia.

Development of age-estimation formula using postmortem oral findings: A pilot study.

The goal of this pilot study was to develop an age-estimation formula and assess its effectiveness after evaluating individual intraoral findings. A total of 198 Japanese adults were included, and intraoral findings were collected from the corpses. To analyze the condition of each tooth, 20 items were established for intraoral findings, and seven tooth states were established. Logistic regression analysis was used to estimate the impact of age on each intraoral finding. Sequentially, linear regression was applied to verify the correlation between age and type of tooth, and multiple regression was used to correlate age-dependent factors. The intraoral findings with age dependency were tooth stump, edentulous jaw, attrition, no caries, dental prostheses, partial dentures, and complete dentures. Tooth stump, attrition, and dental prostheses showed positive multicollinearity. Missing tooth, extant tooth, normal teeth, and untreated lost teeth were age-correlated. Multiple regression analysis included age as the response variable and five factors as the explanatory variables in a new age-estimation formula, resulting in ± 10 years for 86.96% of cases (60-69 years old), 76.47% (70-79 years old), and 61.05% of all cases. The multiple correlation was 0.551, and the contribution rate of the multiple regression formula was 0.304. The accuracy of the proposed age-estimation formula was within ± 10 years for 61.05% of all subjects. However, the accuracy of age estimation in subjects aged 60-79 years was excellent (76.47-86.96%), which showed that this age-estimation formula would be effective for estimating the age of middle-aged to older subjects.

Accurate interpretation of genetic variants in sudden unexpected death in infancy by trio-targeted gene-sequencing panel analysis.

In sudden unexpected death in infancy cases, postmortem genetic analysis with next-generation sequencing potentially can extract candidate genes associated with sudden death. However, it is difficult to accurately interpret the clinically significant genetic variants. The study aim was to conduct trio analysis of cases of sudden unexpected death in infancy and their parents to more accurately interpret the clinically significant disease-associated gene variants associated with cause of death. From the TruSight One panel targeting 4813 genes we extracted candidate genetic variants of 66 arrhythmia-, 63 inherited metabolic disease-, 81 mitochondrial disease-, and 6 salt-losing tubulopathy-related genes in 7 cases and determined if they were de novo or parental-derived variants. Thirty-four parental-derived variants and no de novo variants were found, but none appeared to be related to the cause of death. Using trio analysis and an in silico algorithm to analyze all 4813 genes, we identified OBSCN of compound heterozygous and HCCS of hemizygous variants as new candidate genetic variants related to cause of death. Genetic analysis of these deceased infants and their living parents can provide more accurate interpretation of the clinically significant genetic variants than previously possible and help confirm the cause of death.

Body temperature-dependent microRNA expression analysis in rats: rno-miR-374-5p regulates apoptosis in skeletal muscle cells via Mex3B under hypothermia.

Forensic diagnosis of fatal hypothermia is considered difficult because there are no specific findings. Accordingly, exploration of novel fatal hypothermia-specific findings is important. To elucidate the molecular mechanism of homeostasis in hypothermia and identify novel molecular markers to inform the diagnosis of fatal hypothermia, we focused on microRNA expression in skeletal muscle, which plays a role in cold-induced thermogenesis in mammals. We generated rat models of mild, moderate, and severe hypothermia, and performed body temperature-dependent microRNA expression analysis of the iliopsoas muscle using microarray and quantitative real-time PCR (qRT-PCR). The results show that rno-miR-374-5p expression was significantly induced only by severe hypothermia. Luciferase reporter assay and qRT-PCR results indicated that Mex3B expression was regulated by rno-miR-374-5p and decreased with decreasing body temperature. Gene ontology analysis indicated the involvement of Mex3B in positive regulation of GTPase activity. siRNA analysis showed that Mex3B directly or indirectly regulated Kras expression in vitro, and significantly changed the expression of apoptosis-related genes and proteins. Collectively, these results indicate that rno-miR-374-5p was activated by a decrease in body temperature, whereby it contributed to cell survival by suppressing Mex3B and activating or inactivating Kras. Thus, rno-miR-374-5p is a potential supporting marker for the diagnosis of fatal hypothermia.

Generation of three induced pluripotent stem cell lines from postmortem tissue derived following sudden death of a young patient with STXBP1 mutation.

