Postmortem genetic analysis of 17 sudden cardiac deaths identified nonsense and frameshift variants in two cases of arrhythmogenic cardiomyopathy.

Sudden death, or unexpected natural death of a healthy individual, is a serious problem in all nations. Sudden cardiac death (SCD) mainly due to ischemic heart diseases is the top cause of sudden death. However, there are pathophysiological conditions, referred to as sudden arrhythmic death syndrome, in which no apparent lesion can be identified even after complete conventional or ordinary autopsy. While postmortem genetic analyses have accumulated evidence about underlying genetic abnormality in such cases, the precise relationships between genetic background and the phenotype have been largely elusive. In this study, a retrospective investigation of 17 autopsy cases in which lethal arrhythmia was suspected to be the cause of death was carried out. Genetic analysis focusing on 72 genes reported to be associated with cardiac dysfunctions was performed, in combination with detailed histopathological and postmortem imaging examination, and a family study. As a result, in two cases of suspected arrhythmogenic cardiomyopathy (ACM), we found a nonsense variant in PKP2 and frameshift variant in TRPM4 gene. In contrast, the other 15 cases showed no morphological changes in the heart despite the presence of a frameshift variant and several missense variants, leaving the clinical significance of these variants obscure. The findings of the present study suggest that nonsense and frameshift variants could be involved in the morphological abnormality in cases of SCD due to ACM, while missense variants alone rarely contribute to massive structural changes in the heart.

Acute subdural hematoma caused by rupture of a mycotic aneurysm due to meningitis associated with infectious endocarditis: comparison of autopsy findings with postmortem computed tomography.

Forensic pathologists often encounter cases of acute subdural hematoma (SDH) due to trauma, whereas those attributable to endogenous causes are rare. Here, we report a case of the latter type in a 42-year-old man who was found dead at home after several months of fever and malaise. Postmortem computed tomography (PMCT) and autopsy were undertaken to clarify the cause of death. PMCT images revealed a fatal SDH and a localized hyper-density area in the right parietal lobe; macroscopic and microscopic examinations revealed SDH due to rupture of a mycotic aneurysm (MA) associated with meningitis. The PMCT images also indicated thickening and calcification of the mitral valve, while autopsy demonstrated infective endocarditis (IE). In addition, PMCT demonstrated a low-density area in the spleen, which was shown to be a splenic abscess at autopsy. PMCT also demonstrated tooth cavities. Based on the findings of autopsy, the cause of death was considered to be SDH due to rupture of the MA resulting from meningitis with IE and splenic abscess. Although PMCT was unable to clarify the significance of any individual feature, a retrospective review of the PMCT images might have suggested IE, bacteremia, or ruptured MA leading to SDH. This case suggests that, instead of interpreting individual features demonstrated on PMCT images, integrated interpretation of overall PMCT findings might provide clues for identifying causes of death, despite the fact that PMCT lacks diagnostic accuracy for infectious diseases such as IE and meningitis.

Reduction of blood group A antigen on erythrocytes in a patient with myelodysplastic syndrome harboring somatic mutations in RUNX1 and GATA2.

BACKGROUND: Reduction of blood group ABO antigens on red blood cells (RBCs) is well known in patients with leukemias, and this reduction of ABO expression is strongly associated with DNA methylation of the ABO promoter. Previously, we reported a two-nucleotide deletion in RUNX1 encoding an abnormally elongated protein lacking the trans-activation domain in a patient with myelodysplastic syndrome (MDS) showing A-antigen loss on RBCs. This prompted us to investigate the underlying mechanism responsible for A-antigen reduction on RBCs in another patient with MDS. STUDY DESIGN AND METHODS: Screening of somatic mutations was carried out using a targeted sequencing panel with genomic DNA from peripheral blood mononuclear cells from the patient and eleven MDS controls without A- or B-antigen loss. DNA methylation of the ABO promoter was examined by bisulfite genomic sequencing. Transient transfection assays were performed for functional evaluation of mutations. RESULTS: Screening of somatic mutations showed missense mutations in RUNX1 and GATA2 in the patient, while no mutation was found in exons of those genes in the controls. There was no significant difference in ABO promoter methylation between the patient and the controls. Transient transfection experiments into COS-7 and K562 cells suggested that the amino acid substitutions encoded by those mutations reduced or lost the trans-activation potential of the ABO expression. CONCLUSION: Considering the discrepancy between the variant frequencies of these mutations and the ratios of the RBCs with A-antigens loss, the antigen reduction might be associated with these somatic mutations and hypermethylation of the ABO promoter.

