The B allele with a 5·8 kb deletion in intron 1 of the ABO gene is the major allele in Japanese individuals with Bm and A1 Bm phenotypes.

Bm and A1 Bm phenotypes are the most frequent ABO variants in the Japanese population. The B antigen on Bm red blood cells is only detectable by adsorption and elution tests, and plasma B-transferase activity is usually detected at half or less levels compared with that of common B. Recently, a B allele lacking an erythroid cell-specific transcription enhancer in intron 1 of the ABO gene was identified from individuals with Bm and A1 Bm phenotypes, which could explain the unique serologic properties of Bm . In the Japanese Red Cross Society, eight Blood Centers tested blood samples from donors throughout Japan and collected blood samples from 888 Bm and 415 A1 Bm individuals. DNA analysis revealed that 1300 of 1303 (99·77%) individuals had the B allele with a 5·8 kb deletion (c.28 + 5110_10889del), which included the enhancer element.

Prevalence of RHD alleles in Japanese individuals with weak D phenotype: Identification of 20 new RHD alleles.

We identified 46 different RHD alleles from 226 Japanese individuals with weak D phenotype, 26 of which had been previously described and 20 that were novel. Among these weak D individuals, the alleles with c.960G>A, c.845G>A (RHD*15) or c.1013T>C (RHD*01W.24) mutations were most prevalent with relative occurrences of 36·7%, 15·9% and 9·7%, respectively. These findings demonstrate that the prevalence of common weak D alleles in the Japanese population significantly differs from that of Caucasian populations.

Presence of nucleotide substitutions in the ABO promoter in individuals with phenotypes A3 and B3.

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.

Production of human monoclonal anti-Jk3, recognising an epitope including the Jk(a) /Jk(b) polymorphic site of the Kidd glycoprotein.

BACKGROUND AND OBJECTIVES: The Kidd blood group system consists of polymorphic antigens, Jk(a) (JK1) and Jk(b) (JK2), and a high-incidence antigen, Jk3. Anti-Jk3 is often observed in immunised Jk(a-b-) individuals. In this study, we aimed to establish a human hybridoma cell line secreting monoclonal anti-Jk3 (HIRO-294). MATERIALS AND METHODS: Peripheral blood lymphocytes of a Filipino woman with the Jk(a-b-) phenotype having anti-Jk3 were transformed with Epstein-Barr virus and then hybridised with the myeloma cell line JMS-3 using the polyethylene glycol (PEG) method. The reactivity and specificity of the anti-Jk3 were examined by serology and flow cytometry. RESULTS: Four hybridoma clones secreting anti-Jk3 were established and the antibody from one of these clones, HIRO-294, was examined. The reactivity of HIRO-294 was positive with 227 Jk(a+b-) red blood cells (RBCs), 298 Jk(a-b+) RBCs, and 1043 Jk(a+b+) RBCs, but was negative with 21 Jk(a-b-) RBCs. Eluates from Jk(a+b-) RBCs and Jk(a-b+) RBCs sensitised with the anti-Jk3 were cross-reacted with Jk(a-b+) RBCs and Jk(a+b-) RBCs, respectively. The reactivity of HIRO-294 was enhanced by the treatment of RBCs with ficin, trypsin, pronase and α-chymotrypsin, but was not changed by their treatment with neuraminidase, dithiothreitol and ethylenediaminetetraacetic acid (EDTA) glycine acid (GA). The RBCs sensitised by the anti-Jk3 were not agglutinated with the commercial reagents of anti-Jk(a) and anti-Jk(b) by saline test, whereas the nonsensitised RBCs or those sensitised by monoclonal anti-D [HIRO-3, immunoglobulin G (IgG) class] were agglutinated with those reagents. CONCLUSIONS: We established a human hybridoma cell line secreting monoclonal anti-Jk3 (HIRO-294). This antibody had unique specificity, recognising the Kidd glycoprotein including the Jk(a) /Jk(b) polymorphic site.

