Identification of Four Novel Mutations in Three Patients with Glanzmann’s Thrombasthenia.

Glanzmann’s Thrombasthenia (GT) is a rare autosomal recessive bleeding syndrome with an incidence of 1 in 1,000,000, which is characterised by severely reduced or absent platelet aggregation. Platelets from patients with GT are known to be lacking one of the glycoproteins found in human platelet membranes (α_IIb^β₃ integrin receptor). Sites of bleeding are clearly defined in GT patients and haemorrhage occurs primarily in mucocutaneous tissues. Spontaneous unprovoked bleeding is rare. The occurrence of haemorrhagic episodes is more frequent in childhood, and symptoms appear to diminish with age. There has been no correlation found between genotype and phenotype. Causative mutations have been found in the GPIIb (ITGA2B) and GPIIIa (ITGB3) genes which encode the two subunits of the α_IIb^β₃ receptor. The GPIIb gene is composed of 30 exons ranging in size from 44bp to 243bp, and encodes a 1039 residue protein. The GPIIIa gene comprises 15 exons ranging from 66bp to 425bp in size and encodes a 788 amino acid protein.

Three patients with GT were studied and all exons of the GPIIb and GPIIIa genes were amplified and analysed by dHPLC analysis. Exons showing abnormal retention patterns were then sequenced using the dye-terminator method. Four novel mutations in the GPIIb gene were discovered: 2 splicing defects, which are currently being investigated at the RNA level, a missense mutation and a 1bp insertion.

Patient 1 has a missense mutation in exon 4 of the GPIIb gene, and a splicing mutation in the conserved donor sequence at IVS27+1. Samples from family members were taken to confirm that these mutations are on different alleles. 100 alleles were genotyped for the missense mutation to determine whether this sequence alteration was present in the general population as a polymorphism, but it was not identified in any sample other than patient 1.

Patient 2 had a 1bp insertion in exon 4, resulting in a frameshift, and a splicing mutation in the consensus sequence of the splice acceptor site at IVS1-3. 100 alleles were genotyped for this mutation but it was absent. Due to the close proximity of these two mutations, allele specific oligonucleotides were designed to determine whether these mutations were in cis or trans. Results confirmed that the mutations were located on different alleles.

Patient 3 was shown to be homozygous for a known missense mutation in exon 18 of the GPIIb gene.

The novel missense mutation in exon 4 and insertion in exon 2 were both located in a region of the gene which encodes part of the protein that is responsible for interacting with the GPIIIa subunit. Dimerisation has been shown to be essential for function of the α_IIb^β₃ integrin receptor, and these mutations could disrupt the folding of the protein in such a manner that interactions could be altered or impossible. One of the splicing defects is also located within this area.

To date there have been less than 60 mutations identified in the GPIIb gene, so the discovery of 4 more is of scientific interest to groups studying this disease. Of the mutations so far identified, there appears to be a hotspot for mutations in exon 4, indicating that this region is important for the functioning of the encoded protein. However, all other mutations are evenly distributed throughout the gene.