[A case of locally advanced rectal carcinoma treated with preoperative chemoradiotherapy resulting in pathologically complete response].

An 84-year-old woman presented with the chief complaint of melena. Colonoscopy revealed a type 2 tumor that circumferentially occupied the lumen of the lower rectum about 6 cm from the anal verge. A biopsy specimen was obtained from the tumor and the patient was diagnosed with moderately differentiated adenocarcinoma. Computed tomography revealed that the rectal cancer had invaded the marginal fatty tissue, accompanied by several regional lymph node metastases with no distant metastasis. On the basis of this evidence, the tumor was staged as cT4a, cN2b, cM0 according to the TNM Classification of Malignant Tumors (7th Edition, UICC). Preoperative radiotherapy combined with an oral chemopreventive agent (RT 1.8 Gy × 25 frames; total 45 Gy, S-1 80 mg/day) was administered with trivial adverse effects. Laparoscopy-assisted low anterior resection with Japanese D3 dissection was performed successfully. The patient is doing well without recurrence after 14 months of surgery. Histological examination revealed that both the primary lesion and regional lymph nodes had no residual cancer; that is, the histological effect of the preoperative chemoradiotherapy was a pathologically complete response (pCR).

Usefulness of virtual reassembly of the skull and spine in cases of fragmentation due to high-energy trauma: A feasibility study.

With the widespread use of postmortem computed tomography (PMCT) beside forensic autopsies for investigation of causes of death, three-dimensional (3D) reconstruction and fusion imaging using PMCT data are now becoming common. In the present study, the applicability of virtual reassembly from PMCT data was investigated in three cases involving fragmentation of the skull or spine due to high-energy trauma, as in such cases it is sometimes difficult to obtain detailed information on fractures using macroscopic observation alone. In the first case, virtual reassembly of the skull provided more information about the fractures than conventional reconstruction with adhesive. In the second case, although the skull was severely fractured and could not be examined macroscopically, virtual reassembly allowed detailed visualization of the fractures. In the last case, virtual reassembly of the spine helped to clarify that the 6th-8th thoracic vertebrae had been run over by a vehicle at the scene. Thus, virtual reassembly was shown to be useful for assessment of injury patterns, and event reconstruction.

Hypothermic death resulting from extreme freezing with characteristic postmortem computed tomography findings: A case report and review of the literature.

We report a case of hypothermic death that resulted from extreme freezing, with characteristic postmortem computed tomography (PMCT) findings. A 75-year-old man died in a deeply frozen state. In PMCT, there was a lack of increase in the bilateral lung-field attenuation. Urinary retention, with a hypodense area of frozen urine, was observed in the bladder. Changes that appeared to involve the crystallization of serum in frozen blood were observed in the aorta. Based on the scene and his circumstances, it was speculated that he died of hypothermia. Present case and our review revealed that although PMCT findings from hypothermic death that resulted from deep freezing are very rare, the characteristic PMCT findings may help determine the cause of death.

Emergence of an erythroid cell-specific regulatory region in ABO intron 1 attributable to A- or B-antigen expression on erythrocytes in Hominoidea.