Differences in ABO antibody levels among blood donors: a comparison between past and present Japanese, Laotian, and Thai populations.

Passively transfused blood group antibodies cause clinical problems. High titers of anti-A and anti-B seem to be one reason for hemolytic transfusion reactions and for ABO HDN. In Japan, anti-A and anti-B titers notably decreased in the 15 years between 1986 and 2001. At present, titers of more than 100,as measured using the saline method, are rare. Differences in the level of anti-A and anti-B among ethnic populations have been reported; these differences were found to be the result of environmental factors rather than hereditary factors. In the present study, the anti-A and anti-B titers of random donors in three Asian populations are compared. In Thailand, the IgM anti-A and anti-B titers are low and are similar to the Japanese titers reported in 2001, but the IgG anti-A and anti-B titers are high and are similar to the Japanese titers reported in 1986. In the Lao People's Democratic Republic, both the IgM and the IgG anti-A and anti-B titers are high and are similar to those reported in Japan in 1986. In addition, anti-A and anti-B titers of different sex donors and of various age groups were also compared. High titers were found in 8.8 percent of the female donors in the younger than 30 age group and in 36.7 percent of the female donors in the older than 50 age group.

Two cases of mosaic RhD blood-group phenotypes and paternal isodisomy for chromosome 1.

We encountered a 22-year-old man (case 1) and a 23-year-old woman (case 2), both unrelated and healthy. They were mosaic for the Rh blood group phenotype: one erythrocyte population was D-positive and the other was D-negative. Flow cytometric analysis of density profile of RhD antigen in their erythrocytes, and cytogenetic analysis including in situ hybridization using an RHD/RHCE-containing PAC clone, excluded a deletion of the RHD/RHCE gene complex, but suggested the presence of cells with uniparental disomy for chromosome 1 (UPD1). Microsatellite marker analysis was performed in both probands and their family members. In case 1, the analysis with markers spanning the chromosome 1 revealed both maternal and paternal alleles in his peripheral blood leukocytes (PBL), Epstein-Barr virus-transformed lymphoblastoid cells (EBL), and buccal mucosal cells. However, only paternal alleles were detected in all of 50 individual pieces of his hair or hair-roots and all of five monoclonal cell lines cloned from his established EBL. There was no direct evidence of heterozygous, biparental alleles in these two tissues. The presence of maternal isodisomy 1 was not absolutely ruled out in other tissues examined in case 1. Similar results were obtained in case 2, showing biparental, disomic patterns in her PBL and in 15 of 20 pieces of her hair roots, and showing monoallelic patterns in the remaining five pieces of hair roots. Analysis with markers for other autosomes confirmed their biparental inheritance. These findings indicated that both cases had at least two cell populations, one population having paternal UPD1 (isodisomy 1), and another heterozygous, biparental disomy 1. We emphasize that isodisomy for chromosome 1 is not infrequent and may cause unusual RhD phenotype, as seen in cases we described.

Recombination and gene conversion-like events may contribute to ABO gene diversity causing various phenotypes.

We identified five different alleles, tentatively named ABO*O301, *0302, *R102, *R103, and *A110, in Japanese individuals possessing the blood group O phenotype. These alleles lack the guanine deletion at nucleotide position 261 which is shared by a majority of O alleles. Nucleotide sequence analysis revealed that *0301 and *0302 had single nonsynonymous substitutions compared with *A101 or *A102 responsible for the A1 phenotype. Analysis of intron 6 at the ABO gene by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing revealed that *R102 and *R103 had chimeric sequences of A-02 and B-02, respectively, from exons 6 to 7. In the analysis of five other chimeric alleles detected in the same manner, we identified a total of four different recombination-breakpoints within or near intron 6. When 510 unrelated Japanese were examined, the frequency of the chimeric alleles generated by recombination in intron 6 or exon 7 was estimated to be 1.7%. In addition, we found that *O301, *A110, *C101, *A111, and 35% of *A102 had a unique A-B-A chimeric sequence at intron 6, presumed to originate from a gene conversion-like event. We had previously established that *A110 also had an A-O2-A chimeric sequence around nucleotide position 646 in exon 7. Thus this allele has an A-B-A-O2-A chimeric sequence from intron 6 to exon 7 probably generated by two different gene conversions. Similar patchwork sequences around nucleotide position 646 in exon 7 were observed in two other new alleles responsible for the Ax and B3 phenotypes. Thus, the site is presumably a hotspot for gene conversion. These results indicate that both recombination and gene conversion-like events play important roles in generating ABO gene diversity.

