Treatment with plasma exchange of a pregnant woman with anti-PP1Pk alloimmunization: A case report.

The histo-blood group antigens P, P1 and Pk are a closely related set of glycosphingolipid structures expressed by red blood cells and other tissues. None of these three characters is expressed on p cells, a null phenotype that arises in the context of homozygous mutation of the A4GALT gene. Subjects with p phenotype spontaneously develop a natural alloantibody named anti-PP1Pk, which is a mixture of IgG and IgM against P1, P and Pk. While anti-P1 is a weak cold antibody with poor clinical significance, anti-P and anti-Pk antibodies are potent haemolysins responsible for severe hemolytic transfusion reactions. The rare anti-PP1Pk alloantibodies are associated with recurrent spontaneous abortion in the first trimester of gestation. P and Pk antigens are expressed at high levels on the placenta and antibodies directed against both these structures are deleterious to placental trophoblasts. Here we describe the use of plasma exchange (PEX) in a nulliparous 39-year-old woman with anti-PP1Pk antibodies and a history of repeated spontaneous early abortions and hypofertility. The patient underwent apheresis starting from the third week throughout the pregnancy and a healthy child was delivered by cesarean section at 35 WG. The newborn required only phototherapy within a few days of life. We can state that an early treatment with the only PEX has proven to be effective and safe in the management of a fetomaternal P-incompatibility caused by a high anti-PP1Pk titer (256).

A systematic study of single-nucleotide polymorphisms in the A4GALT gene suggests a molecular genetic basis for the P1/P2 blood groups.

BACKGROUND: The molecular mechanism for the formation of the P1/P2 blood groups remains unsolved. It has been shown that the P1/P2 polymorphism is connected to the different A4GALT gene expression levels in P1 and P2 red blood cells. STUDY DESIGN AND METHODS: The present investigation conducted a pilot investigation that involved the detailed and stepwise screening of single-nucleotide polymorphisms (SNPs) in the A4GALT gene, followed by a larger-scale association study. The transcription-inducing activity by the different genotypes of SNPs was analyzed using reporter assays. RESULTS: A total of 416 different SNP sites in the A4GALT genes from four P1 and four P2 individuals were analyzed in the pilot investigation, and 11 SNP sites, distributed in the A4GALT Intron 1 region, exhibited an association with the P1/P2 phenotypes. In the follow-up association study, the genotypes at the 11 SNPs of a total of 338 individuals across four different ethnic populations were determined, and the results show that two SNPs, rs2143918 and rs5751348, are consistently associated with the P1/P2 phenotypes. Reporter assays demonstrated significantly higher transcription-inducing activity by the SNPs bearing the P(1)-allele genotype than by the SNPs bearing the P(2)-allele genotype and that the difference in transcriptional activity was determined by the different genotypes at SNP rs5751348. CONCLUSION: The results of this investigation demonstrate a consistent association of A4GALT SNPs rs2143918 and rs5751348 with the P1/P2 phenotypes and suggest that SNP rs5751348 may lead to allelic variations in A4GALT gene expression and consequently leads to the formation of the P1/P2 phenotypes.

P1/P2 genotyping of known and novel null alleles in the P1PK and GLOB histo-blood group systems.

BACKGROUND: The rare but clinically important null phenotypes of the P1PK and GLOB blood group systems are due to alterations in A4GALT and B3GALNT1, respectively. A recently identified single-nucleotide polymorphism in Exon 2a of A4GALT predicts the common P1 and P2 phenotypes but rare variants have not been tested. STUDY DESIGN AND METHODS: The aim of this study was to analyze 84 p, P1 (k) , and P2 (k) samples, with special emphasis on unknown alleles and the P(1) /P(2) marker. Of these, 27 samples came from individuals not previously investigated genetically and were therefore subjected to sequencing of A4GALT or B3GALNT1, and a subset was tested by flow cytometry. RESULTS: The P(1) /P(2) genotyping linked 20 p-inducing mutations in A4GALT to P(1) or P(2) allelic background. Eight p alleles remain unlinked due to compound heterozygosity. For 23 of 25 P(k) samples, concordant results were observed: P1 (k) samples had at least one P(1) allele while P2 (k) had P(2) only. The two remaining samples typed as P1+ and P1+(w) but were genetically P(2) /P(2) . A tendency toward higher P(k) antigen expression was observed on P1 (k) cells compared to P2 (k) . In total, six previously unknown null mutations were found and characterized in A4GALT while four new changes were revealed in B3GALNT1. CONCLUSION: For the first time, p alleles were shown to occur on both P(1) and P(2) allelic backgrounds. Furthermore, P(1) /P(2) genotyping predicted the P1 (k) versus P2 (k) phenotype in more than 90% of globoside-deficient samples. The number of GLOB-null alleles was increased by 50% and several P1PK-null alleles were identified.

