Postmortem Identification of Genetic Variations Associated with Sudden Unexpected Death in Young People.

Sudden unexpected death in the young (SUDY) is a traumatic occurrence for their family; however, information on the genetic variations associated with the condition is currently lacking. It is important to carry out postmortem genetic analyses in cases of sudden death to provide information for relatives and to allow appropriate genetic counselling and clinical follow-up. This study aimed to investigate the genetic variations associated with the occurrence of SUDY in Japan, using next-generation sequencing (NGS). The study included 18 cases of SUDY (16 males, 2 females; age 15-47 years) who underwent autopsy, including NGS DNA sequencing for molecular analysis. A total of 168 genes were selected from the sequencing panel and filtered, resulting in the identification of 60 variants in cardiac disease-related genes. Many of the cases had several of these genetic variants and some cases had a cardiac phenotype. The identification of genetic variants using NGS provides important information regarding the pathogenicity of sudden death.

Bleeding-Source Exploration in Subdural Hematoma: Observational Study on the Usefulness of Postmortem Computed Tomography Angiography.

In a few cases, postmortem computed tomography angiography (PMCTA) is effective in postmortem detection of cortical artery rupture causing subdural hematoma (SDH), which is difficult to detect at autopsy. Here, we explore the usefulness and limitations of PMCTA in detecting the sites of cortical arterial rupture for SDH. In 6 of 10 cases, extravascular leakage of contrast material at nine different places enabled PMCTA to identify cortical arterial rupture. PMCTA did not induce destructive arterial artifacts, which often occur during autopsy. We found that, although not in all cases, PMCTA could show the site of cortical arterial rupture causing subdural hematoma in some cases. This technique is beneficial for cases of SDH autopsy, as it can be performed nondestructively and before destructive artifacts from the autopsy occur.

PROS1 variant in sudden death case of pulmonary embolism caused by calcification in the inferior vena cava: The importance of postmortem genetic analysis.

A Japanese man in his 30s died suddenly. Postmortem computed tomography and autopsy revealed a pulmonary embolism from an organizing thrombus in the inferior vena cava as the cause of death. Genomic analysis of congenital thrombophilia-related genes (i.e., SERPINC1, PROC, PROS1, F2, F5, PLG, and MTHFR) revealed a heterozygous variant of PROS1 (p.A139V), which has been reported in patients with congenital protein S deficiency. After a genetic conference that included forensic pathologists, molecular scientists, genetic researchers, genetic clinicians, and clinical physicians, the results of the genetic analysis were explained to the family. Biochemical analyses of protein S (PS) activity and total PS antigen levels were performed with samples from the deceased's family and genetic analysis was not performed until clinical symptoms appear. Herein we demonstrate the importance of genetic background in cases of a sudden death due to pulmonary embolism.

Detection and Morphological Analysis of Micro-Ruptured Cortical Arteries in Subdural Hematoma: Three-Dimensional Visualization Using the Tissue-Clearing Clear, Unobstructed, Brain/Body Imaging Cocktails and Computational Analysis Method.

One of the causes of bleeding in subdural hematoma is cortical artery rupture, which is difficult to detect at autopsy. Therefore, reports of autopsy cases with this condition are limited and hence, the pathogenesis of subdural hematoma remains unclear. Herein, for the detection and morphological analysis of cortical artery ruptures as the bleeding sources of subdural hematoma, we used the tissue-clearing CUBIC (clear, unobstructed, brain/body imaging cocktails and computational analysis) method with light-sheet fluorescence microscopy and reconstructed the two-dimensional and three-dimensional images. Using the CUBIC method, we could clearly visualize and detect cortical artery ruptures that were missed by conventional methods. Indeed, the CUBIC method enables three-dimensional morphological analysis of cortical arteries including the ruptured area, and the creation of cross-sectional two-dimensional images in any direction, which are similar to histopathological images. This highlights the effectiveness of the CUBIC method for subdural hematoma analysis.

The usefulness of postmortem computed tomography angiography for subdural hematoma caused by rupture of the cortical artery: A report of two autopsy cases and a literature review.

