S. Simsek

SEQUENCE ANALYSIS OF CDNA DERIVED FROM RETICULOCYTE MRNAS CODING FOR RH POLYPEPTIDES AND DEMONSTRATION OF E/E AND C/C POLYMORPHISMS

RNA derived from enriched reticulocytes of Rh‐phenotyped donors was isolated, reversely transcribed into cDNA and amplified with Rh‐specific primers by polymerase chain reaction. Nucleotide sequence analyis of the entire coding region of the Rh cDNAs was carried out. Four types of cDNAs were identified, tentatively designated as RhSCI, RhSCII, RhSCIII and RhSCIV. Comparison of RhSCII with RhSCI (identical to the previously reported RhIXb/30A cDNA), showed a single base pair difference. Since RhSCI and RhSCII were found to be related to the presence of E or e antigen, respectively, the P226A amino acid polymorphism appears to be the genetic basis of the E/e polymorphism. RhSCIII was demonstrated to be a transcript derived from the RhD gene, with 35 amino acid substitutions as compared to RhSCI. RhSCIV was found to be present only in RhC‐positive individuals, indicating that RhSCIV encodes a polypeptide carrying the C antigen. Six nucleotide changes, resulting in four amino acid substitutions W16C, L60I, N68S and P103S, were observed between RhSCII and RhSCIV, probably representing the C/c polymorphism.

The genetic basis of a new partial D antigen: DDBT

The Rh system, the most polymorphic system on red cells, is genetically controlled by two different but highly homologous genes on chromosome 1. The RHCE gene encodes different RhCcEe polypeptides and the RHD gene encodes D antigens. It is well established that in D negative individuals the RHD gene is either absent or grossly deleted. The D antigen comprises at least nine serologically defined D epitopes. The D antigen can be divided into different partial D categories, reflecting a different pattern of specific D epitopes.

Cys209 Ser mutation in the platelet membrane glycoprotein ibα gene is associated with Bernard-Soulier syndrome

Summary Molecular genetic analysis has been performed on a patient with Bernard‐Soulier syndrome (BSS). The patient had characteristically giant platelets and was deficient in the glycoprotein (GP) Ib/IX/V complex, the von Wllebrand factor (vWf) receptor on platelets. Previous studies with monoclonal antibodies directed against GP Ibα (CD 42b) and GP IX (CD 42a) demonstrated the absence of GP Ibα and presence of small amounts of GP IX on the surface of the patients platelets. In ths study the presence of GP V (CD 42d) is also demonstrated. This indicates a defect in the α‐subunit of glycoprotein Ib. Therefore polymerase chain reaction (PCR)‐amplification Ib. Therefore polymerase chan reaction (PCR)‐amplification of the genomic DNA coding for GP Ibα was performed. Nucleotide sequence analysis of the entire coding region of GP Ibα revealed a homozygous single base pair mutation T A, leading to a single amino acid substitution cysteine serine at position 209 of the mature protein. We took advantage of the Mse I target site in the mutant allele, created by the T A mutation, to analyse all available family members. PCR‐ASRA (allele‐specific restriction enzyme analysis) using the restriction enzyme Mse I, revealed the heterozygosity of the mother and the two children of the patient, whereas homozygosity of the patient for the Cys209Ser mutation was confirmed. The sister of the patient was not found to the a carrier of the mutant allele.

Genetic analysis of patients with leukocyte adhesion deficiency: genomic sequencing reveals otherwise undetectable mutations.

OBJECTIVE The aim of this study was to analyze mutations in DNA from patients with leukocyte adhesion deficiency (LAD), an immunodeficiency caused by absence of the beta(2) subunit (CD18) of the leukocyte integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and CR4 (CD11d/CD18). METHODS We developed genomic DNA PCR sequencing to detect mutations not only in exons but also in introns. RESULTS Eight LAD patients were analyzed, of which five had homozygous mutations, i.e., a 0.8-kb deletion, a branchpoint mutation in intron 5 causing mRNA missplicing, a nonsense mutation, and two missense mutations. Four of these mutations are novel. We cotransfected the two mutant CD18 proteins with normal CD11a, b, or c in COS cells. This resulted in absence of all three beta(2) integrins on the surface of cells transfected with CD18(252Arg). However, CD18(593Cys) supported some LFA-1 and p150,95 formation in COS cells. The other three patients were compound heterozygotes in which only one allele had previously been characterized, because the other alleles were undetectable at the cDNA level. We identified the unknown mutations as a novel two-nucleotide deletion, a nonsense mutation, and a single nucleotide deletion. CONCLUSION Our method allows identification of mutations in CD18 from genomic DNA. This opens the possibility of early prenatal diagnosis of LAD and reliable carrier detection.

