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Deciphering the Complexity of the 4Q and 10Q Subtelomeres by Molecular Deciphering the complexity of the 4q and 10q subtelomeres by molecular combing in healthy individuals and patients with facioscapulohumeral dystrophy Karine Nguyen, Natacha Broucqsault, Charlene Chaix, Stéphane Roche, Jérôme Robin, Catherine Vovan, Laurene Gerard, André Mégarbané, Jon Andoni Urtizberea, Rémi Bellance, et al. To cite this version: Karine Nguyen, Natacha Broucqsault, Charlene Chaix, Stéphane Roche, Jérôme Robin, et al.. Deci- phering the complexity of the 4q and 10q subtelomeres by molecular combing in healthy individuals and patients with facioscapulohumeral dystrophy. Journal of Medical Genetics, BMJ Publishing Group, In press, 10.1136/jmedgenet-2018-105949. hal-02140159 HAL Id: hal-02140159 https://hal.archives-ouvertes.fr/hal-02140159 Submitted on 5 Jun 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Diagnostics ORIGINAL ARTICLE J Med Genet: first published as 10.1136/jmedgenet-2018-105949 on 22 April 2019. Downloaded from Deciphering the complexity of the 4q and 10q subtelomeres by molecular combing in healthy individuals and patients with facioscapulohumeral dystrophy Karine Nguyen,1,2 Natacha Broucqsault,2 Charlene Chaix,1 Stephane Roche,2 Jérôme D Robin,2 Catherine Vovan,1 Laurene Gerard,1 André Mégarbané,3 Jon Andoni Urtizberea,4 Remi Bellance,5 Christine Barnérias,6,7 Albert David,8 Bruno Eymard,9 Melanie Fradin,10 Véronique Manel,11 Sabrina Sacconi,12,13 Vincent Tiffreau,14 Fabien Zagnoli,15 Jean-Marie Cuisset,16 Emmanuelle Salort-Campana,2,17 Shahram Attarian,2,17 Rafaëlle Bernard,1,2 Nicolas Lévy,1,2 Frederique Magdinier 2 ► Additional material is ABSTRact at subtelomeres is higher than in the rest of the published online only. To view Background Subtelomeres are variable regions genome and subtelomeric variation contributes to please visit the journal online (http:// dx. doi. org/ 10. 1136/ between telomeres and chromosomal-specific regions. genome variability and to a wide range of diseases jmedgenet- 2018- 105949). One of the most studied pathologies linked to from acquired and common ones to rare genet- subtelomeric imbalance is facioscapulohumeral dystrophy ically inherited syndromes.1–3 In addition, these For numbered affiliations see (FSHD). In most cases, this disease involves shortening telomere-adjacent DNA sequences are crucial for end of article. of an array of D4Z4 macrosatellite elements at the 4q35 telomere regulation and integrity.4 Subtelomeres locus. The disease also segregates with a specificA -type contain both coding and non-coding transcripts and Correspondence to Dr Frederique Magdinier, haplotype containing a degenerated polyadenylation promoters for the non-coding telomeric repeat-con- Marseille Medical Genetics signal distal to the last repeat followed by a repetitive taining RNA transcribed from the subtelomere U1251, Aix-Marseille Universite array of β-satellite elements. This classification applies into the (TTAGGG)n telomeric tract.5 Composi- Faculte de Medecine, Marseille, to most patients with FSHD. A subset of patients called tion of subtelomeric regions in terms of sequence France; FSHD2 escapes this definition and carries a mutation in frederique. magdinier@ univ- (haplotype) and DNA copy number contribute to amu. fr the SMCHD1 gene. We also recently described patients the higher-order chromatin organisation of these carrying a complex rearrangement consisting of a chromosomal regions, abutting telomeres, and KN and NB contributed equally. cis-duplication of the distal 4q35 locus identified by regulation of genes in close proximity by telomere http://jmg.bmj.com/ molecular combing. 6–9 Received 17 December 2018 position effect or at a long distance, in a discon- Revised 28 February 2019 Methods Using this high-resolution technology, we tinuous manner through formation of long-distance Accepted 24 March 2019 further investigated the organisation of the 4q35 region loops.10–13 linked to the disease and the 10q26 locus presenting Facioscapulohumeral dystrophy (FSHD) is one with 98% of homology in controls and patients. of the most puzzling genetic diseases linked to Results Our analyses reveal a broad variability in subtelomeric imbalance. In the majority of patients on 5 June 2019 by guest. Protected copyright. size of the different elements composing these loci (FSHD1, 95%), this autosomal dominant neuro- highlighting the complexity of these subtelomeres and muscular disorder ranked as the most common the difficulty for genomic assembly. Out of the 1029 neuromuscular