[Show abstract][Hide abstract] ABSTRACT: Recently, the involvement of mutation and deletion of transcription regulatory elements in the Bm , Am , A3 and B3 phenotypes has been reported. In the present study, we carried out genetic analysis of individuals with A3 and B3 using peptide nucleic acid-clamping PCR to exclude amplification of O alleles. Two single-point mutations, -76G>C and -68G>T, were found in the ABO promoter on the A-allele in three A3 individuals and on the B allele in a B3 individual, respectively. Transient transfection of luciferase reporter plasmids carrying the same mutations into K562 cells revealed decreased luciferase activity in comparison with that carrying the wild-type promoter. These observations suggest that the mutations downregulate the promoter activity, leading to reduction in A- or B-antigen expression on red blood cells in individuals with the A3 and B3 phenotypes.
[Show abstract][Hide abstract] ABSTRACT: A 40-year-old mentally retarded Japanese man was admitted at rehabilitation facility for handicapped persons and found dead in his bed. His neonatal period was complicated by seizures, and he had a medical history of schizophrenia. A postmortem computed tomography scan suggested an intestinal obstruction, but the cause was unknown. To clarify the cause of death, a medicolegal autopsy was carried out. The gastrointestinal tract was found to contain copious amounts of cloth pieces. A diagnosis of intestinal obstruction secondary to pica of clothes was made. Despite still being an essentially neglect condition; mental retardation is cause to significant burden to the patient, his relatives and caregivers and the whole society. Moreover, people with mental retardation may be at increased risk for potentially self-injury due to ingestion of non-eating substance or incongruent intake of eating substances, which may on turn lead to severe or even life-threatening medical and surgical complications as herein reported. Specific attention also to pica in mentally-retarded patients with sudden, severe, gastrointestinal events, should therefore be placed in order to prevent potential death or otherwise severe chronic consequences, ideally aiming at enhancing the early recognition and multi-disciplinary management of those psychological stressors or triggers potentially responsible for pica too.
Annals of General Psychiatry 07/2015; 14(1):22. DOI:10.1186/s12991-015-0060-4 · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed a sequence-specific primer PCR (SSP-PCR) for detection of a 5·8-kb deletion (Bm5·8) involving an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene. Using this SSP-PCR, we performed genetic analysis of 382 individuals with Bm or ABm. The 5·8-kb deletion was found in 380 individuals, and disruption of the GATA motif in the regulatory element was found in one individual. Furthermore, a novel 3·0-kb deletion involving the element (Bm3·0) was demonstrated in the remaining individual. Comparisons of single-nucleotide polymorphisms and microsatellites in intron 1 between Bm5·8 and Bm3·0 suggested that these deletions occurred independently.
[Show abstract][Hide abstract] ABSTRACT: Background and Objectives
Previously, a weak phenotype Am or Bm was assumed to be caused by a reduction of A or B gene expression in bone marrow cells, but not in mucus-secreting cells. However, ABO expression has not been examined in erythroid progenitor cells of Am or Bm individuals.Materials and Methods
We carried out in vitro erythroid differentiation of CD34+ cells from peripheral blood of a Bm individual harbouring a 3·0-kb deletion including an erythroid cell-specific regulatory element, named the +5·8-kb site, in intron 1 of the human ABO blood group gene.ResultsDuring the in vitro differentiation of CD34+ cells from this Bm individual into erythroid cells, B-antigens were not detectable on the cultured cells by flow cytometric analysis, and allele-specific RT-PCR consistently detected the transcripts from the O allele, but not from the B allele. Moreover, chromatin immunoprecipitation assay demonstrated that both RUNX1 and GATA-2 or GATA-1 were bound to the +5·8-kb site in cultured erythroid cells expressing ABO.Conclusion
It is likely that the +5·8-kb site enhances transcription from the ABO promoter in erythroid cells through binding of RUNX1 and GATA-2 or GATA-1.
[Show abstract][Hide abstract] ABSTRACT: Along with time after death, postmortem computed tomography (PMCT) of the brain can reveal sequential changes. In the present study, we investigated the relationship between brain rigidity and advanced postmortem changes such as intravascular gas production, cerebral settling or cerebral liquefaction on PMCT. We then examined the findings of PMCT as an indicator of successful macroscopic examination of arbitrary brain slices at classical autopsy. The association between these advanced postmortem changes and the validity of macroscopic brain examination was investigated in 149 cases that were examined by PMCT at our department prior to autopsy in the period from September 2011 to December 2013. We found that the postmortem changes, classified into four stages, generally reflected the fragility of the brain. Thus, it is likely that PMCT findings of advanced postmortem changes are able to indicate decreased brain rigidity ahead of autopsy. These findings support the idea that PMCT could be used as a guide by forensic pathologists for suitable handling of a fragile brain, thus enhancing the quality of autopsy.
