#57
DEVELOPMENT OF A SNP-BASED METHOD FOR GENOTYPING KILLER IMMUNOGLOBULIN-LIKE RECEPTORS (KIR) USING MALDI-TOF MASS SPECTROMETRY.
Kathleen A. Houtchens, PhD, Hannah E. Boal, BS, Robert J. Nichols, BS and Elizabeth A. Trachtenberg, PhD. Oakland CA, USA, Children
s Hospital and Research Center at Oakland, 94609, HLA Laboratory.
Recent evidence suggests that interactions between an individual’s Killer Immunoglobulin-like Receptor repertoire and Human Leukocyte Antigen (HLA) ligands may play an important role in stem cell transplant outcome (Ruggerri et al., Science 2002; Gagne et al., Human Immunology 2002). If these results are replicated, it may be necessary to assess KIR genotypes as part of the donor selection process. Like the HLA genes, the KIR genes are highly polymorphic. As the characterization of the diversity of these genes is still in its infancy, novel variants are reported frequently. We are developing a high-throughput, single nucleotide polymorphism (SNP)-based KIR genotyping methodology. In this assay, the mass of single and double base pair nucleic acid products generated in a SNP-based primer extension reaction (Sequenom™) is measured by a matrix-assisted laser desorption/ ionization time-of-flight mass spectrometer (MALDI-TOF). The extension product is then identified by base-calling software. The method uses 384 array micro chips, and is highly accurate and rapid. As little as 30 ng DNA is necessary for complete locus-specific profiling, an important criterion in a transplant setting where sample is limited. The method will identify polymorphisms determined to be informative for distinguishing the various loci and is also capable of identifying novel polymorphisms. When fully developed the method will be two-tiered i.e., capable of resolving KIR genotypes with locus-specificity or at an allele-specific level of resolution. Using previously characterized DNA samples, we demonstrate that the method is capable of defining KIR genetic profiles with 30 ng of sample DNA.