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Glanzmann's thrombasthenia (GT) is a recessive autosomal bleeding disorder characterized by abnormal platelet aggregation due to a qualitative or quantitative defect of the glycoprotein (GP) IIb-IIIa complex (integrin aIIbß3). We describe a new mutation in the GPIIIa gene responsible for type I GT in a consanguineous Algerian family. A discordance between phenotyping and genotyping of the GPIIIa-related HPA-1 platelet alloantigen system in three family members heterozygous for the disease suggested a genetic defect in the GPIIIa gene and a normal GPIIb gene. Sequence analysis of amplified genomic DNA fragments showed a 6-bp deletion in exon 7 of the GPIIIa gene resulting in the amino acid deletion/substitution (Ile325Pro326Gly327 ? Met) and creating a new BspHI restriction site. Expression of the mutated integrin ß3 subunit cDNA in Chinese hamster ovary cells showed that the cDNA gene was transcribed into a full-length ß3 protein with an apparent molecular weight identical to wild-type ß3 and accumulated as a single-chain molecule in the cell cytoplasm. The absence of heterodimeric complex formation of the mutant ß3 protein with endogeneous av was shown by immunoprecipitation experiments, intracellular immunofluorescent labeling, and a semiquantitative enzyme-linked immunosorbent assay using the avß3 complex-specific monoclonal antibodies LM609 and 23C6. Substitution of the methionine residue by a proline, present at position 326 of wild-type ß3, did not restore the ability of the recombinant mutant ß3 protein to associate with av, suggesting that the Ile-Pro-Gly motif is located in a ß3 domain important for integrin subunit interaction. The association of a BspHI restriction site with this newly identified mutation has allowed allele-specific restriction analysis of Algerian GT individuals and the identification of two new unrelated type I patients exhibiting the same mutation, suggesting that the described mutation might be significant in this population and that BspHI restriction analysis will provide a useful screening assay for antenatal diagnosis and genetic counselling.

Glanzmann's thrombasthenia is a rare inherited bleeding disorder caused by a qualitative or quantitative defect of platelet αIIb, β3. We describe here a new mutation that is the molecular genetic basis of Glanzmann's thrombasthenia in two gypsy families. Our investigation was focused on the αIIbgene as a result of biochemical and immunologic analysis of patients' platelets showing undetectable αIIbbut residual β3levels. The entire αIIbcDNA was polymerase chain reaction (PCR) amplified using patients platelet RNA. Sequence analysis showed an 8-bp deletion located at the 3' end of exon 15. This deletion causes a reading-frame shift leading to a premature stop codon and the synthesis of a severely truncated form of αIIb.Genomic DNA study showed a G → A substitution, the Gypsy mutation, at the splice donor site of intron 15. This mutation results in an abnormal splicing occurring at an alternative donor site located 8 bp upstream from the mutation. Based on those results, an allele-specific PCR analysis was developed to allow a rapid identification of the mutation in patients and potential carriers of the gypsy community. This PCR analysis can also be used for genetic counseling and antenatal diagnosis.

GFI1B is a transcription factor important for erythropoiesis and megakaryocyte development, but previously unknown to be associated with human disease. We have identified a family with an autosomal dominant bleeding disorder associated with thrombocytopenia, red cell anisopoikilocytosis and platelet dysfunction. The severity of bleeding is variable with some affected individuals experiencing spontaneous bleeding while other family members only exhibit abnormal bleeding with surgery. All affected individuals had a moderate thrombocytopenia ranging from 72-149 x109/L with an elevated mean platelet volume. Platelet function was perturbed with prolonged collagen/epinephrine closure times on the PFA-100 (294 vs 125 sec, P<0.001). Platelet aggregation studies were abnormal, with an absent collagen response and primary aggregation only with ADP, arachidonic acid and adrenaline. Genetic linkage analysis and massively parallel sequencing were performed on 16 family members to localize the mutation causing the disease phenotype to chromosome 9. A single nucleotide insertion was identified in GFI1Bthat predicts a frameshift mutation in the fifth zinc finger DNA-binding domain. This mutation alters the transcriptional activity of the protein as determined by luciferase production from the TGFBR3and GFI1Bpromoters. Aberrant transcriptional activity was associated with a marked reduction in platelet alpha-granule content as measured by electron microscopy (1.3 vs 3.1 alpha-granules per 1µm2, P<0.001) with similar changes in platelet protein expression. Proteomic analysis of platelet lysates demonstrated altered levels of cytoskeletal proteins, including talin, vinculin and myosin light chain 6. GFI1B mutation represents a novel human bleeding disorder and the described phenotype identifies GFI1Bas a critical regulator of platelet number and function.