VP.01 Array comparative genomic hybridisation and droplet digital PCR uncover recurrent copy number variation of the titin segmental duplication region

Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain such segmental duplication regions. Using our custom Comparative Genomic Hybridization array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variants, which is surprisingly common. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by Magnus Ehrnrooth foundation, Finska Laekaresaellskapet, Svenska Kulturfonden, Stiftelsen Dorothea Olivia, Karl Walter och Jarl Walter Perklens minne, Folkhaelsan Research Center. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Ethics Committee of the Children's Hospital of the University of Helsinki, Finland gave ethical approval for this work (approval number 6/E7/05). The Ethics Review Board of the University of Helsinki, Finland, renewed the ethical approval in 2021. Samples were obtained according to the declaration of Helsinki of 1975 and written consent was obtained from subjects, or data was analysed pseudonymously where appropriate. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All relevant data in the present work are contained in the manuscript and its supporting information. Raw data produced in the present study are available upon reasonable request to the authors.