A phenylalanine-55 to serine amino-acid substitution in the human glycoprotein IX leucine-rich repeat is associated with Bernard-Soulier syndrome.

The platelet membrane glycoprotein (GP) Ib-IX-V complex, the major von Willebrand factor receptor on platelets, is absent or dysfunctional in patients with the Bernard-Soulier syndrome (BSS). The four single subunits of the GPIb-IX-V complex (GPIb alpha, Ib beta, IX and V) are molecular products of different genes. Several point mutations and deletions affecting the GPIb alpha gene have been identified as the cause of BSS, whilst in four BSS families a GPIX gene defect has been reported. Moreover, a single case of BSS has been associated with a genetic defect of GPIb beta. We investigated the molecular basis of another case of BSS with a deficient expression of GPIX, as detected by immunofluorescence studies. After amplification of the entire GPIX coding region, nucleotide sequence analysis showed a homozygous single point mutation predicting a phenylalanine to serine substitution at position 55 of the mature GPIX within its unique leucine-rich repeat. By allele-specific oligonucleotide hybridization we confirmed the homozygosity of the patient as well as the carrier state of two out of three of his children studied. Although the parents of the patient, who were first cousins, were no longer alive and thus not available for study, we speculate that the molecular defect observed in the proband was inherited from both parents, who probably were heterozygous for this GPIX gene defect. This study confirms that BSS may be caused by many different subtle molecular defects that often prevent the assembly and expression of a functional GPIb-IX-V complex

Max(a), a new low-frequency platelet-specific antigen localized on glycoprotein IIb, is associated with neonatal alloimmune thrombocytopenia.

We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no known human platelet antigen (HPA)-system was involved. In the monoclonal antibody (MoAb)-specific immobilization of platelet antigens (MAIPA) assay, the new antigen was located on the platelet membrane glycoprotein (GP) IIb-IIIa complex, but immunoprecipitation and immunoblot experiments to further localize the antigen failed. However, in the MAIPA assay, the binding of the anti-Max(a) antibodies from the maternal serum was blocked by two anti-GPIIb MoAbs. Thus, the antigen appeared to be located on GPIIb. Analysis of the family lead to the identification of six additional Max(a+) individuals. Three of these six individuals and the father were tested in the platelet aggregation test and were found to be normal. In the analysis of normal donors, three of 500 were typed positive for the new platelet-specific antigen, indicating a phenotype frequency of 0.6% in the normal population. Platelet RNA was isolated from the newborn's Max(a+) father and from a healthy donor phenotyped as Max(a-), reverse-transcribed, and the entire GPIIb coding region was amplified by polymerase chain reaction. Subsequent nucleotide sequence analysis showed a single G --> A substitution at position 2,603, predicting a valine --> methionine amino acid substitution at position 837 of the mature glycoprotein. This mutation abolished a BsiYI restriction site at the cDNA level and a BstNI restriction site at genomic DNA level, respectively. The genetic association between the new antigen and this point mutation was confirmed by allele-specific restriction analysis on cDNA and on genomic DNA, as well as by allele-specific primer amplification on genomic DNA. The new mutation is 19 bp upstream of the mutation underlying the HPA-3 system. Therefore, we also evaluated the association between Max and the HPA-3 polymorphism. So far, all Max(a+) individuals were also found to be HPA-3b, whereas 50 HPA-3a individuals were all Max(a-). This may indicate that Max(a) is a variant of the HPA-3b allele