Functional characterization of the SHOX2 coding variants. A Luciferase activity of BMP4 (left) or ISL1 (right) luciferase reporter constructs co-expressed with SHOX2 wild type (Wt) or SHOX2 mutants (p.G81E; p.H283Q) in HEK293 cells (human embryonic kidney cells), determined 24 h after transfection. All values are reported as fold changes of luciferase activity normalized to the empty vector (pGL3 basic) co-transfected with the respective expression constructs. Data are expressed as mean ± SEM of four independent experiments performed in triplicate. p values were determined by a paired t test (*p < 0.05; **p < 0.01; ***p < 0.001). B Injection of shox2 antisense morpholino (MO), which leads to pericardial edema and pericardial blood congestion due to reduced heart rates in zebrafish can not be rescued by cardiomyocyte-specific overexpression of shox2 Mut p.H227Q (corresponds to human SHOX2 p.H283Q mutation) compared to shox2 Wt (wild type) 72 h post fertilization (hpf). C The mean heart rate of control embryos 72 hpf amounts to 149 beats per minute (bpm). After shox2 knockdown, the heart rate is significantly reduced to 87 bpm. While cardiomyocyte-specific overexpression of Wt shox2 rescues the MO-mediated bradycardia (137 bpm), overexpression of shox2 Mut p.H227Q does not alter the reduced heart rate significantly at 72 hpf (95 bpm). Data are expressed as mean ± SEM of 3 independent experiments. p values were determined by one-way ANOVA with Sidak’s multiple comparisons test (****p < 0.0001; ns not significant; n = 10-16 per condition per experiment)