Review of ABO Expression and Variations based on Transcriptional Regulation of the ABO Blood Group Gene.

Background and Summary: We review the transcriptional regulation of ABO expression and discuss variants in the promoter and erythroid cell-specific regulatory region in individuals with weak ABO phenotypes such as Bm, Am, B3, and A3. We also review the molecular mechanisms responsible for variations in ABO expression in development and disease including the cell type-specific expression of ABO during erythroid cell differentiation, and reduction of A- or B-antigens in cancer cells or on red blood cells in patients with leukemia. Although the relationship between ABO blood group antigens and diseases has been characterized, the physiological significance of the ABO blood group system remains unclear. Key Messages: This review discusses accumulated knowledge of the ABO gene regulation and potential reasons for conservation of ABO during evolution.

A novel genotyping method for rapid identification of the Le gene to select patients for diagnosis with CA19-9.

BACKGROUND: The antigenic determinant of CA19-9 is synthesized by the α1,3/4fucosyltransferase encoded by the Le gene in the Lewis blood group system. Accordingly, a diagnosis with CA19-9 is not appropriate forLe-negative patients who possess the Le gene-mutated le alleles homozygously. METHODS: A Le gene-specific PCR was undertaken to determine c59T>G by using a set of tag-sense and biotin-labeled anti-sense primers and a peptide nucleic acid-le-clamp which bound to G59 in the le alleles. Following mixing with streptavidin-coatedbluelatex beads, the PCR products were developed on a strip on which the complementary tag oligonucleotide to theLe gene-specific amplicon was immobilized. RESULTS: When the PCR products were developed on the strip, a clear line was rapidly observed in Le-positive but not in Le-negative individuals. In contrast, a significant number of cancer patients with Lewis-negative phenotype were found to possess CA19-9, while they were specifically genotyped asLe/-. No contradictory results were observed in cancer patients (n = 315) with respect to their Lewis genotypes and CA19-9 levels. CONCLUSIONS: c59T>G occurred commonly in the le alleles could be specifically and rapidly identified by the present method. This method appeared to be relevant forselecting cancer patientsto bediagnosed with CA19-9.

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.

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 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.

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.

Reduced-intensity haploidentical peripheral blood stem cell transplantation using low-dose thymoglobulin for aggressive adult T cell leukemia/lymphoma patients in non-complete remission.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) has been accepted as a treatment option for aggressive (acute or lymphoma type) adult T cell leukemia/lymphoma (ATLL) patients with a poor prognosis, when a suitable HLA-matched donor is not available. However, haplo-HSCT carries a potential risk of treatment-related mortality including severe graft-versus-host disease (GVHD). Therefore, we conducted a prospective pilot study in order to evaluate the efficacy and safety of reduced-intensity haploidentical peripheral blood stem cell transplantation (haplo-PBSCT) with low-dose thymoglobulin (2.5 mg/kg only on day -2), fludarabine, melphalan, and total body irradiation 4 Gy for aggressive ATLL. Three consecutive acute type ATLL patients, who were ineligible for conventional myeloablative conditioning due to advanced age or comorbidities, were enrolled. One patient received pretransplant mogamulizumab therapy. All the patients were not in complete remission (CR) at the time of transplantation. Our transplantation protocol was safely carried out. CR was achieved in all the patients after transplantation. HTLV-I viral loads became undetectable after transplantation. No severe adverse events such as grade III-IV GVHD or viral/fungal diseases were observed. At a follow-up of 2 years, they were still in CR. However, T cell receptor repertoire diversities were low 1 year after transplantation in next-generation sequencing. Our results show encouraging therapeutic benefits of this pilot approach using reduced-intensity haplo-PBSCT with low-dose thymoglobulin for aggressive ATLL patients.

RUNX1 mutation in a patient with myelodysplastic syndrome and decreased erythrocyte expression of blood group A antigen.