Different alleles cause an imbalance in A2 and A2B phenotypes of the ABO blood group.

BACKGROUND AND OBJECTIVES: In several populations, including the Japanese, the frequency of the A2B phenotype is significantly higher than expected based on the A2 phenotype frequency. To understand the genetic basis of this 'excess' of A2B, we examined ABO alleles in individuals with A2-related phenotypes. MATERIALS AND METHODS: ABO alleles were identified by means of polymerase chain reaction single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses. RESULTS: The frequencies of A2-related alleles (*A105, *A106, *A107, *A111 and *R101) were clearly different between the A2 and A2B phenotypes. In particular, a putative recombinant allele, *R101, was uncommon in the A2 but common in the A2B phenotype individuals. This allele was also detected in 4 of 401 (1%) unrelated A1 phenotype (AO genotype) individuals. CONCLUSION: *R101 is presumably expressed as phenotype A1 in *R101/*O heterozygous individuals, but as phenotype A2 in *R101/*B heterozygotes, thus giving rise to a high A2B phenotype frequency.

A new solid-phase assay system using magnetic force on blood group serology.

A new solid-phase assay system has been developed for the detection of irregular antibodies in plasma and serum specimens. By means of a magnetic indicator cell, we have established a sensitive and easy-to-handle antihuman globulin (AHG) test involving magnetic-mixed passive hemagglutination (M-MPHA). Comparative studies of M-MPHA with conventional AHG tests demonstrated that this is sufficiently sensitive and specific to detect irregular antibodies to red blood cells. Elimination of centrifugation in this test seems to open the way toward new standardization of solid-phase systems.

Molecular genetic analysis of variant phenotypes of the ABO blood group system.

ABO is clinically the most important blood group system in transfusion medicine and includes many variant phenotypes. To understand the molecular genetic basis of this polymorphic system, we have analyzed genomic DNAs obtained from Japanese individuals possessing variant ABO phenotypes including A2, Ax, Ael, cis-AB, Bx, and Bel. By polymerase chain reaction-single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses, we identified 11 different alleles. These alleles had nucleotide sequences different from those of the previously described 13 different alleles responsible for the common ABO phenotypes. Analysis of the nucleotide sequences of the alleles responsible for those variant phenotypes showed that the amino acid residues at position 266 and 268 may be crucial for transferase specificity, whereas those at positions 214, 216, 223, 291, and 352 may be critical for the activity level. Nine of the 11 alleles, responsible for the A2, Ax, Ael, cis-AB, Bx, and Bel phenotypes, were presumed to be generated from common ABO alleles by single nucleotide mutations such as nonsynonymous substitution, deletion, or insertion. Two other alleles, responsible for the A2 and Ael phenotypes, may have originated by recombination, gene conversionlike events or accumulation of nucleotide substitutions. Our data indicate that different alleles could cause the same ABO variant phenotypes, and that these alleles do not necessarily belong to a single evolutionary lineage.

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes.

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A1 (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *B101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.

Use of standardized protease enzymes for antibody screening of blood donor samples with the microplate system AutoAnalyzer.

