Double heterozygosity of the GPIIb gene in a Swiss patient with Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) results from a qualitative or quantitative defect of GPIIb–IIIa complexes (integrin α_IIbβ₃)_, the fibrinogen receptor on platelets. This integrin plays a critical role in platelet aggregation. In this report we describe the molecular abnormalities of a patient with clinical and laboratory findings typical of type I Glanzmann's thrombasthenia. SDS-PAGE with Western blotting revealed an absence of GPIIb but small amounts of normally migrating GPIIIa in his platelets. A non-radioactive PCR-SSCP procedure and direct sequence analysis of PCR-amplified DNA fragments showed the patient to be a compound heterozygote for mutations in the GPIIb gene. A single point mutation (G to A) at nucleotide 1064 of the cDNA derived from the mother's allele led to a Glu³²⁴ to Lys amino acid substitution in GPIIb. It was responsible for a MscI restriction site in exon 12 of the GPIIb gene. This amino acid substitution changes the electric charge between the second and third Ca⁺⁺-binding domains of GPIIb. The second mutation was inherited from his father and is in exon 18 of the GPIIb gene. It was a T → C base transition at position 1787 of GPIIb cDNA and results in a Ile⁵⁶⁵ to Thr substitution. The two GPIIb mutations identified in this study will provide new information on GPIIb–IIIa structure and biosynthesis.     

Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.     

A 1063G-->A mutation in exon 12 of glycoprotein (GP)IIb associated with a thrombasthenic phenotype: mutation analysis of [324E]GPIIb

We report the molecular, genetic and functional analysis of a case of thrombasthenic phenotype. The proband showed absence of platelet glycoprotein (GP)IIb and very low content of GPIIIa, and both his parents showed a marked reduction in the levels of platelet GPIIb-IIIa. Single-stranded conformational polymorphism-polymerase chain reaction (SSCP-PCR) analysis and direct sequencing of PCR-amplified GPIIb exon-12 revealed the presence of a G-->A transition at position 1063 with the expected substitution of glutamate 324 with lysine (K). This mutation did not alter the level of GPIIb mRNA. Co-expression of normal or mutant [324K] GPIIb with normal human GPIIIa in Chinese hamster ovary (CHO) cells failed to show surface exposure of [324K]GPIIb-IIIa complexes. Pulse-chase and immunoprecipitation analysis demonstrated that [324K]GPIIb cDNA was translated into proGPIIb, but neither mutant GPIIb heavy chain (GPIIbH) nor [324K]GPIIb-GPIIIa complexes were detected, suggesting that this mutation is the underlying molecular basis for the thrombasthenic phenotype. Mutation analysis demonstrated that 324E of GPIIb could be replaced by other negatively charged or polar amino acids (AAs) without impairing the surface expression of GPIIb-IIIa. However, substitution of 324E of GPIIb for a positively charged AA other than K prevented the expression of GPIIb-IIIa complexes. These observations suggest that a domain encompassing 324E of GPIIb is essential for heterodimerization with GPIIIa and its substitution for a positively charged residue precludes normal subunit association.     

A novel (288delC) mutation in exon 2 of GPIIb associated with type I Glanzmann's thrombasthenia

This work reports the molecular genetic analysis of two patients who suffer mucocutaneous haemorrhages, prolonged bleeding time and failure of platelets to aggregate, either spontaneously or in response to agonists. The absence of platelet surface glycoprotein (GP)IIb-IIIa complexes confirmed the clinical diagnosis of Glanzmann's thrombasthenia (GT). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of exon 2 of GPIIb showed polymorphic bands caused by the homozygous deletion of a cytosine at position 288 relative to the translation start site. causing a shifting of the reading frame and appearance of a premature termination codon. The heterozygous relatives showed a reduced platelet content of GPIIb-IIIa, and a correlation was found between the levels of GPIIb mRNA and surface expression of GPIIb-IIIa complexes. Unlike other mRNAs carrying a nonsense mutation, (288Cdel)GPIIb does not force alternative splicing of GPIIb mRNA. As expected, co-transfection of Chinese hamster ovary (CHO) cells with cDNAs encoding GPIIIa and (288delC)GPIIb failed to enhance the surface exposure of GPIIIa. It is concluded that the (288delC)GPIIb mutation is responsible for the thrombasthenic phenotype of the patients. In addition, it has also been determined that heterodimerization of GPIIb-IIIa requires the integrity of exons 2 and 3 of GPIIb.     