BACKGROUND: Loss of blood group ABO antigens on red blood cells (RBCs) is well known in patients with leukemias, and such decreased ABO expression has been reported to be strongly associated with hypermethylation of the ABO promoter. We investigated the underlying mechanism responsible for A-antigen reduction on RBCs in a patient with myelodysplastic syndrome. STUDY DESIGN AND METHODS: Genetic analysis of ABO was performed by PCR and sequencing using peripheral blood. RT-PCR were carried out using cDNA prepared from total bone marrow (BM) cells. Bisulfite genomic sequencing was performed using genomic DNA from BM cells. Screening of somatic mutations was carried out using a targeted sequencing panel with genomic DNA from BM cells, followed by transient transfection assays. RESULTS: Genetic analysis of ABO did not reveal any mutation in coding regions, splice sites, or regulatory regions. RT-PCR demonstrated reduction of A-transcripts when the patient's RBCs were not agglutinated by anti-A antibody and did not indicate any significant increase of alternative splicing products in the patient relative to the control. DNA methylation of the ABO promoter was not obvious in erythroid cells. Targeted sequencing identified somatic mutations in ASXL1, EZH2, RUNX1, and WT1. Experiments involving transient transfection into K562 cells showed that the expression of ABO was decreased by expression of the mutated RUNX1. CONCLUSION: Because the RUNX1 mutation encoded an abnormally elongated protein without a transactivation domain which could act as dominant negative inhibitor, this frame-shift mutation in RUNX1 may be a genetic candidate contributing to A-antigen loss on RBCs.

Postmortem computed tomography evaluation of fatal gas embolism due to connection of an intravenous cannula to an oxygen supply.

An 84-year-old man who had suffered from chronic obstructive pulmonary disease accompanied by moderate pneumonia as well as gastric cancer with liver metastasis was found dead by a nurse, who noticed that the patient's intravenous catheter in the left forearm had been erroneously connected to an oxygen supply in his hospital room, leading to infusion of oxygen into a vein. Postmortem CT scanning demonstrated multiple accumulations of gas in the pulmonary artery, the right atrium and ventricle, as well as the left subclavian and brachiocephalic veins, corresponding to the route that the infused gas would have taken to the heart and pulmonary artery. Conventional autopsy revealed the presence of gas in the right ventricle. These findings suggested that the immediate cause of death was a gas embolus due to oxygen that had entered the cardiopulmonary circulation via the intravenous catheter. This case highlights the usefulness of postmortem imaging as an aid to conventional autopsy for demonstrating gas embolism.

Epithelial Expression of Human ABO Blood Group Genes Is Dependent upon a Downstream Regulatory Element Functioning through an Epithelial Cell-specific Transcription Factor, Elf5.

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. The ABO system is composed of complex carbohydrate structures that are biosynthesized by A- and B-transferases encoded by the ABO gene. However, the mechanisms regulating ABO gene expression in epithelial cells remain obscure. On the basis of DNase I-hypersensitive sites in and around ABO in epithelial cells, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays and histone modifications indicated a novel positive regulatory element, designated the +22.6-kb site, downstream from ABO, and this was shown to enhance ABO promoter activity in an epithelial cell-specific manner. Expression of ABO and B-antigen was reduced in gastric cancer KATOIII cells by biallelic deletion of the +22.6-kb site using the CRISPR/Cas9 system. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the site bound to an epithelial cell-specific transcription factor, Elf5. Mutation of the Ets binding motifs to abrogate binding of this factor reduced the regulatory activity of the +22.6-kb site. Furthermore, ELF5 knockdown with shRNA reduced both endogenous transcription from ABO and B-antigen expression in KATOIII cells. Thus, Elf5 appeared to be involved in the enhancer potential of the +22.6-kb site. These results support the contention that ABO expression is dependent upon a downstream positive regulatory element functioning through a tissue-restricted transcription factor, Elf5, in epithelial cells.

Combination of postmortem mass spectrometry imaging and genetic analysis reveals very long-chain acyl-CoA dehydrogenase deficiency in a case of infant death with liver steatosis.

CASE HISTORY: A 3-month-old infant was found dead in his bed. A postmortem computed tomography (CT) scan suggested fatty attenuation in the liver parenchyma, but no other potentially fatal changes were found. To clarify the cause of death, a medicolegal autopsy was carried out. AUTOPSY FINDINGS: Internal examination confirmed the presence of liver steatosis as well as hepatomegaly. There were no other significant findings including encephalitis or brain edema. MASS SPECTROMETRY ANALYSIS: To clarify the mechanism underlying lipid accumulation in the liver, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) analysis was conducted. This indicated a significant accumulation of C14:1 acylcarnitine in the liver of the deceased, suggesting very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. GENETIC ANALYSIS: To find the cause of the VLCAD deficiency, genetic analysis of the responsible gene, acyl-CoA dehydrogenase, very long chain (ACADVL), was performed. This revealed two novel mutations that may have accounted for the disease. CONCLUSION: A combination of these data revealed that the liver steatosis in this case might have been caused by VLCAD deficiency based on genetic mutations of ACADVL. Thus, the deceased might have been vulnerable to energy crisis and sudden infant death. The present findings show that MALDI-IMS analysis as well as genetic analysis can be useful for elucidating the cause of death.

