A Leu7Pro mutation in the signal peptide of platelet glycoprotein (GP)IX in a case of Bernard–Soulier syndrome abolishes surface expression of the GPIb–V–IX complex

Abstract

Summary. This paper describes the molecular defect of the second case of Bernard–Soulier syndrome, initially reported in 1957. Analysis of the patient's platelets by flow cytometry and Western blotting failed to detect surface expression of any of the four subunits of the glycoprotein (GP)Ib–V–IX complex and revealed small amounts of intracellular GPIbα, GPIbβ and GPV but no GPIX. DNA sequencing revealed a novel missense mutation in the GPIX gene which replaced Leu (CTG) by Pro (CCG) at position 7 of the signal peptide. This mutation is, to date, the only known example of a leader sequence defect in Bernard–Soulier syndrome. The change occurred in a prototypic alpha‐helical hydrophobic core region, typically enriched in leucine and devoid of proline residues. Co‐transfection of GPIXPro7 with normal GPIbα and GPIbβ into Chinese hamster ovary cells reproduced the platelet phenotype, resulting in no detectable GPIX, low intracellular levels of GPIbα and GPIbβ, and an absence of surface expression. This mutation presumably leads to an abnormal conformation and, hence, incorrect insertion of GPIX into the endoplasmic reticulum and/or to defective signal peptide cleavage, both of which are required for correct transport to the cell membrane. This provides further evidence for a critical role of GPIX in controlling biosynthesis of the GPIb–IX complex.

Familial hypercatabolic hypoproteinemia caused by deficiency of the neonatal Fc receptor, FcRn, due to a mutant β2-microglobulin gene

“…The mutation of alanine to proline does not affect the net charge or hydrophobicity of the ␣-helix but presumably disturbs the secondary structure as a result of its helix-breaking properties, thus leading to compromised interaction with the signal recognition protein and abnormal insertion into the endoplasmic reticulum membrane (11). Similar proline mutations in signal sequences have been described in specific diseases (12)(13)(14)(15).…”

Compound heterozygosity for a novel nine-nucleotide deletion and the Asn45Ser missense mutation in the glycoprotein IX gene in a patient with Bernard-Soulier syndrome

“…It has been suggested that GPIX gene mutations affecting cysteine residues [13,14,21] prevent the formation of intramolecular disulfide-loop structures that are critical for the interaction of the polypeptide with the GPIbb subunit. A mutation involving the GPIX signal peptide was recently shown to drastically affect the biosynthesis of both GPIba and GPIX in affected platelets [23]. While it is not clear how point mutations in the GPIX gene not affecting cysteine residues give rise to the BSS phenotype, it has been postulated that these alter the configuration of the GPIX subunit, thus preventing its normal interaction with other constituents of the complex.…”

Novel Mutation in Bernard-Soulier Syndrome

“…The perfect Kozak sequence consists of gccRccAUGG with AUG as start codon, Sandrock/Knöfler/Greinacher/Fürll/Gerisch/ Schuler/Gehrisch/Busse/Zieger sis of patient's platelets revealed GPIX deficiency by using a GPIX antibody. Within the N-terminal part of GPIX, two different single amino acid substitutions were described causing BSS, one in the signal peptide [21] and one at amino acid 8 [22], both leading to the absence of the GPIb/IX/V complex at the platelet surface. We conclude that the GPIX deletion in the described patient prevents proper formation of the GPIb/ IX/V complex.…”