Classification of mutations at the HLA-A locus by use of the polymerase chain reaction.
We investigated whether the polymerase chain reaction (PCR) could be used to determine the mechanism of mutation in lymphocyte clones mutated at the HLA-A locus. Three polymorphisms, at Factor XIIIA, D6S109, and intron 3 of the HLA-A gene, were used to study a series of clones previously characterised by Southern blotting (SB) at multiple loci on chromosome 6. For detection of loss of heterozygosity, the results of PCR and SB were concordant in 140 of 141 clones when polymorphism in the Factor XIIIA region was studied and in 144 of 145 clones when polymorphism in the HLA-A gene was studied. For classification of the mechanism of mutation, PCR and SB gave the same result in 88 of 92 clones (96%) when a combination of the HLA-A and Factor XIIIA polymorphisms was used and in 46 of 47 clones (98%) when a combination of the HLA-A and D6S109 polymorphisms was used. The results indicate that PCR provides a simple and reliable method for categorising mutations at the HLA-A locus as arising from mitotic recombination, deletion, or from presumptive minor changes within the gene. Rare events such as gene conversion, nondisjunction, or large deletions extending to the telomere will be misclassified. However, such events are rare for mutations at this locus.
Joseph
G
G
Department of Haematology, Flinders University and Medical Centre, Bedford Park, Australia.
Grist
S
S
Firgaira
F
F
Turner
D
D
Morley
A
A
D001483:Base Sequence; D015139:Blotting, Southern; D002896:Chromosomes, Human, Pair 6; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D005785:Gene Conversion; D017353:Gene Deletion; D005805:Genes, MHC Class I; D015234:HLA-A Antigens / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D008214:Lymphocytes / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D011503:Transglutaminases / Q000235:genetics
D017931:DNA Primers; D015234:HLA-A Antigens; D011503:Transglutaminases