Papain, bromelin and ficin can be standardized by the casein method for use in red blood cell antibody screening tests. The minimal and optimal enzyme activity for detecting blood group antibodies in donors, using the new Automated Pre-Transfusion Blood Testing System Olympus PK7200 by the two-stage method for all three enzymes, is one casein unit. One casein unit of proteinase activity changed the red blood cell surface charge to a low plateau value as measured by electrophoretic mobility and sialic acid content, and removed sterically inhibiting structures of surface protein as detected by SDS-PAGE.

[Biochemical differences in middle ear effusions between pediatric and adult patients, (I). The lipid composition].

Middle ear effusions (MEEs) from adult were usually serous in nature, but those from child were mucous. MEEs contain the substances secreted by the epithelial cells of the middle ear and Eustachian tube to regulate surface tensions as well as those produced as the result of inflammation, but the biochemical bases of serous and mucous MEEs have not been clearly established. To clarify the biochemical differences giving rise to the characteristic physicochemical properties of MEE from children and from adults, all lipid components of MEEs from 7 children and 7 adults by thin-layer and gas liquid chromatographies were analyzed, and also they were compared with those from sera. Although no significant difference in lipid composition was observed between adult and pediatric sera, the amounts of phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS) in pediatric MEE were higher than those in adult MEE. The relative concentration of PE in the total phospholipids of pediatric MEE was 26.5%, significantly higher than that in adult MEE (9.2%), and pediatric (6.1%) and adult (3.1%) sera. A similar high concentration of PS was also observed in pediatric MEE. Since phospholipids were the major components of surfactants secreted from the epithelial cells of the middle ear, significantly high concentrations of the charged phospholipids, PE and PS, might be responsible in part for the characteristics of pediatric MEE. To compare physical properties in different phospholipid compositions between pediatric and adult MEEs, polarization value was measured with liposomes in each phospholipid compositions. The polarization value in pediatric phospholipid composition liposomes was higher than in adult. It seemed that the microviscosity in pediatric MEE was lower than in adult.

[Biochemical differences in middle ear effusions between pediatric and adult patients, (II). The protein composition].

To clarify the biochemical differences between pediatric and adult middle ear effusions (MEEs) in secretory otitis media with effusion (OME), we analyzed the protein components from MEEs of 3 children and 3 adults by electrophoresis. High molecular protein components (280 and 265 kDa proteins), which contained in sera in the concentrations less than 2.5%, were not detected in MEEs even in trace amounts. The protein components which molecular weights were less than 260 kDa are transferred from sera into MEEs in a same proportion as in sera. Mucin type glycoprotein was comprised about 30% of the total protein in pediatric mucous MEEs and the weight ratio of mucin to albumin in pediatric mucous MEEs was 2.69, but serous MEEs from adult patients did not contain mucin. Immunoglobulin (IgA, IgG and IgM) concentrations in pediatric mucous MEEs were same as in adult serous MEEs. The concentrations of IgA and IgM in MEEs were higher than in sera. Lysozyme concentration in MEEs was also higher than in sera, and that in pediatric mucous MEEs was nearly 2 times higher than serous MEEs from adult patients.

Characteristic differences in the lipid composition of middle ear effusions in adult and pediatric patients: phosphatidylethanolamine and phosphatidylserine levels.

Middle ear effusions (MEEs) from adult patients with otitis media with effusion are usually serous in nature, but those from pediatric patients younger than 8 years old are frequently mucous in consistency. MEEs contain substances secreted by the epithelial cells of the middle ear and eustachian tube to regulate surface tensions as well as those produced as the result of inflammation. Since the biochemical bases of serous and mucous MEEs have not been clearly established, we analyzed all lipid components of MEEs from seven children and seven adults by thin-layer and gas-liquid chromatography, and also compared them with those from sera. Although no significant difference in the lipid composition was observed between adult and pediatric sera, the relative concentration of phosphatidylethanolamine (PE) in the pediatric MEEs was 26.5%, while that in the adult MEEs was 9.2% and was significantly different. A similar high concentration of phosphatidylserine (PS) was also observed in the pediatric MEE. Since phospholipids are major components of surfactants secreted from the epithelial cells of the middle ear, significantly high concentrations of both PE and PS as charged phospholipids may be responsible in part for the mucoid characteristics seen in pediatric MEEs.

