Bahram Hosseini-Maaf

Increased levels of cell-free hemoglobin, oxidation markers, and the antioxidative heme scavenger alpha(1)-microglobulin in preeclampsia.

Preeclampsia is a major cause of morbidity and mortality during pregnancy. To date, the pathogenesis of the disease is not fully understood. Recent studies show that preeclampsia is associated with overexpression of the hemoglobin genes alpha2 and gamma and accumulation of the protein in the vascular lumen of the placenta. Hypothesizing that cell-free hemoglobin leaks from the placenta into the maternal circulation and contributes to the endothelial damage and symptoms by inducing oxidative stress, we analyzed fetal and adult hemoglobin (HbF, HbA), haptoglobin, oxidation markers, and the heme scavenger and antioxidant alpha(1)-microglobulin in plasma, urine, and placenta in preeclamptic women (n=28) and women with normal pregnancy (n=27). The mean plasma concentrations of HbF, HbA, protein carbonyl groups, membrane peroxidation capacity, and alpha(1)-microglobulin were significantly increased in preeclamptic women. The levels of total plasma Hb correlated strongly with the systolic blood pressure. The plasma haptoglobin concentrations of women with preeclampsia were significantly depressed. Increased amounts of alpha(1)-microglobulin mRNA and protein were found in placenta from preeclamptic women, and the levels of plasma and placenta alpha(1)-microglobulin correlated with the plasma Hb concentrations. The heme-degrading form t-alpha(1)-microglobulin was significantly increased in urine in preeclampsia. These results support the idea that hemoglobin-induced oxidative stress is a pathogenic factor in preeclampsia.

ABO exon and intron analysis in individuals with the AweakB phenotype reveals a novel O1v-A2 hybrid allele that causes four missense mutations in the A transferase

BackgroundSince the cloning in 1990 of cDNA corresponding to mRNA transcribed at the blood-group ABO locus, polymorphisms due to ethnic and/or phenotypic variations have been reported. Some subgroups have been explained at the molecular level, but unresolved samples are frequently encountered in the reference laboratory.ResultsABO blood grouping discrepancies were investigated serologically and by ABO genotyping [duplex polymerase-chain-reaction (PCR) – restriction-fragment-length-polymorphism (RFLP) and PCR – allele-specific-primer (ASP) across intron 6] and DNA sequencing of the ABO gene and its proposed regulatory elements. Blood samples from five individuals living in Portugal, Switzerland, Sweden and the USA were analysed. These individuals were confirmed to be of Black ethnic origin and had the unusual AweakB phenotype but appeared to have the A2B genotype without previously reported mutations associated with weak A or B expression. Sequencing of this A allele (having 467C>T and 1061delC associated with the common A2 [A201] allele) revealed three mutations regularly encountered in the O1v[O02] allele: 106C>T (Val36Phe), 188G>A (Arg63His), 220C>T (Pro74Ser) in exons 3, 4 and 5, respectively. The additional presence of 46G>A (Ala16Thr) was noted, whilst 189C>T that normally accompanies 188G>A in O1vwas missing, as were all O1v-related mutations in exons 6 and 7 (261delG, 297A>G, 646T>A, 681G>A, 771C>T and 829G>A). On screening other samples, 46G>A was absent, but two new O alleles were found, a Jordanian O1 and an African O1vallele having 188G>A but lacking 189C>T. Sequencing of introns 2, 3, 4 and 5 in common alleles (A1 [A101], A2, B [B101], O1, O1vand O2 [O03]) revealed 7, 12, 17 and 8 polymorphic positions, respectively, suggesting that alleles could be defined by intronic sequences. These polymorphic sites allowed definition of a breakpoint in intron 5 where the O1v-related sequence was fused with A2 to form the new hybrid. Intron 6 has previously been sequenced. Four new mutations were detected in the hybrid allele and these were subsequently also found in intron 6 of A2 alleles in other Black African samples.ConclusionsA novel O1v-A2 hybrid was defined by ABO exon/intron analysis in five unrelated individuals of African descent with the AweakB blood group phenotype.

New and unusual O alleles at the ABO locus are implicated in unexpected blood group phenotypes.

BACKGROUND:  In the ABO blood group system mutations in the A gene may lead to weak A subgroups owing to a dysfunctional 3‐α‐N‐acetylgalactosaminyltransferase.

Blood grouping discrepancies between ABO genotype and phenotype caused by O alleles

Purpose of reviewIn the modern transfusion service, analysis of the ABO allele underlying a donor or recipients A or B subtype phenotype is becoming a mainstream adjunct to the serological investigation. Although an analysis of the ABO gene can be helpful in establishing the nature of the subtype phenotype, numerous confounding factors exist that can lead to a discrepancy between the genotype and the observed phenotype. Recent findingsAlthough the most common group O alleles share a common crippling polymorphism, a growing number of alleles feature other polymorphisms that render their protein nonfunctional yet are similar enough to the consensus A allele that an errant phenotype would be predicted from the genotype, if the genotyping method was not specifically designed for their detection. Some of these O alleles might actually encode a protein with weak and variable A antigen synthetic ability. SummaryABO genotyping can be a powerful asset in the transfusion service, but a thorough knowledge of the confounding factors that can lead to genotype/phenotype discrepancies is required.

