Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 1 Non-recurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair Running title: Genomics of MECP2 duplications Marijke Bauters,1,2,1 Hilde Van Esch,3,1 Michael %. Frie(,4 Odile Boespflug- ,anguy,5 Martin .enker,6 Angela M. Vianna-Morgante,0 1arla 2osenberg,0 %aakko Ignatius,8 Martine 2aynaud,5 6aren Hollanders,1,2 6aren 7ovaerts,1,2 6ris Vandenreijt,1,2 Florence Niel,5 8ierre Blanc,5 2oger E. Stevenson,4 %ean-8ierre Fryns,3 8eter Marynen,1,2 1harles E. Schwart(,4 and 7uy Froyen1,2,11 1Human Genome Laboratory, Dept. for Molecular and Developmental Genetics, IB, Leuven, Belgium$ 2Human Genome Laboratory, Dept. of Human Genetics, %.U.Leuven, Leuven, Belgium$ 3Dept. of Human Genetics, University Hospital Gasthuisberg, Leuven, Belgium$ 4JC Self ,esearch Institute of Human Genetics, Greenwood Genetic Center, Greenwood, SC$ .Centre Hospitalier Universitaire, Clermont-0D, G1n1ti2ue Humaine, Clermont-0errand, 0rance$ 3Institute of Human Genetics, University of Erlangen- Nuremberg, Erlangen, Germany$ 7Dept. Genetics and Evolutionary Biology, Institute of Biosciences, University of S6o Paulo, Bra7il$ 8Dept. Clinical Genetics, Oulu University Hospital and Oulu University, 0inland$ 9Centre Hospitalier Universitaire de Tours, Service de G1n1ti2ue, Tours, 0rance Ke words: MECP2, duplication, recombination model, breakpoint anal sis 1 ,hese two authors contributed equally to this work. Bauters_ms4.doc 14-3-2008 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 2 111orresponding author. E-mail guy.froyen@ med.kuleuven.be; fax: +32-16-340166 Address for correspondence: Dr. Gu Fro en, (uman Genome Laborator , Dept. for Molecular and Developmental Genetics, ,IB, K...Leuven, Gasthuisberg 0121, (erestraat 43, P0 bo5 602, B-3000 Leuven, Belgium. Phone: 732-16-330130. Bauters_ms4.doc 14-3-2008 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 3 Recurrent submicroscopic genomic cop number changes are the result of non-allelic homologous recombination 82A(R9. 2on-recurrent aberrations however, can result from different non-e5clusive recombination-repair mechanisms. : e previousl described small microduplications at Xq28 containing MECP2, in four male patients with a severe neurological phenot pe. (ere, we report on the fine-mapping and breakpoint anal sis of 16 unique microduplications. The size of the overlapping cop number changes varies between 0.3 and 2.3 Mb and FIS( anal sis on three patients demonstrated a tandem orientation. Although 8 of the 32 breakpoint regions coincide with low-cop repeats 8LCRs), none of the duplications are the result of 2A(R. Bioinformatics anal sis of the breakpoint regions demonstrated a 2.A-fold higher frequency of Alu interspersed repeats as compared to control regions, as well as a ver high GC content 8A3B9. .ne5pectedl , we obtained the Cunction in onl one patient b long-range PCR, which revealed non- homologous end Coining 82(EE9 as the mechanism. Breakpoint anal sis in two other patients b inverse PCR and subsequent arra -CG( anal sis, demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one cop was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed b breakage-induced replication 8BIR9 with strand invasion of the normal sister chromatid. 0ur results indicate that the mechanism b which cop number changes occur in regions with a comple5 genomic architecture, can ield comple5 rearrangements. FSupplemental material is available online at www.genome.org.G Bauters_ms4.doc 14-3-2008 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 4 Introduction : ith the introduction of arra -CG(, high-resolution detection of microdeletions and microduplications became possible. This resulted in the identification of man disease- associated genomic submicroscopic aberrations 8Pinkel and Albertson 200AH ,issers et al. 200AH Lockwood et al. 20069. In our screen of a large cohort of patients with X-linked mental retardation 8XLMR9 b full coverage X-chromosome-specific arra -CG( 8Fro en et al. 200I9 and real time quantitative PCR 8qPCR9 we identified small duplications at Xq28 in four unrelated male patients with severe to profound mental retardation and additional clinical features 8,an Esch et al. 200A9, referred to as the L.BS X-linked Mental Retardation syndrome 8XLMRLH 0MIM 3002609 8http://www.ncbi.nlm.nih.gov/0MIM/9 8Lubs et al. 13339. Delineation of the minimal critical region and detection of a two-fold increased e5pression of MECP2 mR2A in the patient-derived cell lines compared to controls, pointed to an increased dosage of MECP2 as the cause of the MR phenot pe, thereb demonstrating a new disease mechanism in mental retardation 8,an Esch et al. 200A9. Subsequentl , other groups reported 8del Gaudio et al. 2006H Friez et al. 2006H Lu et al. 200IH