hereditary disorder with a preva- DNA samples analysed in our centre in the last 7 years, lence of 1:8000–1:20 000 is not linked to a muta- we also identified 54 cases clinically diagnosed with tion affecting the coding sequence of a protein FSHD carrying complex genotypes. This includes mosaic involved in muscle function but to a deletion of an patients, patients with deletions of the proximal 4q integral number of repetitive D4Z4 macrosatellites region and 23 cases with an atypical chromosome 10 14 15 at the 4q35 chromosome end. Clinically, symp- pattern, infrequently found in the control population and toms usually arise between the age of 20–40 years never reported before. © Author(s) (or their with a typical asymmetrical weakness of facial, Conclusion Overall, this work underlines the complexity employer(s)) 2019. No scapular girdle, upper limb and lower extremities of these loci challenging the diagnosis and genetic commercial re-use. See rights muscles.16 and permissions. Published counselling for this disease. by BMJ. The locus linked to the pathology is located in the subtelomeric region of the 4q arm.17 18 In the To cite: Nguyen K, control population, the number of repeated units Broucqsault N, Chaix C, et al. J Med Genet Epub ahead of INTRODUCTION of this 3.3 kb GC-rich element is between 11 and print: [please include Day Subtelomeres are highly variable DNA sequences 150 copies (eg, >35 kb and up to an estimated size Month Year]. doi:10.1136/ lying at the interface between telomeres and chro- of 495 kb) whereas in patients with FSHD1, one jmedgenet-2018-105949 mosome-specific regions. The rate of recombination of the D4Z4 arrays carried by the 4q35 allele is Nguyen K, et al. J Med Genet 2019;0:1–12. doi:10.1136/jmedgenet-2018-105949 1 Diagnostics contracted and contains between 1 and 10 units with a threshold Statement of objectives J Med Genet: first published as 10.1136/jmedgenet-2018-105949 on 22 April 2019. Downloaded from size <35 kb.19 Distal to D4Z4, two main sequences have been Exploit the resolution provided by MC and bar coding of the described, termed qA and qB haplotypes.20 The 4qA sequence is 4q35 and 10q26 subtelomeric loci to uncover the complexity characterised by the presence of a 260 bp sequence called pLAM of these regions in individuals affected with FSHD and in the containing a degenerated polyadenylation signal for the DUX4 general population. retrogene encoded by D4Z4. The pLAM is followed by an esti- mated array of 6.2 kb containing tandem copies of 68 bp ß-sat- Materials and methods 20–22 ellites. The 4qB sequence is 92% homologous to 4qA but Materials and Methods are detailed in the online supplementary contains a LINE sequence. The 10q26 region is approximately information section. 98% identical to the 4qA end in the 40 kb proximal to D4Z4 and at least 10 kb distal to the repeat.21 23 24 On this chromo- some, the number of D4Z4 is variable. In addition, SSLP anal- RESULTS AND DISCUSSION yses of the 4qter region revealed the existence of microsatellites Analysis strategy Since the validation of MC for the molecular diagnosis of of different sizes upstream of D4Z4 with at least three different FSHD,27 we have processed 1029 blood samples from index haplotypes for the 4qA allele and six haplotypes for 4qB.24 The cases or relatives (figure 1A–D). A number of cases were referred current model explaining the pathogenesis of the disease postu- to our Medical Genetics Department for FSHD molecular diag- lates that in FSHD1, chromatin relaxation linked to shortening nosis and exclusion diagnosis or familial segregation studies. of the D4Z4 array or to mutation in SMCHD1 in FSHD2 causes After initial steps of validation of the technique,27 we have used overexpression of the DUX4 transcript encoded by D4Z4. DUX4 the combing methodology to test in priority patients for whom is transcribed from the last D4Z4 repeat and through the distal ambiguous results were obtained by SB (either positive or nega- pLAM sequence containing a degenerated polyadenylation site tive). The major advantage of MC is the direct visualisation of required for stabilisation of the transcript and production of the the haplotypes allowing a straightforward interpretation, espe- DUX4 protein. cially in complex situations. For SB, we considered unequivocal In most routine laboratories, FSHD diagnosis is based on a blotting results where four alleles (two signals for 4q and 10q Southern blot (SB) technique after digestion of genomic DNA alleles) were clearly visible and additional alleles were absent. with
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