Family study and frequency of blood group with strong B transferase accompanied by decreased A and H antigens.

Subgroups of type A blood, named A1, A2, and A1-A2 intermediate (Aint), are specifically characterized by their peculiar A alleles and have their own A1-, A2- or Aint-forms of alpha-N-acetyl-D-galactosaminyltransferase (A-transferase). It is known, however, that certain type A2B persons exhibit A1-transferase. The reason may be an unusual alpha-galactosyltransferase (B-transferase). This strong B-transferase competes with A-transferase for the substrate, H antigen, so as to decrease the A and H antigens on the red cells. We studied this blood group over three generations and found that the strong B-transferase is, in fact, inherited with the B gene and is dominant over normal B-transferase. In AB blood groups in Tokyo, the frequency of people with a strong B-transferase is 5% for A1B and 22% for A2B. This enzyme does not always cause weak H or A antigens.

[Otitis media with effusion in patients with cleft palate and congenital velopharyngeal insufficiency].

The incidences of otitis media with effusion (OME), otoscopic findings of the ear drum, hearing acuity and tympanometric findings were evaluated in 56 cases of cleft palate (CP), 33 cases of submucous cleft palate (SMCP) and 25 cases of congenital velopharyngeal insufficiency without cleft (CVPI). In all the cases, the incidences of OME, the pathological findings in otoscopy and the hearing test were far poorer in the group 8 years old and up than in the younger group. In the younger age group, the incidence of OME was 69% among the CP group, 62% in the SMCP group and showed significantly less incidence in the CVPI group (28%). The incidence of pathological findings in the ear drum was significantly less in the CVPI group than in either the CP group or the SMCP group. Hearing impairment was more frequent in the CP group than in both the SMPC and CVPI groups, while fewer incidences of abnormal tympanogram were found in the CVPI group than in the other two groups. Nearly 14% of the younger CP group developed pathological findings after surgery for velopharyngeal improvement. Most cases were found after push back operations, but rare after pharyngeal flap operations.

Atrial natriuretic peptide in human neuroblastoma.

To clarify the presence of atrial natriuretic peptide (ANP) in neural tissue, extracts from human neuroblastoma which is considered of neural crest origin were analyzed using a specific radioimmunoassay (RIA) for ANP. High concentrations of immunoreactive ANP ranging from 2.7 to 18.4 ng per mg of protein were demonstrated in the tissue. Furthermore, high performance gel permeation chromatography (HPGPC) coupled with the RIA revealed that the immunoreactive ANP found in the tissue consisted of only one component with molecular weight of 12,000 to 13,000 daltons, corresponding to gamma-human ANP (hANP). These results could be direct evidence for generation of ANP intrinsic of human neuronal tissue, and also suggest that neuroblastoma can be used as a model for investigation of mechanism of ANP formation within the neuronal tissues.

Atrial natriuretic factor (ANF) increases urinary protein excretion in patients with essential hypertension: a possible role of ANF for renal handling of protein.

Low dose iv infusion (0.01 and 0.03 micrograms/kg per min, for 30 min each) of alpha-human atrial natriuretic factor (alpha-hANF) produced a significant increase (+300%) in urinary protein excretion in patients with essential hypertension but not in normotensive controls, when their renal function was normal. The major component of excreted proteins induced by alpha-hANF infusion was presumed to be albumin on the basis of molecular weight (69,000) analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Urine output and sodium and potassium excretion rates were increased dose-dependently by alpha-hANF infusion in the hypertensive patients in a similar fashion to those in the controls. Glomerular filtration rate (GFR) remained unchanged in the controls but was slightly increased in the patients (+33%) during the infusion. These results suggest that besides its previously recognized physiological functions such as natriuresis and diuresis, ANF plays an important role in the regulation of renal handling of proteins in patients with essential hypertension.