Rapid Detection of Rare Mutations in Multiple Genetic Disorders Using DHPLC. Z. Zhou, R.G. Pace, K.J. Friedman, M. Zariwala, P.G. Noone, M.R. Knowles, L.M. Silverman. Dept Pathology & Lab Medicine and Dept Medicine, Univ North Carolina, Chapel Hill, NC.
   Denaturing HPLC (DHPLC) is a highly sensitive method for detecting sequence variants in heterozygotes. Previous studies have shown detection rates ranging from 90% to 100%. We tested Varian's ProStar Helix DHPLC system for sensitivity and specificity. Each of the 39 known variants (37 point mutations and 2 small deletions) evaluated possessed a distinct absorption pattern compared with negative controls and other variants nearby. The sensitivity was 100% when more than one melting temperature was used for some samples. There were no false positives. To facilitate clinical diagnoses and carrier identification, we applied DHPLC to screening for rare CFTR mutations in patients with cystic fibrosis (CF). To date, there are 915 reported mutations scattered throughout the gene (27 exons). Patients with only one or no mutation are common. Full gene sequencing is both time-consuming and costly. Screening of samples with DHPLC followed by sequencing of the DNA fragments with altered DHPLC patterns is an effective and economical way to identify new mutations. With this strategy, we identified numerous rare mutations rapidly at low cost in CF patients with only one mutation identified by routine clinical tests of 6-70 mutations. We also used DHPLC to screen carriers for known mutations or variants in other genes, e.g., 3 variants (Z, S, and M3) in the a1-antitrypsin gene, mitochondrial mutations associated with MELAS and MERRF, and mutations in the medium chain acyl-CoA dehydrogenase (ACADM) gene. Each variant produced a distinct and highly reproducible DHPLC pattern. More recently, we have discovered 2 novel mutations (W468S and W468X) in the dynein IC78 gene in patients with primary ciliary dyskinesia (PCD). After screening 125 normal controls for these mutations, we identified one W468X carrier by DHPLC with confirmation by sequencing. We conclude that rapid sample processing, low cost, and automated operation make DHPLC an ideal tool of detecting heterozygosity for rare mutations in patients and for screening carriers for known mutations, especially in a large patient volume.