A 13 base pair deletion in the GPIbβ gene in a second unrelated Bernard–Soulier family due to slipped mispairing between direct repeats

Abstract

Bernard–Soulier syndrome (BSS) is a congenital recessive disorder characterized by a prolonged bleeding time, macrothrombocytopenia and defective adhesion of platelets to the subendothelium [1,2]. Quantitative deficiencies of the GPIb–V– IX complex, a receptor for von Willebrand factor, are mostly responsible for this syndrome, although a-non-functional complex is present in rare cases. Genetic defects have been identified on the genes coding for theGPIba, GPIbb andGPIX subunits but not on the GPV gene. Typically, lack of a single subunit profoundly decreases the surface expression of the whole complex. Up until 2004, 45 separate genetic defects associated with BSS had been reported (http://www.orpha.net) [2]. The majority is due to mutations in GPIba, which is the larger subunit and bears the von Willebrand binding site, the others being equally distributed between GPIbb and GPIX. These defects can be divided into three major classes: (i) missense mutations or short in frame deletions which give rise to normal or slightly lower expression of a dysfunctional receptor or to an abnormal/unstable complex with decreased surface expression; (ii) nonsense mutations giving rise to shorter subunits typically lacking the transmembrane domain; and (iii) insertions or deletions leading to a frame shift, a novel polypeptide sequence and often a premature stop. In very rare cases, as in certain GPIba and GPIX mutants, a single mutationmainly affects the function of the receptor, which is at least partially expressed, although still in giant platelets [3]. The GPIX Asn45Ser mutant is the most common defect and has been reported in several families from different countries [4–6]. The GPIba Ala156Val mutation is frequent in the Italian population and gives rise to a disorder limited to macrothrombocytopenia in heterozygotes [7]. In certain mutations combining BSS and a DiGeorge/Velo–cardio–facial syndrome, defective GPIbb is found on one allele while the other has the large chromosomal deletion at 22q11.2 typical of DiGeorge syndrome, which encompasses the GPIbb gene [8,9]. GPIbb defects were reported rarely in the early studies but have since been documented in 13 cases (as compared to 20 defects in the larger GPIba gene). With the exception of the Tyr88 fi Cys mutation, which was found in several Japanese families, these GPIbb defects were isolated [10]. The mutants include missense and nonsense mutations (seven and three cases) and deletion/ insertion defects leading to translation frame shifts (three cases). Among the latter, two related Japanese patients were described recently who have a 13 base pair deletion of the signal peptide coding sequence leading to premature termination [11]. In the present report, we describe a third case of the same defect in a separate kindred. The propositus was a 2-year-old boy from Saudi Arabia presenting the cardinal characteristics of BSS: a bleeding tendency (epistaxis), a platelet count of 37 · 10 mL and platelet volume of 20.4 fL, an absence of ristocetin-induced platelet agglutination and decreased prothrombin consumption. The father and mother, who were direct cousins, had moderately increased platelet volumes (13 and 11.8 fl, respectively), platelet counts in the low normal range (148 and 129 · 10 mL, respectively), decreased ristocetin-induced agglutination and a normal skin bleeding time. Flow cytometry revealed an almost complete absence ofGPIba in the patient’s platelets (0.3 arbitrary fluorescence units (AFU) vs. 20 AFU for the control withmAb SZ2) and half the normal content of GPIba in the parents’ cells (11 AFU for the father and 11.6 AFU for the mother). Sequence analyzes of the GPIba, GPIbb and GPIX genes revealed a 13 nucleotide deletion in GPIbb on both alleles in the patient and as a single copy in the parents (Fig. 1a). This deletion occurs within the signal peptide coding sequence and is identical to that reported by Watanabe et al. [11]. The presence of a rearrangement with exactly the same boundaries in separate families suggests that it has occurred through a common mechanism, probably during replication. Inspection of the sequences flanking the deletion showed the presence of a four nucleotide GCCC direct repeat flanking a putative short stem-loop secondary structure (Fig. 1b). This hairpin structure would prompt a replication Correspondence: INSERM U311-Etablissement Francais du Sang, 10 rue Spielmann/BP 36, Strasbourg Cedex, France. Tel.: +33 388 21 25 25; fax: +33 388 21 25 21; e-mail: francois.lanza@ efs-alsace.fr