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Abstract

The sinoatrial node (SAN) is the natural pacemaker of the heart and initiates the rhythmic contractions of this organ. Its unique genetic profile is mediated by a network of transcriptional regulators. Among them is the homeodomain transcription factor SHOX2, which plays a major role in maintaining the phenotypic border between the SAN and the surrounding tissue. Mutations in this gene have been associated with early-onset and familial forms of Atrial Fibrillation (AF). AF is the most common cardiac rhythm disorder, affecting 1-2% of the general population. In the clinical context, it often co-exists with malfunctions of the sinus node (sinus node dysfunction, SND), however, it is unknown if both diseases interact, perpetuate, or initiate each other. In the first part of this project, a candidate gene study was combined with functional analyses to identify a causal relationship between novel SHOX2 gene variants and the development of AF and SND. Screening 98 SND patients and 450 individuals with AF led to the identification of four heterozygous variants in SHOX2 (p.P33R in the SND cohort and p.G77D, p.L129=, p.L130F, p.A293= in the AF cohort). We selected mutations based on their in silico predicted pathogenic potential and overexpressed them in embryonic zebrafish hearts. A dominant-negative effect leading to bradycardia and pericardial edema was detected for p.G77D, while no effect was revealed for the p.P33R and p.A293= variants. A significantly impaired transactivation activity for both missense variants p.P33R and p.G77D was demonstrated by in vitro reporter assays. Moreover, upon overexpression of the p.P33R mutant in zebrafish hearts, a reduced Bmp4 target gene expression was revealed. This study demonstrated for the first time a genetic link between SND and AF involving SHOX2. Patient-specific human induced pluripotent stem cells (iPSCs) harboring putative disease-causing variants offer unprecedented opportunities for the investigation of cardiovascular diseases. We generated and characterized iPSCs from patients with previously identified heterozygous SHOX2 mutations (SHOX2 c.849C>A and SHOX2 c.*28T>C). To establish an isogenic control, we developed a novel strategy for the scarless correction of heterozygous mutations. Patient-derived iPSCs were gene-edited with the CRISPR/Cas system and subdivided into small cell pools (sib-selection). We quantified wildtype and mutant alleles via digital PCR and next generation sequencing to detect shifts in the wildtype/mutant allele ratio that indicated the presence of gene-corrected cells. Using this method, we managed to enrich our target cells 8-10-fold before generating a monoclonal cell population via single-cell cloning. The recharacterization of the new lines confirmed a preserved pluripotency and a normal karyotype. Future electrophysiological and molecular analysis will give further insights into the contribution of SHOX2 to onset and progression of AF.