Acute subdural hematoma (SDH) occurs following severe head trauma with brain contusion or rupture of bridging veins. Conversely, SDH caused by rupture of a cortical artery without trauma or with minor trauma is also possible. Although over 180 cases of the latter SDH have been reported, they were predominantly diagnosed only during surgery, and therefore, no adequate histological evaluation has been performed. Therefore, essential etiology of this SDH type has remained unclear. In addition, the scarcity of autopsy cases may be attributed to arterial rupture being missed if the microscopic findings are too minimal to detect during autopsy. Here, we describe two autopsy cases of SDH of cortical artery origin. Extravasation on postmortem computed tomography angiography and arterial leakage on macroscopic observation during autopsy facilitated detection of the ruptured artery and allowed detailed histological evaluation of the ruptured artery and adjacent dura mater. The etiology of arterial rupture is briefly described on the basis of histopathological findings in this study and the available literature.

High-performance liquid chromatographic determination of chlorhexidine in whole blood by solid-phase extraction and kinetics following an intravenous infusion in rats.

This paper presents the extraction and analysis of chlorhexidine (CHX) from whole blood using solid-phase extraction (SPE) together with high-performance liquid chromatography (HPLC). Blood samples, spiked with chlorpromazine used as an internal standard, were fortified with sodium acetate buffer and purified with Bakerbond C(18) SPE columns. The columns were washed, dried, and eluted with experimental optimized solvent systems. The HPLC was performed using a Capcell Pak C(18) MG column (4.6 x 250-mm) and monitored at 260 nm, using a UV detector. A mobile phase consisting of acetonitrile/water (40:60 v/v), containing 0.05% trifluoroacetic acid, 0.05% heptafluorobutyric acid, and 0.1% triethylamine, was employed. The assay was linear over the range of 0.05 to 2.0 microg/g and the limit of detection was 0.01 microg/g for CHX in whole blood. At the concentration range of 0.05 to 2.0 microg/g, the recoveries ranged from 72% to 85%, and the intra- and interday precision, expressed as coefficient of variation, were less than 11% and 13%, respectively. Kinetic characteristics following an intravenous infusion of a CHX product, Maskin solution, at a dose of 15 mg/kg in rats were evaluated using the present method. The kinetic profiles of CHX conformed to a two-compartment model with an alpha half-life (of distribution) at 0.05 h and a beta half-life (of elimination) at 0.55 h in rats. The method is simple and reliable for the determination of CHX in blood samples and could be expected to apply to forensic and clinical specimens.

Ascaris lumbricoides found in ashore corpses from Korean peninsula to Japan.

Yearly, several reports of unknown boats and corpses brought by the Tsushima Current are found ashore Japanese coast. Niigata prefecture had the highest number of the drifting ashore corpses in Japan with 45.7% (16/35) in 2017. Corpses from North Korea, confirmed by documents and photos were autopsied and in 3/16 was possible to recover worms full of eggs, morphologically identified as ascarids. Further molecular analysis of ITS1, 5.8S rDNA and ITS2 sequences confirmed all specimens were Ascaris lumbricoides. The contamination level by Ascaris lumbricoides eggs in the coast, the health impact and consequences of the epidemiological bridging produced by this forced migration in public health should be investigated. Moreover, control of helminthiases might be a necessary task in North Korea.

Postmortem volume change of the spleen and kidney on early postmortem computed tomography: comparison with antemortem computed tomography.

PURPOSE: To clarify an early postmortem change, we investigated the volume changes of the spleen and kidney on postmortem CT compared with antemortem CT in the same patients. MATERIALS AND METHODS: We retrospectively evaluated the volumes of 56 spleens (56 cases) and 50 kidneys (25 cases) using antemortem and postmortem CT, which were performed within 168 min after death. We divided the cases of spleen analysis into a hemorrhagic group (n = 12) and a non-hemorrhagic group (n = 44). RESULTS: The volumes of the organs before and after death were 101.0 ± 70.9 (cm3, mean ± standard deviation) and 81.1 ± 57.8 in spleens, 120.3 ± 49.2 and 109.2 ± 39.2 in kidneys, respectively. Both spleens and kidneys shrank after death (p < 0.05). The volumes of spleens before and after death were 111 ± 66.5 and 67.5 ± 27.7 in the hemorrhagic group, and 98.2 ± 72.5 and 84.9 ± 63.3 in the non-hemorrhagic group, respectively. The median value of the ratio of postmortem splenic volume to antemortem volume in the hemorrhagic group (65.0%) was smaller than the one in the non-hemorrhagic group (90.5%) (p < 0.05). CONCLUSION: We demonstrated that spleens and kidneys significantly reduced in size after death. The rate of shrinkage of spleens in the hemorrhagic group significantly became larger than the one in the non-hemorrhagic group.