Novel point mutation in the leucine-rich motif of the platelet glycoprotein IX associated with Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder which is caused by a qualitative or quantitative abnormality of the platelet glycoprotein (GP) Ib/IX/V complex. We examined a patient with BSS to find a molecular basis for the defect underlying this disease. The propositus was a 39-year-old Japanese female with life-long bleeding diathesis. Sequence analysis of the GPIX gene revealed a T → C point mutation at nucleotide 1856 (EMBL, M80478), resulting in Phe⁵⁵(TTT) → Ser(TCT) replacement. This substitution created a new Mnl I restriction site in the mutant allele. Restriction analysis revealed that the propositus was homozygous for this sequence, and the same mutation was not detected in 57 unrelated Japanese subjects. Since this mutation is located in the leucine-rich motif (LRM) of the GPIX polypeptide, the Phe⁵⁵ → Ser substitution may result in an alteration of the LRM which leads to the impaired surface expression of GPIb/IX/V complex, a characteristic of BSS.     

A Val193Met mutation in GPIIIa results in a GPIIb/IIIa receptor with a constitutively high affinity for a small ligand

We have identified a patient designated as (GTa) with Glanzmann's Thrombasthenia (GT) diagnosed on the basis of a prolonged bleeding time and failure of the patient's platelets to aggregate. The number of glycoprotein (GP)IIb/IIIa receptors on the platelet surface was 37% of normal and those receptors displayed a defect in soluble fibrinogen binding. Nevertheless, GTa platelets showed increased adhesion to solid-phase fibrinogen and binding affinity for the RGD-mimetic (3)H-SC52012, a non-peptide GPIIb/IIIa antagonist. Dithiothreitol (DTT) and ADP enhanced the affinity for [(3)H]-SC52012 in normal platelets, but had little effect in GTa platelets. These findings suggested that GTa platelets were locked in an altered affinity state. Genetic analysis showed that GTa was a compound heterozygote for the GPIIIa gene. One allele showed a deletion at the 3' end of exon 3 resulting in a premature stop codon. The second GPIIIa allele had a G to A transition at nucleotide 577, resulting in a Val193Met substitution. HEK 293T cells transfected with mutant GPIIb/IIIaV193M bound [(3)H]-SC52012 with a higher affinity than wild-type GPIIb/IIIa, and this was not increased by DTT. The mutant receptor distinguishes between platelet adhesion and aggregation, and demonstrates the phenotype that may be expected when platelet aggregation alone is inhibited.     

A Cys374Tyr homozygous mutation of platelet glycoprotein IIIa (beta 3) in a Chinese patient with Glanzmann's thrombasthenia

A 20-year-old woman from a consanguineous family in the Hunan Province of the People's Republic of China was diagnosed as having Glanzmann's thrombasthenia based on (1) nearly a lifelong history of epistaxis, gum bleeding, petechiae, and purpura; (2) severe menorrhagia resulting in anemia and need for whole-blood transfusion; (3) normal coagulation assays; (4) prolonged bleeding time; (5) absent clot retraction; (6) decreased glass bead retention; (7) absent platelet aggregation in response to adenine diphosphate, epinephrine, and collagen; and (8) normal initial slope of platelet aggregation in response to ristocetin, but with a diminished maximal extent. The patient's platelets had a decreased level of platelet fibrinogen, but the deficiency was not as severe as in other Glanzmann's thrombasthenia patients. As judged by monoclonal antibody binding studies, surface glycoprotein (GP) IIb/IIIa (alpha IIb beta 3) expression was less than 15% of normal and alpha v beta 3 vitronectin receptor expression was 15% to 19% of normal, suggesting that the defect was in GPIIIa (beta 3). Immunoblotting of platelet lysates demonstrated decreased levels of GPIIb (approximately 30% to 35% of normal) and GPIIIa (approximately 10% of normal), and the GPIIb had undergone normal maturational processing into GPIIb heavy and light chains. Sequence analysis of the patient's GPIIIa RNA identified a G to A mutation at nucleotide 1219, predicting a Cys to Tyr substitution at residue 374. The patient's parents, who are first cousins, are asymptomatic and have only minor reductions in platelet aggregation. Direct sequencing of polymerase chain reaction-amplified cDNA and GPIIIa exon VIII indicated that the patient is homozygous and her parents are heterozygous for the mutation. Transient transfection studies in Chinese hamster ovary cells indicated that the mutation results in an 85% to 90% reduction in GPIIb/IIIa surface expression, but these cells retain the ability to mediate adhesion to immobilized fibrinogen. The relative preservation of platelet fibrinogen despite the very low level of platelet surface GPIIb/IIIa expression in this patient raises some interesting questions regarding the mechanism of fibrinogen uptake and the pathophysiology of Glanzmann's thrombasthenia.     

Reactivity of autoantibodies from chronic ITP patients with recombinant glycoprotein Ilia peptides

Summary Chronic immune thrombocytopenia is an autoimmune disorder characterized by destructive thrombocytopenia due to the formation of autoantibodies against platelet-associated antigens. Most antiplatelet autoantibodies react with either the platelet glycoprotein IIb/IIIa or Ib/IX complex, whereas some plasma autoantibodies react with glycoprotein IIIa. Previous studies from our laboratory suggested that most platelet-associated autoantibodies to platelet GPIIb/IIIa, which bind to the intact complex, bind much less avidly to the EDTA-dissociated complex, suggesting that the epitopes were complex-dependent. To evaluate this further we have studied the binding of platelet-associated autoantibody and plasma auto- and alloantibody eluates to large recombinant GPIIIa peptides: peptide 1 (GPIIIa Gly¹-Val²⁰⁰); peptide 2 (GPIIIa Arg¹⁵⁰-Glu⁴⁰⁰); peptide 3 (GPIIIa Lys³⁵⁰-Asp⁵⁵⁰); peptide 4 (GPIIIa Asn⁴⁵⁰-Val⁷⁰⁰) and peptide 5 (GPIIIa Trp⁷¹⁵-Thr⁷⁶², cytoplasmic fragment). Of the 33 platelet-associated antibody eluates tested, all bound avidly to the GPIIb/IIIa complex, but only one showed significant binding (>3 SD above control values) to one of the immobilized peptides (peptide 3). Conversely, antibodies known to bind to specific regions of GPIIIa (murine monoclonal antibody, anti-LIBS2; plasma autoantibody against the GPIIIa cytoplasmic fragment and anti-P1^A1 antibody) all bound avidly to the GPIIIa peptide containing the appropriate epitope. Based on these and our previous results, we conclude that platelet-associated antibodies from chronic ITP patients rarely bind to epitopes localized to GPIIIa alone.     

Prenatal diagnosis of Glanzmann thrombasthenia using the polymorphic markers BRCA1 and THRA1 on chromosome 17

Glanzmann thrombasthenia is an autosomal recessive bleeding disorder caused by mutations in the genes encoding platelet GPIIb or GPIIIa. Both genes map to chromosome 17q21 and polymorphisms within this chromosomal region have been identified. In the current study, prenatal diagnosis was performed for a family that already had one affected child, patient 1, who had a compound heterozygous mutation in GPIIb. At the time of prenatal diagnosis, the maternal GPIIb mutation had been identified but the paternal GPIIb mutation was unknown. By sequence analysis, the fetus was identified as a carrier of the mother's mutation. To determine the probability of the fetus inheriting the father's mutation, haplotype analysis of DNA samples from the fetus, mother, father and affected child were performed using polymorphic markers on chromosome 17q12-q21. These markers included polymorphisms within the thyroid hormone receptor α1 gene (THRA1), the breast cancer gene (BRCA1), GPIIb, GPIIIa, and an anonymous marker D17S579. Heterozygosity within the THRA1, BRCA1 and GPIIIa polymorphic markers predicted that the fetus carried the father's normal allele. Based on genetic linkage studies, no recombination was identified with any of the informative markers, and from the map distance between GPIIb and BRCA1 the accuracy of diagnosis was predicted to be >98%. The father's mutation was subsequently identified and direct sequence analysis of fetal DNA confirmed that the fetus did not inherit the fathers' mutant allele.     

Competition between normal [674C] and mutant [674R] subunits: role of the molecular chaperone BiP in the processing of GPIIb-IIIa complexes

This work aimed at investigating the function of the [C674R] mutation in GPIIb that disrupts the intramolecular 674 to 687 disulfide bridge. Individuals heterozygous for this mutation show a platelet GPIIb-IIIa content approximately 30% of normal controls, which is less than expected from one normal functioning allele. Coexpression of normal [674C]GPIIb and mutant [674R]GPIIb with normal GPIIIa produced a [674R]GPIIb concentration-dependent inhibition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary (CHO) cells, suggesting that [674R]GPIIb interferes with the association and/or intracellular trafficking of normal subunits. Mutation of either 674C or 687C had similar effects in reducing the surface exposure of GPIIb-IIIa. However, substitution of 674C for A produced a much lesser inhibition than R, suggesting that a positive-charged residue at that position renders a less efficient subunit conformation. The mutant [674R]GPIIb but not normal GPIIb was found associated with the endoplasmic reticulum chaperone BiP in transiently transfected CHO cells. BiP was also found associated with [674R]GPIIb-IIIa heterodimers, but not with normal GPIIIa or normal heterodimers. Overexpression of BiP did not increase the surface exposure of [674R]GPIIb-IIIa complexes, indicating that its availability was not a limiting step. Platelets from the thrombasthenic patient expressing [674R]GPIIb-IIIa were found to bind soluble fibrinogen in response to physiologic agonists or dithiothreitol treatment. Thus, the [674R]GPIIb mutation leads to a retardation of the secretory pathway, most likely related to its binding to the molecular chaperone BiP, with the result of a defective number of functional GPIIb-IIIa receptors in the cell surface.     

Severe coagulation factor V deficiency caused by a 4 bp deletion in the factor V gene

Factor V (FV) deficiency (parahaemophilia) is an autosomal recessive bleeding disorder with an incidence of 1:10⁶. We have studied a young girl with very mild bleeding symptoms and undetectable levels of plasma factor V antigen and activity (<0.3% and <1.6% of normal, respectively). Both parents showed plasma levels of factor V activity of about 50% of normal. Sequence analysis of the 5'- and 3'-untranslated, coding and adjacent regions of the factor V gene revealed the presence of a 4 bp deletion in exon 13. Subsequent screening of members of the family for the mutation showed that both parents were heterozygous for the mutation, that one healthy sister carried only normal alleles, and that the patient was homozygous for the mutated allele. The mutation introduced a frameshift and a novel premature stop codon in codon 1303, and would predict the synthesis of a truncated factor V molecule that lacks part of the B domain and the complete light chain. However, no factor V heavy chain could be detected in the plasma of the patient. Furthermore, factor V activity could not be detected in the patients' platelets. This is the first reported mutation in the factor V gene that predicts a type I quantitative factor V deficiency. Surprisingly, the patient, who is homozygous for the mutation, so far has only a very mild bleeding tendency.     

Single-strand conformation polymorphism analysis is a rapid and effective method for the identification of mutations and polymorphisms in the gene for glycoprotein IIIa

Glanzmann thrombasthenia (GT) is the most common inherited disorder of platelets. Most of the molecular defects previously identified in GT have been caused by point (or other small) mutations in the genes for glycoprotein (GP) IIb or GPIIIa. We have used single-strand conformation polymorphism (SSCP) analysis to rapidly identify single- base changes in the GPIIIa gene. Using genomic DNA from normal individuals and patients with GT, each GPIIIa exon and a short stretch of flanking intronic sequence was amplified, heat-denatured, and separated in nondenaturing acrylamide gels. Only those fragments with an abnormal migration pattern were isolated and the nucleotide sequence determined. Using SSCP, we detected the polymorphism in the HPA-1 (P1A) system and all three known silent polymorphisms in the GPIIIa gene. Screening 14 GPIIIa exons from 5 patients with GT, one mutant allele was identified. The nucleotide sequence of the abnormal 240-bp SSCP fragment was determined and a G-->A substitution in the splice donor site of exon iv was identified. Analysis of platelet RNA resulting from this mutation showed two mRNA species: one contained a deletion of exon iv, whereas the other had a 27-bp addition to exon iv due to the use of a cryptic splice site in the downstream intron. Single-base substitutions are the most common mutation in GT and often result in abnormal mRNA splicing. SSCP is a rapid and sensitive technique for identifying mutations or polymorphisms in the GPIIIa gene.     

Factor V Arg306 → Thr (factor V Cambridge) and factor V Arg306 → Gly mutations in venous thrombotic disease

We investigated the prevalence of two reported mutations of the factor V gene (factor V Arg³⁰⁶ → Thr, or factor V Cambridge, and factor V Arg³⁰⁶ → Gly) in 104 relatively young patients with verified venous thrombosis and in 208 age-, sex- and race-matched controls, in order to establish whether the two mutations are associated with increased predisposition for venous thrombosis. PCR amplification followed by Bst NI and Msp I digestion was employed to determine the genotypes, and each mutation was confirmed by DNA sequencing. Among the controls, one individual was found to be heterozygous for the factor V Arg³⁰⁶ → Thr mutation and one heterozygous for the factor V Arg³⁰⁶ → Gly mutation; none of the patients carried either mutation. Our findings do not support factor V Cambridge and factor V Arg³⁰⁶ → Gly as risk factors for venous thrombosis.     

Glanzmann's thrombasthenia associated with deletion-insertion and alternative splicing in the glycoprotein IIb gene

Glanzmann's thrombasthenia is a bleeding disorder characterized by a decrease or absence of the functional platelet membrane glycoprotein (GP) complex, GPIIb/IIIa (alpha IIb beta 3). We describe a new deletion- insertion mutation in the GPIIb gene causing type I Glanzmann's thrombasthenia in two siblings of a consanguineous Iranian-Jewish family. The proband's platelets bound more antibodies against the vitronectin receptor-alpha V beta 3 than normal platelets, suggesting a normal GPIIIa (beta 3) gene and a defect in the GPIIb gene. Sequencing of amplified cDNA and genomic DNA fragments showed a 6-bp deletion and 31-bp insertion in exon 25 of the GPIIb gene. The predominant platelet GPIIb mRNA of the proband was a product of the splicing of exon 24 to a cryptic AG acceptor site in the insertion and encoded for deletion of amino acids Leu817-Asn826 and insertion of eight different amino acids. Cotransfection of COS-7 cells with expression vectors containing wild- type GPIIIa cDNA and the mutated GPIIb cDNA failed to produce detectable amounts of GPIIb/IIIa on the surface of the cells. Allele- specific restriction analysis of genomic DNA of family members showed homozygosity for the mutation in the affected siblings, heterozygosity in the parents, and homozygosity for the normal allele in an unaffected sibling. The observed mutation is in a region that is conserved from rodents to humans and has been suggested to be involved in the interaction between GPIIb and GPIIIa when these GPs are complexed in solution.     

Identification of a nonsense mutation at amino acid 584–Arginine of platelet glycoprotein IIb in patients with type I Glanzmann thrombasthenia

Using Southern blot, the restriction digests of genomic DNAs in 11 patients with Glanzmann thrombasthenia from 10 unrelated kindreds were probed with a full-length GPIIb cDNA. An addiitonal 2.3 kb Taq I fragment and two 1.65 kb and 0.65 kb fragments with reduced band intensity were found in the genes of two affected siblings from a family originating from the city of Huang Yan in the Zhejiang province. The Taq I digest of the abnormal gene was further probed with three portions of GPIIb cDNA, revealing that the heterozygous mutation was present in the region around exons 15-17 of the GPIIb gene. Two primers for polymerase chain reaction (PCR) were then designed, and a 394 bp PCR product was generated and sequenced, indicating that a stop codon (TGA) was substituted for an Arg codon (CGA) at amino acid position 584 of GPIIb, and resulted in a premature termination of translation and production of a shortened protein. The Western blot analysis showed that GPIIIa at the platelet surface was apparently deficient, it may be ascribed to the rapid turn-over of GPIIIa uncomplexed with the truncated GPIIb. The abnormal 2.3 kb Taq I fragment was used as a specific genetic marker to detect the carrier status of the patient family. The abnormal allele was proved to be derived from the mother, the two affected siblings are double heterozygotes, and one clinically unaffected daughter has also inherited this defective allele, while the father carries another recessive abnormal allele unidentified.     

