Rie Sano

Expression of ABO blood-group genes is dependent upon an erythroid cell-specific regulatory element that is deleted in persons with the B(m) phenotype.

The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase I-hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cell-specific manner. Electrophoretic mobility-shift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element. Furthermore, we found that a partial deletion in intron 1 involving the element was associated with B(m) phenotypes. Therefore, it is plausible that deletion of the erythroid cell-specific regulatory element could down-regulate transcription in the B(m) allele, leading to reduction of B-antigen expression in cells of erythroid lineage, but not in mucus-secreting cells. These results support the contention that the enhancer-like element in intron 1 of ABO has a significant function in erythroid cells.

Mutation of the GATA site in the erythroid cell–specific regulatory element of the ABO gene in a Bm subgroup individual

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 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes.

Use of postmortem computed tomography to reveal an intraoral gunshot injuries in a charred body

A 53-year-old man was found dead after a fire at his residence had been extinguished. Although a pistol was recovered beside the body, external examination was unable to indicate any gunshot wound because of severe charring of the body. Postmortem computed tomography (CT) scan performed prior to autopsy suggested an entrance gunshot wound in the posterior pharynx with loss of soft tissue and an internal bullet path through the right anterior and posterior parts of the occipital bone. Autopsy revealed an entrance gunshot wound with hemorrhage in the soft tissue of the posterior pharynx, massive contusion of the right occipital lobe, and subarachnoid hemorrhage in the right temporal lobe, both occipital lobes and the superior surface of the left cerebellar hemisphere, thus being consistent with the findings of postmortem CT. A carboxyhemoglobin concentration of 5% in blood from the cadaver was consistent with the lack of soot deposition from the larynx to the bronchus. These observations confirmed that death had been caused by an intraoral gunshot resulting in severe brain damage, before the body had been burned.

Deletion of the RUNX1 binding site in the erythroid cell-specific regulatory element of the ABO gene in two individuals with the Am phenotype.

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.

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.

Evaluation of all non‐synonymous single nucleotide polymorphisms (SNPs) in the genes encoding human deoxyribonuclease I and I‐like 3 as a functional SNP potentially implicated in autoimmunity

The objectives of this study were to evaluate all the non‐synonymous single nucleotide polymorphisms (SNPs) in the DNase I and DNase I‐like 3 (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 three 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 Biol42 (2010) 1216–1225; Ueki et al. Electrophoresis32 (2012) 1465–1472], only four and one, 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 two activity‐abolishing and five 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 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.

Use of postmortem computed tomography to reveal acute subdural hematoma in a severely decomposed body with advanced skeletonization

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.

Presence of nucleotide substitutions in transcriptional regulatory elements such as the erythroid cell-specific enhancer-like element and the ABO promoter in individuals with phenotypes A3 and B3, respectively

An 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.

A 3·0-kb deletion including an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene in an individual with the Bm phenotype

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.

A case of fatal drug intoxication showing a high-density duodenal content by postmortem computed tomography

A 22-year-old woman was found dead in her bed, and subsequent postmortem examination was performed using ordinary methods such as external examination, Triage®, and computed tomography (CT) scan which demonstrated a high-density content of the duodenum. Autopsy and quantitative analysis of drugs present in the GI tract showed that high amounts of radiopaque psychotic agents such as fluvoxamine maleate, carbamazepine, and zolpidem tartrate had been responsible for the high-density profile of the duodenum. Postmortem quantitative analysis of drugs in the blood suggested that death had been caused by fatal intoxication with fluvoxamine maleate. Thus, postmortem CT could offer an opportunity to suspect drug intoxication due to radiopaque psychotic agents such as chloral hydrate, phenothiazine, bromovaleryl urea, fluvoxamine maleate, and probably zolpidem tartrate, although it is neither a specific nor a quantitative test for drugs. Therefore, postmortem CT happened to provide clues to investigation of drug intoxication in the present case.