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.

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

PURPOSE OF REVIEW: In the modern transfusion service, analysis of the ABO allele underlying a donor or recipient's 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 FINDINGS: Although 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. SUMMARY: ABO 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.

Investigation into A antigen expression on O2 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. STUDY DESIGN AND METHODS: Donor samples from Pittsburgh and Lund were ABO typed by automated methods. DNA was extracted from 779 group O donors whose red blood cells (RBCs) were available for transfusion. ABO genotyping identified those with O2 alleles. The following tests were performed on randomly selected O2 samples (number): adsorption-elution with anti-A (3), flow cytometry (15), plasma enzyme activity (4), and attempts to convert group O RBCs to A (2) with O2 plasma and titration of plasma anti-A/-A1 (3). RESULTS: Forty O2-heterozygous donors were identified (5.1%). Adsorption-elution and sensitive flow cytometry did not reveal A antigens on O2 RBCs. Plasma enzyme analysis failed to show GTA activity above baseline; O2 plasma was unable to add measurable A antigens to O RBCs. Titers of anti-A/-A1 appeared reduced in O2 plasma but did not cause ABO typing discrepancies. No immediate hemolysis or adverse reactions were reported following transfusion of O2 RBCs to six evaluable group O recipients. CONCLUSIONS: Other than lower plasma anti-A titers, GTA activity was not found in these O2 samples. Neither automated blood grouping discrepancies nor clinical problems related to transfusing these O2 units were observed.

An extensive polymerase chain reaction-allele-specific polymorphism strategy for clinical ABO blood group genotyping that avoids potential errors caused by null, subgroup, and hybrid alleles.

BACKGROUND: ABO genotyping is complicated by the remarkable diversity at the ABO locus. Recombination or gene conversion between common alleles may lead to hybrids resulting in unexpected ABO phenotypes. Furthermore, numerous mutations associated with weak subgroups and nondeletional null alleles should be considered. All known ABO genotyping methods, however, risk incorrect phenotype predictions if any such alleles are present. STUDY DESIGN AND METHODS: An extensive set of allele-specific primers was designed to accomplish hybrid-proof multiplex polymerase chain reaction (PCR) amplification of DNA fragments for detection of ABO alleles. Results were compared with serologic findings and ABO genotypes defined by previously published PCR-restriction fragment length polymorphism/PCR-allele-specific polymorphism (ASP) methods or DNA sequencing. RESULTS: Phenotypically well-characterized samples from blood donors with common blood groups and rare-subgroup families were analyzed. In addition to the commonly encountered alleles (A1, A1(467C>T), A2, B, O1, O1v, and O2), the new method can detect hybrid alleles thanks to long-range amplification across intron 6. Four of 12 PCR-ASP procedures are used to screen for multiple infrequent subgroup and null alleles. This concept allows for a low-resolution typing format in which the presence of, for example, a weak subgroup or cis-AB/B(A) is indicated but not further defined. In an optional high-resolution step, more detailed genotype information is obtained. CONCLUSION: A new genotyping approach has been developed and evaluated that can correctly identify ABO alleles including nondeletional null alleles, subgroups, and hybrids resulting from recombinational crossing-over events between exons 6 and 7. This approach is clinically applicable and decreases the risk for erroneous ABO phenotype prediction compared to previously published methods.

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. STUDY DESIGN AND METHODS: Individuals or families having serologically weak B antigen on their red cells were studied. Alleles were characterized by sequencing of exons 1 through 7 in the ABO gene. Single crystal X-ray diffraction, three-dimensional-structure molecular modeling, and enzyme kinetics showed the effects of the B allele mutations on the glycosyltransferases. RESULTS: Seven unrelated individuals with weak B phenotypes possessed seven different B alleles, five of which are new and result in substitution of highly conserved amino acids: M189V, I192T, F216I, D262N, and A268T. One of these (F216I) was due to a hybrid allele resulting from recombination between B and O(1v) alleles. The two other alleles were recently described in other ethnic groups and result in V175M and L232P. The first crystal-structure determination (A268T) of a subgroup glycosyltransferase and molecular modeling (F216I, D262N, L232P) indicated conformational changes in the enzyme that could explain the diminished enzyme activity. The effect of three mutations could not be visualized since they occur in a disordered loop. CONCLUSION: The genetic background for B(w) phenotypes is very heterogeneous but usually arises through seemingly random missense mutations throughout the last ABO exon. The targeted amino acid residues, however, are well conserved during evolution. Based on analysis of the resulting structural changes in the glycosyltransferase, the mutations are likely to disrupt molecular bonds of importance for enzymatic function.

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 (Fuc alpha(1-2)Gal beta-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 (211)DVD(213) motif that coordinates to a Mn(2+) ion shown to be critical in donor binding and catalysis. Three GTB mutants, M214V, M214T, and M214R, with alterations adjacent to the (211)DVD(213) motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the B(el) variant expressing very low levels of B antigens, whereas those with M214T and M214V mutations give rise to A(weak)B phenotypes. Kinetic analysis of recombinant mutant GTB enzymes revealed that M214R has a 1200-fold decrease in k(cat) compared with wild type GTB. The crystal structure of M214R showed that DVD motif coordination to Mn(2+) 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 Mn(2+). 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.

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-alpha-N-acetylgalactosaminyltransferase. STUDY DESIGN AND METHODS: Blood and DNA were investigated to correlate weak A phenotypes with genotype, and an overrepresentation of the infrequent O2 allele was observed. Consequently, 57 available O2 alleles were examined in detail. RESULTS: Two new O2 alleles were identified having mutations resulting in Gly229Asp with or without Arg217Cys. A recently described O2 variant (488C>T; Thr163Met) was also found. Surprisingly, both the original and the variant O2 alleles were associated with either O or Aweak phenotypes. Three novel O alleles surfaced in six other samples with suspected A subgroups. These were A1-like alleles having nonsense mutations causing premature truncation at codons 56, 107, or 181. A second example of the rare O3 allele was also identified. A newly described O1 allele having 768C>A was found to be the third most frequent O allele among Swedish donors. Of the five novel O alleles, three were incorrectly interpreted as A1 following routine ABO genotyping. CONCLUSION: Apparent O alleles lacking 261delG may cause weak A expression on red blood cells and/or inhibit anti-A production. A hypothesis that exchange of genetic material between principally dissimilar O alleles during mitosis ("autologous chimerism") restores glycosyltransferase activity in some cells would explain this interesting phenomenon.

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.

BACKGROUND: Since 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. RESULTS: ABO 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 O1v was 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 O1v allele 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. CONCLUSIONS: A novel O1v-A2 hybrid was defined by ABO exon/intron analysis in five unrelated individuals of African descent with the AweakB blood group phenotype.