A4GALT Research Articles

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).

Proteomic analysis unveils Gb3-independent alterations and mitochondrial dysfunction in a gla−/− zebrafish model of Fabry disease

BackgroundFabry disease (FD) is a rare lysosomal storage disorder caused by mutations in the GLA gene, resulting in reduced or lack of α-galactosidase A activity. This results in the accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids in lysosomes causing cellular impairment and organ failures. While current therapies focus on reversing Gb3 accumulation, they do not address the altered cellular signaling in FD. Therefore, this study aims to explore Gb3-independent mechanisms of kidney damage in Fabry disease and identify potential biomarkers.MethodsTo investigate these mechanisms, we utilized a zebrafish (ZF) gla−/− mutant (MU) model. ZF naturally lack A4GALT gene and, therefore, cannot synthesize Gb3. We obtained kidney samples from both wild-type (WT) (n = 8) and MU (n = 8) ZF and conducted proteome profiling using untargeted mass spectrometry. Additionally, we examined mitochondria morphology and cristae morphology using electron microscopy. To assess oxidative stress, we measured total antioxidant activity. Finally, immunohistochemistry was conducted on kidney samples to validate specific proteins.ResultsOur proteomics analysis of renal tissues from zebrafish revealed downregulation of lysosome and mitochondrial-related proteins in gla−/− MU renal tissues, while energy-related pathways including carbon, glycolysis, and galactose metabolisms were disturbed. Moreover, we observed abnormal mitochondrial shape, disrupted cristae morphology, altered mitochondrial volume and lower antioxidant activity in gla−/− MU ZF.ConclusionsThese results suggest that the alterations observed at the proteome and mitochondrial level closely resemble well-known GLA mutation-related alterations in humans. Importantly, they also unveil novel Gb3-independent pathogenic mechanisms in Fabry disease. Understanding these mechanisms could potentially lead to the development of innovative drug screening approaches. Furthermore, the findings pave the way for identifying new clinical targets, offering new avenues for therapeutic interventions in Fabry disease. The zebrafish gla−/− mutant model proves valuable in elucidating these mechanisms and may contribute significantly to advancing our knowledge of this disorder.Graphical

Serological characteristics and molecular mechanism of an individual with p phenotype

To analyze the serological characteristics and molecular mechanism for an individual with p phenotype. An individual with p phenotype upon blood group identification at Jiaxing Blood Center in May 2021 was analyzed. ABO, RhD and P1PK blood groups and irregular antibodies in her serum were identified using conventional serological methods. The encoding region of α1, 4-galactosyltransferase gene (A4GALT) encoding P1 and Pk antigens was analyzed by polymerase chain reaction-sequence-based typing (PCR-SBT). The individual was A group, RhD positive and had a p phenotype of the P1PK blood group system. Anti-PP1Pk was discovered in her serum. Sequencing analysis revealed that she has harbored a homozygous c.343A>T variant of the A4GALT gene. The homozygous c.343A>T variant of the A4GALT gene probably underlay the p phenotype in this individual.

Association analysis between novel polymorphism in A4GALT gene and milk production traits in crossbred cattle of Kerala

Association analysis between novel polymorphism in A4GALT gene and milk production traits in crossbred cattle of Kerala

Comparative analysis of antigen coding genes in 15 red cell blood group systems of Yunnan Yi nationality in China: A cross-sectional study.

IntroductionThere are few analyses of the 15 red blood group system antigen coding genes found in the Yunnan Yi nationality. This has caused many poteintial dangers relating to clinical blood transfusion. In this report, the coding genes and distribution of 15 blood group antigens system in the Yi nationality were tested and compared with those of Han nationality and other ethnic minorities.MethodsThe samples came from the healthy subjects in the first people's Hospital of Qujing, Yunnan Province. Two hundred and three Yunnan Yi and 197 Han nationality individuals were included. Thirty‐three blood group antigens with a low frequency from the 15 blood group systems of Yunnan Yi blood donors were genotyped and analyzed by PCR‐SSP. Sanger sequencing was used to detect A4GALT from the Yunnan Yi nationality. The χ 2 test was used to analyze observed and expected values of gene distribution to verify conformation to the Hardy‐Weinberg equilibrium law. Fisher's exact test was used to analyze gene frequency distribution, and the statistical significance was set at p < 0.05.ResultsThe ABO blood group examination results for the Yi nationality and the local Han nationality in Qujing City, Yunnan Province, showed the majority were type A and type O, while the least prevalent was type AB. RhD+ accounts for more than 98% of the Yi and Han populations. There was a significant difference in ABO blood group antigen distribution between these two nationalities (p < 0.05), but there was no significant difference in the composition ratio of D antigen in the Rh blood group system (p > 0.05). Compared with Tibetan (Tibet), Zhuang (Nanning), and Dong (Guangxi), the gene distribution frequencies of Rh blood group system phenotype CC were significantly lower in the Yunnan Yi nationality (p < 0.05). There were significant differences in six erythrocyte phenotypic antigens in the Yi nationality in Yunnan compared with Han nationality, such as LW(a−b−), JK(a−b+), MMSs, Di(a−b+), Wr(a−b−), and Kp(a−b+) (p < 0.05). There were gene phenotypes with a low frequency in the four rare blood group systems: LW, MNS, Wright, and Colton. Several different mutation types occurred in the P1PK blood group system's A4GALT gene.ConclusionYunnan Yi nationality has a unique genetic background. There are some significantly different distributions of blood group system genes with a low frequency in different regions and groups in China. Multiple mutations in the A4GALT gene of the P1PK blood group system may be related to their environment and ethnic evolution.

