MICA allele-level typing by sequence-based typing with computerized assignment of polymorphic sites and short tandem repeats within the transmembrane region.

Abstract

MICA genes are located close to, but are structurally distinct from, HLA class I genes and have been found to be associated with some diseases and with immune responses to transplants. We have developed a MICA typing method based on polymerase chain reaction (PCR)/sequence-based typing and a computer program that determines the polymorphisms and distinguishes the GCT repeats in exon 5. One PCR amplification was performed to obtain templates of 2.2 kb including exons 2, 3, 4, and 5 of MICA to be sequenced with two forward and two reverse primers. Overlay of nucleotide sequencing signals resulting from presence of different GCT repeats in exon 5 antisense from two different MICA alleles can be identified by a computer-based analysis of the combined signal string containing the 35 bases. Eighteen reference samples from the 10th International Histocompatibility Workshop with known MICA alleles, as more recently determined, were tested and the concordance was 100% with the previous typing. In addition, 46 samples from kidney or heart transplant recipients and donors were analyzed for their MICA typing by this approach. Results demonstrated that the majority of samples were MICA heterozygous. The most common allele was MICA*00801/A5.1 (44.7%), which was consistent with previous reports. Three samples manifested ambiguous results, either because of polymorphism in exon 6 which was not tested or because the combination of two alleles gives the same pattern as the other two. The procedure was relatively simple and fast and is presently our method of choice for high-resolution clinical MICA typing.