Assessment of a simple method of heart weight estimation by postmortem computed tomography.

BACKGROUND: Measurement of heart weight is important when investigating cause of death, but there is presently no satisfactory method of heart weight estimation by postmortem computed tomography (PMCT). METHOD: We investigated 33 consecutive cases that underwent both PMCT and autopsy between February 2008 and June 2014. Heart and left ventricular (LV) weights were calculated by PMCT morphometry. We used a simple method to estimate LV weight: We assumed that LV was an ellipsoid and multiplied its volume on PMCT with myocardial specific gravity. We then compared the various heart and LV weights using linear regression. The calculated and estimated LV weights on PMCT were also compared. RESULTS: It was not possible to predict heart weight at autopsy from PMCT (R2 = 0.53). However, heart weight at autopsy could be accurately predicted from LV weight calculated by PMCT (R2 = 0.77). In addition, there was a strong correlation between the estimated and calculated LV weights by PMCT (R2 = 0.92). Heart weight at autopsy could also be accurately predicted using the PMCT-estimated LV weight (R2 = 0.72). CONCLUSION: Heart weight at autopsy could be accurately predicted using a simple method in which LV volume was assumed to be an ellipsoid on PMCT.

An autopsy case of peliosis hepatis with X-linked myotubular myopathy.

This report describes the autopsy case of a 4-year-old boy who died from hepatic hemorrhage and rupture caused by peliosis hepatis with X-linked myotubular myopathy. Peliosis hepatis is characterized by multiple blood-filled cavities of various sizes in the liver, which occurs in chronic wasting disease or with the use of specific drugs. X-linked myotubular myopathy is one of the most serious types of congenital myopathies, in which an affected male infant typically presents with severe hypotonia and respiratory distress immediately after birth. Although each disorder is rare, 12 cases of pediatric peliosis hepatis associated with X-linked myotubular myopathy have been reported, including our case. Peliosis hepatis should be considered as a cause of hepatic hemorrhage despite its low incidence, and it requires adequate gross and histological investigation for correct diagnosis.

Cytochrome P450 2J2*7 polymorphisms in Japanese, Mongolians and Ovambos.

Human cytochrome P450 2J2 (CYP2J2) is abundant in cardiovascular tissue and active in the metabolism of arachidonic acid to eicosanoids that have potent vasodilatory properties. Variability of the CYP2J2 gene is highly constrained except for its proximal promoter: there is a relatively common and functionally relevant single nucleotide polymorphism, indicated by -50G > T polymorphism (CYP2J2*7). Although genetic variation is known among ethnic groups, data for allele frequency are limited to a few Caucasian, Asian, and one African populations. In the present study, genotype distribution of CYP2J2*7 polymorphisms was investigated using polymerase chain reaction and restriction fragment length polymorphism assay in Japanese (n = 338), Mongolian (n = 118), and Ovambo (n = 186) populations and the findings compared with other populations. The mutant (CYP2J2*7) frequencies in the Japanese, Mongolians, and Ovambos were 0.0621, 0.0339, and 0.0672, respectively. Except for the Taiwanese, a general uniformity in the polymorphism in the Asian populations was observed. The mutation frequency of Ovambos was relatively lower than that of the African-American population. This study is the first to investigate the distribution of the CYP2J2*7 gene polymorphisms in Japanese, Mongolians, and Ovambos. These data will be informative and facilitate genetic association studies, in Asian and African populations for CYP2J2-related diseases such as cardiovascular disorders.

Three single nucleotide polymorphisms leading to non-synonymous amino acid substitution in the human ribonuclease 2 and angiogenin genes exhibit markedly less genetic heterogeneity in six populations.