A- and B-antigens are present on red blood cells (RBCs) as well as other cells and secretions in Hominoidea including humans and apes such as chimpanzees and gibbons, whereas expression of these antigens on RBCs is subtle in monkeys such as Japanese macaques. Previous studies have indicated that H-antigen expression has not completely developed on RBCs in monkeys. Such antigen expression requires the presence of H-antigen and A- or B-transferase expression in cells of erythroid lineage, although whether or not ABO gene regulation is associated with the difference of A- or B-antigen expression between Hominoidea and monkeys has not been examined. Since it has been suggested that ABO expression on human erythrocytes is dependent upon an erythroid cell-specific regulatory region or the + 5.8-kb site in intron 1, we compared the sequences of ABO intron 1 among non-human primates, and demonstrated the presence of sites orthologous to the + 5.8-kb site in chimpanzees and gibbons, and their absence in Japanese macaques. In addition, luciferase assays revealed that the former orthologues enhanced promoter activity, whereas the corresponding site in the latter did not. These results suggested that the A- or B-antigens on RBCs might be ascribed to emergence of the + 5.8-kb site or the corresponding regions in ABO through genetic evolution.

Post-traumatic cerebral infarction caused by thrombus in the middle cerebral artery.

A woman in her 80s was found unconscious after being hit by a car while crossing a road. After admission to hospitals, computed tomography (CT) scans revealed traumatic brain injury (TBI), and the patient was treated symptomatically. However, despite improvement of TBI in CT images, she died unexpectedly. Postmortem CT demonstrated cerebral infarction in the territory of the right middle cerebral artery (MCA). Histopathological examination revealed lumen-obstructing thrombosis and intimal injury upstream of the thrombosis in the right MCA. These findings suggested that the intimal injury in the MCA had led to thrombus formation, and thromboembolism in the region distal to the injury leading to post-traumatic cerebral infarction (PTCI). Both postmortem CT and autopsy were able to reveal the final condition of the deceased, which had not been fully anticipated by the clinicians who had treated her after the accident. The longitudinal antemortem to postmortem course revealed by multiple CT images and the histopathological examination provided crucial clues to the pathogenesis of PTCI in this case.

Usefulness of a tissue optical clearing technique for forensic autopsy.

Forensic pathologists are required to investigate lethal trauma or disease at autopsy. In addition to massive contusions of various organs, a number of small features with potentially fatal implications also need to be sought. Since such lesions may need microscopic examinations for detailed evaluation, it is important to select suitable anatomic locations for tissue sampling. For practical screening of small lesions, we have developed a tissue optical clearing (TOC) technique for forensic autopsy. The technique involves clearing with a non-toxic organic solvent, ethyl cinnamate, which renders excised organs transparent, while hemorrhages or blood-containing vessels remain opaque. Using this technique, tiny hemorrhages in the spinal cord were able to be identified by gross examination, allowing proper selection of locations for tissue sampling. Subsequent histopathological evaluation was successfully performed with no apparent artifacts related with the TOC procedure. In addition, a combination of TOC and targeted CT angiography allowed feasible examination of the arterial occlusive lesion in the superior mesenteric artery, and when combined with micro-CT scanning it was useful for evaluating the lumen of the coronary artery with stent implantation. The results obtained so far indicated that TOC could complement routine forensic autopsy procedures when detailed evaluation of small lesions is required.

Superimposed CT imaging using fusion function to visualize the relationship between the knife and the wound path in a stabbing victim.

With the increasing use of postmortem computed tomography (PMCT) in medicolegal autopsies, three-dimensional (3D) models of injured areas can now be generated from multislice computed tomography images. However, since PMCT has low sensitivity for detecting injuries in solid organs in the absence of contrast administration, it has been difficult to demonstrate the tracks of stab wounds leading to solid organ injury using 3D reconstruction. Here, we report one homicide case with two stab wounds. On the skin surface, the stab wounds were located on the neck and anterior chest wall. A medicolegal autopsy revealed that one stab wound in the neck had penetrated the wall of the right pleural cavity and the upper portion of the right lung whereas the other stab wound in the anterior chest wall had penetrated the right diaphragm and the heart. To illustrate the tracks of the stab wounds, superimposed CT images of the body, the excised organ, and a knife model were constructed to obtain a 3D model. This allowed clear and concise visualization of the complex relationship of the knife to the heart incision and the stab wound on the chest surface.

