EP.62A series of unfortunate events: familial case of DMD, two different mutational events and skewed X chromosome inactivation in a pregnant woman

Abstract

Duchenne muscular dystrophy (DMD) is a neuromuscular X-linked recessive disease caused by mutations in DMD gene. Here, we present a family with a DMD symptomatic pregnant woman and two affected boys. One of them had a previous multiplex PCR study showing a 45-54 exon deletion. Interestingly, during the prenatal diagnosis another mutation was discovered in the pregnant woman and her fetus, a 38-43 exon duplication. On the basis of this finding, we reanalyzed the initially studied boy, discovering both mutations in his gDNA. In order to unravel this riddle, we performed a complete molecular analysis in family members, applying the following techniques: MLPA, STRs segregation, Humara Assay, CGH Array, Sanger sequencing and WGS. As expected, the Humara assay revealed that the symptomatic female has skewed X-chromosome Inactivation (XCI), while an asymptomatic carrier showed a random XCI. Given the inheritance pattern of the rearrangements, only the affected child carried the del/dup, we could deduce that the deletion was the second mutational event. Furthermore, STRs segregation allowed the detection of a recombinant event in the affected boy, which could be related to the generation of the deletion. We were able to characterize the deletion breakpoints NC_000023.10:g.31664475_32111223del, suggesting the involvement of the non-homologous end joining mechanism. On the other hand, we limited the borders of the duplication up to NC_000023.10: g.(32245444_32247193)_(32380996_32385390)dup by CGH array. However, a thorough characterization of the duplication is currently being done. The profound analysis of complex structural variants, such as the presented one, would allow to detect predisposing mutagenic sequences and widen the understanding on the molecular events that takes place in DMD gene. Finally, this study highlights the importance of retesting patients with identified deletions by PCR, in order to reduce the probability of missing out other rearrangements which could affect the effectiveness of mutation-dependent therapies.