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme’s promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galβ1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

Two Thai Burmese descendants with A4GALT*01N.21, p phenotype, and anti-PP1Pk

Anti-PP1Pk is produced by p individuals without prior red blood cell alloimmunization. This antibody can react over a wide thermal amplitude, has the potential to bind complement, and has caused hemolytic transfusion reaction, hemolytic disease of the fetus and newborn, and a high rate of spontaneous abortions. This report of two cases describes the genetic basis of p phenotype underlying anti-PP1Pk production and the development of a semi-nested polymerase chain reaction (PCR) assay for screening this observed mutation among Thai blood donors. Antibody detection and confirmation were examined by serologic testing. Genomic DNA was extracted from two Thai Burmese descendants with the p phenotype and a history of spontaneous abortions caused by anti-PP1Pk; the entire coding region of the A4GALT gene of each was sequenced and analyzed. Additionally, a semi-nested PCR assay of the observed mutation was developed and used for screening the genomic DNA of 1502 Thai blood donors. Anti-PP1Pk was identified and the p phenotype was confirmed in the two Thai individuals of Burmese descent. A single-base duplication (c.201dupC in exon 3) in the A4GALT gene was detected in both p patients. The duplication is consistent with the A4GALT*01N.21 allele associated with the p phenotype and anti-PP1Pk production. A semi-nested PCR assay was developed and subsequently used for mass screening for this mutation. The mutation was not found among the 1502 Thai blood donors tested with this newly developed assay.

The differential expression of the blood group P1 -A4GALT and P2 -A4GALT alleles is stimulated by the transcription factor early growth response 1.

The P1 /P2 phenotypic polymorphism is one of the earliest blood groups discovered in humans. These blood groups have been connected to different levels of expression of the A4GALT gene in P1 and P2 red blood cells; however, the detailed molecular genetic mechanism that leads to these two phenotypes has not been established. After our previous identification of an association between the single-nucleotide polymorphisms (SNPs) rs2143918 and rs5751348 in A4GALT gene and the P1 /P2 phenotype, we conduct a survey of transcription factors that might connect these SNPs with the differential expression of the P1 -A4GALT and P2 -A4GALT alleles. An in silico analysis of potential transcription factor binding motifs within the polymorphic SNPs rs2143918 and rs5751348 genomic regions was performed, and this was followed by reporter assays examining the candidate transcription factors, gene expression profiling, electrophoretic mobility shift assays, and P1 -A4GALT and P2 -A4GALT allelic expression analysis. The results revealed that the differential binding of transcription factor early growth response 1 to the SNP rs5751348 genomic region with the different genotypes in the A4GALT gene leads to differential activation of P1 -A4GALT and P2 -A4GALT expression. The present investigation, together with our previous study (Lai et al., Transfusion 2014;54:3222-31), have elucidated the molecular genetic details associated with the P1 /P2 blood groups.

A novel mutation in A4GALT was identified in a Chinese individual with p phenotype

A novel mutation in <i>A4GALT</i> was identified in a Chinese individual with p phenotype

Human Gb3/CD77 synthase reveals specificity toward two or four different acceptors depending on amino acid at position 211, creating Pk, P1 and NOR blood group antigens

Human Gb3/CD77 synthase (α1,4-galactosyltransferase, Pk synthase), encoded by A4GALT gene, is known for synthesis of Gal(α1-4)Gal moiety in globotriaosylceramide (Gb3Cer, CD77, Pk blood group antigen), a glycosphingolipid of the globo series. Recently, it was shown that c.631C > G mutation in A4GALT, which causes p.Q211E substitution in the open reading frame of the enzyme, broadens the enzyme specificity, making it able also to synthesize Gal(α1-4)GalNAc moiety, which constitutes the defining terminal disaccharide of the NOR antigen (carried by two glycosphingolipids: NOR1 and NOR2). Terminal Gal(α1-4)Gal disaccharide is also present in another glycosphingolipid blood group antigen, called P1, which together with Pk and NOR comprises the P1PK blood group system. Despite several attempts, it was never clearly shown that P1 antigen is synthesized by Gb3/CD77 synthase, leaving open an alternative hypothesis that there are two homologous α1,4-galactosyltransferases in humans. In this study, using recombinant Gb3/CD77 synthase produced in insect cells, we show that the consensus enzyme synthesizes both the Pk and P1 antigens, while its p.Q211E variant additionally synthesizes the NOR antigen. This is the first direct biochemical evidence that Gb3/CD77 synthase is able to synthesize two different glycosphingolipid antigens: Pk and P1, and when p.Q211E substitution is present, the NOR antigen is also synthesized.

