Isabelle Marey1, Rabah Ben Yaou2,3, Nathalie Deburgrave1, Aurélie Vasson1, Juliette Nectoux1,4, France Leturcq1,2, Bruno Eymard3, Pascal Laforet3, Anthony Behin3, Tanya Stojkovic3, Michèle Mayer5, Vincent Tiffreau6, Isabelle Desguerre7, François Constant Boyer8, Aleksandra Nadaj-Pakleza9, Xavier Ferrer10, Karim Wahbi11, Henri-Marc Becane3, Mireille Claustres12,13, Jamel Chelly1,4, Mireille Cossee12,13. 1. Service de Biochimie et Génétique Moléculaire, HUPC Hôpital Cochin, Paris, France. 2. UPMC-Paris 6, UM 76, INSERM, U974, CNRS, UMR 7215, Center of Research in Myology, Institut de Myologie, Paris, France. 3. AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Centre de Référence de Pathologie Neuromusculaire Paris-Est, Paris, France. 4. INSERM, U1016, Institut Cochin, CNRS UMR8104, Université Paris Descartes, Paris, France. 5. AP-HP, Hôpital Armand TROUSSEAU, Centre de référence de pathologie neuromusculaire Paris-Est, Paris, France. 6. Université de Lille 2, EA 4488, Centre de référence des maladies neuromusculaires du CHRU de Lille, Service de médecine physique et réadaptation, Hôpital Swynghedauw, Lille, France. 7. AP-HP, Hôpital Necker-Enfants Malades, Service de Neuropédiatrie, Centre de référence de pathologie neuromusculaires Garches-Necker-Mondor-Hendaye, Paris, France. 8. Service de Médecine Physique et Réadaptation, Centre de référence de pathologie neuromusculaires, Hôpital Sébastopol, CHU de Reims, Reims, France. 9. Service de neurologie, Centre de référence de pathologie neuromusculaires Pays de Loire, Hôpital Larrey, CHU d'Angers, Angers, France. 10. Service de neurologie, Centre de référence de pathologie neuromusculaires Aquitaine, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France. 11. APHP, service de cardiologie, Hôpital Cochin, Paris, France. 12. CHRU Montpellier, Laboratoire de Génétique moléculaire, Montpellier, France. 13. Université de Montpellier, Laboratoire de Génétique de Maladies rares, EA 7402, Montpellier, France.
Abstract
BACKGROUND: Dystrophinopathies are mostly caused by copy number variations, especially deletions, in the dystrophin gene (DMD). Despite the large size of the gene, deletions do not occur randomly but mainly in two hot spots, the main one involving exons 45 to 55. The underlying mechanisms are complex and implicate two main mechanisms: Non-homologous end joining (NHEJ) and micro-homology mediated replication-dependent recombination (MMRDR). OBJECTIVE: Our goals were to assess the distribution of intronic breakpoints (BPs) in the genomic sequence of the main hot spot of deletions within DMD gene and to search for specific sequences at or near to BPs that might promote BP occurrence or be associated with DNA break repair. METHODS: Using comparative genomic hybridization microarray, 57 deletions within the intron 44 to 55 region were mapped. Moreover, 21 junction fragments were sequenced to search for specific sequences. RESULTS: Non-randomly distributed BPs were found in introns 44, 47, 48, 49 and 53 and 50% of BPs clustered within genomic regions of less than 700bp. Repeated elements (REs), known to promote gene rearrangement via several mechanisms, were present in the vicinity of 90% of clustered BPs and less frequently (72%) close to scattered BPs, illustrating the important role of such elements in the occurrence of DMD deletions. Palindromic and TTTAAA sequences, which also promote DNA instability, were identified at fragment junctions in 20% and 5% of cases, respectively. Micro-homologies (76%) and insertions or deletions of small sequences were frequently found at BP junctions. CONCLUSIONS: Our results illustrate, in a large series of patients, the important role of RE and other genomic features in DNA breaks, and the involvement of different mechanisms in DMD gene deletions: Mainly replication error repair mechanisms, but also NHEJ and potentially aberrant firing of replication origins. A combination of these mechanisms may also be possible.
BACKGROUND: Dystrophinopathies are mostly caused by copy number variations, especially deletions, in the dystrophin gene (DMD). Despite the large size of the gene, deletions do not occur randomly but mainly in two hot spots, the main one involving exons 45 to 55. The underlying mechanisms are complex and implicate two main mechanisms: Non-homologous end joining (NHEJ) and micro-homology mediated replication-dependent recombination (MMRDR). OBJECTIVE: Our goals were to assess the distribution of intronic breakpoints (BPs) in the genomic sequence of the main hot spot of deletions within DMD gene and to search for specific sequences at or near to BPs that might promote BP occurrence or be associated with DNA break repair. METHODS: Using comparative genomic hybridization microarray, 57 deletions within the intron 44 to 55 region were mapped. Moreover, 21 junction fragments were sequenced to search for specific sequences. RESULTS: Non-randomly distributed BPs were found in introns 44, 47, 48, 49 and 53 and 50% of BPs clustered within genomic regions of less than 700bp. Repeated elements (REs), known to promote gene rearrangement via several mechanisms, were present in the vicinity of 90% of clustered BPs and less frequently (72%) close to scattered BPs, illustrating the important role of such elements in the occurrence of DMD deletions. Palindromic and TTTAAA sequences, which also promote DNA instability, were identified at fragment junctions in 20% and 5% of cases, respectively. Micro-homologies (76%) and insertions or deletions of small sequences were frequently found at BP junctions. CONCLUSIONS: Our results illustrate, in a large series of patients, the important role of RE and other genomic features in DNA breaks, and the involvement of different mechanisms in DMD gene deletions: Mainly replication error repair mechanisms, but also NHEJ and potentially aberrant firing of replication origins. A combination of these mechanisms may also be possible.
Authors: Mengling Qi; Peter D Stenson; Edward V Ball; John A Tainer; Albino Bacolla; Hildegard Kehrer-Sawatzki; David N Cooper; Huiying Zhao Journal: Hum Mutat Date: 2022-02-01 Impact factor: 4.700