Abstract
The lack of readily available, patient-derived materials for molecular genetic testing of many heterozygous or rare disorders creates a major impediment for laboratory proficiency and quality control procedures. The paucity of clinically derived mutation-positive samples could be surmounted if it were possible to construct artificial samples containing mutations of interest that would sufficiently resemble natural human samples. Such samples could then function as acceptable and realistic performance evaluation challenges and quality control reagents for recipient laboratories. Using the cystic fibrosis gene (CFTR) as a prototype, we have devised and executed experiments designed to generate unique DNA samples that could be used for these purposes. We used site-directed mutagenesis to generate mutations of interest in plasmid DNA derived from common bacterial artificial chromosome sources containing the cystic fibrosis transmembrane conductance receptor gene. CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population-screening panel of 25 mutations. DNA samples containing predetermined concentrations and ratios of wild-type and mutated plasmids, bacterial artificial chromosomes of interest, and nonhuman genomic carrier DNA were characterized and tested in-house and in a group of nine pilot testing laboratories using a variety of technical platforms. The results indicate that these constructs, containing CFTR mutations in heterozygous and homozygous states, can serve as valid and accessible materials for quality assurance, including performance evaluation, proficiency testing, and assay quality control.