[Serological and genetic study of a pedigree featuring a rare p phenotype].

OBJECTIVE: To explore genetic background of a pedigree with a rare p phenotype from Guangdong province. METHODS: The rare p phenotype was identified by a conventional serologic method. With genomic DNA of proband and family members extracted, exon 3 of alpha-(1,4)galactosyltransferase (A4GALT) gene was amplified with PCR and analyzed by direct sequencing. The mutation found in the pedigree was screened in a normal population using direct sequencing. RESULTS: The proband and 4 family members with the rare p phenotype have all carried a point mutation c.100G>A (p.Val34Ile) in combination with a deletion-insertional mutation c.418_428del11ins34(p.Gln139Trpfs*72), which renders a compound mutation of A4GALT gene. One family member with P2 phenotype has carried a same heterozygous mutation. Of the 100 healthy donors, 5 have carried a heterozygous point mutation c.100G>A, and none carried the deletion-insertional mutation c.418_428del11ins34. CONCLUSION: The rare p phenotype of the pedigree has resulted from a compound mutation of the A4GALT gene, which is in keeping with a recessive inheritance pattern of the p phenotype.

Functional characterisation of a complex mutation in the α(1,4)galactosyltransferase gene in Taiwanese individuals with p phenotype.

BACKGROUND AND OBJECTIVE: Individuals with p phenotype lack P1, P(k) and P antigens on red blood cells, presumably as a result of deficiency in the enzyme α(1,4)galactosyltransferase (A4GALT). The aim of this study was to explore the molecular background of a Taiwanese family with p phenotype. MATERIALS AND METHODS: Blood samples from two p siblings and seven family members were investigated. The coding region of the A4GALT gene was analysed by polymerase chain reaction and direct sequencing. The wild- and mutant-complementary DNAs (cDNAs) of A4GALT were cloned into an expression vector and transfected to Chinese hamster ovary (CHO) cells. P(k) expression on the transfected cells was analysed by flow cytometry and the activities of A4GALT were measured by high-performance liquid chromatography. RESULTS: The two individuals with p phenotype were homozygous for the complex mutation, which was caused by a combined deletion and insertion between nt 418 and 428. No expression of P(k) and no enzyme activity were observed in cells transfected with the mutant construct. CONCLUSION: The first case of p phenotype in Taiwan was caused by a non-functional allele resulting from a homozygous complex mutation of A4GALT gene.

Identification of a novel A4GALT exon reveals the genetic basis of the P1/P2 histo-blood groups.

The A4GALT locus encodes a glycosyltransferase that synthesizes the terminal Galα1-4Gal of the P(k) (Gb3/CD77) glycosphingolipid, important in transfusion medicine, obstetrics, and pathogen susceptibility. Critical nucleotide changes in A4GALT not only abolish P(k) formation but also another Galα1-4Gal-defined antigen, P1, which belongs to the only blood group system for which the responsible locus remains undefined. Since known A4GALT polymorphisms do not explain the P1-P(k)+ phenotype, P(2), we set out to elucidate the genetic basis of P(1)/P(2). Despite marked differences (P(1) > P(2)) in A4GALT transcript levels in blood, luciferase experiments showed no difference between P(1)/P(2)-related promoter sequences. Investigation of A4GALT mRNA in cultured human bone marrow cells revealed novel transcripts containing only the noncoding exon 1 and a sequence (here termed exon 2a) from intron 1. These 5'-capped transcripts include poly-A tails and 3 polymorphic sites, one of which was P(1)/P(2)-specific among > 200 donors and opens a short reading frame in P(2) alleles. We exploited these data to devise the first genotyping assays to predict P1 status. P(1)/P(2) genotypes correlated with both transcript levels and P1/P(k) expression on red cells. Thus, P(1) zygosity partially explains the well-known interindividual variation in P1 strength. Future investigations need to focus on regulatory mechanisms underlying P1 synthesis.

