Sadahiko Iwamoto

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.

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.

Genetic variation of FUT3 in Ghanaians, Caucasians, and Mongolians

BACKGROUND: The FUT3 gene regulates the expression of Lewis blood group antigens. Several lines of evidence suggest association between expression of these antigens and Helicobacter pylori infection or susceptibility to cardiovascular diseases. Single‐nucleotide polymorphisms (SNPs) responsible for the Lewis‐negative phenotype have been reported, but systematic sequence analyses have not yet been performed.

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.

Detection of Rh23 in the partial D phenotype associated with the DVa category

The Rh blood group system, which is the most polymorphic of the blood group systems, is of major importance in transfusion medicine. The partial D phenotypes are classified into different categories according to the absence of one or more D epitopes. Most partial D phenotypes are generated from gene conversion events between the RHD and the RHCE genes. The low-frequency antigen, Rh23 (Dw), is characteristic of the DVa category.1 The DVa red cell (RBC) is also characterized as being negative when tested with monoclonal antibodies (MoAbs) to epitopes D1 and D5 in the 9epitope model.2 The molecular basis for the DVa category has been reported, in which either the entire RHD exon 5 (DVa Hus) or a portion of it (DVa Kou) was replaced by the RHCE equivalents.3 Recently, we reported the presence of a new RHD variant (DVa-like) that closely resembles the DVa category both structurally and serologically.4 In that study, we attempted to detect the expression of Rh23 on the DVa-like RBC membrane by using a polyclonal anti-Rh23 from a donor of blood group A. After the absorption test, the antibody reacted with a control DVa category RBC in an indirect antiglobin test, showing a higher titer, and was negative with normal RBCs. Rh23 was detected on the RBC membrane in three DVa and six DVa-like individuals. These results showed that the DVa-like sample was categorized as DVa category (Table 1). Various amino acid (aa) substitutions existed on the fourth loop of the Rh polypeptides in the DVa category. Detection of Rh23 on the RBC membrane is necessary for classification of the DVa category. The molecular structures of the mutated RHD genes from the DVa (Jpn) and the International Society of Blood Transfusion (ISBT) 49 categories were very similar.4,5 Both phenotypes exhibited four aa substitutions as compared to the aa sequence in the intact D polypeptide: Phe223Val, Glu233Gln, Val238Met, and Val245Leu. However, one aa difference (Ala226Pro) between these two phenotypes was found on the fourth external loop of the D polypeptide. It is known that Pro226 and Ala226 are the E/e polymorphism in the CE polypeptide. This result indicated that the hybrid RHD-CE-D genes of DVa ( Jpn) and ISBT 49 generated through a gene-conversion event were derived from the RHe and RHE genes, respectively. Avent et al.5 also described ISBT 49 as negative when tested with some epitope D8 MoAbs. We threorized that DVa (Jpn) was different from ISBT 49. A previous study3 proposed that the aa residing at position 233 was probably involved in the Rh23 epitope, because the difference in the extracellular region between the D polypeptides and those of the DVa category was only at aa 233 (Glu/Gln), while the other aa substitution at 223 (Phe/Val) was located in the intramembrane domain in all DVa RBCs. In our previous study,4 one aa substitution was detected at 233 on the fourth loop of the Rh polypeptide in S.M. and H. K. individuals. In this serologic study, the S.M. sample with the Glu233Gln substitution was serologically positive for Rh23, while the H.K. and M.I. samples with the Glu233Lys substitution were negative (Table 1). These results suggested that Rh23, which can be expressed on the RBC membrane, results from only one aa substitution (Glu233Gln) in the D polypeptide. We also described the reactivity of the partial D RBCs (in 9-epitope model) with MoAbs (Table 2). The DVa (Jpn) category was confirmed by the lack of epitopes D1 and D5 in this study. Then, the DVa (S.M.) RBCs were negative with MoAbs to epitope D1 and with one MoAb (P3X35 [Table 2]) to epitope D5. This result may indicate that the substitution Glu233Gln was associated not only with the expression of Rh23 but also with the loss of epitopes D1 and D5 in the DVa (S.M.) sample. We also showed that one substitution, Glu233Lys in new partial D phenotype, DHK, results in the lack of various RhD epitopes (Table 2). Negative reactions were observed with MoAbs to epitopes D1, D4, D5, D6/7 (a part), and D9 (a part). This information will allow further study of the epitopes of D.

