ABSTRACT
We here describe the identification of the new allele HLA-B*4431, which was found in three members of a Turkish family. Sequencing of the new allele following haplotype-specific PCR amplification revealed that exon 2 is identical to HLA-B*4402, whereas exon 3 resembles a HLA-B*40 variant with the exception of position 572, where a single nucleotide transversion (C > G) leads to an amino acid exchange (Trp162Ser). The generation of the 3' part of B*4431 may be best explained by a separate recombination between B*40 and B*07. Although B*4431 consists of B44 in its alpha1 domain and of B60(40) in its alpha2 domain; the new allele only displayed B44 seroreactivity, which demonstrates that epitopes crucial for B60(40) specificity must be located in the alpha1 domain.
Subject(s)
HLA-B Antigens/genetics , HLA-B7 Antigen/genetics , Alleles , Amino Acid Sequence , Bone Marrow Transplantation , Cytotoxicity Tests, Immunologic , Female , Genetic Markers/genetics , Genetic Predisposition to Disease/genetics , HLA-B40 Antigen , HLA-B44 Antigen , Haplotypes , Histocompatibility Testing , Humans , Molecular Sequence Data , Myelodysplastic Syndromes/diagnosis , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/therapy , Polymorphism, Single Nucleotide/geneticsABSTRACT
The identification of the new allele HLA-A*6813, which was found in a woman of Syrian origin and her son, is described. In the sequence analysis the new allele differs from A*68011 by positions 259 (A>G) and 261 (C>G) in exon 2. As the structure is thus identical to the HLA-A consensus sequence it is likely that the new allele originated by gene conversion. At the protein level, the new allele has one amino acid difference from A*6801 (Asn63Glu), which results in a distinct banding pattern in one dimensional-isoelectric focusing. Amino acid residue 63 contributes to the formation of pocket A and B and is thus important for peptide binding. A*6813 was serologically detectable only by two of six polyclonal, but by three monoclonal antisera. The restricted serological A68 activity may be explained by altered peptide binding as presented peptides can affect the serological recognition of major histocompatibility complex (MHC) class I molecules. Moreover, our findings suggest that a possible mismatch with the other known A*68 variants may impair clinical outcome of bone marrow transplantation.
Subject(s)
Alleles , Bone Marrow Transplantation , HLA-A Antigens/genetics , Base Sequence , DNA, Complementary , Female , Humans , Male , Molecular Sequence Data , SyriaABSTRACT
Variable amounts of non-specific amplification may occur in HLA PCR-SSP typing, and this can be significantly reduced by the use of AmpliTaq Gold. In an effort to achieve an optimal balance between specificity and efficiency of the PCR amplification for both HLA alleles and the internal control, we designed a system of timed-release activity by combining two different Taq DNA polymerases. The reaction was started at a relatively low level of enzyme activity and as thermal cycling progressed more and more Ampli-Taq Gold was slowly activated for the reaction to continue. We applied this system to all routine HLA PCR-SSP typing. The number of repeat typings due to non-specific amplification and/or amplification failure of the internal control was remarkably reduced.
Subject(s)
Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class I/genetics , Histocompatibility Testing , Polymerase Chain Reaction/methods , Taq Polymerase/metabolism , DNA Primers , Histocompatibility Antigens Class I/classification , Histocompatibility Antigens Class II/classification , Reagent Kits, Diagnostic , Time FactorsABSTRACT
OBJECTIVES: Human platelet alloantigen (HPA) typing has potential clinical relevance in a variety of contexts. We can improve methods for HPA genotyping by complementing our knowledge of the DNA sequence polymorphisms of HPA genes and experience with various DNA-based HPA typing techniques. METHODS: A newly available DNA polymerase, AmpliTaq Gold (Perkin Elmer), provided in an inactive state and activated by heat, makes it possible to perform a hot start polymerase chain reaction (PCR) in order to prevent nonspecific amplification during the setup of PCR. To establish a practical procedure for HPA-1, 2, 3 and 5 genotyping, we applied the AmpliTaq Gold for a hot start PCR and employed 8 pairs of published sequence-specific primers (SSP). A simple simultaneous genotyping of these 4 HPA systems could be rapidly achieved with high specificity. RESULTS: The HPA gene frequencies observed in 126 randomly selected German blood donors were 0.82 and 0.18 for HPA-1a and 1b, 0.92 and 0.08 for HPA-2a and 2b, 0.63 and 0.37 for HPA-3a and 3b and 0.90 and 0.10 for HPA-5a and Sb, respectively. CONCLUSION: Using our hot start PCR-SSP procedure with AmpliTaq Gold a simple, rapid and reproducible genotyping for HPA-1, 2, 3 and S systems could be achieved.
Subject(s)
Antigens, Human Platelet/genetics , Polymerase Chain Reaction/methods , Alleles , DNA-Directed DNA Polymerase , Gene Frequency , Genotype , Germany , Hot Temperature , HumansABSTRACT
DRB1*08 haplotypes have not been known to carry a DRB3 gene. We have found a patient suffering from liver disease who has a novel HLA haplotype of DRB1*0801 with DRB3*0202 as established by family segregation. These two genes were confirmed by sequencing. DR8 and DR52 antigen specificities were serologically detected, indicating expression of these genes.