Identification and characterization of a novel genetic mutation with prolonged QT syndrome in an unexplained postoperative death.

INTRODUCTION: The human ether-à-go-go-related gene (hERG) encodes the α-subunit of a cardiac potassium channel. Various mutations of hERG, including missense mutations, have been reported to cause long QT syndrome (LQTS) and severe arrhythmic disorders such as sudden cardiac death. We identified a novel hERG frameshift mutation (hERG(ΔAT)) in the S5-pore region from a LQTS patient who died suddenly and analyzed its genetic profile and the molecular and electrophysiological behaviors of the protein product to assess the pathogenicity of hERG(ΔAT). METHODS AND RESULTS: We performed direct sequencing of hERG and evaluated its transcript level by using a whole blood sample from the patient. We performed immunoblotting, immunocytochemistry, and patch-clamp recordings of HEK-293 T cells transfected with hERG(ΔAT), wild-type hERG (hERG(WT)), or both. The patient demonstrated an AT deletion (c.1735_1736del) in hERG and a decrease in hERG mRNA transcripts. HEK-293 T cells showed lower production and cell surface expression of hERG(ΔAT) compared with hERG(WT) protein. In addition, the hERG(∆AT) protein failed to form functional channels, while the activation kinetics of functional channels, presumably consisting of hERG(WT) subunits, were unaffected. CONCLUSION: The ΔAT mutation may decrease the number of functional hERG channels by impairing the posttranscriptional and posttranslational processing of the mutant product. This decrease may partly explain the cardiac symptoms of the patient who was heterozygous for hERG(ΔAT).

Genetic and expression analysis of SNPs in the human deoxyribonuclease II: SNPs in the promoter region reduce its in vivo activity through decreased promoter activity.

Five SNPs in the human DNase II gene have been reported to be associated with rheumatoid arthritis (RA). Genotype and haplotype analysis of 14 SNPs, nine SNPs of which reported in the NCBI dbSNP database in addition to these five SNPs, was performed in healthy subjects. The enzymatic activities of the amino acid substituted DNase II corresponding to each SNP and serum DNase II in healthy Japanese, and promoter activities derived from each haplotype of the RA-related SNPs were measured. Significant correlations between genotype in each RA-related SNP and enzymatic activity levels were found; alleles associated with RA exhibited a reduction in serum DNase II activity. Furthermore, the promoter activities of each reporter construct corresponding to predominant haplotypes in three SNPs in the promoter region of the gene exhibited significant correlation with levels of serum DNase II activity. These findings indicate these three SNPs could alter the promoter activity of DNASE2, leading to a decline in DNase II activity in the serum through gene expression. Since the three SNPs in the promoter region of the DNase II gene could affect in vivo DNase II activity through reduction of the promoter activity, it is feasible to identify these SNPs susceptible to RA.

Nonsynonymous single-nucleotide polymorphisms of the human apoptosis-related endonuclease--DNA fragmentation factor beta polypeptide, endonuclease G, and Flap endonuclease-1--genes show a low degree of genetic heterogeneity.

DNA fragmentation factor beta (DFFB) polypeptide, endonuclease G (EndoG), and Flap endonuclease-1 (FEN-1) are responsible for DNA fragmentation, a hallmark of apoptosis. Although the human homologs of these genes show three, four, and six nonsynonymous single-nucleotide polymorphisms (SNPs), respectively, data on their genotype distributions in populations worldwide are limited. In this context, the objectives of this study were to elucidate the genetic heterogeneity of all these SNPs in wide-ranging populations, and thereby to clarify the genetic background of these apoptosis-related endonucleases in human populations. We investigated the genotype distribution of their SNPs in 13 different populations of healthy Asians, Africans, and Caucasians using novel genotyping methods. Among the 13 SNPs in the 3 genes, only 3 were found to be polymorphic: R196K and K277R in the DFFB gene, and S12L in the EndoG gene. All 6 SNPs in the FEN-1 gene were entirely monoallelic. Although it remains unclear whether each SNP would exert any effect on endonuclease functions, these genes appear to exhibit low degree of genetic heterogeneity with regard to nonsynonymous SNPs. These findings allow us to conclude that human apoptosis-related endonucleases, similarly to other human DNase genes, revealed previously, are well conserved at the protein level during the course of human evolution.

