ABSTRACT
BACKGROUND: Twenty-four low-frequency human platelet antigens (LFHPAs) have been implicated as immunogens in neonatal alloimmune thrombocytopenia (NAIT). We performed studies to define more fully how often these antigens trigger maternal immunization leading to NAIT. STUDY DESIGN AND METHODS: In a Phase 1 study, fathers of selected NAIT cases not resolved by serologic testing but thought to have a high likelihood of NAIT on clinical and serologic grounds were typed for LFHPAs by DNA sequencing. In a Phase 2 study, high-throughput methods were used to type fathers of 1067 consecutive unresolved NAIT cases for LFHPAs. Mothers of 1338 unresolved cases were also typed to assess the prevalence of LFHPAs in a population racially/ethnically similar to the fathers. RESULTS: In Phase 1, LFHPAs were identified in 16 of 244 fathers (6.55%). In Phase 2, LFPAs were found in only 28 of 1067 fathers (2.62%). LFHPAs were identified in 27 of 1338 maternal samples (2.01%). HPA-9bw was by far the most common LFHPA identified in the populations studied and was the only LFHPA that was significantly more common in fathers than in mothers of affected infants (p = 0.02). CONCLUSIONS: Maternal immunization against recognized LFHPAs accounts for only a small fraction of the cases of apparent NAIT not resolved by standard serologic testing. Typing of the fathers of such cases for LFHPAs is likely to be rewarding only when a maternal antibody specific for a paternal platelet glycoprotein is demonstrated and/or there is compelling clinical evidence for NAIT.
Subject(s)
Antigens, Human Platelet/genetics , Thrombocytopenia, Neonatal Alloimmune/etiology , Alleles , Female , High-Throughput Screening Assays , Humans , Male , Polymorphism, Single NucleotideABSTRACT
BACKGROUND: Maternal immunization against low-frequency, platelet (PLT)-specific antigens is being recognized with increasing frequency as a cause of neonatal alloimmune thrombocytopenia (NAIT). STUDY DESIGN AND METHODS: Serologic and molecular studies were performed on PLTs and DNA from two families in which an infant was born with severe thrombocytopenia not attributable to maternal immunization against known PLT-specific alloantigens. RESULTS: Antibodies reactive only with paternal PLTs were identified in each mother using flow cytometry and solid-phase assays. Unique mutations encoding amino acid substitutions K164T in glycoprotein (GP)IIb (Case 1) and R622W in GPIIIa (Case 2) were identified in paternal DNA and in DNA from the affected infants. Each maternal antibody recognized recombinant GPIIb/IIIa mutated to contain the polymorphisms identified in the corresponding father. None of 100 unselected normal subjects possessed these paternal mutations. CONCLUSIONS: Severe NAIT observed in the affected infants was caused by maternal immunization against previously unrecognized, low-frequency antigens created by amino acid substitutions in GPIIb/IIIa (α(IIb) /ß(3) integrin). A search should be conducted for novel paternal antigens in cases of apparent NAIT not explained on the basis of maternal-fetal incompatibility for known human PLT antigens.
Subject(s)
Antigens, Human Platelet/immunology , Platelet Membrane Glycoproteins/genetics , Polymorphism, Genetic , Thrombocytopenia, Neonatal Alloimmune/etiology , Female , Humans , Infant, Newborn , Male , Mutation , PregnancyABSTRACT
BACKGROUND: Recent reports suggest that maternal immunization against low-frequency, platelet (PLT)-specific glycoprotein (GP) polymorphisms is a more common cause of neonatal alloimmune thrombocytopenia (NATP) than previously thought. STUDY DESIGN AND METHODS: Serologic and molecular studies were performed on PLTs and DNA from three families in which an infant was born with apparent NATP not attributable to maternal immunization against known PLT-specific alloantigens. RESULTS: Antibodies reactive only with paternal PLTs were identified in each mother. In Cases 2 (Kno) and 3 (Nos), but not Case 1 (Sta), antibody recognized paternal GPIIb/IIIa in solid-phase assays. Unique mutations encoding amino acid substitutions in GPIIb (Case 2) or GPIIIa (Cases 1 and 3) were identified in paternal DNA and in DNA from two of the affected infants. Antibody from all three cases recognized recombinant GPIIIa (Case 1 [Sta] and Case 3 [Nos]) and GPIIb (Case 2, Kno) mutated to contain the polymorphisms identified in the respective fathers. None of 100 unselected normal subjects possessed the paternal mutations. Enzyme-linked immunosorbent assay and flow cytometric studies suggested that failure of maternal serum from Case 1 (Sta) to react with paternal GPIIIa in solid-phase assays resulted from use of a monoclonal antibody AP2, for antigen immobilization that competed with the maternal antibody for binding to the Sta epitope. CONCLUSION: NATP in the three cases was caused by maternal immunization against previously unreported, low-frequency GP polymorphisms. Maternal immunization against low-frequency PLT-specific alloantigens should be considered in cases of apparent NATP not resolved by conventional serologic and molecular testing.
Subject(s)
Antigens, Human Platelet/genetics , Integrin alpha2/genetics , Integrin beta3/genetics , Polymorphism, Genetic , Thrombocytopenia, Neonatal Alloimmune/genetics , Adult , Animals , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Antigens, Human Platelet/immunology , CHO Cells , Cricetinae , Cricetulus , Epitopes/genetics , Epitopes/immunology , Female , Gene Frequency , Humans , Immunoglobulin G/immunology , Infant, Newborn , Integrin alpha2/immunology , Integrin beta3/immunology , Isoantibodies/immunology , Male , Maternal-Fetal Exchange , Models, Molecular , Pregnancy , Recombinant Fusion Proteins/immunology , Thrombocytopenia, Neonatal Alloimmune/immunologyABSTRACT
Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbß3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Sec(a)) residing on αIIbß3. The location of Sec(a) on αIIbß3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal ß3 revealed a single nucleotide exchange (G(1818)T) in exon 11 of the ß3 gene (ITGB3), changing Lys(580) (wild-type) to Asn(580) (Sec(a)). Two additional members of the family Sec were typed Sec(a) positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn(580), but not Lys(580) αIIbß3, bound anti-Sec(a), which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn(580) variant compared to wild-type αIIbß3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Sec(a) positive platelets, which, however, are heterozygous for the Lys(580)Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbß3. Analysis with mutant transfected cells showed that the Lys(580)Asn mutation responsible for the formation of the Sec(a) antigenic determinant affects αIIbß3 receptor function.