Precise Correction of Heterozygous SHOX2 Mutations in hiPSCs Derived from Patients with Atrial Fibrillation via Genome Editing and Sib Selection.

Patient-specific human induced pluripotent stem cells (hiPSCs) offer unprecedented opportunities for the investigation of multigenic disease, personalized medicine, and stem cell therapy. For heterogeneous diseases such as atrial fibrillation (AF), however, precise correction of the associated mutation is crucial. Here, we generated and corrected hiPSC lines from two AF patients carrying different heterozygous SHOX2 mutations. We developed a strategy for the scarless correction of heterozygous mutations, based on stochastic enrichment by sib selection, followed by allele quantification via digital PCR and next-generation sequencing to detect isogenic subpopulations. This allowed enriching edited cells 8- to 20-fold. The method does not require antibiotic selection or cell sorting and can be easily combined with base-and-prime editing approaches. Our strategy helps to overcome low efficiencies of homology-dependent repair in hiPSCs and facilitates the generation of isogenic control lines that represent the gold standard for modeling complex diseases in vitro.

Functional Characterization of Rare Variants in the SHOX2 Gene Identified in Sinus Node Dysfunction and Atrial Fibrillation.

Sinus node dysfunction (SND) and atrial fibrillation (AF) often coexist; however, the molecular mechanisms linking both conditions remain elusive. Mutations in the homeobox-containing SHOX2 gene have been recently associated with early-onset and familial AF. Shox2 is a key regulator of sinus node development, and its deficiency leads to bradycardia, as demonstrated in animal models. To provide an extended SHOX2 gene analysis in patients with distinct arrhythmias, we investigated SHOX2 as a susceptibility gene for SND and AF by screening 98 SND patients and 450 individuals with AF. The functional relevance of the novel mutations was investigated in vivo and in vitro, together with the previously reported p.H283Q variant. A heterozygous missense mutation (p.P33R) was identified in the SND cohort and four heterozygous variants (p.G77D, p.L129=, p.L130F, p.A293=) in the AF cohort. Overexpression of the pathogenic predicted mutations in zebrafish revealed pericardial edema for p.G77D and the positive control p.H283Q, whereas the p.P33R and p.A293= variants showed no effect. In addition, a dominant-negative effect with reduced heart rates was detected for p.G77D and p.H283Q. In vitro reporter assays demonstrated for both missense variants p.P33R and p.G77D significantly impaired transactivation activity, similar to the described p.H283Q variant. Also, a reduced Bmp4 target gene expression was revealed in zebrafish hearts upon overexpression of the p.P33R mutant. This study associates additional rare variants in the SHOX2 gene implicated in the susceptibility to distinct arrhythmias and allows frequency estimations in the AF cohort (3/990). We also demonstrate for the first time a genetic link between SND and AF involving SHOX2. Moreover, our data highlight the importance of functional investigations of rare variants.

Murine transgenic embryonic stem cell lines for the investigation of sinoatrial node-related molecular pathways.

The elucidation of molecular mechanisms that restrict the potential of pluripotent stem cells and promote cardiac lineage differentiation is of crucial relevance, since embryonic stem cells (ESCs) hold great potential for cell based heart therapies. The homeodomain transcription factor Shox2 is essential for the development and proper function of the native cardiac pacemaker, the sinoatrial node. This prompted us to develop a cardiac differentiation model using ESC lines isolated from blastocysts of Shox2-deficient mice. The established cell model provides a fundamental basis for the investigation of molecular pathways under physiological and pathophysiological conditions for evaluating novel therapeutic approaches.

Comparative expression analysis of Shox2-deficient embryonic stem cell-derived sinoatrial node-like cells.

The homeodomain transcription factor Shox2 controls the development and function of the native cardiac pacemaker, the sinoatrial node (SAN). Moreover, SHOX2 mutations have been associated with cardiac arrhythmias in humans. For detailed examination of Shox2-dependent developmental mechanisms in SAN cells, we established a murine embryonic stem cell (ESC)-based model using Shox2 as a molecular tool. Shox2+/+ and Shox2-/- ESC clones were isolated and differentiated according to five different protocols in order to evaluate the most efficient enrichment of SAN-like cells. Expression analysis of cell subtype-specific marker genes revealed most efficient enrichment after CD166-based cell sorting. Comparative cardiac expression profiles of Shox2+/+ and Shox2-/- ESCs were examined by nCounter technology. Among other genes, we identified Nppb as a novel putative Shox2 target during differentiation in ESCs. Differential expression of Nppb could be confirmed in heart tissue of Shox2-/- embryos. Taken together, we established an ESC-based cardiac differentiation model and successfully purified Shox2+/+ and Shox2-/- SAN-like cells. This now provides an excellent basis for the investigation of molecular mechanisms under physiological and pathophysiological conditions for evaluating novel therapeutic approaches.

A large-scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function.

MicroRNAs (miRNAs) are important regulators of neuronal development, network connectivity, and synaptic plasticity. While many neuronal miRNAs were previously shown to modulate neuronal morphogenesis, little is known regarding the regulation of miRNA function. In a large-scale functional screen, we identified two novel regulators of neuronal miRNA function, Nova1 and Ncoa3. Both proteins are expressed in the nucleus and the cytoplasm of developing hippocampal neurons. We found that Nova1 and Ncoa3 stimulate miRNA function by different mechanisms that converge on Argonaute (Ago) proteins, core components of the miRNA-induced silencing complex (miRISC). While Nova1 physically interacts with Ago proteins, Ncoa3 selectively promotes the expression of Ago2 at the transcriptional level. We further show that Ncoa3 regulates dendritic complexity and dendritic spine maturation of hippocampal neurons in a miRNA-dependent fashion. Importantly, both the loss of miRNA activity and increased dendrite complexity upon Ncoa3 knockdown were rescued by Ago2 overexpression. Together, we uncovered two novel factors that control neuronal miRISC function at the level of Ago proteins, with possible implications for the regulation of synapse development and plasticity.

A coding-independent function of an alternative Ube3a transcript during neuronal development.

The E3 ubiquitin ligase Ube3a is an important regulator of activity-dependent synapse development and plasticity. Ube3a mutations cause Angelman syndrome and have been associated with autism spectrum disorders (ASD). However, the biological significance of alternative Ube3a transcripts generated in mammalian neurons remains unknown. We report here that Ube3a1 RNA, a transcript that encodes a truncated Ube3a protein lacking catalytic activity, prevents exuberant dendrite growth and promotes spine maturation in rat hippocampal neurons. Surprisingly, Ube3a1 RNA function was independent of its coding sequence but instead required a unique 3' untranslated region and an intact microRNA pathway. Ube3a1 RNA knockdown increased activity of the plasticity-regulating miR-134, suggesting that Ube3a1 RNA acts as a dendritic competing endogenous RNA. Accordingly, the dendrite-growth-promoting effect of Ube3a1 RNA knockdown in vivo is abolished in mice lacking miR-134. Taken together, our results define a noncoding function of an alternative Ube3a transcript in dendritic protein synthesis, with potential implications for Angelman syndrome and ASD.