The CCAAT/enhancer (C/EBP) family of basic-leucine zipper (bZIP) transcription factors is a multifaceted highly-regulated system for gene regulation.

The C/EBP family of proteins represents an important group of bZIP transcription factors that are key to the regulation of essential functions such as cell cycle, hematopoiesis, skeletal development, and host immune responses. They are also intimately associated with tumorigenesis and viral disease. These proteins are regulated at multiple levels, including gene induction, alternative translational initiation, post-translational modification, and protein-protein interaction. This review attempts to integrate recent reports with more than 20 years of previous effort focused on this fascinating collection of regulators.

The 3' flanking region of the human ABO histo-blood group gene is involved in negative regulation of gene expression.

Gene expression is driven by promoters, enhancers, silencers, and other cis-regulatory elements upstream and downstream of the gene. Previous studies of the regulation of human ABO gene transcription have focused mainly on the 5' region, including the core promoter and the region proximal to it. However, as the involvement of the 3' flanking region in transcriptional regulation has not yet been examined, we focused on this issue. The 3' region approximately 2.2kb downstream of the ABO gene was PCR-amplified and inserted into a cloning vector, followed by sequence determination and preparation of luciferase reporter vectors. Transient transfections into KATOIII and K562 cells were performed using various reporter plasmids containing the 3' region. The 3' region of the ABO gene, which was characterized by a high degree of sequence repetition, was effectively cloned by a single-copy cloning method. Transfections in KATOIII and K562 cells showed that negative elements were demonstrable within the 3' region. These observations suggest that negative regulatory elements seem to be present in the 3' region of ABO in both epithelial and erythroid lineages. As we had observed a negative region just upstream of the ABO promoter, transcription from ABO could be negatively regulated by repressive regions just upstream of the promoter and downstream of the gene. Further studies of the enhancer will be required for elucidating the molecular basis of ABO gene expression.

11-Tungstophosphate with iron(II) and hydrogen peroxide efficiently detached bacterial biofilm.

An attempt was made to detach bacterial biofilm, formed by Staphylococcus epidermidis, by using hydrogen peroxide (H(2)O(2)) and tungsten compounds. When iron(II) (Fe(2+)) was mixed with undecatungstophosphate ([PW(11)O(39)](7-)) and then H(2)O(2), the resulting mixture was able to totally remove the biofilm probably because of co-generation of (1)O(2) and .OH. A mixture of undecatungstosilicate ([SiW(11)O(39)](8-)) and Fe(2+) (or Cu(2+)) also gave a good result, but their catalytic activities for producing .OH (or (1)O(2)) were rather weak. An electron microscopic study showed that almost nothing was visible on the surface of a biofilm-coated glass after treatment with 1mM [PW(11)O(39)](7-)+1 mM Fe(2+) and 500 mM H(2)O(2) (incubated for 1 h at 37 degrees C).

An infant with asymptomatic hepatic granuloma probably caused by bacillus Calmette-Guérin (BCG) vaccination found incidentally at autopsy: a case report.

INTRODUCTION: Bacillus Calmette-Guérin (BCG) is an attenuated strain of Mycobacterium bovis. Usually, systemic complications due to BCG vaccination are quite rare. However, since BCG is a live vaccine, there is still a possibility that it may cause an infection. CASE PRESENTATION: Hepatic granuloma was found incidentally in an asymptomatic 5-month-old infant who was found dead in his bed. The probable cause of death was asphyxia due to milk aspiration into the lungs. The granuloma was composed of epithelioid histiocytes with frequent multinucleated Langhans-type giant cells and a small number of lymphocytes. CONCLUSION: The cause of the asymptomatic granuloma was not identified, but was considered likely due to BCG vaccination.

Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1.

High mobility group box 1 protein (HMGB1), originally described as a non-histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte-specific E26 transformation-specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31-kDa proIL-1beta protein. The -131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S-transferase-pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix-turn-helix DNA-binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1-binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1-deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes.