Eiji Kajii

The RHD gene is highly detectable in RhD-negative Japanese donors.

Recent molecular studies on the Rh blood group system have shown that the Rh locus of each haploid RhD-positive chromosome is composed of two structural genes: RHD and RHCE, whereas the locus is made of a single gene (RHCE) on each haploid RhD-negative chromosome. We analyzed the presence or absence of the RHD gene in 130 Japanese RhD-negative donors using the PCR method. The RhD-negative phenotypes consisted of 34 ccEe, 27 ccee, 17 ccEE, 26 Ccee, 19 CcEe, 1 CcEE, and 6 CCee. Among them, 36 (27.7%) donors demonstrated the presence of the RHD gene. Others showed gross or partial deletions of the RHD gene. These results were confirmed by Southern blot analysis. Additionally, the RHD gene detected in the RhD-negative donors seemed to be intact through sequencing of the RhD polypeptide cDNA and the promoter region of RHD gene. The phenotypes of these donors with the RHD gene were CC or Cc, but not cc. It suggested that there is some relationship between the RHD gene and the RhC phenotypes in RhD-negative individuals. In Caucasian RhD-negative individuals, the RHD gene has not been found outside of the report of Hyland et al. (Hyland, C.A., L.C. Wolter, and A. Saul. 1994. Blood. 84:321-324). The discrepant data on the RHD gene in RhD-negative donors between Japanese and Caucasians appear to be derived from the difference of the frequency of RhD-negative and RhC-positive phenotypes. Careful attention is necessary for clinicians in applying RhD genotyping to clinical medicine.

Isolation of a new cDNA clone encoding an Rh polypeptide associated with the Rh blood group system

The polymerase chain reaction (PCR) was used to amplify Rh-related cDNAs from erythroid cells cultured by the selective two-phase liquid culture system for human erythroid progenitors in peripheral blood. Direct sequencing based on PCR presents heterozygous bands. Two Rh polypeptide cDNAs have been isolated from the PCR products and tentatively designated RhPI cDNA and RhPII cDNA. Both cDNA clones have an open reading frame composed of 1251 nucleotides. The RhPI cDNA clone shows a single nucleotide substitution with no amino acid substitution compared with the published sequence. The RhPII cDNA clone, on the other hand, differs from the above by 41 nucleotide substitutions with the open reading frame, resulting in 31 amino acid substitutions.

An intronic variable number of tandem repeat polymorphisms of the cold-induced autoinflammatory syndrome 1 ( CIAS1) gene modifies gene expression and is associated with essential hypertension

Cold-induced autoinflammatory syndrome 1 (CIAS1) gene is a member of the NALP subfamily of the CATERPILLER protein family that is expressed predominantly in peripheral blood leukocytes, which is to regulate apoptosis or inflammation through the activation of NF-κB and caspase. Recent genetic analyses suggested an association between inflammation and oxidative stress-related genes in the development of hypertension. This is the first genetic study indicating an association between the CIAS1 gene and susceptibility to essential hypertension (EH). The frequency of subject with the homozygote of 12 repeat allele was significantly higher in patients with hypertension compared with control subjects (987 cases, 924 controls) (P=0.030; odds ratio=1.24) at a novel VNTR polymorphism of CIAS1 intron 4 loci. We also found that the mean of systolic blood pressure of homozygotes of 12 repeat allele was 6.4 mmHg higher than those of homozygotes of non-12 repeat allele in male random population (P=0.009). The frequency of six SNPs spanning of the CIAS1 gene was not significantly between patients and controls. The real-time PCR analysis showed that among healthy young adults, 12-12 subjects expressed CIAS1 mRNA in peripheral leukocytes significantly more abundantly than homozygote of non-12 repeat alleles subjects (P<0.05). Reporter gene assay of the CIAS1-VNTR in HL60 stimulated by lipopolysaccharides showed that the intronic sequence involving 12 repeat increased the expression of luciferase compared with 9, 7, and 6 repeats. Thus, we propose here the CIAS1 is associated with EH through the dominant expression of transcripts, which may depend on the CIAS1-VNTR genotype.

