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

Abstract

Glanzmann's thrombasthenia (GT) results from a qualitative or quantitative defect of GPIIb–IIIa complexes (integrin αIIbβ3), 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 Cys542Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia

“…for 1 h at 37ЊC in a 40 ml reaction mixture (New England buffer 2). The digests were separated by electrophoresis on a 12% (w/v) polyacrylamide gel in Trisborate-EDTA buffer for 16 h, and visualized by ethidium bromide staining (Ruan et al, 1998a). The bands were visualized using UV light and photographed on Polaroid TM film (Poly Labo).…”

“…The concentration was determined at 260 nm and diluted to 50 ng/ml prior to PCR amplification. PCR-SSCP analysis of the exons (and splice sites) was then performed for both the GPIIb and GPIIIa genes as described previously (Ruan et al, 1998a). The oligonucleotides used to amplify exon 11 of the GPIIIa gene were CAGCGGGTCCACCTTCCT (forward) and CCAGCCTCCCGGCTCTCT (reverse).…”

“…Washed platelets were isolated from acid-citrate-dextrose anticoagulated blood, and samples prepared for SDS-polyacrylamide gel electrophoresis as previously described (Ruan et al, 1998a). Solubilizing buffer was 10 mM Tris-HCl, pH 7·2, 130 mM NaCl, 5 mM EDTA, 1% w/v SDS, 5 mM N-ethylmaleimide and contained the protease inhibitors phenylmethylsulphonyl fluoride, leupeptin and benzamidine.…”

“…PCR amplification products containing exon 11 of the GPIIIa gene were purified by 'PCR preps DNA purification resin' and Wizard TM minicolumns (Ruan et al, 1998a) and then digested with 10 U HinP1 I restriction enzyme (New England Biolabs, Mass.) for 1 h at 37ЊC in a 40 ml reaction mixture (New England buffer 2).…”

Platelet Glycoprotein IIb/IIIa Receptors and Glanzmann’s Thrombasthenia

“…Four missense mutations and 1 in-frame deletion mutation in 7 patients have been identified within and surrounding the calcium-binding domains, which are located within the fourth to seventh blades of the propeller. These mutations affect transport of the GPIIb/IIIa complex to the cell surface and include a G273D(G242D) substitution (patient FLD), 24 which precedes the first calcium-binding domain; E355K(E324K) (patients FL and Swiss) 25,26 and R358H(R327H) (patients KJ and Mila-1) 27,28 substitutions, located between the second and third calcium-binding domains; a G449D(G418D) (patient LM) 29 substitution, which precedes the fourth calcium-binding domain; and a V425D426 (patient LeM) 30 deletion at the beginning of the fourth calcium-binding domain. Another group of mutations is located within the vicinity of the third blade (W3) of the ␤ propeller, which contains a predicted ␤-turn structure that has been implicated in ligand-binding of GPIIb/IIIa and other integrin receptors.…”