Un caso de grupo sanguíneo raro: fenotipo p / A rare blood group: p phenotype

Un individuo con un fenotipo eritrocitario raro carece de uno o varios antígenos presentes en la mayor parte de la población de pertenencia. Cuando presenta el anticuerpo correspondiente, se pueden producir complicaciones perinatales, transfusionales y/o transplantológicas. Se presenta el caso de una embarazada aloinmunizada derivada a nuestro servicio en la semana 12 de su tercera gesta para su evaluación y seguimiento. El diagnóstico inmunohematológico le asignó el excepcional fenotipo "p" (aproximadamente 1/200 000 individuos), asociado con una mayor tasa de abortos espontáneos y a reacciones transfusionales graves cuando se transfunden unidades incompatibles. El estudio del gen A4GALT demostró la presencia de la mutación c.752C > T en doble dosis. Esta mutación lleva a un cambio de una prolina por una leucina en el residuo 251 de la 4-α-galactosiltransferasa. Por parto inducido por sufrimiento fetal, nace a las 36 semanas una bebé con prueba de antiglobulina (Coombs) directa negativa, eluido reactivo, con ictericia que requirió luminoterapia. Una semana después el neonato fue externado sin secuelas aparentes. Posteriormente, a raíz de una cirugía inminente y la improbabilidad de encontrar sangre compatible, se elaboró un plan para cubrir las posibles demandas. Este caso pone en evidencia la necesidad de contar a nivel nacional con un laboratorio de referencia de inmunohematología y un banco de sangre de grupos raros, que permita resolver con celeridad situaciones que requieran transfundir a estos individuos.

A rare blood group is usually defined as the absence of a high prevalence antigen or the absence of several antigens within a single blood group system. These individuals may develop clinically significant red cell antibodies to the high incidence red cell antigens they lack. A 33-year-old alloimmunized woman was referred to our center at the 12th week of her third pregnancy for evaluation and follow up. The laboratory work-up grouped her as belonging to "p" phenotype, associated with difficulties to find compatible blood for transfusion and a high incidence of recurrent miscarriage. At 36 weeks, a baby girl was born by induced labor due to fetal suffering. With a negative direct antiglobulin test but a positive elution test, she was in the neonatology ward for one week receiving luminotherapy. Homozygosity for a missense mutation at position 752 (c.752C > T) in the A4GALT gene was found to be responsible for the p phenotype. This mutation changes a proline to a leucine at codon 251 of the 4-α-galactosyltransferase. Recently, due to an imminent chirurgical intervention and the impossibility to have compatible blood available for transfusion, an autologous donation plan was designed to satisfy probable demand. This case showed the need for blood bank facilities capable to respond satisfactorily to these situations in Argentina. This would facilitate the storage of cryopreserved blood from individuals with rare blood groups for homologous use or to develop rare blood donors programs.

[A rare blood group: p phenotype]. / Un caso de grupo sanguíneo raro: fenotipo p.

A rare blood group is usually defined as the absence of a high prevalence antigen or the absence of several antigens within a single blood group system. These individuals may develop clinically significant red cell antibodies to the high incidence red cell antigens they lack. A 33-year-old alloimmunized woman was referred to our center at the 12th week of her third pregnancy for evaluation and follow up. The laboratory work-up grouped her as belonging to "p" phenotype, associated with difficulties to find compatible blood for transfusion and a high incidence of recurrent miscarriage. At 36 weeks, a baby girl was born by induced labor due to fetal suffering. With a negative direct antiglobulin test but a positive elution test, she was in the neonatology ward for one week receiving luminotherapy. Homozygosity for a missense mutation at position 752 (c.752C > T) in the A4GALT gene was found to be responsible for the p phenotype. This mutation changes a proline to a leucine at codon 251 of the 4-?-galactosyltransferase. Recently, due to an imminent chirurgical intervention and the impossibility to have compatible blood available for transfusion, an autologous donation plan was designed to satisfy probable demand. This case showed the need for blood bank facilities capable to respond satisfactorily to these situations in Argentina. This would facilitate the storage of cryopreserved blood from individuals with rare blood groups for homologous use or to develop rare blood donors programs.

A single base insertion of the 4-alpha-galactosyltransferase gene led to the deficiency of Gb3 biosynthesis.

cDNAs for alpha 1,4 galactosyltransferase (A4GALT) have been isolated. To explore the molecular basis of the p phenotype in Japanese donors, we analyzed the A4GALT gene sequences of normal and p phenotype samples. The coding region in the A4GALT gene for DNA sequencing was amplified by PCR amplification. A4GALT expression vectors for individual were constructed by PCR amplification of the coding region using primers and subsequent subcloning into an expression vector. The expression of Gb3/CD77 antigen on the cell surface was evaluated by flow cytometry and by immunochemical techniques. All individuals with the p phenotype were found to have a single base insertion (A4GALT/insC) at the same nucleotide position. Neither the transfectant cells with a mutant gene (A4GALT/insC) of donor origin or those with a synthesized mutant gene (A4GALT/insC-Mu) expressed Gb3 antigen indicating that the presence of A4GALT/insC diminished the A4GALT enzyme activity. In addition, an allele-specific PCR (ASP) system was developed in which of the p phenotype with A4GALT/insC can be unambiguously discriminated from normal donors. Based on the finding that a single base insertion (A4GALT/insC) diminishes A4GALT activity, an ASP assay was developed to detect individuals with this particular p phenotype.

