Combining Real-Time COLD- and MAMA-PCR TaqMan Techniques to Detect and Quantify R201 GNAS Mutations in the McCune-Albright Syndrome

Background/Aim: The McCune-Albright syndrome (MAS) is a potentially severe disorder hallmarked by fibrous bone dysplasia, cafe-au-lait skin spots, and endocrine hyperfunction. It is caused by postzygotic activating mutations at the R201 codon of the GNAS gene, leading to a state of somatic mosaicism. Our aim was to improve the mutation detection rate and to quantify the presence of R201 GNAS mutations in different DNA samples from MAS patients. Methods: Realtime COLD-and MAMA-PCR TaqMan techniques were combined to search for R201 mutations in the DNA of blood or affected tissues from 16 previously molecularly characterized MAS patients, from a further 84 subjects with MAS signs who were R201 negative at RFLP analysis, and from 36 controls. The ability of this new method to provide quantitative data was tested in the serial dilution of wild-type, R201H, or R201C cloned plasmid DNA samples; the mutant abundance was measured by spectrophotometry. Results: A linear correlation between the true and the relative mutant abun-dance was observed until 2.5%, indicating a reliable quantification of R201 mutations. The assay's sensitivity was 0.05%, similar to that of previously described molecular methods. Conclusion: The real-time COLD-MAMA-PCR approach is a rapid, efficient, and inexpensive molecular technique for the identification of mutant alleles poorly represented in DNA samples. (C) 2017 S. Karger AG, Basel