Newborn screening of duchenne muscular dystrophy specifically targeting deletions amenable to exon-skipping therapy

Abstract

Duchenne Muscular Dystrophy (DMD) is a lethal progressive muscle-wasting disease. New treatment strategies relying on DMD gene exon-skipping therapy have recently been approved and about 30% of patients could be amenable to exon 51, 53 or 45 skipping. We evaluated the spectrum of deletions reported in DMD registries, and designed a method to screen newborns and identify DMD deletions amenable to exon 51, 53 and 45 skipping. We developed a multiplex qPCR assay identifying hemi(homo)-zygotic deletions of the flanking exons of these therapeutic targets in DMD exons (i.e. exons 44, 46, 50, 52 and 54). We conducted an evaluation of our new method in 51 male patients with a DMD phenotype, 50 female carriers of a DMD deletion and 19 controls. Studies were performed on dried blood spots with patient's consent. We analyzed qPCR amplification curves of controls, carriers, and DMD patients to discern the presence or the absence of the target exons. Analysis of the exons flanking the exon-skipping targets permitted the identification of patients that could benefit from exon-skipping. All samples were correctly genotyped, with either presence or absence of amplification of the target exon. This proof-of-concept study demonstrates that this new assay is a highly sensitive method to identify DMD patients carrying deletions that are rescuable by exon-skipping treatment. The method is easily scalable to population-based screening. This targeted screening approach could address the new management paradigm in DMD, and could help to optimize the beneficial therapeutic effect of DMD therapies by permitting pre-symptomatic care.

Figure 1

(A) Reading frame from exons 41 to 53 of DMD gene (upper frame). Example of the in-frame deletion of exons 45–47, which cannot benefit from exon-45 skipping (central frame). Example of the out-of-frame deletion of exons 46–49, which is amenable to exon-45 skipping (lower frame). (B) Number of patients with a large deletion (≥ 1 exon) covering at least one flanked exon reported in LOVD-DMD database. Deletions involving 35 or more patients are shown. Blue-bars point the deletions rescuable to either single exon-51, exon-53 or exon-45 skipping, red-bars correspond to deletion not rescuable by the aforementioned therapies.

Figure 2

Amplification curves and scattered endpoint fluorescence of control group and carrier group, and DMD patients with a deletion of at least one target exon (deleted group). (A) Amplification curves of a patient without deletion of any target exons. (B) Amplification curves of a patient with a deletion of exons 50 to 54. (C) Normalized Fluorescence Ratio (NFR) of control and carrier group versus deleted group (i.e. DMD patients with a deletion overlapping at least one of the five target exons).