Two previously proposed P1/P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen.

BACKGROUND: The molecular genetics of the P blood group system and the absence of P1 antigen in the p phenotype are still enigmatic. One theory proposes that the same gene encodes for both the P1 and Pk glycosyltransferases, but no polymorphisms in the coding region of the Pk gene explain the P1/P2 phenotypes. We investigated the potential regulatory regions up- and downstream of the A4GALT (Pk) gene exons. RESULTS: P1 (n = 18) and P2 (n = 9) samples from donors of mainly Swedish descent were analysed by direct sequencing of PCR-amplified 5'- and 3'-fragments surrounding the Pk coding region. Seventy-eight P1 and P2 samples were investigated with PCR using allele-specific primers (ASP) for two polymorphisms previously proposed as P2-related genetic markers (-551_-550insC, -160A>G). Haplotype analysis of single nucleotide polymorphisms was also performed with PCR-ASP. In approximately 1.5 kbp of the 3'-untranslated region one new insertion and four new substitutions compared to a GenBank sequence (AL049757) were found. In addition to the polymorphisms at positions -550 and -160, one insertion, two deletions and one substitution were found in approximately 1.0 kbp of the 5'-upstream region. All 20 P2 samples investigated with PCR-ASP were homozygous for -550insC. However, so were 18 of the 58 P1 samples investigated. Both the 20 P2 and the 18 P1 samples were also homozygous for -160G. CONCLUSION: The proposed P2-specific polymorphisms, -551_-550insC and -160G, found in P2 samples in a Japanese study were found here in homozygous form in both P1 and P2 donors. Since P2 is the null allele in the P blood group system it is difficult to envision how these mutations would cause the P2 phenotype. None of the novel polymorphisms reported in this study correlated with P1/P2 status and the P1/p mystery remains unsolved.

The Drosophila melanogaster homologue of the human histo-blood group Pk gene encodes a glycolipid-modifying alpha1,4-N-acetylgalactosaminyltransferase.

Insects express arthro-series glycosphingolipids, which contain an alpha1,4-linked GalNAc residue. To determine the genetic basis for this linkage, we cloned a cDNA (CG17223) from Drosophila melanogaster encoding a protein with homology to mammalian alpha1,4-glycosyltransferases and expressed it in the yeast Pichia pastoris. Culture supernatants from the transformed yeast were found to display a novel UDP-GalNAc:GalNAcbeta1,4GlcNAcbeta1-R alpha-N-acetylgalactosaminyltransferase activity when using either a glycolipid, p-nitrophenylglycoside or an N-glycan carrying one or two terminal beta-N-acetylgalactosamine residues. NMR and MS in combination with glycosidase digestion and methylation analysis indicate that the cloned cDNA encodes an alpha1,4-N-acetylgalactosaminyltransferase. We hypothesize that this enzyme and its orthologues in other insects are required for the biosynthesis of the N5a and subsequent members of the arthro-series of glycolipids as well as of N-glycan receptors for Bacillus thuringiensis crystal toxin Cry1Ac.

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-alpha-galactosyltransferase gene responsible for synthesis of Pk (Gb3) antigen. STUDY DESIGN AND METHODS: Forty samples from individuals of eight different nationalities were investigated by serologic methods and DNA sequencing of the Pk gene. RESULTS: Ten different Pk-null alleles, of which 6 are novel, were encountered. The 29 Swedes were homozygous for M183K or G187D, with the former as the predominant allele. Three Israelis were homozygous for a single-nucleotide deletion at codon 219 that shifts and truncates the reading frame by 5 amino acids. Two Italians were homozygous for a triplet deletion causing F81del, while an English donor was heterozygous for F81del but also carried another allele with a combined deletion and insertion. A Pole was heterozygous for alleles with either a single-base deletion at codon 257 or a mutation causing S97L. A Norwegian person and a Japanese person were homozygous for single-base insertions causing a premature stop at codon 282 or extension of the protein by 92 residues, respectively. In 2 samples no mutations were detected. CONCLUSION: The genetic heterogeneity underlying the p phenotype is further emphasized by this study. To date, 11 p-specific mutations have been found in 14 distinct alleles.