A cell-specific regulatory region of the human ABO blood group gene regulates the neighborhood gene encoding odorant binding protein 2B.

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. ABO transcription is known to be regulated by a constitutive promoter in a CpG island and regions for regulation of cell-specific expression such as the downstream + 22.6-kb site for epithelial cells and a site in intron 1 for erythroid cells. Here we investigated whether the + 22.6-kb site might play a role in transcriptional regulation of the gene encoding odorant binding protein 2B (OBP2B), which is located on the centromere side 43.4 kb from the + 22.6-kb site. In the gastric cancer cell line KATOIII, quantitative PCR analysis demonstrated significantly reduced amounts of OBP2B and ABO transcripts in mutant cells with biallelic deletions of the site created using the CRISPR/Cas9 system, relative to those in the wild-type cells, and Western blotting demonstrated a corresponding reduction of OBP2B protein in the mutant cells. Moreover, single-molecule fluorescence in situ hybridization assays indicated that the amounts of both transcripts were correlated in individual cells. These findings suggest that OBP2B could be co-regulated by the + 22.6-kb site of ABO.

Characteristic postmortem computed tomography findings of ingestion of benzine.

There are some reports investigating the cause of death by examining the contents of the stomach and duodenum using postmortem computed tomography, but most of these have been based on radiopaque contents. Here, we report a case of suicide after ingesting a large amount of benzine. Although the gastric contents were radiolucent, the characteristic postmortem computed tomography imaging findings helped to determine the cause of death.

Histone deacetylase inhibitors suppress ACE2 and ABO simultaneously, suggesting a preventive potential against COVID-19.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide as a pandemic throughout 2020. Since the virus uses angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry, increment of ACE2 would lead to an increased risk of SARS-CoV-2 infection. At the same time, an association of the ABO blood group system with COVID-19 has also been highlighted: there is increasing evidence to suggest that non-O individuals are at higher risk of severe COVID-19 than O individuals. These findings imply that simultaneous suppression of ACE2 and ABO would be a promising approach for prevention or treatment of COVID-19. Notably, we have previously clarified that histone deacetylase inhibitors (HDACIs) are able to suppress ABO expression in vitro. Against this background, we further evaluated the effect of HDACIs on cultured epithelial cell lines, and found that HDACIs suppress both ACE2 and ABO expression simultaneously. Furthermore, the amount of ACE2 protein was shown to be decreased by one of the clinically-used HDACIs, panobinostat, which has been reported to reduce B-antigens on cell surfaces. On the basis of these findings, we conclude that panobinostat could have the potential to serve as a preventive drug against COVID-19.

Investigation of the applicability of virtual gastroscopy based on postmortem computed tomography to detect changes in the stomach, along with reports of three rare cases.

Postmortem computed tomography is now being used more commonly for routine forensic investigation. The use of 3D reconstruction techniques including virtual gastroscopy is effective and also improves the speed of interpretation, recognition, and description of specific clinical conditions. However, it has been unclear whether postmortem virtual endoscopy could be applicable for medicolegal autopsy or whether it could complement pathological examination at autopsy. Here, we investigated the applicability of postmortem virtual gastroscopy by reviewing 295 medicolegal autopsy cases seen at our institution, and found four cases in which the technique had been able to demonstrate features corresponding to changes that were evident at autopsy. Thus,postmortem virtual gastroscopy would have only rarely been effective forvisualizing any change in the stomach in such cases. In addition, we describe in detail three of those cases in which virtual gastroscopy had been able to visualize changes in the stomach, including a gastric ulcer, a polyp, and the presence of foamy fluid, which were all verified at autopsy. In those cases, virtual gastroscopy was useful for understanding features in the stomach of the deceased, which were revealed by axial images of the abdomen, to forensic pathologists who were not familiar with PMCT 2D images. Taken together, our findings suggest that postmortem virtual gastroscopy might help facilitate clear, straightforward sharing of information about PMCT images of complex anatomical structures among radiologists and forensic pathologists, as well as non-medical professionals with a limited knowledge of anatomy and physiology.