We established three iPSC lines from postmortem-cultured fibroblasts derived following the sudden unexpected death of an 8-year-old girl with Lennox-Gastaut syndrome, who turned out to have the R551H-mutant STXBP1 gene. These iPSC clones showed pluripotent characteristics while retaining the genotype and demonstrated trilineage differentiation capability, indicating their utility in disease-modeling studies, i.e., STXBP1-encephalopathy. This is the first report on the establishment of iPSCs from a sudden death child, suggesting the possible use of postmortem-iPSC technologies as an epoch-making approach for precise identification of the cause of sudden death.

Chronic Inflammation in Response to Injury: Retention of Myeloid Cells in Injured Tissue Is Driven by Myeloid Cell Intrinsic Factors.

Chronic inflammation is a hallmark of impaired healing in a plethora of tissues, including skin, and is associated with aging and diseases such as diabetes. Diabetic chronic skin wounds are characterized by excessive myeloid cells that display an aberrant phenotype, partially mediated by stable intrinsic changes induced during hematopoietic development. However, the relative contribution of myeloid cell-intrinsic factors to chronic inflammation versus aberrant signals from the local environmental was unknown. Moreover, identification of myeloid cell intrinsic factors that contribute to chronic inflammation in diabetic wounds remained elusive. Here we show that Gr-1+CD11b+ myeloid cells are retained specifically within the presumptive granulation tissue region of the wound at a higher density in diabetic mice and associate with endothelial cells at the site of injury with a higher frequency than in nondiabetic mice. Adoptive transfer of myeloid cells demonstrated that aberrant wound retention is due to myeloid cell intrinsic factors and not the local environment. RNA sequencing of bone marrow and wound-derived myeloid cells identified Selplg as a myeloid cell intrinsic factor that is deregulated in chronic wounds. In vivo blockade of this protein significantly accelerated wound healing in diabetic mice and may be a potential therapeutic target in chronic wounds and other chronic inflammatory diseases.

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.

Identification of Specific miRNAs in Neutrophils of Type 2 Diabetic Mice: Overexpression of miRNA-129-2-3p Accelerates Diabetic Wound Healing.

Neutrophils are involved in the first stage of acute inflammation. After injury, they are mobilized and recruited to the injured tissue. In diabetes, wound healing is delayed and aberrant, leading to excessive recruitment and retention of neutrophils that fail to promote angiogenesis and prolong inflammation. However, the exact pathological mechanisms of diabetic-derived neutrophils in chronic inflammation remain unclear. Here, miRNA profiling of neutrophils from bone marrow in type 2 diabetic mice was performed using a microarray. miRNAs regulate the posttranscriptional expression of target mRNAs and are important in countering inflammation-related diseases. Our study revealed that miRNAs exhibit differential expression in diabetic-derived neutrophils compared with non-diabetic-derived neutrophils, especially miR-129 family members. miR-129-2-3p directly regulated the translation of Casp6 and Ccr2, which are involved in inflammatory responses and apoptosis. Furthermore, miR-129-2-3p overexpression at the wound site of type 2 diabetic mice accelerated wound healing. These results suggest possible involvement of miR-129-2-3p in diabetic-derived neutrophil dysfunction and that retention kinetics of neutrophils and chronic inflammation may be initiated through miR-129-2-3p-regulated genes. This study characterizes changes in global miRNA expression in diabetic-derived neutrophils and systematically identifies critical target genes involved in certain biological processes related to the pathology of diabetic wound healing.

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.

Early diagnosis using canonical discriminant analysis of innate immune receptor gene expression profiles in a murine infectious or sterile systemic inflammation model.