Structural effects of naturally occurring human blood group b galactosyltransferase mutations adjacent to the DXD motif

Human blood group A and B antigens are produced by two closely related glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) utilizes UDP-GalNAc to extend H antigen acceptors (Fucα(1–2)Galβ-OR) producing A antigens, whereas a galactosyltransferase (GTB) utilizes UDP-Gal as a donor to extend H structures producing B antigens. GTA and GTB have a characteristic 211DVD213 motif that coordinates to a Mn2+ ion shown to be critical in donor binding and catalysis. Three GTB mutants, M214V, M214T, and M214R, with alterations adjacent to the 211DVD213 motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the Bel variant expressing very low levels of B antigens, whereas those with M214T and M214V mutations give rise to AweakB phenotypes. Kinetic analysis of recombinant mutant GTB enzymes revealed that M214R has a 1200-fold decrease in kcat compared with wild type GTB. The crystal structure of M214R showed that DVD motif coordination to Mn2+ was disrupted by Arg-214 causing displacement of the metal by a water molecule. Kinetic characterizations of the M214T and M214V mutants revealed they both had GTA and GTB activity consistent with the serology. The crystal structure of the M214T mutant showed no change in DVD coordination to Mn2+. Instead a critical residue, Met-266, which is responsible for determining donor specificity, had adopted alternate conformations. The conformation with the highest occupancy opens up the active site to accommodate the larger A-specific donor, UDP-GalNAc, accounting for the dual specificity.

Structural basis for red cell phenotypic changes in newly identified, naturally occurring subgroup mutants of the human blood group B glycosyltransferase.

BACKGROUND: Four amino‐acid‐changing polymorphisms differentiate the blood group A and B alleles. Multiple missense mutations are associated with weak expression of A and B antigens but the structural changes causing subgroups have not been studied.

The Abantu phenotype in the ABO blood group system is due to a splice‐site mutation in a hybrid between a new O1‐like allelic lineage and the A2 allele

Background and Objectives  Many phenotypic variations in the expression of blood group A have been explained by variations in gene structure, but unresolved samples are frequently encountered in the reference laboratory. Among ABO subgroups, Abantu has the highest frequency in a specified population. The molecular basis of this phenotype is now described.

A novel B(weak) hybrid allele lacks three enhancer repeats but generates normal ABO transcript levels.

Background and Objectives  Weak expression of A/B histo‐blood group antigens is often explained by single nucleotide substitutions at the ABO locus. However, hybrid alleles containing segments from different ABO alleles can result in unexpected phenotypes and may complicate genotype analysis. We investigated the basis of weak B phenotype in a referred sample.

Investigation into A antigen expression on O-2 heterozygous group O-labeled red blood cell units

BACKGROUND: There are two principal types of group O alleles; deletional alleles feature 261delG leading to nonfunctional truncated protein. Nondeletional alleles have the consensus guanosine at residue 261. The major nondeletional allele, O2, encodes full‐length protein with Gly268Arg. While reports vary, O2 has been proposed to encode weakly functional A‐glycosyltransferase (GTA). The main objective of this study was to evaluate if GTA activity is detectable in O2 donors.

Mixed field reactions in ABO and Rh typing chimerism likely resulting from twin haematopoiesis.

Discrepancies in ABO and Rh typing due to the presence of mixed field reactions may be encountered in the transfusion medicine laboratory1,2. In order to resolve the discrepancy and to provide compatible blood for transfusion, there is a requirement to obtain relevant historical information from the patient and to perform additional laboratory investigations. Often the cause of the mixed field reactions is easily ascertained on history, when the patient is found to have had recent transfusion or stem cell transplantation from a non-group identical donor. Certain ABO subgroups and pathologic or physiologic conditions may also lead to mixed field reactions on ABO typing; however, they should rarely affect Rh typing1,2. Similarly, a change in RhD phenotype has been described in a patient with leukaemia, but this did not affect ABO typing3. One of the potential causes of mixed field reactions on ABO and Rh typing is the presence within an individual of a chimeric state or mosaicism4,5. A chimera is present when two or more distinct cell populations containing genetic material from more than one zygote exist within an individual. Although descriptions highlighting the concept of chimerism as a cause of blood group typing discrepancies have existed for decades, chimerism is uncommon and presents challenges when mixed-field agglutination is encountered on forward or reverse ABO typing during routine pre-transfusion testing6,7. There are several forms of chimerism which have varied manifestations1,4,5. Congenital chimerism refers to the situation where there is either embryo fusion or dizygotic twin-twin blood vessel anastomoses between two dichorionic placentas which results in the exchange of haematopoietic cells. Partial haematopoietic chimerism also exists and is the result of transfusion or stem cell transplantation; in these situations evidence of chimerism is confined to one body compartment (haematopoietic cells). Both serologic and molecular methodologies may be useful in determining the cause of mixed field reactions. In addition, a highly sensitive flow cytometric method may assist in characterizing ABO subgroups through the detection of low levels of A and B antigen on red blood cells (RBCs) or on low fractions of RBCs in a sample8. Here we describe the use of blood group genotyping and flow cytometry to investigate a patient with mixed field reactivity in ABO, RhD, and RhE typing.