Table_1_Functional Characterization of Rare Variants in the SHOX2 Gene Identified in Sinus Node Dysfunction and Atrial Fibrillation.xlsx

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.

窦房结功能障碍（Sinus node dysfunction, SND）与心房颤动（Atrial Fibrillation, AF）常合并存在，但二者关联的分子机制仍未明确。近期研究发现，含同源盒的SHOX2基因的突变与早发性家族性心房颤动相关。Shox2是窦房结发育的关键调控因子，动物模型实验证实其缺陷可导致心动过缓。为对不同心律失常患者开展扩展的SHOX2基因分析，本研究将SHOX2作为窦房结功能障碍与心房颤动的易感基因，对98例窦房结功能障碍患者及450例心房颤动患者进行了基因筛查。结合此前报道的p.H283Q变异体，我们在体内与体外模型中探究了新型突变的功能相关性。在窦房结功能障碍队列中鉴定出1个杂合错义突变（p.P33R），在心房颤动队列中鉴定出4个杂合变异体（p.G77D、p.L129=、p.L130F、p.A293=）。在斑马鱼中过表达预测具有致病性的突变体后发现，p.G77D与阳性对照p.H283Q可引发心包水肿，而p.P33R与p.A293=变异体则无此效应。此外，针对p.G77D与p.H283Q，检测到其可通过显性负效应降低心率。体外报告基因实验显示，与此前报道的p.H283Q变异体类似，p.P33R与p.G77D这两个错义变异体的反式激活活性均显著受损。在过表达p.P33R突变体的斑马鱼心脏中，还观察到靶基因Bmp4的表达水平显著降低。本研究将额外的罕见SHOX2基因变异与不同心律失常的易感性相关联，并可对心房颤动队列中的变异携带频率进行估算（3/990）。我们还首次证实了窦房结功能障碍与心房颤动之间存在涉及SHOX2的遗传关联。此外，本研究数据凸显了对罕见变异开展功能验证的重要性。