Legal Medicine 10/2014; 17(2). DOI:10.1016/j.legalmed.2014.10.008 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Case history
A 3-month-old infant was found dead in his bed. Postmortem 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.
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 significant accumulation of C14:1-acylcarnitine in the liver of the deceased, suggesting very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency.
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.
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.
Forensic Science International 09/2014; 244. DOI:10.1016/j.forsciint.2014.08.031 · 2.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 inhERG 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(ΔAT)
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).
[Show abstract][Hide abstract] ABSTRACT: Background and objectivesAn erythroid cell-specific regulatory element, referred to as the +5.8-kb site, has been identified in the first intron of the human ABO blood group gene. Subsequent studies have revealed involvement of deletion or mutation at the site in phenotypes Am, Bm and ABm. We investigated the molecular mechanisms involved in the A3 and B3 phenotypes. Materials and methodsGenomic DNAs were prepared from peripheral blood of seven A3 individuals and twelve B3 or AB3 individuals, and the nucleotide sequences were investigated using PCR and sequencing. Promoter assays were performed with K562 cells. ResultsTwo single point-mutations at +5893 or +5909 in the site on the A-allele were found in A3 individuals, while promoter assays revealed decreased activity at the site as a result of each substitution. In two B3 individuals, a single point-mutation at −77 in the ABO promoter on the B-allele was found, and the substitution was demonstrated to reduce the promoter activity. Conclusion
Nucleotide substitutions in the transcriptional regulatory elements such as the +5.8-kb site and the ABO promoter appear to decrease transcription from the A- and B-alleles, resulting in reduction in A- and B-antigen expression in A3 and B3, respectively.
[Show abstract][Hide abstract] ABSTRACT: Blood group-related glycans determining ABO and Lewis blood groups are known to function as attachment factors for most of the norovirus (NoV) strains. To identify binding specificity of each NoV, recombinant norovirus-like particles (VLPs) and human saliva samples with different ABO, Lewis phenotypes and secretor status have been commonly applied. When binding specificities of VLPs prepared from 16 different genotypes of NoVs in GI and GII genogroups were characterized in samples of human gastric mucosa compared to human saliva based on blood group phenotypes, considerable differences were observed for several strains. Novel binding specificities determined by an ELISA using preparations from human gastric mucosa were also ascertained by immunohistochemical analyses using human jejunal mucosa, widely believed to be susceptible to NoV infection. Further, A, B and O(H) blood group substances prepared from porcine and squid tissues were found to be effective for preventing ABO blood group-specific binding of VLPs to both saliva and mucosa samples. Therefore, these blood group substances might have potential for the prevention and treatment of NoV infection.
PLoS ONE 02/2014; 9(2):e89071. DOI:10.1371/journal.pone.0089071 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objectives of this study were to evaluate all of the non-synonymous single nucleotide polymorphisms (SNPs) in the DNase 1 and 1L3 genes, potentially implicated in autoimmune diseases, as a functional SNP in terms of alteration of the activity levels. We examined the genotype distributions of the 32 and 20 non-synonymous SNPs in DNASE1 and DNASE1L3, respectively, in the 3 ethnic groups, and the effect of these SNPs on the DNase activities. Among a total of 44 and 25 SNPs including those characterized in our previous studies [Yasuda et al., Int. J. Biochem. Cell Biol. 42 (2010) 1216-1225; Ueki et al. Electrophoresis, 32 (2012) 1465-1472], only 4 and 1, respectively, exhibited genetic heterozygosity in one or all of the ethnic groups examined. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, 11 activity-abolishing and 11 activity-reducing SNPs in DNASE1, and 2 activity-abolishing and 5 activity-reducing SNPs in DNASE1L3 were confirmed as a functional SNP. Phylogenetic analysis showed that all of the amino acid residues in activity-abolishing SNPs were completely or well conserved in animal DNase I and 1L3 proteins. Although almost all of non-synonymous SNPs in both genes that affected the catalytic activity showed extremely low genetic heterogeneity, it seems plausible that a minor allele of 13 activity-abolishing SNPs producing a loss-of-function variant in both the DNase genes would be a direct genetic risk factor for autoimmune diseases. These findings may have clinical implications in relation to the prevalence of autoimmune diseases. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: A 53-year-old man who had suffered from hypertension was found dead on the floor of his room by his roommate on return from work in the summer. The body showed severe postmortem changes with advanced putrefaction and autolysis. No evident injury was detectable anywhere on the body. Postmortem computed tomography (PMCT) scan demonstrated cerebral hemorrhage in the left putamen and infiltration of blood into the third and lateral ventricles, although the brain had undergone putrefactive and autolytic changes including intravascular gas accumulation and partial cerebral settling. Detailed slice examination of the brain at autopsy proved impossible because of its fragility when attempting to remove it from the skull. This case illustrates that PMCT can be useful for demonstrating cerebral hemorrhage even in putrefactive brains, acting as a guide for forensic pathologists when conducting careful examination of a fragile brain.
