GPIbα Gene Research Articles

Molecular and Genetic Analysis of Two Patients with Bernard-Soulier Syndrome – Identification of New Mutations in Glycoprotein Ibα Gene

We investigated two unrelated patients with Bernard-Soulier syndrome (BSS) by performing molecular and genetic analysis. A flow cytometric and immunoblotting analysis showed GP Ib alpha to be absent from the platelet membrane of both patients. Other glycoproteins that formed GP Ib/IX/V complex were present on the platelets, but in decreased amounts. Therefore, GP Ib alpha gene from both cases was sequenced after PCR amplification and subcloning. We identified a homozygous mutation of a dinucleotide deletion within the TGTG repeat at cDNA number 972 to 975 in GP Ib alpha gene from Case 1. In Case 2, compound heterozygosity was demonstrated in GP Ib alpha gene; an insertion of a single base (T) at cDNA number 1,418 in one allele, and a deletion of a single base (A) within the 7-adenine repeat at cDNA number 1,438 to 1,444 in another allele. The three new mutations in both patients appeared to cause a frameshift, which created a new termination codon shortly thereafter, and thus lead to a GP Ib alpha deficiency on the platelet membrane. Truncated mutant proteins could be detected in the plasma and platelets of Case 2, but not of Case 1. According to these findings, it is thus supposed that the properties and conformation of additional COOH-terminal peptides, which were supposedly synthesized as results of the mutations, may have an important role on the processing of mutant GP Ib alpha in megakaryocytes and platelets.

Systematic screening for genetic polymorphism in human platelet glycoprotein Ibalpha.

Glycoprotein Ibalpha (GP Ibalpha; CD 42b; hereafter GPIBA) is a component of the cell surface receptor for the von Willebrand factor (vWf) on platelets. Immunizations against various platelet surface antigens play a major role in neonatal alloimmune thrombocytopenia and in post-transfusion purpura. Only one antigenic polymorphism in GPIBA has thus far been established: the HPA-2 (Ko) alloantigen system. To screen other polymorphisms in GPIBA systematically, we analyzed the whole coding sequence of the GPIBA gene in 50 Finnish blood donors using the single-strand conformation polymorphism method. In addition to the known polymorphisms, we detected three others. Sequencing of the gene segments carrying the new polymorphisms revealed that none of them changed the predicted amino acid sequence. Polymorphism designated RS was located five base pairs upstream from the initiation codon at position 3064 and had the gene frequency of 16% for R and 84% for S, respectively, in the Finnish population, and it was detectable by the restriction enzyme Hae III. The EF polymorphism was at position 3842 (Asn242) and the gene frequencies were 97% for E and 3% for F. The KL polymorphism was at position 4142 (Arg342) and the gene frequencies were 98% for K and 2% for L. The five polymorphic positions in GPIBA formed altogether six different alleles of the gene. The data suggest that there are only a few variable amino acids in GPIBA.

Bernard-Soulier Syndrome with Severe Bleeding: Absent Platelet Glycoprotein lb alpha Due to a Homozygous One-base Deletion

Bernard-Soulier syndrome is a rare congenital platelet disorder that affects a surface membrane adhesion receptor, glycoprotein (GP) Ib-V-IX. Both the genetic defects and the bleeding diatheses associated with the syndrome are heterogeneous due, in part, to the complexity of the involved receptor which consists of four different members, GPs: Ib alpha-Mr 143 K (contains the von Willebrand factor-binding site), Ib beta-Mr 22 K, V-Mr 83 K and IX-Mr 20 K. We studied a kindred that includes a 40 year-old man with severe Bernard-Soulier syndrome: life-threatening gastrointestinal bleeding, thrombocytopenia, giant platelets and absent ristocetin-dependent platelet aggregation. By Southern blotting, PCR amplification/sequencing, hetero-duplex analysis, and allele-specific oligonucleotide hybridization, the Ib-V-IX genes were analyzed, and the molecular genetic defect was defined as a one-base deletion in the GPIb alpha gene, involving an adenine of codon 19. The mutation, K19R, homozygous in the propositus and heterozygous in the available unaffected relatives, leads to a frame shift in codons 19-21 and a premature stop codon after codon 21. No functional GPIb alpha can be produced from the mutant allele, implying that the platelets of the affected patient lack all GPIb alpha. Within the spectrum of Bernard-Soulier syndrome, this patient's disorder exemplifies a severe or "classic" extreme; an "experiment of Nature" that illustrates the effect of a complete deficiency of the ligand-binding chain (GPIb alpha) of the GPIb-V-IX receptor.

Genetic and Structural Characterization of an Amino Acid Dimorphism in Glycoprotein Iba Involved in Platelet Transfusion Refractoriness

AbstractThe platelet-specific alloantigen. Sib®, located within the a-subunit of the glycoprotein (GP) Ib-IX membrane receptor, has been found to be involved in the pathogenesis of platelet transfusion refractoriness. We have identified the existence of a naturally occurring threonine / methionine dimorphism at position 145 of the GPIbα sequence, and determined that the Sib® antigen corresponds to the molecule containing methi-onine145. The diallelic codons can be detected by restriction enzyme analysis of amplified genomic DNA fragments from the GPIbα gene. Evaluation of 61 healthy blood donors showed that the allele frequencies are 89% and 11% for the threonine145 and methionine145 codons, respectively. A posi-tive correlation exists between platelet reactivity with the anti-Sib® antibody and the presence of a methionine145-encoding allele. Moreover, recombinant expression of two soluble GPIbα fragments differing only at residue 145, provided definitive evidence that the human anti-Sib® antibody reacts only with the molecule containing methionine145. These results explain the structural basis of the Sib® human alloantigen system and define screening methodologies useful in transfusion medicine to match donor and recipient platelets accordingly.

The 5′ flanking region and chromosomal localization of the gene encoding human platelet membrane glycoprotein Ibα

The human blood platelet membrane glycoprotein Ib (GPIb) functions as a receptor for von Willebrand factor and thrombin. The gene ( gpIbα) encoding the GPIb α-chain was cloned from a genomic cosmid library. The promoter region of this gene was characterized by sequencing two BamHI fragments including 2.8 kb of the 5′ flanking region where several Alu repeated elements and purine-rich sequences were found. Possible cis-regulatory elements were identified by comparing the gpIbα gene with established consensus sequences known to function as binding sites for transcription factors. To obtain further information on possible megakaryocyte-specific promoter or enhancer sequences, the gpIbα, promoter region was compared with other genes expressed in platelets that are known so far. The gpIbα gene was found to be located on chromosome 17 in region 17p12-ter, by in situ hybridization.