Angiogenin and ribonuclease 2 (RNase 2) are members of the human RNase superfamily. Although three potential single nucleotide polymorphisms (SNPs) in these genes, which could give rise to an amino acid substitution in the protein, have been identified, relevant population data are not available, and accordingly they have not been applied to clinical-genetic analysis. For this purpose, a novel genotyping method for each SNP using the mismatched PCR-restriction fragment length polymorphism technique has been developed. Using this method, the genotype distribution of each SNP was investigated in six populations: Japanese (n = 167), Korean (n = 90), Mongolian (n = 92), Ovambos (n = 86), Turkish (n = 87), and German (n = 70). In all the populations, only one genotype was found in each SNP. Irrespective of differences in ethnic groups, the angiogenin and RNase 2 genes appear to exhibit markedly less genetic heterogeneity with regard to these SNPs.

Two N-linked glycosylation sites (Asn18 and Asn106) are both required for full enzymatic activity, thermal stability, and resistance to proteolysis in mammalian deoxyribonuclease I.

Deoxyribonuclease I (DNase I) is known to be a glycoprotein, and two potential N-linked glycosylation sites (N18 and N106) are known for mammalian enzymes. In the present study, N18 and N106 were mutated in order to investigate the biological role of N-linked glycosylation in three mammalian (human, bovine, and equine) DNases I. The enzyme activities of N18Q and N106Q were lower than that of the wild type, and that of the double mutant (N18Q/N106Q) was lower than those of the single mutants, in accord with the sugar moiety contents in the three mammals. In addition, all mutant enzymes were unstable to heat, suggesting that both sites are required for heat stability. Moreover, in human and equine enzymes, the N18Q and N106Q mutant enzymes were less resistant to trypsin, while N18Q/N106Q was the most sensitive to trypsin. As for bovine DNase I, the trypsin resistance of N18Q and N106Q was similar to that of the wild type, but that of N18Q/N106Q decreased in a time-dependent manner. On the other hand, N-linked glycosylation was not related to pH sensitivity. The results of the present study suggest that N18 and N106 are both necessary for (i) full enzymatic activity, (ii) heat-stability, and (iii) trypsin resistance.

Two deoxyribonuclease I gene polymorphisms and correlation between genotype and its activity in Japanese population.

Deoxyribonuclease I (DNase I) plays important roles for DNA fragmentation and degradation during programmed cell death. The single nucleotide polymorphism (SNP) at DNase I, designated as DNASE1, in exon 8 (A2317G) is considered to be one of the susceptibility genes for gastric and colorectal carcinoma and myocardial infarction. Recent research has shown the presence of a novel 56-bp variable number of tandem repeat (VNTR) polymorphism in intron 4 at DNase I, designated as HumDN1. In the present study, DNASE1 and HumDN1polymorphisms and serum DNase I activities in each different genotype were investigated in 137 Japanese populations. The allele frequencies of A and G in DNASE1 were 0.5839 and 0.4161, respectively. The allelic frequencies of alleles 2, 3, 4, and 5 in HumDN1 were 0.0219, 0.5803, 0.2226, and 0.1752, respectively. In the DNASE1 polymorphism, the activities of genotypes GG and AG were significantly higher than that of AA. As for the HumDN1 polymorphism, the activity of genotype 55 was significantly higher than the activities of 33 and 34. In addition, a significant difference was observed between haplotypes AA/33 and GG/55. The analysis of the correlation between genotype and DNase I activity may be potentially useful for clinical purposes.

Quantitative change in mitochondrial DNA content in various mouse tissues during aging.

In order to systematically characterize age-related changes in the mtDNA content of various tissues during aging, we analyzed the mtDNA content of eight tissues from mice at five different ages from young to senescent by quantitative real-time PCR analysis. Obvious variations of mtDNA content among the tissues were detected: There was a 20-fold range in 2-week-old mice and a 50-fold range in 15-month-old mice. The mtDNA contents of the heart, lung, kidney, spleen and skeletal muscle increased gradually with age, whereas those of bone marrow and brain showed no age-related pattern. The expression patterns of mitochondrial transcription factor A (mtTFA) and mitochondrial single-strand DNA binding protein (mtSSB), possible regulatory factors of the mtDNA copy number, were not necessarily linked with the age-related pattern of the mtDNA content, suggesting the existence of other factors that affect the mtDNA content. The Western blot analysis of mtDNA-encoded cytochrome c oxidase subunit III (MTCO3) demonstrated that the expression levels of this protein in the heart and skeletal muscle increase with age in parallel with the mtDNA content. These findings confirm that the mtDNA content of tissues changes during aging.

