An αIIb mutation in patients with Glanzmann thrombasthenia located in the N-terminus of blade 1 of the ß-propeller (Asn2Asp) disrupts a calcium binding site in blade 6.

BACKGROUND: Studies of Glanzmann thrombasthenia (GT)-causing mutations has generated invaluable information on the formation and function of integrin αIIbß(3). OBJECTIVE: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. METHODS AND RESULTS: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the ß-propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbß(3) was found in patients' platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT ß(3). Although the αIIbß(3) was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbß(3) crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbß(3). Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbß(3) surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbß(3), αVß(3) harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVß(3) to calcium chelation compared with αIIbß(3). CONCLUSION: The new GT causing mutation highlights the importance of calcium binding domains in the ß-propeller for intracellular trafficking of αIIbß(3). The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.

A 13-bp deletion in alpha(IIb) gene is a founder mutation that predominates in Palestinian-Arab patients with Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by lack or dysfunction of alpha(IIb)beta3 in platelets. GT is relatively frequent in highly inbred populations. We previously identified a 13-bp deletion in the alpha(IIb) gene that causes in-frame deletion of six amino acids in three Palestinian GT patients. In this study, we determined the molecular basis of GT in all known Palestinian patients, examined whether Jordanian patients harbor the same mutations, analyzed whether there is a founder effect for the 13-bp deletion, and determined the mechanism by which the 13-bp deletion abolishes alpha(IIb)beta3 surface expression. Of 11 unrelated Palestinian patients, eight were homozygous for the 13-bp deletion that displayed common ancestry by haplotype analysis, and was estimated to have occurred 300-600 years ago. Expression studies in baby hamster kidney cells showed that substitution of Cys107 or Trp110 located within the deletion caused defective alpha(IIb)beta3 maturation. Substitution of Trp110, but not of Cys107, prevented fibrinogen binding. The other Palestinian patients harbored three novel mutations: G2374 deletion in alpha(IIb) gene, TT1616-7 deletion in beta3 gene, and IVS14: -3C --> G in beta3 gene. The latter mutation caused cryptic splicing predicting an extended cytoplasmic tail of beta3 and was expressed as dysfunctional alpha(IIb)beta(3). None of 15 unrelated Jordanian patients carried any of the described mutations.

Type I Glanzmann thrombasthenia: most common subtypes in North Indians.

The expression of GPIIb/IIIa on the platelet surface was assessed in 10 patients with Glanzmann thrombasthenia and their families by flow cytometry to determine the common subtype in North Indians. Glanzmann thrombasthenia was diagnosed in patients with bleeding manifestations accompanied by absent/reduced platelet aggregation, secondary to ADP, ADR, arachidonic acid, and collagen. Flow cytometry revealed variable GPIIb/IIIa expression by CD61 and CD41 in patients with Glanzmann thrombasthenia on the basis of CD61 levels, six patients were subtyped as type I because they had absent GPIIb/IIIa, three patients were subtyped as type II because their GPIIb/IIIa levels varied from 7.72% to 20.40%, and one patient was diagnosed as type III, because his clot retraction was 60% and GPIIb/IIIa was 46.0% of normal. Four fathers, three mothers, and five siblings were found to have GPIIb/IIIa levels less than 35% of normal. It is possible that low GPIIb/IIIa levels in family members may reflect their carrier status. It is postulated that flow cytometric estimation of GPIIb/IIIa in parents/siblings may detect carrier status in Glanzmann thrombasthenia.

A Gln747-->Pro substitution in the IIb subunit is responsible for a moderate IIbbeta3 deficiency in Glanzmann thrombasthenia.

To clarify a molecular defect responsible for moderate alphaIIb beta3 deficiency, we examined two unrelated patients, MT and MS, suffering from type II and type I Glanzmann thrombasthenia (GT), respectively. Sequence analysis of polymerase chain reaction (PCR) fragments derived from platelet mRNA showed a single A-->C substitution at nucleotide (nt) 2334 leading to a Gln747--> Pro in alphaIIb in both patients. Allele-specific restriction enzyme analysis (ASRA) of genomic DNA demonstrated that patient MT was homozygous for the Gln747-->Pro substitution and patient MS was compound heterozygous for this substitution and for an RNA splice mutation at the consensus sequence of the splice acceptor site of exon 18 (AG-->AA). Furthermore, ASRA showed that, among 17 unrelated Japanese GT patients, this Gln747-->Pro substitution was detected in 4 patients, including MT and MS (homozygous, 2 patients; heterozygous, 2 patients). Cotransfection of Pro747alphaIIb and beta3 constructs into 293 cells resulted in moderate reduction in the amount of alphaIIb beta3 within the transfected cells as well as on the cell surface. However, Pro747alphaIIb beta3 bound the ligand mimetic monoclonal antibody (MoAb) PAC-1 after activation of alphaIIb beta3 by the MoAb PT25-2, suggesting that the mutant alphaIIb beta3 possesses the ligand-binding function. The association between the mutant proalphaIIb and beta3 was not disturbed. Surface labeling and pulse chase study showed that the Gln747-->Pro substitution moderately impaired both intracellular transport of the alphaIIb beta3 heterodimers to the Golgi apparatus and endoproteolytic cleavage of proalphaIIb into heavy and light chains. By contrast, replacement of Gln747 with Ala by mutagenesis did not impair alphaIIbbeta3 expression on the cell surface. These results suggest that the presence of Pro, rather than the absence of Gln, at amino acid residue 747 on alphaIIb is responsible for moderate alphaIIbbeta3 deficiency.

Glanzmann thrombasthenia caused by an 11.2-kb deletion in the glycoprotein IIIa (beta3) is a second mutation in Iraqi Jews that stemmed from a distinct founder.

Glanzmann thrombasthenia (GT) is a rare bleeding disorder resulting from mutations in either glycoprotein (GP) IIb or GPIIIa genes. The disease is relatively frequent in highly inbred populations such as Iraqi Jews. The molecular basis of GT in 6 unrelated Iraqi-Jewish patients was previously identified as an 11-bp deletion in exon 12 of the GPIIIa gene. We now describe a second mutation found in 3 unrelated Iraqi-Jewish families that consists of an 11.2-kb deletion between an Alu repeat in intron 9 and exon 13 of the GPIIIa gene. The mutant DNA is transcribed into mRNA in which exons 10 through 13 are absent. Splicing of exon 9 directly to exon 14 leads to a shift in the reading frame resulting in a stop codon. The predicted protein is truncated in the middle of the third cysteine-rich domain before the transmembrane domain. Simple DNA-based methods were devised for identification of both mutations in Iraqi Jews for the purpose of carrier detection and prenatal diagnosis enabling prevention of GT. A survey of the general Iraqi-Jewish population for the first 11-bp deletion and the second 11.2-kb deletion disclosed that the allele frequency of the first mutation was 0.0043, whereas none of 700 individuals examined bore the second mutation (allele frequency <0.0007). Among 40 GT patients of Iraqi-Jewish origin 31 were homozygous for the first mutation, 4 were compound heterozygotes for the first and second mutations, and 2 were homozygous for the second mutation. Haplotype analyses using 4 polymorphic markers in the GPIIIa gene showed that each mutation originated in a distinct founder.

Molecular genetics aspects of factor XI deficiency and Glanzmann thrombasthenia.

Factor XI deficiency and Glanzmann thrombasthenia are among the hereditary disorders frequently encountered in Israel. Factor XI deficiency is particularly frequent in Ashkenazi (European) Jews with 1:190 individuals affected by the severe deficiency and 8.1% of the population being heterozygotes. So far 4 mutations causing factor XI deficiency have been identified of which the type II (a non-sense mutation) and type III (a missense mutation) are predominant and type I and IV observed only in 5 families. Recently, the type II mutation was observed in Iraqui-Jews as well with 3.7% of 400 unrelated subjects being heterozygotes and with the type III mutation completely absent. Since Iraqui-Jews represent the original gene pool of Jews who lived in Babylon 2500 years ago we hypothesize that the type II mutation is ancient and that the type III mutation occurred more recently, after the divergence of the original Babylonian Jews into Ashkenazi, Sephardic (Spanish) and Middle Eastern Jews. Preliminary data on factor XI intragenic polymorphic markers indeed indicate that type II and type III mutations reside on chromosomes each characterized by a different specific haplotype. Fifty living patients with type I Glanzmann thrombasthenia (28 families) have been observed in Israel. Most of them are Iraqui-Jewish and the rest are Arabs (5 families) and one Iranian Jewish. All Iraqui-Jewish patients have an IIbp deletion within exon 12 of the glycoprotein (GP) IIIa resulting in a shift of the reading frame that leads to premature termination of the GPIIIa synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of the Glanzmann's thrombasthenia mutations in Arab and Iraqi-Jewish patients by polymerase chain reaction and restriction analysis of blood or urine samples.

Severe Glanzmann's thrombasthenia is relatively frequent in Iraqi-Jews and Arabs residing in Israel. We have recently described the mutations responsible for the disease in Iraqi-Jews--an 11 base pair deletion in exon 12 of the glycoprotein IIIa gene, and in Arabs--a 13 base pair deletion at the AG acceptor splice site of exon 4 on the glycoprotein IIb gene. In this communication we show that the Iraqi-Jewish mutation can be identified directly by polymerase chain reaction and gel electrophoresis. With specially designed oligonucleotide primers encompassing the mutation site, an 80 base pair segment amplified in healthy controls was clearly distinguished from the 69 base pair segment produced in patients. Patients from 11 unrelated Iraqi-Jewish families had the same mutation. The Arab mutation was identified by first amplifying a DNA segment consisting of 312 base pairs in controls and of 299 base pairs in patients, and then digestion by a restriction enzyme Stu-1, which recognizes a site that is absent in the mutant gene. In controls the 312 bp segment was digested into 235 and 77 bp fragments, while in patients there was no change in the size of the amplified 299 bp segment. The mutation was found in patients from 3 out of 5 unrelated Arab families. Both Iraqi-Jewish and Arab mutations were detectable in DNA extracted from blood and urine samples. The described simple methods of identifying the mutations should be useful for detection of the numerous potential carriers among the affected kindreds and for prenatal diagnosis using DNA extracted from chorionic villi samples.

Immunoblot analysis of platelet glycoprotein IIb in patients with Glanzmann thrombasthenia in Israel.

Recent studies have indicated that severe ('type I') Glanzmann thrombasthenia is a heterogeneous hereditary disorder caused by quantitative and/or qualitative abnormalities of platelet membrane glycoproteins (GP) IIb and IIIa. Immunoblot analysis of sodium dodecyl sulphate (SDS)-solubilized platelets was carried out on controls and 18 patients (12 Iraqi-Jews, two Iranian Jews and four Arabs) employing three antibodies (one monoclonal and two polyclonal) directed at different sites on GPIIb. Nonreduced control platelet samples contained a major Mr approximately 140k immunoreactive protein that was split into an Mr approximately 120k (alpha) and an Mr approximately 25k (beta) band after reduction with mercaptoethanol. The nonreduced samples from all 18 patients tested had trace amounts of Mr approximately 140k band corresponding to normal GPIIb; the intensity of this band was estimated to be less than 1% of the normal amount. Unlike the control samples, however, this Mr approximately 140k band did not change electrophoretic mobility following reduction. Since GPIIb originates from a single chain precursor molecule of Mr approximately 140k that comprises both the alpha and beta chains, and which does not change mobility with reduction, our data suggest that the platelets of these patients contain small amounts of this precursor.