Journal of Forensic Radiology and Imaging 10/2013; 1(4):212–214. DOI:10.1016/j.jofri.2013.06.003
[Show abstract][Hide abstract] ABSTRACT: An erythroid cell-specific regulatory element, referred to as the +5·8-kb site, had been identified in the first intron of the human ABO blood group gene. Subsequent studies revealed that either a 5·8-kb deletion including the +5·8-kb site or disruption of a GATA factor binding motif at the site was present in all Bm and ABm individuals examined. We investigated the molecular mechanism of the Am phenotype, which is analogous to the Bm phenotype.
Genomic DNAs were prepared from peripheral blood of two Am individuals, and the nucleotide sequences were investigated using PCR and direct sequencing. Electrophoretic mobility shift assay (EMSA) and promoter assay with K562 cells were carried out.
A novel 23-bp nucleotide deletion was found at the +5·8-kb site in both individuals. EMSAs demonstrated binding of the transcription factor RUNX1 to the nucleotides within the deletion. Promoter assays showed that the deletion reduced the transcriptional activity of the +5·8-kb site.
Deletion of the 23-bp nucleotides including the RUNX1 binding site decreases transcription of the A allele, resulting in the reduction in A antigen expression in the Am phenotype.
[Show abstract][Hide abstract] ABSTRACT: Background
The ABO blood group is important in blood transfusion. Recently, an erythroid cell-specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell-specific regulatory activity of the element was dependent upon GATA-1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 B-m and AB(m) individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes. Study Design and Methods
In this study, further investigation was performed to reveal the underlying mechanism responsible for reduction of B antigen expression in the exceptional B-m individual. Peptide nucleic acid-clamping polymerase chain reaction was carried out to amplify the B-related allele, followed by sequence determination. Electrophoretic mobility assays and promoter assays were performed to examine whether a nucleotide substitution reduced the binding of a transcription factor and induced loss of function of the element. ResultsSequence determination revealed one point mutation of the GATA motif in the element. The electrophoretic mobility shift assays showed that the mutation abolished the binding of GATA transcription factors, and the promoter assays demonstrated complete loss of enhancer activity of the element. Conclusion
These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the B-m individual.
[Show abstract][Hide abstract] ABSTRACT: 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.
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).
Deutsche Zeitschrift für die gesamte gerichtliche Medizin 04/2013; 128:105-115. DOI:10.1007/s00414-013-0853-4 · 2.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An 81-year-old man was found dead 1 month after he had disappeared following a visit to a hot spring resort in early autumn. The body showed severe postmortem changes with advanced skeletonization from the head to the abdomen as well as putrefactive and autolytic changes in the remaining tissues. The thoracic and abdominal organs had been lost. Naked eye examination revealed soft tissue injuries accompanied by ragged edges and characteristic punctures with no signs of vitality, suggesting that these injuries had been due to postmortem animal scavenging. However, bruises were prominent on the anterior parts of both lower extremities. Postmortem computed tomography (PMCT) scan demonstrated subdural hematoma over the right cerebral hemisphere, although the brain itself had undergone putrefactive and autolytic changes. Subsequent autopsy confirmed the presence of a 140 g acute subdural hematoma, which would likely have been fatal. This case illustrates that PMCT is able to yield important information about possible cause of death, even in a partially skeletonized body.
Legal Medicine 01/2013; 15(1):32–34. DOI:10.1016/j.legalmed.2012.05.005 · 1.24 Impact Factor