Résumé :
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Facio-scapulo-humeral dystrophy (FSHD), including muscle and non-muscle features, is usually associated with the contraction of a repeat array (D4Z4) in the subtelomeric region of chromosome 4q (4q35). Because of a non-pathogenic variant of 4q and of the presence of a homologous array on 10q26, it is critical that the copy number of D4Z4 be counted within its chromosomal context. Moreover, translocations between 4q and 10q are frequent, as well as recombination events making challenging the exploration of the FSHD critical region. To solve this diagnostic hurdle we developed a Molecular Combing test for FSHD and transferred the technology in the diagnostics laboratory. This approach allows, in a unique experiment, to discriminate between pathogenic and non pathogenic 4q35 and 10q26 haplotypes in FSHD patients carrying a contracted D4Z4 array. The ongoing clinical validation not only showed a perfect correlation with previous diagnostics with a better accuracy, but also allowed to identify unreported haplotypes and to precisely visualize and define mosasms, deletions and complex rearrangements. However, beside explorations for diagnostic purposes are currently performed and despite important and recent advances in understanding the pathophysiological mechanism underlying FSHD, it is not yet possible to dispose of a "unified" mechanistic view for FSHD. Recent reports have underlined the crucial role of the double homeobox transcription factor DUX4 in FSHD. Meanwhile, the direct and mechanistic process by which DUX4 expression causes FSHD remains unclear. In particular, the identified - mRNA stabilizing - SNP in the 3'UTR region of DUX4 is present in a large cohort of FSHD and unaffected individuals. To further unravel the pathophysiology of FSHD, we considered clinically typical FSHD patients without clear D4Z4 contractions on 4qA haplotype and hypothesized that the disease in these patients could be associated to a genomic defect at the 4q35 locus. We thus performed large scale genomic analyses based on high throughput genomic approaches. A custom design CGH-chip (135K) covering the entire FSHD associated 4q35 locus (12.8 Mb, tiling 10bases to 100bases) including the gene's desert regions, was created and tested on 21 FSHD patients including 10 FSHD1 and 11 FSHD2. This allowed detecting duplications and deletions in coding as well as non-coding regions. Beside, we set up the capture of the full set of coding regions from the telomere up to 4 Mb. Among 84 identified variants absent in the SNPs databases on the same set of Patients, 12 were found in coding genes including 7 predicted pathogenic variants. All together, our results support the hypothesis for FSHD being associated to a complex molecular and pathophysiological mechanism, combining DUX4 pathogenic implication together with another potential FSHD associated master gene.
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