Prehypertension and the risk for cardiovascular disease in the Japanese general population: the Jichi Medical School Cohort Study

Background Prehypertension is associated with an increased risk of the development of hypertension and subsequent cardiovascular disease. However, it is unclear whether the increased risk of cardiovascular disease associated with prehypertension varies by duration of follow-up (i.e., the first 5 years vs. second 5 years) or varies between nonelderly and elderly individuals. Methods We enrolled 11 000 community dwelling persons (6739 women and 4261 men, aged 18–90 years) from the Japanese general population, followed them for an average of 10.7 ± 2.4 years (117 517 person-years) and evaluated the incidence of cardiovascular events (including both stroke and myocardial infarction). Results In the full cohort, prehypertension was associated with a 45% higher risk of cardiovascular events than normal blood pressure after adjusting for traditional cardiovascular risk factors (hazard ratio = 1.45, P = 0.03). The risk of cardiovascular events with prehypertension during the second 5-year period was elevated in the nonelderly subgroup (<65 years) (hazard ratio = 2.13, P = 0.01), but not in the elderly subgroup (≥65 years) (hazard ratio = 0.93, P = 0.82) (P = 0.054 for the difference in hazard ratio). The elevated risk with prehypertension during the first 5-year period was not significant in either the nonelderly (hazard ratio = 1.60, P = 0.36) or elderly (hazard ratio = 1.19, P = 0.63) group. However, the risks with prehypertension were not statistically different between the first and second 5-year period. Conclusion Prehypertension is associated with an increased 10-year risk of cardiovascular disease; the provocative finding that this risk may be especially elevated during the second 5-year period in the nonelderly requires confirmation in a larger cohort.

A novel missense mutation of the tissue-nonspecific alkaline phosphatase gene detected in a patient with hypophosphatasia

AbstractHypophosphatasia is a rare heritable inborn error of metabolism characterized by abnormal bone mineralization associated with a deficiency of alkaline phosphatase. The clinical expression of hypophosphatasia is highly variable, ranging from death in utero to pathologic fractures first presenting in adulthood. We investigated the tissue-nonspecific alkaline phosphatase (TNSALP) gene from a Japanese female patient with hypophosphatasia. By a quantitative polymerase chain reaction (PCR) method, the amount of TNSALP mRNA appeared to be almost equal to that in normal individuals. Gene analysis clarified that the hypophosphatasia originated from a missense mutation and a nucleotide deletion. The missense mutation, a C → T transition at position 1041 of cDNA, results in an amino acid change from Leu to Phe at codon 272, which has not yet been reported. The previously reported deletion of T at 1735 causes a frame shift mutation downstream from Leu at codon 503. Family analysis showed that the mutation 1041T and the deletion 1735T had been inherited from the probands father and mother, respectively. An expression experiment revealed that the mutation 1041T halved the expression of alkaline phosphatase activity. Using homology analysis, the Leu-272 was confirmed to be highly conserved in other mammals.

Characterization of autoantibodies in mixed-type autoimmune hemolytic anemia

Abstract. Three of 46 patients with autoimmune hemolytic anemia (AIHA) satisfied the diagnostic criteria for mixed‐type AIHA in which both warm‐type and cold‐type autoantibodies against red blood cells (RBCs) are present. The specificities of these autoantibodies were analyzed. All of the warm‐type autoantibodies were IgG‐x, and the specificities were not serologically classified. The autoantibody of patient 1 reacted to the 41‐ and 80‐kD peptides on immunoblotting, and the epitope corresponding to it was papain sensitive. Two warm‐type autoantibodies from patients 2 and 3 resembled each other in serological analyses and reacted with a protease‐ and neuraminidase‐resistant antigen. However, the antigen corresponding to the autoantibody of patient 3 was located on the 37‐kD peptide by immunoprecipitation. All of the cold‐type autoantibodies were IgM‐x and showed high titer and high thermal amplitude. According to the reaction pattern with untreated and enzyme‐treated RBCs, the cold‐type autoantibodies of patients 2 and 3 were revealed to be anti‐Om and anti‐I, respectively. In patient 1, the cold‐type autoantibody was characterized as having high affinity for autologous RBCs, but its specificity was unclassified. The antigens corresponding to the cold‐type autoantibodies were not located by immunoblotting and immunoprecipitation. These serological and immunochemical approaches to autoantibodies in mixed‐type AIHA revealed that the warm and cold components recognized the different antigens.

