Noninvasive Prenatal Diagnosis of Duchenne Muscular Dystrophy: Comprehensive Genetic Diagnosis in Carrier, Proband, and Fetus.

BACKGROUND: Noninvasive prenatal diagnosis of monogenic disorders using maternal plasma and targeted massively parallel sequencing is being investigated actively. We demonstrated previously that comprehensive genetic diagnosis of a Duchenne muscular dystrophy (DMD) patient is feasible using a single targeted sequencing platform. In the present study, we demonstrate the applicability of this approach to carrier detection and noninvasive prenatal diagnosis. METHODS: Custom solution-based target enrichment was designed to cover the entire DMD region. Targeted massively parallel sequencing was performed using genomic DNA from four mother and proband pairs to test whether carrier status could be detected reliably. Maternal plasma DNA at varying gestational weeks from the same families was collected from two pregnant carrier mothers who also participated in the preceding carrier testing, and the DNA wasand sequenced using the same targeted-platform to predict the inheritance of the DMD mutation by their fetus. Overrepresentation of an inherited allele was determined by comparing the average read fractionallele fraction of two phased haplotypes after examining and correcting for the recombination event. RESULTS: The carrier status of deletion/duplication mutations was detected reliably through coverage plotting and breakpoint analysis. De novo nonsense mutations were identified correctly in one family. Whether the fetus had inherited the DMD mutation was predicted correctly in all fourtwo families as early as 6 weeks of gestation. In one of these, detection of the recombination event and reconstruction of the phased haplotype produced a correct diagnosis. CONCLUSIONS: Noninvasive prenatal diagnosis of DMD is feasible using a single targeted massively parallel sequencing platform with tiling design.