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.