ERBB2 R599C variant is associated with left ventricular outflow tract obstruction defects in human

Background and aims Non-syndromic congenital heart defects (CHD) are occasionally familial and left ventricular out flow tract obstruction (LVOTO) defects are among the subtypes with the highest hereditability. The aim of this study was to evaluate the pathogenicity of a heterozygous ERBB2 variant R599C identified in three families with LVOTO defects. Methods Variant detection was done with exome sequencing. Western blotting, digital PCR, mass spectrometry (MS), MS-microscopy and flow cytometry were used to study the function of the ERBB2 variant R599C. Cardiac structure and function were studied in zebrafish embryos expressing human ERBB2 WT or R599C. Patient-derived human induced pluripotent stem cell cardiomyocytes (hiPS-CM) and endothelial cells (hiPS-ECs) were used for transcriptomic analyses. Results While phosphorylation of the ERBB2 R599C receptor was not altered, the variant affected dramatically the binding partners of the protein and lead to mislocalization of ERBB2 from plasma membrane to ER and mitochondria. Expression of human ERBB2 R599C in zebrafish embryos resulted in cardiomyocyte hypertrophy, increased cardiac wall thickness, and impaired fractional shortening, demonstrating that the mutant receptor induces functional and structural defects during heart development. Transcriptomic analyses of hiPS-ECs and hiPS-CMs from a patient with the R599C variant indicated aberrant expression of genes related to cardiovascular system development and abnormal response to oxidative stress in both cell types. Conclusion The heterozygous variant ERBB2 R599C leads to abnormal cellular localization of the ERBB2 receptor inducing structural changes and dysfunction in the zebrafish embryo heart. This evidence suggests ERBB2 as a novel disease gene for CHD. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study (EH) has been funded by the Academy of Finland (331405), the Finnish Medical Foundation, Foundation for Pediatric Research, The Finnish Cultural Foundation, Finnish Foundation for Cardiovascular Research, Sigrid Juselius Foundation, University of Helsinki, Helsinki University Hospital, and Orion Research Foundation. RK has been funded by Academy of Finland (297245) and Finnish Foundation for Cardiovascular Research. MA has been funded by Orion Research Foundation, Paivikki and Sakari Sohlberg Foundation and Aarne Koskelo Foundation. SS has been funded by the Finnish Medical Foundation. Exome sequencing and data analysis were provided by the University of Washington Center for Rare Disease Research (UW-CRDR) with support from NHGRI grants U01 HG011744, UM1 HG006493 and U24 HG011746. ### 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: Ethical committee of Helsinki University Hospital District gave ethical approval for this work 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, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced are available online at GEO and MassIVE (MSV000093153).