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.