Single novel mutation in transmembrane region of glycoprotein (GP) IX affects GP Ib‐ IX complex expression and causes Bernard–Soulier syndrome

Bernard–Soulier syndrome (BSS) is a rare, inherited platelet disorder characterized by prolonged bleeding time, thrombocytopenia, giant platelets, and decreased ristocetin-induced agglutination. This disorder is caused by dysfunction or absence of the platelet glycoprotein (GP) Ib-IX–V complex, which consists of four components: GP Iba, GP Ibb, GP IX and GP V. So far, at least 50 different BSS genetic mutations have been reported in the genes coding these components (Salles et al, 2008). Here, we report a novel mutation in the transmembrane (TM) region of GP IX, which to our knowledge, it is the first mutation identified in this region that has definite BSS-causing effects. The patient bearing this novel mutation was a 6-year-old girl, who was diagnosed with BSS, according to the criteria of this syndrome (detailed in Appendix S1). Surface expression of GP Iba and GP IX on the patient’s platelets was not detectable by flow cytometry, while the normal control tested positive for these proteins (Fig 1). As the antibody used for GP Iba only recognizes this peptide when it is in combination with GP Ibb (McMichael et al, 1981), a negative result indicated that the GP Ib polypeptide (GP Iba plus GP Ibb) had been abolished from the platelet surface. The patient’s GP Ib-IX complex tested negative. GP V, GP IIb and GP IIIa expression in the patient’s platelets were higher than that of the normal control, which might be caused by the increased surface area of the patient’s platelets. Complete sequencing of genomic DNA of the four genes (GP1BA, GP1BB, GP9 and GP5) revealed multiple nucleotide alterations existed in the patient’s GP1BA, GP5 and GP9 genes (Table I) (detailed information is available in GeneBank, GI 264683459, GI 269819655, and GI 269819653, respectively), while no alteration was identified in GP1BB. Further analysis of the resultant effects of these alterations disclosed two meaningful mutations (see Appendix S1, Fig S2 for sequences), which were all located in the TM region of the GP IX protein. One of these mutations (1402G>A; Ala140 fi Thr) has been reported (Wang et al, 2004), whereas the second mutation (1388G>A; Trp134 fi stop) is novel. Because the 1388G>A; Trp134 fi stop mutation results in translation termination from the mutation point and is located upstream from 1402G>A; Ala140 fi Thr, the latter mutation could not function. The 1388G>A; Trp134 fi stop mutation deleted nearly all the TM region and all of the intracellular residues of the GP IX protein. Given that deletion of the TM region is known to reduce the GP Ib-IX complex’s surface expression on platelets (Luo et al, 2007), the 1388G>A; Trp134 fi stop muation caused the BSS in our patient. None of the 18 members of the patient’s family that were included in this study claimed to have a bleeding disorder. Their platelet morphology smears were normal and the blood counts were within normal ranges (Appendix S1). Flow cytometric analysis detected slightly decreased expressions of GP Iba (equal to GP Ib polypeptide as mentioned above) and GP IX in the mother and in an older cousin of the patient (Appendix S1). The mother of the patient was heterozygous for both Trp134 fi stop and Ala140Thr, while the older cousin of the patient was heterozygous for Trp134 fi stop but homozygous for Ala140Thr. GP IX is composed of a signal peptide of 16 amino acids and the mature proteins of 161 amino acids, the latter contains an N-terminal extracellular domain (1–131aa), TM domain (132– 153aa), and intracellular residues (154–161aa) (Luo et al, 2007). Recently, studies in transfected cells have disclosed the important role and molecular basis of GP IX’s TM domain for the assembly and expression of GP Ib–IX–V complex on platelets. Notably, Asp153 (interacting with Gln129 of GPIbb) and several leucine residues (Leu136, Leu138, Leu148, Leu149, Leu153), which form leucine-zipper-like sequences and thus interact with GP Ibb, are important for normal assembly and surface expression of the GP Ib–IX complex (Luo et al, 2007). Based on this knowledge, it is easy to understand why the mutation Trp134 fi stop is responsible for BSS. This mutation nearly abolishes the TM domain of GP IX (leaving only two residues), deleting all the residues in this domain that are important for formation and expression of the GP Ib–IX complex. Nevertheless, in homozygote or heterozygote, this mutation had no reducing effect on GP V’s platelet surface expression, which agrees with some previous reports that GP IX mutation had little effect on GP V (Drouin et al, 2005; Hayashi & Suzuki, 2000; Suzuki et al, 2000). An important question remains regarding the patient’s family: all 12 older brothers and sisters of the patient died of bleeding (see the case report in Appendix S1), but the exact causes of death are not certain. Because it is not possible for us to discover the cause(s) of death we must speculate. If they did indeed all suffer from BSS, there are two possibilities: they were all homozygous for the identified mutation, or some of them were heterozygous yet had BSS. The chances of the first possibility are quite slim, and thus the second possibility seems most probable. In support of the second hypothesis, the Correspondence