STUDY OF MICA IN 201 AFRICAN AMERICANS BY FLOW CYTOMETRY–BASED SINGLE NUCLEOTIDE EXTENSION: A NEW PLATFORM FOR HIGH

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TITLE: STUDY OF MICA IN 201 AFRICAN AMERICANS BY FLOW CYTOMETRY–BASED SINGLE NUCLEOTIDE EXTENSION: A NEW PLATFORM FOR HIGH–THROUGHPUT DNA TYPING

Y. Zhang,¹ M. Han,¹ C. Giang,¹ R. Vorhaben,¹ B. Lavingia,¹ P. Stastny.¹

¹Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX

We have developed a rapid, high–throughput method for MICA genotyping by using single nucleotide extension (SNE) and flow cytometric analysis of an array of fluorescent microspheres. This robust technique employs a PCR–derived target DNA containing all the polymorphic sites of MICA, a synthetic complementary primer, a fluorescent reporter molecule (streptavidin–phycoerythrin), and a thermophilic DNA polymerase. Genomic DNA was amplified by MICA locus–specific primers. SNE reactions were carried out in thermosequenase buffer in the presence of 33 SNE primers, one biotin–labeled ddNTP, three non–labeled ddNTPs and DNA template. An array of fluorescent microspheres was hybridized to the SNE reaction products and sequestered them for flow cytometric analysis. The SNE reaction was used to assay polymorphisms in exon 2, 3, and 4 of the MICA gene in a 96–well format. 20 HLA homozygous typing cell lines representing MICA*001–*019 were used as reference material. The gene frequencies among 201 African American unrelated donors were determined by the SNE assay. Of 51 previously known alleles, 18 were observed in African Americans, compared to 16 that were found in North American Caucasians and 9 in South American Indians, suggesting a more diversified allelic distribution in African Americans. MICA*00201 and MICA*00801 were the two most frequent alleles in African Americans, with gene frequencies of 27.9% and 26.9%, respectively, followed by MICA*004 (18.7%), *00802 (5.5%), *00901 (4.2%). The gene frequencies of the remaining alleles, MICA*001, *007, *0902, *010, *011, *012, *015, *017, *018, *019, *027, *030, *041, were lower than 3%. The concordance between SNE typing and SSOP analysis was 99%. The reproducibility was 100% in 50 samples typed on two separate occasions. No false–positive or false–negative typing results were obtained in the homozygous cell lines or in the heterozygous individuals investigated. The methodology described here has also been applied to type other HLA region genes. It offers a powerful new approach to DNA typing of the MICA alleles.