Advantages of ddPCR in detection of PLP1 duplications

Pelizaeus-Merzbacher disease (PMD) is an X-linked, recessively inherited disorder associated with hypomyelination in the brain white matter. Mutations involving the proteolipid protein 1 gene (PLP1) located on Xq22.2 are responsible for PMD. PLP1 duplication is the major genetic abnormality in PMD patients. In this study, we utilized droplet-digital polymerase chain reaction (ddPCR) as a potential method to detect PLP1 duplications. Samples from four PMD patients and one of their mothers were used as positive controls. They had been previously diagnosed as having an additional PLP1 copy by chromosomal microarray testing. Genomic copy number of PLP1 was analyzed in triplicate experiments and compared with reference genes XIST and AR on the X-chromosome, and RPP30 and RPPH1 on the autosomes. As a result, precise results were obtained for each triplicate procedure. Thus, we concluded that triplicate experiments are no longer necessary. Compared to other methods, including fluorescence in-situ hybridization, multiplex ligation-dependent probe amplification, chromosomal microarray testing, and quantitative PCR, we were able to establish ddPCR results rapidly with very small amounts of DNA. In conclusion, we showed that ddPCR can be a potential diagnostic tool to confirm genomic copy number as a routine clinical application, including in prenatal diagnostic settings.