An autopsy case of prolonged asphyxial death caused by the impacted denture in the esophagus.

A foreign body impacted in the esophagus is not a rare incident among adults or children. In adults, a dental prosthesis is prone to become impacted in the esophagus. The diagnostic difficulty of this often causes a delay in its removal, which can lead to serious complications, including death. This report describes the autopsy case of a man who died of prolonged asphyxiation induced by the delayed removal of an impacted denture, which was misdiagnosed on his first visit notwithstanding that a part of the denture could be seen on X-rays. Cases in which an impacted denture led to death have rarely been reported in contrast to numerous papers about recovered cases.

CpG-DNA-induced IFN-alpha production involves p38 MAPK-dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors.

Human plasmacytoid or CD4(+)CD11c(-) type 2 dendritic cell precursors (PDC) were identified as natural type I interferon (IFN)-producing cells in response to viral and bacterial infection. They represent effector cells of innate immunity and link it to the distinct adaptive immunity by differentiating into mature DC. It has been reported that oligodeoxyribonucleotides containing unmethylated CpG motifs (CpG DNA) stimulate PDC to produce IFN-alpha, but the molecular mechanisms involved remain unknown. We found that CpG-DNA-induced IFN-alpha production in PDC was completely impaired by the inhibitor of the p38 mitogen-activated protein kinase (MAPK) pathway. Expression of IFN regulatory factor (IRF)-7 was enhanced by CpG-DNA treatment, which was preceded by the phosphorylation of signal transducer and activator of transcription (STAT)1 on Tyr-701, as well as its enhanced phosphorylation on Ser-727. All of these events were also suppressed by the p38 MAPK inhibitor. STAT1, STAT2, and IRF-9, components of IFN-stimulated gene factor 3 (ISGF3), were recognized in the nuclear fraction of CpG-DNA-treated cells. Neither anti-IFN-alpha/beta antibodies (Ab) nor anti-IFNAR Ab suppressed STAT1 phosphorylation, enhancement of IRF-7 expression, or IFN-alpha production in the early phase of the culture. These results suggest that CpG DNA induces p38 MAPK-dependent phosphorylation of STAT1 in a manner independent of IFN-alpha/beta, which may cause ISGF3 formation to increase the transcription of the IRF-7 gene, thereby leading to IFN-alpha production in human PDC.

Improvement of a multiplex PCR system for DYS441, DYS442, DYS443, DYS444 and DYS445, and a population study in 340 Japanese males.

A multiplex PCR system for five Y-STRs (DYS441, DYS442, DYS443, DYS444 and DYS445) has been improved to increase the probability of obtaining a DNA typing result from aged samples. Newly designed PCR primers for amplification of the DYS441 and DYS442 loci and optimization of PCR conditions enabled successful typing from blood and semen stains that had been stored for more than seven years at room temperature. Analysis of 340 Japanese males revealed 7, 5, 6, 5 and 4 alleles at the DYS441, DYS442, DYS443, DYS444 and DYS445 loci, respectively, yielding 122 haplotypes with a cumulative haplotype diversity of 0.97.

A novel multiplex PCR system consisting of Y-STRs DYS441, DYS442, DYS443, DYS444, and DYS445.

We have developed a new sensitive multiplex PCR system consisting of five male-specific and polymorphic tetranucleotide STRs--DYS441 (GDB: 10013873), DYS442 (GDB: 10030304), DYS443 (GDB: 10807127), DYS444 (GDB: 10807128), and DYS445 (GDB: 10807129) on the Y chromosome. Fifty pg DNA per 10 microL reaction volume was required for the correct typing of five STRs. Using this system, the five Y-STRs were correctly typed from blood and semen stains that had been stored for several years at room temperature.

A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H)1 immunity.

BACKGROUND: CpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated T(H)1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. METHODS: T(H)1 and T(H)2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination. RESULTS: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T(H)1, but not T(H)2-type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. CONCLUSIONS: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T(H)1 immunity.