The Arg633His substitution responsible for the private platelet antigen Groa unravelled by SSCP analysis and direct sequencing

We have previously described the private or family platelet antigen, Groa, which was identified in a case of neonatal alloimmune thrombocytopenia. The Groa antigen was found to be located on the GP IIIa (β3) subunit of the GP IIb/IIIa complex, the most prominent fibrinogen receptor of platelets. Initial experiments to characterize the Groa antigen at the molecular genetic level were unsuccessful. We therefore decided to use a different strategy to unravel the molecular basis of this antigen. Platelet GP IIIa mRNA of a Gro(a+) and a Gro(a−) donor was amplified with suitable primers in a reverse transcriptase–polymerase chain reaction (RT‐PCR) and subjected to single‐strand conformational polymorphism (SSCP) analysis. Three regions of the amplified GP IIIa cDNA derived from the Gro(a+) donor showed a different SSCP pattern when compared to that of the Gro(a−) donor. Direct nucleotide sequence analysis of these three segments revealed that two of them contained silent substitutions, A1163C, A1553G and G1565A. The first and the latter changes were described previously. In the third segment a G1996A mutation was found, predicting an arginine  →  histidine substitution at position 633 of the mature glycoprotein. PCR‐ASRA (allele‐specific restriction enzyme analysis) performed on cDNA as well as on genomic DNA with the restriction enzyme MaeIII showed that the His633 form of GPIIIa is restricted to the Gro(a+) phenotype. The observed mutation is three amino acids upstream of the mutation underlying the HPA‐8/Sr system (Arg636Cys), suggesting this region of GP IIIa to be susceptible for mutations. Moreover, the presence of a silent mutation and two low‐frequency forms of the silent polymorphisms strongly suggests that the G1996A mutation did not occur in a direct ancestral allele.

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 (GPIbα, Ibβ, IX and V) are molecular products of different genes. Several point mutations and deletions affecting the GPIbα 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 GPIbβ. 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.

A New Private Platelet Antigen, Groa, Localized on Glycoprotein IIIa, Involved in Neonatal Alloimmune Thrombocytopenia

The serum of a Caucasian woman who gave birth to a child with neonatal alloimmune thrombocytopenia contained antibodies directed against a platelet antigen of the newborn. There was no incompatibility for the known platelet alloantigens HPA‐1 to HPA‐7 or for the private or low‐frequency antigens Sraand Vaa, between the platelets of the parents. However, crossmatching with the serum of the mother and the platelets of the child and the father was strongly positive, suggesting a new platelet antibody specificity. To investigate the inheritance of the ‘Groa’ antigen involved, the available family members were tested in the platelet immunofluorescence test (PIFT) and the monoclonal antibody‐specific immobilization of platelet antigens (MAIPA) assay. The Groaantigen was found to be inherited in an autosomal‐codominant fashion. In the MAIPA, we localized the Groaantigen on the glycoprotein IIb/IIIa complex (αIIbβ3). The GP IIb/IIIa localization was confirmed in immunoprecipitation studies. In Western blotting experiments, we further localized the Groaantigen on the GP IIIa (β3) subunit of the GP IIb/IIIa complex. Until now we have tested approximately 400 unrelated donors. None of these appeared to be positive for the Groaantigen, suggesting a phenotype frequency in the Dutch population of less than 0.01.

Human platelet antigen4 (Br) genotyping by ASPA: allele‐specific primer amplification (PCR‐SSP)

Summary. We have developed a PCR assay named ASPA (allele‐specific primer amplification) to determine the HPA‐5a and −5b genotypes. It consists of two PCR‐reactions. One primer of each priomer set has a 3’‐end nucleotide which is specific for A or G at position 505 of the nHPA‐5b or −5a allele respectively. The HPA‐5 genotypes determined in this way were strictly concordant with the genotypes established by the PCR‐ASRA and with the phenotypes established using MAIPA. The ASPA is a rapid and reliable technique and can be used for the determination of alleles which code for platelet antigen allotypes.

The human platelet alloantigens, HPA‐5(a+ b‐) and HPA‐5(a‐ b+), are associated with a Glu505/Lys505 polymorphism of glycoprotein Ia (the α2 subunit of VLA‐2)

Summary. GP Ia/IIa (also called VLA‐2 or α2β1) is the primary receptor for collagen on platelets. The human platelet alloantigens HPA‐5a(Brb) and HPA‐5b(Bra) have been found to reside on the platelet GP Ia/IIa complex. In order to establish the molecular basis of the HPA‐5 system, platelet RNA was isolated from HPA‐5 (a+ b‐) and HPA‐5(a b+) individuals. After reverse transcription, cDNA coding for glycoprotein Ia (GP Ia) was amplified by the polymerase chain reaction (PCR). Nucleotide sequence analysis of the PCR products revealed an A·G polymorphism at base pair 1648 of the coding region of the mature protein, resulting in a substitution of lysine (AAG) in HPA‐5b(Bra) by glutamic acid (GAG) in HPA‐5a (Brb) at amino acid 505. Subsequent PCR‐ASRA (allele‐specific restriction enzyme analysis) with Mnl I using cDNA derived from three HPA‐5 (a+b‐), one HPA‐5 (a+b+) individuals demonstrated that HPA‐5a and −5b alleles are distinguishable by DNA typing.

Molecular Genetics of Human Platelet Antigens

The purpose of this article is to review the molecular genetics of human platelet antigens, the application of molecular biological techniques to detect mutations underlying polymorphisms and the importance of these techniques for clinical medicine of immune-mediated platelet destruction. Review articles, original papers and preliminary (unpublished) observations from our own laboratory are the main source for this article. The nomenclature and phenotype frequency of the platelet alloantigens in different ethnic groups are described. Recent molecular biological advances are also reviewed. It appears that the human platelet antigen systems are due to single base pair substitutions. These mutations create or are responsible for the loss of a target site for a restriction enzyme in one of the alleles. Thus, DNA typing by polymerase chain reaction and subsequently allele-specific restriction enzyme analysis (PCR-ASRA) can be performed.