ESPE Abstracts

Background: McCune-Albright syndrome (MAS; OMIM#174800) is a rare disorder hallmarked by the triad fibrous osseous dysplasia, cafè-au-lait skin spots and endocrine hyperfunctions, usually peripheral precocious puberty. It is caused by post-zygotic activating mutations at R201 codon of the GNAS gene, which lead to a somatic mosaic state; the clinical manifestations of MAS are highly heterogeneous due to variability of mutation abundance among affected tissues.

Objective and hypotheses: To improve the mutational detection rate and to quantify the presence of R201 GNAS mutation in different DNA samples from MAS patients.

Method: COLD and MAMA-PCR real time taqman techniques have been combined to search for the R201 mutation in the genome DNA from blood or affected tissues of previously molecular characterized MAS patients and controls. The ability of this new method in providing quantitative data was tested in a serial dilution of wildtype, R201H or R201C cloned plasmid DNA samples; the mutant abundance was then measured by spectrophotometry.

Results: A linear correlation between true mutant abundance and relative mutation abundance (proportion of sequence reads containing the mutation) was observed until 2.5%, indicating reliable quantification of both R201H and R201C mutations (0.984 and 0.987, respectively). The assay sensitivity was determined by the lowest standard dilution consistently detectable in replicates at a frequency of 100% and it was found to be 0.05% for both mutations, similar to previously described molecular methods (PNA 1%, NGS 0.03% and for PNA-NGS 0.01%).

Conclusion: Our results indicate that the COLD-MAMA-PCR real time approach is an efficient method for the enrichment of unknown mutant alleles poorly represented in DNA samples and that it can be applied to identify the degree of the R201 change distribution in different tissues from MAS patients for which the clinical course of the disease could correlate with the mutation abundance in each affected tissue.