BACKGROUND: Infection in patients with systemic inflammation is difficult to diagnose with a single biomarker. We aimed to clarify the time course of change in the gene expression profile of innate immune receptors in infectious or sterile inflammation and to establish an early diagnostic method using canonical discriminant analysis (CDA) of the gene expression profile. METHODS: To compare infectious and sterile inflammation, we used cecal ligation and puncture (CLP) and 20% full-thickness burn injury (Burn) models. C57BL/6 mice underwent sham treatment (n = 9 × three groups), CLP (n = 12 × three groups), or Burn (n = 12 × three groups) injury. Mice were killed at 6, 12, and 24 hours after injury, and total RNA was extracted from whole blood. We used quantitative real-time polymerase chain reaction to investigate gene expression of innate immune receptors Toll-like receptor 2 (TLR2), TLR4, TLR9, NLRP3 (nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3), and retinoic acid-inducible gene I. To evaluate all gene expression together as patterns, each value was standardized, and CDA was performed at each time point. RESULTS: Gene expression of TLR2 and TLR4 was already significantly increased in both CLP and Burn compared with sham mice at 6 hours after injury (p < 0.05). Gene expression of TLR9 was significantly decreased in CLP compared with sham and Burn mice at 12 hours and 24 hours after injury (p < 0.05) but not at 6 hours. Gene expression of NLRP3 was significantly increased in CLP and Burn compared with sham mice at 6 hours and 24 hours after injury (p < 0.05). In the CDA, each group showed distinctive gene expression patterns at only 6 hours after injury. Each group was clearly classified, and the classification error rates were 0% at all of the time points. CONCLUSIONS: Canonical discriminant analysis of the gene expression profile of innate immune receptors could be a novel approach for diagnosing the pathophysiology of complicated systemic inflammation from the early stage of injury.

Temporal expression of chitinase-like 3 in wounded murine skin.

In forensic practice, it is important to diagnose wound age accurately. We analyzed the proteome of injured murine skin to identify a novel protein marker of wound age after recent injury. We used samples from 3 days after injury, with 0 days as the control. The proteins were separated with two-dimensional electrophoresis. Using mass spectrometry, we identified a protein, chitinase-like 3 (Chil3). Chil3 messenger RNA (mRNA) expression showed temporal changes, which included a peak increase at 2 days after injury. Next, we produced an anti-Chil3 antibody and confirmed its specificity with western blotting. Similar to the mRNA results, an analysis of temporal changes in Chil3 protein expression revealed a peak at 2 days after injury. We also investigated the time course of changes in Chil3 tissue localization using immunohistochemistry. Chil3 signals remained in the wounded area for up to 9 days. However, Chil3-positive cells were observed in the scab, the edge of the dermal layer, and neogenetic granulation tissue between 1 and 3 days. Thus, wound age can be histologically determined using the localization of Chil3 but not its general existence. Additionally, double-labeled fluorescent immunohistochemistry revealed that the Chil3-expressing cells were mainly neutrophils. These data show that Chil3 is expressed in neutrophils during the early stage of wound healing in mice; thus, Chil3 is a potential histological marker of 1-3-day-old wounds.

Postmortem genetic analysis of sudden unexpected death in infancy: neonatal genetic screening may enable the prevention of sudden infant death.

Tandem mass screening has recently been started in Japan, but genetic screening has yet to be widely performed in neonates and many unexpected deaths are still being reported. We previously reported two cases of sudden infant death that may have been prevented had newborn screening been performed. In this study, we retrospectively reviewed 71 cases of sudden infant death for 66 arrhythmia- and 63 metabolic disease-related genes to identify how many cases of sudden infant death may have been prevented had mass screening been performed. Next-generation sequencing revealed that six cases had arrhythmia-related gene variants and five cases had metabolic disease-related gene variants. Had genetic screening been performed in addition to biochemical and physiological screening during the neonatal period to identify those at risk of arrhythmia or metabolic disease, these infants could have been diagnosed and treated, preventing their deaths. As such, screening of newborns may prevent sudden infant death.

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.

Diabetes Inhibits Gr-1+ Myeloid Cell Maturation via Cebpa Deregulation.

Recruitment of innate immune cells from the bone marrow (BM) to an injury site is required for effective repair. In diabetes, this process is altered, leading to excessive recruitment and retention of dysfunctional myeloid cells that fail to promote angiogenesis, prolong inflammation, and block healing. The aberrant myeloid phenotype is partially mediated by stable intrinsic changes to developing cells in the BM that are induced by the diabetic (db) environment, but the exact mechanisms remain largely unknown. Here, we show that the db-derived Gr-1(+)CD11b(+) immature myeloid population has widespread misexpression of chromatin-remodeling enzymes and myeloid differentiation factors. Crucially, diabetes represses transcription of the key myeloid transcription factor CEBPA via diminished H3 Lys 27 promoter acetylation, leading to a failure in monocyte and granulocyte maturation. Restoring Cebpa expression by granulocyte colony-stimulating factor reverses the db phenotype and rescues myeloid maturation. Importantly, our data demonstrate a possible link between myeloid cell maturation and chronic inflammation.

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.

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.