Whole genome amplification increases the efficiency and validity of buccalcell genotyping in pediatric populations

The collection of buccal cells provides a noninvasive method for obtaining DNA fbr genetic studies. Here we report the results on buccal cell genotypi ng from our ongoing study of childhood leukemia in Northern California. We have collected buccal samples from children ranging in age front 4 months t o 15 years using an interviewer- or nurse-administered protocol using a cyt ology brush. Initial results of the genotyping, including the glutathione S -transferase mu, glutathione S-transferase theta, NAD(P)H:quinone oxidoredu ctase, and methylenetetrahydrofolate reductase polymorphisms, were disappoi nting because many specimens contained little DNA, failed repeated attempts at PCR amplification, and produced unreliable results. Here we evaluate a solution to the problem that involves whole genome amplification using the improved primer extension preamplification methodology, Sixty cases of pedi atric acute leukemia were studied; five PCR-based genotypes were attempted using buccal cell DNA and whole genome amplified (WGA) buccal DNA. Results were compared with genotyping results using: DNA isolated from peripheral w hole blood or bone marrow for each child. The standard buccal protocol fail ed to yield successful PCR reactions in 30-57% of specimens, whereas WGA-bu ccal was markedly more efficient (25% failed PCR), A success rate of 100% w as achieved with one repeat test of the failed WGA-PCR reactions, Misclassi fication of genotype was common for the glutathione S-transferase theta mar ker using the standard buccal procedure. The WGA-buccal protocol, however, produced genotyping results fully concordant with the referent blood or bon e marrow DNA results for all five loci. DNA yields were increased by WGA to allow for similar to 900 PCR reactions/brush. WGA is very useful for impro ving the efficiency and validity of PCR-based genotyping in pediatric popul ations.