Introduction: McCune-Albright syndrome (MAS) is caused by somatic mosaic mutations in the GNAS gene. Detection of the GNAS mutations is usually difficult because of the low frequency mosaicism. Droplet digital polymerase chain reaction (ddPCR) is a new technology that enables to measure absolute quantities of target nucleic acids in a sample by counting nucleic acid molecules encapsulated in discrete water-in-oil droplet partitions.
Objective: To investigate usefulness of ddPCR for detection of low-frequency mosaic GNAS mutations in patients with MAS.
Materials and Methods: We recruited a patient already diagnosed with MAS in this study. Sanger sequencing using DNA extracted from the thyroid grand revealed a somatic p.R201H mutation in GNAS in the patient. We performed ddPCR using DNA obtained from peripheral blood leukocytes. FAM- and HEX-labeled probes were designed for the mutant and wild-type alleles, respectively. Mixture of DNA and reaction reagents were generated into 20,000 droplets by using Droplet Generator, and amplified as manufacturer's protocol. After the amplification, each droplet was measured for fluorescence intensities one by one by Droplet Reader. The droplets including the mutant or wild-type allele indicate strong amplitude of FAM or HEX, respectively. The mutant allele frequency was calculated by comparing the number of FAM-positive droplets from the total number of either FAM- or HEX-positive droplets. Next, the detection sensitivity of ddPCR was examined by diluting patient's DNA with control DNA. In order to further improve the detection sensitivity, we performed a nested PCR method using PNA (peptide nucleic acid) prior to ddPCR.
Results: The ddPCR assay demonstrated that frequency of the p.R201H mutation was 9.4% in peripheral leukocytes DNA in the patient. The detection limit of ddPCR was calculated to 0.2% by the diluting method. The detection limit of ddPCR was decreased to 0.005% after performing the nested PCR assay.
Conclusion: The present study provided evidence that the ddPCR is a powerful tool saving cost and time for detection of low-frequency mosaic mutations.
19 - 21 Sep 2019
European Society for Paediatric Endocrinology