Madrigal et al. 200I9 or communicated on additional patients with a gain of the MECP2 locus. Since all reported duplications seem to be different in size and location, this duplication entit is defined as a non-recurrent event. (owever, the mechanism b which this apparent frequent rearrangement occurs has not been resolved so far and potential mechanisms deduced from breakpoint studies of other non-recurrent rearrangements are still speculative. Recurrent rearrangements are mediated b non-allelic homologous recombination 82A(R9 between LCRs, also referred to as segmental duplications, or between highl Bauters_ms4.doc 14-3-2008 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press A similar Alu repeats. Such an event can result in deletions, duplications or inversions of the intermediate genomic segments, generating aberrations of equal size and location 8Shaw and Lupski 2004H Lupski 20069. In non-recurrent rearrangements on the other hand, the breakpoints are scattered throughout a genomic region and thus the aberrations are variable in size. Although the precise underl ing mechanism8s9 remain8s9 elusive, genomic architectural features have been associated with the generation of these cop number differences 8Shaw and Lupski 2004H Lupski 20069. Studies of non- recurrent PLP1 duplications and deletions at Xq22 in patients with Pelizaeus- Merzbacher Disease 8PMD9 implied that the presence of several LCRs and other smaller repeats seems to render the region unstable and thus, more susceptible to rearrangements 8: oodward et al. 200AH Lee et al. 20069. In such cases, the D2A repair mechanism is not alwa s a simple event. Several groups recentl reported comple5 rearrangements that at first glance seem to be generated in a mechanisticall simple wa but after detailed molecular anal sis revealed more comple5 rearrangements potentiall due to alternative D2A repair mechanisms 8Potocki et al. 200IH Balciuniene et al. 200IH Gotter et al. 200IH Sheen et al. 200I9 or replication errors 8Lee et al. 200I9. : e present a comprehensive anal sis of 16 unique duplications at Xq28. : e found the region to be highl repetitive, which likel contributed to chromosomal breakage at one or more locations, and subsequent D2A mis-repair. Anal sis of the Cunctions demonstrated that the recombination in two patients resulted from an insertion of a non- contiguous neighboring region preceding the MECP2 duplication event. Bauters_ms4.doc 14-3-2008 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 6 2esults Identification of male patients with MECP2 duplications In collaboration with several European groups, we screened for duplications of the MECP2 gene b qPCR in patients selected based on the clinical features of our initiall reported patients with MECP2 duplications. In addition to the 4 males reported earlier 8,an Esch et al. 200A9, we identified 4 new positive patients, 2 from France 8E316, X049, one from German 832603I9, and one sporadic patient from Belgium 8(T9. Additionall , we utilized D2A from previousl published male patients: I from Greenwood, SC 8K8210, K8300, K831A, K322I, K3228, K3244 and K34239 8Friez et al. 20069, and one from Brazil 81A3829 8Rosenberg et al. 20069. Together with the 4 initiall reported b our group 8,an Esch et al. 200A9 we anal zed the breakpoints of MECP2 microduplications in a total of 16 patients. Clinical details of 12 of these are described elsewhere 8,an Esch et al. 200AH Rosenberg et al. 2006H Friez et al. 20069. 8osition and orientation of the duplications : e investigated the position and orientation of the duplication b FIS( in four patients from whom we had an Epstein Barr virus-transformed peripheral blood l mphoc te- derived 8EB,-PBL9 cell line 8L36, T33, T88 and K32289, using the differentiall labeled adCacent BAC clones RP11-314B3 81A2.63 Mb9 and RP11-113A22 81A2.30 Mb9. Positions on the X chromosome are based on the .CSC genome browser build hg18 8http://genome.ucsc.edu/9. For L36, T33 and T88, both BAC clones are present within the duplication as indicated in Fig. 1A. : ith all samples tested the green and red probe Bauters_ms4.doc 14-3-2008 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press I signals were detected on metaphase spreads at the ver end of the Xq arm and not at other positions in the genome 8Supplemental Fig. S1A-C9 demonstrating that the duplication occurred at Xq28. 0n interphase nuclei of these patients, alternating green- red-green-red signals were detected 8Supplemental Fig. S1A-C9, a pattern strongl suggestive for a tandem duplication event. For patient K3228, a green-red-red order was obtained because RP11-314B3 is not within the duplication 8Fig. 1B9. (owever, the signals on metaphase spreads showed that the duplication also occurred at Xq28 8Supplemental Fig.