Molecular basis for an individual with rare p phenotype in P1Pk blood group system

To explore the molecular basis for an individual with rare p phenotype in the P1Pk blood group system. Erythrocyte blood group antigens and antibodies in serum were identified in the proband and five family members with a serological method. Coding regions and flanking untranslated regions of the α1,4-galactosyltransferase gene (A4GALT) encoding P1Pk antigens were amplified with polymerase chain reaction and directly sequenced. The haplotypes of A4GALT in the parents of the proband were also analyzed by cloning sequencing. The proband was found with a rare p phenotype with anti-Tja antibody in his serum by serological method. The other family members all had a common P2 phenotype. The results of DNA sequencing showed that a cytosine was inserted at nucleotide position 1026 to 1029 (1026_1029insC) of both alleles of the A4GALT gene in the proband. The mutation has caused a reading frame shift and formed a mutant protein by extending 92 amino acid residues. The other family members were either heterozygous for the insertion or of the wild type at above position. The 1026_1029insC mutation of the A4GALT gene is probably responsible for the p phenotype identified for the first time in Chinese population. The individual with the p phenotype possesses anti-Tja antibody.

Pedigree investigation and genetic analysis of a case with p blood group

To explore the molecule basis of a p blood group in a patient with gastric carcinoma. The p phenotype was determined with serological method. Inheritance of the p phenotype was investigated by pedigree analysis. Sequence of α-1,4- galactosyltransferase (A4GALT) gene was determined by Sanger method. The proband and his younger brother were both determined to have a p phenotype. Two homozygous variations, c.343A>T (AAA>TAA) and c.903C>G (CCC>CCG), have been detected in exon 3 of the A4GALT gene. Among these, c.343 A>T (AAA>TAA) was a novel mutation, which has resulted in a termination codon, with which no normal product of the gene can be produced. c.903C>G was determined to be a polymorphism. A novel c.343A>T mutation in the A4GALT gene probably underlies the p phenotype, to which a Genbank access number KC202808 has been assigned.

A rare p phenotype caused by a 26-bp deletion in α 1,4-galactosyltransferase gene

To delineate serological features and genetic basis for a rare p phenotype of P1Pk blood group system found in a Chinese individual. Serological assaying was carried out for a proband with unexpected antibody found in his serum using specific antibodies and panel cells. Coding regions and flanking introns of α 1,4-galactosyltransferase gene (A4GALT) associated with the p phenotype were screened with polymerase chain reaction and DNA sequencing. A rare p phenotype of the P1Pk blood group system has been identified with red blood cells from the proband, whose serum contained anti-Tja antibody which can agglutinate and hemolyze with other common red blood cells. Other members of the proband's family were all normal with P1 or P2 phenotype. DNA sequencing has identified in the proband a homozygous 26 bp deletion at position 972 to 997 of the A4GALT gene. The deletion has caused a shift of the reading frame, resulting in a variant polypeptide chain with additional 83 amino acid residues compared with the wild-type protein. Other family members were either heterozygous for above deletion or non-deleted. A 26 bp deletion at position 972 to 997 of the A4GALT gene has been identified in a Chinese individual with p phenotype.

A Single Point Mutation in the Gene Encoding Gb3/CD77 Synthase Causes a Rare Inherited Polyagglutination Syndrome

Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR(int), and NOR2, in which Gal(α1-4), GalNAc(β1-3)Gal(α1-4), and Gal(α1-4)GalNAc(β1-3)Gal(α1-4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1-4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1-4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1-4)Gal and Gal(α1-4)GalNAc moieties.

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

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. 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. 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. The first case of p phenotype in Taiwan was caused by a non-functional allele resulting from a homozygous complex mutation of A4GALT gene.

Sequence variation in the 5′ untranslated region of the human A4GALT gene is associated with, but does not define, the P1 blood‐group polymorphism

The gene responsible for the P1 polymorphism of the P blood-group system remains unidentified, although the A4GALT gene, whose product is responsible for the production of P(k), has been implicated. No coding differences in A4GALT account for the P1 polymorphism, but homozygosity for two polymorphisms (-551_-550insC and -160A>G) in the 5' untranslated region of the gene has been reported to be unique to Japanese P1- individuals. This study aimed to confirm this correlation in a larger number of British individuals. Serologically confirmed P1+ (n = 35) and P1- (n = 15) individuals were genotyped for polymorphisms in the 5' untranslated region of A4GALT. In addition to those previously reported, a further polymorphism, -164C>T, was identified. All P1- individuals were homozygous for -551_-550insC and -160G as compared with 10 of 35 (29%) P1+ individuals (P = 0.000003, two-tailed Fisher's exact test). Allele frequencies for all polymorphisms and estimated haplotype frequencies across the region differed significantly between P1+ and P1- groups. Homozygosity for the A4GALT-551_-550insC and -160G allele is significantly associated with, but not restricted to, the P1- phenotype. No single A4GALT genotype or haplotype was unique to P1- individuals. Thus, A4GALT cannot be unequivocally confirmed as the gene responsible for the P1 phenotype.

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

BackgroundThe 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.ResultsP1 (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 ~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 ~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.ConclusionThe 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.