Plasma autoantibodies against platelet glycoprotein IIb/IIIa from patients with autoimmune thrombocytopenic purpura may recognize different antigenic determinants

Abstract: Autoantibodies against platelet glycoprotein (GP) GPIIb/IIIa have been demonstrated in patients with autoimmune thrombocytopenic purpura. Recently, it has been shown that plasma autoantibodies from some patients bind to the cytoplasmic domain of GPIIIa. Our aim was to evaluate further the binding specificity of these plasma autoantibodies. From 7 patients with detectable plasma antibodies against intact GPIIb/IIIa, 1 showed strong antibody binding to a synthetic C-terminal peptide of GPIIIa. Ig class analysis of affinity purified anti-GPIIb/IIIa autoantibodies from this patient revealed an IgM antibody that reacted with intact GPIIb/IIIa as well as with recombinant GPIIb/IIIa lacking the C-terminal domains, and an IgG antibody that bound to intact GPIIb/IIIa but not to GPIIb/IIIa lacking the C-terminal region. These data indicate that this patient has at least 2 autoantibodies, an IgG directed against the cytoplasmic domain of GPIIIa and an IgM reacting with the extracellular part of GPIIIa. This may support the hypothesis that plasma IgG antibodies directed against the C-terminal domain of GPIIIa may be due to the exposition of cytoplasmic epitopes of GPIIIa as a result of increased cell lysis by IgM autoantibodies.     

Molecular pathology of inherited Glanzmann's thrombasthenia. Report of 11 cases]

Glycoprotein IIb-IIIa (GPIIb-IIIa) concentration was studied in 11 patients with Glanzmann's thrombasthenia (GT) with sensitive Western blotting technique. 7 patients with severe GPIIb-IIIa deficiency (less than 10% of the normal) were designated as type I (64% of patients), 2 patients with moderate GPIIb-IIIa deficiency (10-25% of the normal) as type II (18%) and 2 patients with GPIIb-IIIa 40-100% of the normal as variants (18%). Southern Blotting was used to analyze the GPIIb and GPIIIa genes in the 11 patients. The results showed that there were no major deletions or insertions in either the GPIIb or GPIIIa genes. However, a small change in GPIIb gene was demonstrated in two sibling patients and the abnormality of GPIIIa gene was found in another two patients. These observations combined with those from literature provide a basis for discussing the molecular pathology of Glanzmann's thrombasthenia.     

Family screening for a Glanzmann's thrombasthenia mutation using PCR-SSCP

Genetic counselling is often requested in Glanzmann's thrombasthenia, but measurements of GPIIb-IIIa density on platelets are often too inconclusive to allow a precise assessment of whether prospective parents are obligate heterozygotes for this disease by this measure alone. The recent application of PCR technology to Glanzmann's thrombasthenia has resulted in the identification of a large number of mutations, i.e. insertions/ deletions, splicing defects, in the genes for both GPIIb and GPIIIa. Among the reported abnormalities is an intronic G-->A substitution at the splice donor site of intron 15 in the GPIIb gene of a European gypsy tribe. This gives rise to an abnormal splicing, of an 8-bp deletion located at the 3' end of exon 15, a reading-frame shift and a premature stop codon in the mRNA for GPIIb. In applying PCR-SSCP to the elucidation of the genetic defects of a series of Glanzmann's patients, we have found the above-cited abnormality in three more gypsy families in France. The presence of the mutation was initially established by sequencing the amplified fragment, and its presence in family members was confirmed by both PCR-SSCP and HphI restriction analysis. Evaluation of the intronic G-->A mutation enabled genetic counselling to prospective parents within these families.