Molecular basis of the globoside-deficient P(k) 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, P(k), 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 P(1)(k) and P(2)(k) lack the capability to synthesize P antigen identified as globoside, the cellular receptor for Parvo-B19 virus and some P-fimbriated Escherichia 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 P(k) phenotype we analyzed the full coding region of a candidate gene reported in 1998 as a member of the 3-beta-galactosyltransferase family but later shown to possess UDP-N-acetylgalactosamine:globotriaosylceramide 3-beta-N-acetylgalactosaminyltransferase or globoside synthase activity. Homozygosity for different nonsense mutations (C(202) --> T and 538insA) resulting in premature stop codons was found in blood samples from two individuals of the P(2)(k) phenotype. Two individuals with P(1)(k) and P(2)(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 P(k) phenotype.

Three-base deletion and one-base insertion of the alpha(1,4)galactosyltransferase gene responsible for the P phenotype.

BACKGROUND: Recently, an alpha(1,4)galactosyltransferase gene that is responsible for synthesis of P(k) (Gb3) was isolated. The P individuals who did not express the P(k), P, and P(1) antigens on RBC membranes were shown to lack the P(k) (Gb3) synthase activity because of multiple distinct mutations in the alpha(1,4)galactosyltransferase gene. STUDY DESIGN AND METHODS: DNA sequences of the P(k) (Gb3) synthase gene in three Japanese individuals with the p phenotype were analyzed. RESULTS: One individual was found to be homozygous for an allele containing a three-base deletion of CTTCTTC to CTTC from bases 237 through 243 in the coding region. The other two individuals were found to be homozygous for an allele containing a single cytosine insertion in a cytosine repeat at positions 1026 through 1029, resulting in a reading frame shift. CONCLUSION: The P blood group phenotype is due to several distinct nonfunctional alleles without any predominant allele.

Blood group, red cell, and serum protein variation in the Cabecar and Huetar, two Chibchan Amerindian tribes of Costa Rica.

Genetic variation, using blood groups and red cell and serum proteins, was surveyed in the Cabecar of Chirripo and the Huetar of Quitirrisi, Costa Rica. Thirty-nine loci were screened in a sample of 91 Cabecars and 40 loci in 45 Huetars. Twenty-seven loci were monomorphic in the Cabecar and 30 in the Huetar. The proportions of polymorphic loci (P), out of 34 studied by electrophoresis, were 0.235 and 0.177, respectively. Estimated gene diversities (H) of the polymorphic loci were 0.050 in the Cabecar and 0.053 in the Huetar. Two polymorphisms, reported until now in Costa Rican and Panamanian Chibchan groups only, occurred at very high frequencies: TF*DGUA = 0.357 in the Cabecar, the highest frequency ever reported, and 0.033 in the Huetar; and PEPA*F, which reached 0.26 in the Cabecar and 0.29 in the Huetar. Nei's genetic distances and trees (two methods) were used to compare them to seven other Chibchan tribes of Costa Rica. The results placed both the Cabecar of Chirripo and the Huetar closer to the Talamancan Tribes (Bribri and Cabecar). This was an unexpected result for the Huetar, since linguistic studies suggested a closer relationship to the Guatuso. GST, DST, RST, and Dm for three Cabecar subpopulations (Atlantic, Chirripo, and Pacific) doubled their values compared to estimates based on comparison of only two subpopulations: Atlantic and Pacific. Total genetic diversity considering just the three Cabecar subpopulations resembled that obtained including them plus six other Chibchan populations of Costa Rica.

Molecular basis for the p phenotype. Identification of distinct and multiple mutations in the alpha 1,4-galactosyltransferase gene in Swedish and Japanese individuals.

p phenotype individuals lack both P(k) (Gb3) and P (Gb4) glycolipid antigens of the P blood group system. To explore the molecular basis for this phenotype, DNA sequences of Gb3 synthase (alpha1, 4-galactosyltransferase; alpha1,4Gal-T) in six p phenotype individuals from Japan and Sweden were analyzed. A missense mutation P251L and a nonsense mutation W261stop in three and one Japanese indivuiduals, respectively, and missense mutations M183K and G187D in one each of two Swedish p individuals were found, indicating that p individuals from Japan and Sweden have distinct and multiple homozygous point mutations in the coding region. In the function analysis of the mutated alpha1,4Gal-Ts by the transfection of the expression vectors, P251L and M183K mutations showed complete loss of enzyme function, and W261stop and G187D mutations resulted in the marginal activity. BLAST analysis of homologous sequences of alpha1, 4Gal-T revealed that three residues, Met(183), Gly(187), and Pro(251), at which missense mutations were found, were highly conserved among all species examined, suggesting their importance for the function of alpha1,4Gal-T.