Molecular characterization of weak D phenotypes by site-directed mutagenesis and expression of mutant Rh–green fluorescence protein fusions in K562 cells

Mutations detected in 161 weak D samples from Caucasians have been classified into 16 types. Because flow cytometry using monoclonal anti‐D antibodies (mAbs) has shown that weak D red cells display type‐specific antigen density, these mutations in transmembranous regions have been assigned weak D phenotypes. The present study attempts to confirm or refute this assignment.

A new mutation detected in RhAG of a Japanese family with Rhmod syndrome may form a longer RhAG protein

We have read with interest the article on bacterial contamination of whole blood by de Korte et al.1 In the study, the authors found bacterial contamination in 0.34 percent of the whole-blood units tested. Others have reported a wide range of bacterial contamination rates for different blood components.2-4 We are concerned that the differences may depend more on test methods than on the type of blood component analyzed. For whole blood, the percentage of positive results ranges from 0.34 percent, as reported by de Korte et al., to 2.2 percent, as reported in the study of Bruneau et al.2 In a recent review on bacterial contamination in platelet concentrates, the percentage of positive results ranged from 0.08 to 0.8 percent.3,4 We conducted a prospective study by culturing 9232 units of random-donor platelet concentrates using an automated microbe detection system (Bact/Alert, Organon Teknika, Boxtel, The Netherlands).5 Similar to Goldman and Blajchman,3 we focused on careful donor arm disinfection and aseptic sampling, and we used a confirmation algorithm (Table 1). All samples that were reactive in the Bact/Alert were confirmed by routine culture. Samples that did not react were considered negative for bacteria. Each reactive sample with bacteria growth on the routine culture was subcultured for identification of the bacteria, and a second sample from the same blood component was cultured (retest). A reactive result with the detection system (Bact/Alert) was interpreted in three different ways. First, the result could be a false positive, reported when the subculture gave a negative result. This was usually caused by machine failure. The second possibility, an unconfirmed positive result, was reported when the subculture showed bacterial growth and the species was identified but the retest was negative or a different bacteria species was identified. An unconfirmed positive result was likely to be due to contamination during sampling. The third possibility, a confirmed positive result, was reported when the subculture showed bacterial growth and the bacterial species was identified, with the retest showing growth and identical species identification. In our study, we found a confirmed positive result rate of 0.03 percent, similar to that of Goldman and Blajchman.3 All together, our rate of initially reactive samples (Bact/Alert) was between 0.4 and 0.6 percent. The confirmed positive result rate was five times greater when 5 units of random-donor platelets were cultured in the same culture bottle (pool testing). The rate was 1.4 percent during the first 12 weeks of the study, which showed a characteristic learning curve for the technologists who performed the sampling. We believe that the higher rates observed with pooling and during the beginning of our study were due to contamination of the samples during the study. De Korte et al.1 reported careful arm disinfection and sampling methods, but the method used to identify contamination caused by sampling was not well defined. Thus, the rate of true positive results could be lower than the rate reported. We think that because contamination during sample manipulation is not completely avoidable, a confirmatory algorithm should be used. We understand that samples that are truly positive could test negative in the retest, but the use of a confirmatory algorithm would allow reporting of a more complete picture of the true prevalence of bacterial contamination of blood components and permit a better comparison among the results of different studies. Emma Castro, MD e-mail: ecastro@cdscruzroja.infonegocio.com José L. Bueno, MD Centro de Donación de Sangre Cruz Roja Española C/Juan Montalvo 3, bajo 28040 Madrid, Spain

Entire sequence of a mouse chromosomal segment containing the gene Rhced and a comparative analysis of the homologous human sequence.

The mouse genomic sequence of the region containing the gene Rhced, the orthologue to the human gene RH30, was determined to elucidate the structure of Rhced and its flanking regions and to compare these with the corresponding human genomic region. Two genes, Smp1 and AK003528 (an orthologue of FLJ10747), flank Rhced. Neither sequences homologous to the characteristic nucleotide elements flanking the RHD gene in humans (rhesus boxes) nor an additional Rh gene were found within the mouse region sequenced. This result and that of a previous report demonstrate that this chromosomal region of the mouse comprises five genes (FLJ10747-RHCE-SMP1-NPD014-P29) that exhibit syntenic homology with the corresponding human region, which suggests that the RHD gene and rhesus boxes were inserted later. Evaluations of tissue distribution and subcellular localization of these genes indicate that the SMP1 orthologue has a ubiquitous tissue distribution and cytoplasmic localization, whereas AK003528 is expressed slightly higher in testis with a strong subcellular localization in the nucleus. Despite the steady improvements in the draft sequence of the human genome, this study demonstrates the continuing benefits of comparative genetic analyses in increasing our understanding of human genomic structure.