INTRODUCTION
Dystrophinopathies are X-linked recessive diseases caused by pathogenic
variants in DMD gene (OMIM ID: 300377). Under this term are
included different clinical features, covering a spectrum of muscle
disease ranging from mild to severe that includes Duchenne Muscular
Dystrophy (DMD), Becker Muscular Dystrophy (BMD) and DMD-associated
Dilated Cardiomyopathy (DCM)
(Brandsema & Darras,
2015; Darras et al., 2000). DMD is the most prevalent pediatric form of
muscular dystrophy, with an incidence of 1:3500-5000 male births
(Mendell et al., 2012). The
disease has a de novo mutation rate of ∼33%, that is why there
exist many families without previous history of the disease
(Haldane, 2004). DMD is
mainly generated by a complete absence of the dystrophin protein, which
produces early muscle degeneration, leading to increase serum levels of
Creatine Kinase (CK) (Emery,
1977).
Despite Dystrophinopathies are X-linked recessive diseases with a
complete penetrance in males, in heterozygous females carriers the
penetrance varies and may depend on X-chromosome inactivation patterns
(XIP) (Darras et al.,
2000). Pegoraro et al, have showed that more than 90% of
heterozygous females with skewed XIP develop signs of muscular dystrophy
(Pegoraro et al., 1995).
This correlation was also observed by Giliberto et al and
Viggiano et al(Giliberto et
al., 2014; Viggiano et al., 2016, 2017).
DMD pathogenic variants comprise mainly Structural Variants
(SVs), such as deletions (∼68%) or duplications (∼11%) of one or more
exons, and small mutations (∼20%)
(Aartsma-Rus et al., 2016).
Precise deleterious variant identification and accurate molecular
diagnosis are crucial for Dystrophinopathy patients not only to access
to the specific and optimal standard of care and to achieve family
planning, but also provides information on eligibility for
mutation-specific treatments.
SVs are defined by the novel sequence generated at the breakpoints
junctions. Different mutational mechanisms have been hypothesized to
explain their origin. Among them can be highlighted: Double Strand
Breaks (DSB) repair pathways independent of DNA synthesis, such as
Non-Homologous End Joining (NHEJ) or Microhomology-Mediated End Joining
(MMEJ); exchanges between highly homologous sequences which can take
place in meiosis or in DSB repair by Non-Allelic Homologous
Recombination (NAHR); replication dependent DSB repair pathways such as
Break-Induced Replication (BIR) or Fork Stalling and Template Switching
(FoSTeS); and, retroposition of mobile elements
(Hastings
et al., 2009; Lee et al., 2007; McEachern & Haber, 2006; McVey & Lee,
2008; Moore & Haber, 1996; Quinlan & Hall, 2012; Szostak et al.,
1983).
The analysis of the surrounding breakpoints sequences, i.e. screening of
DNA instability and DSB stimulators motifs, provide hints about which
molecular mechanisms may be involved in the origin of SVs. Several and
heterogenous motifs have been described in literature, among them:
repetitive elements such as Alu, Long Terminal Repeats (LTRs) and Long
Interspersed Nuclear Elements (LINEs), which stimulate DSB to initiate
their transposition into elsewhere on the genome
(Brouha et
al., 2003; Deininger & Batzer, 1999; Kazazian, 2004; Rüdiger et al.,
1995); Non-B DNA, such as tetraplex DNA, cruciform DNA, bent DNA,
Z-DNA, and all sort of secondary structures
(Bacolla & Wells,
2004; Wang & Vasquez, 2014); Short Tandem Repeats (STRs), which were
found enriched at DSBs and in DMD intron breakpoint hotspots
(Luce et al., 2016;
Zavodna et al., 2018); and finally, Alu/LINE specific
retro-transposition target sequences
(Been et
al., 1984; Jurka, 1997; Spitzner & Muller, 1988; Weaver & DePamphilis,
1982).
Here, we present a familial case of DMD with a symptomatic pregnant
woman and a thorough analysis of the complex SVs (cxSVs) found. Were our
aims to carry out precise prenatal diagnosis and to hypothesize the
molecular mechanisms underlying the origin of the SV, implementing a
multi-technique molecular algorithm.