A splicing mutation of the RHAG gene associated with the Rhnull phenotype

Rhnull is a syndrome serologically characterized by the deficiency of all Rh antigens on human red blood cells. Rhnull is divided into two types: regulator and amorph. Recently, Cherif‐Zahar et al. proposed that the RHAG gene encoding the Rh50 glycoprotein is a candidate for inducing regulator type Rhnull. We investigated both the RH and RHAG genes in an Rhnull individual. The reticulocytes from the propositus had RHD, RHcE, and RHCe transcripts without any mutation. However, the sequence analysis of RHAG cDNA showed a deletion of 122 bp from nucleotide 946 to 1067. This deletion was revealed to be due to a homozygous splicing mutation, which is a single base substitution at the consensus sequence of the splicing acceptor site (AG→AT). The mutation appeared to break the ‘GT‐AG’ splicing rule and to cause 122 bp exon skipping accompanied by a frameshift. This study confirms that the RHAG gene is the most likely candidate for the ‘regulator’ gene of Rhnull cases.

Dinucleotide repeat in the 3' flanking region provides a clue to the molecular evolution of the Duffy gene.

Abstract The Duffy blood group system consists of three alleles, FYA, FYB, and FY. To study the molecular evolution of the three alleles, we established the polymorphism of a dinucleotide (GT) repeat sequence (designated FyGT/ C) in the 3′ flanking region of the Duffy gene, and studied the relationship between FyGT/C and Duffy polymorphism in Japanese, people of African origin, and chimpanzee. By single-strand conformation polymorphism and sequence analysis, five and two alleles were identified in Japanese and Africans, respectively. In 110 random Japanese, the FyGT/C genotypes observed were in agreement with Hardy-Weinberg law. From the sequence of the chimpanzee Duffy gene, including both flanking regions, FYB was identified as the ancestral gene of the human alleles. The FyGT/C sequences associated with the FY allele of Africans were distinct from those of Duffy positives, whereas the FYB and FYA alleles shared common FyGT/C sequences. Thus, it is suggested that the first split took place between the FYB and FY alleles, and the second between the FYB and FYA alleles.

Autoimmune hemolytic anemia in patients with de novo acute myelocytic leukemia

Autoantibody against erythrocytes has occasionally been observed in patients with de novo acute myelocytic leukemia (AML). However, it is not clear whether this autoantibody in AML patients induces frank hemolysis (autoimmune hemolytic anemia, AIHA), as seen in lymphoid neoplasms. We present two de novo AML patients who showed hemolysis due to antiglobulin test-positive and test-negative AIHA, respectively. AIHA should be considered as one cause of anemia in de novo AML patients, and blood transfusions should be given carefully in such cases to avoid harmful hemolysis.

Intricate combinatorial patterns of exon splicing generate multiple Rh-related isoforms in human erythroid cells

The Rhesus (Rh) blood group system shows complex polymorphisms in the human. Some of the heterogeneity may be generated by alternative RNA splicing. For a systematic analysis of Rh-related mRNA isoforms expressed in reticulocytes, we isolated mRNA, which was then reverse transcribed and amplified by the polymerase chain reaction (PCR) to give Rh-related cDNAs of two segments of 704 bp and 975 bp. The PCR amplification of the 5′-region yielded a single PCR product, whereas a complex electrophoretic pattern of PCR bands was derived from the 3′-region. A highly reproducible ladder of multiple additional bands migrated below the PCR products corresponding to the full-size cDNAs for RhPI and RhPII and encoding two different Rh polypeptides. Eleven and five truncated isoforms of the RhPI and RhPII cDNAs, respectively, were identified in the PCR products. These isoforms appear to be generated by combinatorial splicing of six RhPI and three RhPII exons. Our results suggest that the Rh-related polypeptides consist of a mixture of RhPI and RhPII polypeptide isoforms differing at the C terminus. Multiple RNA splicing pathways are thus operative in the two Rh-related genes even within a single cell lineage of human erythroid cells.