Åsa Hellberg

The human P-k histo-blood group antigen provides protection against HIV-1 infection

Several human histo-blood groups are glycosphingolipids, including P/P1/P(k). Glycosphingolipids are implicated in HIV-host-cell-fusion and some bind to HIV-gp120 in vitro. Based on our previous studies on Fabry disease, where P(k) accumulates and reduces infection, and a soluble P(k) analog that inhibits infection, we investigated cell surface-expressed P(k) in HIV infection. HIV-1 infection of peripheral blood-derived mononuclear cells (PBMCs) from otherwise healthy persons, with blood group P(1)(k), where P(k) is overexpressed, or blood group p, that completely lacks P(k), were compared with draw date-matched controls. Fluorescence-activated cell sorter analysis and/or thin layer chromatography were used to verify P(k) levels. P(1)(k) PBMCs were highly resistant to R5 and X4 HIV-1 infection. In contrast, p PBMCs showed 10- to 1000-fold increased susceptibility to HIV-1 infection. Surface and total cell expression of P(k), but not CD4 or chemokine coreceptor expression, correlated with infection. P(k) liposome-fused cells and CD4(+) HeLa cells manipulated to express high or low P(k) levels confirmed a protective effect of P(k). We conclude that P(k) expression strongly influences susceptibility to HIV-1 infection, which implicates P(k) as a new endogenous cell-surface factor that may provide protection against HIV-1 infection.

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.

Molecular basis of the globoside-deficient Pk blood group Phenotype.Identification of four inactivating mutations in the UDP-N-acetylgalactosamine:globotriaosylceramide 3-beta-N-acetylgalactosaminyltransferase gene

The biochemistry and molecular genetics underlying the related carbohydrate blood group antigens P, Pk, and LKE in the GLOB collection and P1 in the P blood group system are complex and not fully understood. Individuals with the rare but clinically important erythrocyte phenotypes P1 k and P2 klack the capability to synthesize P antigen identified as globoside, the cellular receptor for Parvo-B19 virus and some P-fimbriatedEscherichia coli. As in the ABO system, naturally occurring antibodies, anti-P of the IgM and IgG class with hemolytic and cytotoxic capacity, are formed. To define the molecular basis of the Pk phenotype we analyzed the full coding region of a candidate gene reported in 1998 as a member of the 3-β-galactosyltransferase family but later shown to possess UDP-N-acetylgalactosamine:globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase or globoside synthase activity. Homozygosity for different nonsense mutations (C202 → T and 538insA) resulting in premature stop codons was found in blood samples from two individuals of the P2 k phenotype. Two individuals with P1 k and P2 k phenotypes were homozygous for missense mutations causing amino acid substitutions (E266A or G271R) in a highly conserved region of the enzymatically active carboxyl-terminal domain in the transferase. We conclude that crucial mutations in the globoside synthase gene cause the Pk phenotype.

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.

Additional molecular bases of the clinically important p blood group phenotype

BACKGROUND: The purpose of this study was to explore the molecular basis of the p phenotype by analysis of the recently cloned 4‐α‐galactosyltransferase gene responsible for synthesis of Pk (Gb3) antigen.

Blood group genotype analysis for the quality improvement of reagent test red blood cells.

Background and Objectives  Reagent red blood cells (RBCs) for antibody detection should express certain important antigens as a double dose, that is, the donors must be homozygous for the corresponding alleles. Traditionally, dose is determined by serological typing and known allele frequencies. However, RHD zygosity cannot be predicted serologically owing to the absence of an antithetical antigen, and FY zygosity is confounded by two variant haplotypes, FY*0 and FY*X. Furthermore, lack of reagents hampers our ability to type for some clinically important antigen pairs such as Doa/Dob.

Genetic heterogeneity at the glycosyltransferase loci underlying the GLOB blood group system and collection

The aim of this study was to further explore the molecular genetic bases of the clinically important but rare blood group phenotypes p, P1k and P2k by analysis of the 4‐α‐galactosyltransferase (Pk) and 3‐β‐N‐acetylgalactosaminyltransferase (P) genes responsible for synthesis of the related Pk (Gb3) and P (Gb4) antigens respectively. Lack of these glycolipid moieties is associated with severe transfusion reactions and recurrent spontaneous abortions but also offers immunity against certain infectious agents. Blood samples from 20 p and 11 P1k or P2k individuals of different geographic and ethnic origin were investigated. DNA sequencing by capillary electrophoresis was performed following amplification of the coding regions in the Pk or P genes. In the Pk gene, nine novel and five previously described mutations were detected. One of the newly found mutations introduced an immediate stop, five shifted the reading frame introducing premature stop codons and three were missense mutations causing amino acid substitutions in conserved regions of the transferase. Four new and two previously described mutations in the P gene were found. Three of the novel alleles reported here carried nonsense mutations whilst the fourth allele had a missense mutation. The finding of 13 novel mutations in 14 alleles emphasizes further the genetic heterogeneity at the glycosyltransferase loci underlying the GLOB blood group system and collection.

Weak A phenotypes associated with novel ABO alleles carrying the A2‐related 1061C deletion and various missense substitutions

BACKGROUND: The 1061delC single‐nucleotide polymorphism (SNP) has been reported mostly in the context of the common A2[A201] allele and typically produces an A2 phenotype. This study evaluated new Aweak alleles, each containing 1061delC.

Novel glycolipid variations revealed by monoclonal antibody immunochemical analysis of weak ABO subgroups of A

Background and Objectives  The chemical basis of the subgroups of A is largely unknown. We used thin‐layer chromatography immunochemical staining techniques together with a range of characterized monoclonal reagents to analyse glycolipids isolated from a variety of weak subgroups.