Discussion
We molecularly characterized a novel contiguous 1.18Mbp duplication inDMD including its 5’UTR up to exon 7 in a two-month-old male with mildly elevated CK levels (324 U/L). The duplication was maternally inherited and initially discovered during prenatal carrier screening; the prenatal analysis reported this rearrangement to be a pathogenic exon 1-7 duplication, with the child being at a 50% risk of developing DMD. Follow-up diagnostic analyses of proband in our laboratory included MLPA, CMA, and Sanger sequencing; we showed this duplication to be in direct tandem orientation within Xp21.1, extending further away from exon 1 and including DMD ’s 5’UTR as well as the DP427c alternative promoter/exon1 (Figure 2).
Sanger sequencing showed the duplication to encompass Xp21.1:32,741,022-33,928,069. There are ~570Kb of intervening sequence separating DMD ’s DP427c isoform start (neuronal and retinal isoform)[5] and the duplication end, and ~698Kb separating the duplication end from Dp427m, which is the main isoform produced in muscle and is involved in DMD and BMD (Figure 2)[5]. While we cannot rule out splicing defects in the normal DMD transcript caused by the presence of the duplicatedDMD exons 1-7, the intervening ~570-698Kb of sequence between the duplication and the two main DMD promoters led us to hypothesize this duplication to be likely benign, as no promoter or regulatory regions were separated from the main gene body, and any potentially truncated DMD product would likely undergo nonsense mediated decay (NMD). Our hypothesis was further supported by the observation that, at thirteen months of age, new measurements of CK levels in proband showed a reduction to 211 U/L (original measure was 324 U/L). In addition, proband remains asymptomatic and shows normal development without any noticeable neuromuscular issues or motor delays. Splicing analysis remains to be performed in future experiments, as we are currently unable to complete protein truncation testing (PTT) in proband due to the requirement of a muscle biopsy which is not recommended in an asymptomatic young child [6]. Family studies are being pursued, including testing of males II-8, II-9, III-9, III-11, and IV-4 (Figure 1), to further clarify the significance of the duplication.
At the sequence level, the duplication described herein overlaps the duplication hotspot previously described for DMD, encompassing exons 2-20 [4]. The majority of DMD duplications have different sizes and are non-recurrent events. These findings support non-homologous end-joining as a possible mechanism of generation. Junction analysis of the 1.18Mbp duplication in proband revealed the presence of microhomology, which also suggests a possible origin through microhomology-mediated mechanisms such as fork stalling and template switching (FoSTeS)[7] and microhomology-mediated break-induced replication (MMBIR)[8]. Microhomology tracts have been previously reported in the analysis of complex DMD rearrangements [9], suggesting that several mechanisms can participate in DMD and BMD pathogenesis.
We surmise the exon 1-7 duplication was given a pathogenic classification by the prenatal screening laboratory based on a previous study that had reported an exon 1-7 duplication in an individual with the Duchenne phenotype [10]. Moreover, the mild CK elevation in proband could have prompted such overdiagnosis. Currently most laboratories use a Gaussian distribution of 0-200 IU/L as the normal CK range in white individuals, with affected males having ranges >504 IU/L; however, it has been shown that CK levels in healthy populations can be skewed toward higher values, which can lead to overdiagnosis of mild abnormal values [11]. Said issue poses a dilemma to the correct diagnosis of asymptomatic infants with variants of uncertain significance (VUS) in DMD and mild CK elevations. In such instances the comprehensive characterization of the identified VUSs, particularly duplications, is paramount to the correct prediction and diagnosis of dystrophinopathies.