[A case of type 4 gastric cancer with peritoneal dissemination complicating venous thromboembolism treated effectively by combination of S-1 and warfarin].

A 49-year-old female patient was admitted to our hospital for a type 4 gastric cancer with peritoneal dissemination. Two courses of paclitaxel (PTX), and eight courses of S-1 were carried out. Although a partial response was obtained, she had complications with a deep venous thromboembolism (DVT) and pulmonary embolism (PE) during the treatment. Heparin, followed by warfarin, was useful to treat the embolism. After the venous thromboembolism (VTE) disappeared, combination therapy with S-1 and warfarin were started, and the quality of life (QOL) of this patient was maintained for about one year. Fine monitoring of the international normalized ratio (INR) was required in order to prevent side effects of blood coagulation by S-1 and warfarin coadministration. This case suggests that the combination therapy of S-1 and warfarin may be a safe and effective treatment able to prolong time to progression against a type 4 gastric cancer with VTE.

Greater omentum gastrointestinal stromal tumor with PDGFRA-mutation and hemoperitoneum.

Although gastrointestinal stromal tumor (GIST) occurs generally in the digestive tract, omental GIST is very rare. We report the first case of an adult greater omental GIST with a new platelet-derived growth factor receptor α gene (PDGFRA)-mutation with hemoperitoneum. A 43-year-old man was admitted to our hospital complaining of acute abdominal pain. Abdominal contrast-enhanced computed tomography revealed a huge mass in the right abdominal cavity, and a large accumulation of fluid in the pelvic cavity, suggesting hemoperitoneum. We diagnosed the rupture as an intra-abdominal tumor, and an emergency tumorectomy was performed with resection of the greater omentum. This tumor was located in the distal right side of the greater omentum, and showed no continuity with the gastric wall. The tumor occurred primarily in the greater omentum. The resected tumor was about 19 cm × 12 cm × 14 cm in diameter, and weighed 1529 g. Histologically, the tumor was composed of epithelioid-shaped cells with high cellularity, and was positive for CD117 and CD34, and negative for S-100, α-smooth muscle actin. The mitosis was 6/50 under high power field. This case showed exon 18 mutation of PDGFRA with 846 (Asp to Glu) substitution, 848 (Asn to Lys) substitution. This is the first report of this PDGFRA mutation in omental GIST, and this might play an important role in the tumorigenesis of this case. Based on these findings, the tumor was diagnosed as high risk GIST primarily occurring in the greater omentum. The patient was treated with imatinib at a dose of 400 mg/d as adjuvant chemotherapy, and has been followed up for 24 mo with no evidence of recurrence.

Synovial sarcoma after chemotherapy for osteosarcoma: a case report.

A 23-year-old man had eight cycles of adjuvant chemotherapy including doxorubicin, cisplatin, methotrexate, and ifosfamide and radical surgery for biopsy-proved osteosarcoma of the right fibular shaft. Two years after the initial diagnosis, he noticed a mass in the medial aspect of his right knee. Magnetic resonance imaging scans revealed a soft tissue tumor measuring 2 x 2 cm in the pericapsular region of the right knee. Histologically, this soft tissue tumor was composed of spindle cells with occasional atypical mitoses and without matrix formation. Immunohistochemically, the tumor cells were positive for vimentin, cytokeratin, and epithelial membrane antigen, and negative for alpha smooth muscle actin. A fusion gene, SYT-SSX was detected with reverse transcription-polymerase chain reaction. From the results, the secondary tumor was diagnosed as a synovial sarcoma. The current case of double sarcomas is rare. Both sarcomas were diagnosed accurately using immunohistochemical and molecular procedures. This case suggests a positive association between a second tumor and chemotherapy including intraarterial perfusion of doxorubicin.