Denaturing high-performance liquid chromatography is a suitable method for PMM2 mutation screening in carbohydrate-deficient glycoprotein syndrome type IA patients

The phosphomannomutase 2 gene (PMM2; MIM 601785) has been identified as the carbohydrate-deficient glycoprotein syndrome type 1A gene (CDGS type 1A; MIM 212065), The gene spans 8 exons and 741 bp of coding DNA, Previously, we have identified 20 different mutations in the PMM2 gene using mutation screening with single-stranded conformation polymorphism (SSCP) and sequencing of DNA from 61 CDGS type 1A patients. Because eight of these could not be detected by SSCP, we were not satisfied with the sensitivity of the mutation detection technique used. Thus, we wanted to investigate if denaturing high-performance liquid chromatography (DHPLC) was a more suitable mutation screening method for PMM2. DHPLC was set up for PMM2 by optimizing eight different PCR fragments, one for each exon, The mutation detection was optimized empirically with PCR fragments from controls. First, control samples were run at a universal gradient and after modification and shortening of the gradient, also run at 10 different temperatures, 50-70 degreesC with 2-degree intervals, to enable setting of the temperature with the highest resolution. Then, PCR products with known mutations from the previous study were analyzed, and the results were compared to the control chromatograms for aberrations. We detected 19/20 mutations with DHPLC, and several mutations not detected by earlier screening techniques were readily detected by DHPLC. We conclude that DHPLC is a suitable detection technique for a rapid and reliable first scan of CDGS type 1A patients.