Abstract：
Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. Our understanding of the genetic bases and pathogenic mechanisms underlying HCM has improved significantly in the recent past, but the combined effect of various pathogenic gene variants and the influence of genetic modifiers in disease manifestation are very poorly understood. Here, we set out to investigate genotype-phenotype relationships in 2 siblings with an extensive family history of HCM, both carrying a pathogenic truncating variant in the MYBPC3 gene (p.Lys600Asnfs*2), but who exhibited highly divergent clinical manifestations.
We used a combination of induced pluripotent stem cell (iPSC)-based disease modeling and CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing to generate patient-specific cardiomyocytes (iPSC-CMs) and isogenic controls lacking the pathogenic MYBPC3 variant.
Mutant iPSC-CMs developed impaired mitochondrial bioenergetics, which was dependent on the presence of the mutation. Moreover, we could detect altered excitation-contraction coupling in iPSC-CMs from the severely affected individual. The pathogenic MYBPC3 variant was found to be necessary, but not sufficient, to induce iPSC-CM hyperexcitability, suggesting the presence of additional genetic modifiers. Whole-exome sequencing of the mutant carriers identified a variant of unknown significance in the MYH7 gene (p.Ile1927Phe) uniquely present in the individual with severe HCM. We finally assessed the pathogenicity of this variant of unknown significance by functionally evaluating iPSC-CMs after editing the variant.
Our results indicate that the p.Ile1927Phe variant of unknown significance in MYH7 can be considered as a modifier of HCM expressivity when found in combination with truncating variants in MYBPC3. Overall, our studies show that iPSC-based modeling of clinically discordant subjects provides a unique platform to functionally assess the effect of genetic modifiers.
We used a combination of induced pluripotent stem cell (iPSC)-based disease modeling and CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing to generate patient-specific cardiomyocytes (iPSC-CMs) and isogenic controls lacking the pathogenic MYBPC3 variant.
Mutant iPSC-CMs developed impaired mitochondrial bioenergetics, which was dependent on the presence of the mutation. Moreover, we could detect altered excitation-contraction coupling in iPSC-CMs from the severely affected individual. The pathogenic MYBPC3 variant was found to be necessary, but not sufficient, to induce iPSC-CM hyperexcitability, suggesting the presence of additional genetic modifiers. Whole-exome sequencing of the mutant carriers identified a variant of unknown significance in the MYH7 gene (p.Ile1927Phe) uniquely present in the individual with severe HCM. We finally assessed the pathogenicity of this variant of unknown significance by functionally evaluating iPSC-CMs after editing the variant.
Our results indicate that the p.Ile1927Phe variant of unknown significance in MYH7 can be considered as a modifier of HCM expressivity when found in combination with truncating variants in MYBPC3. Overall, our studies show that iPSC-based modeling of clinically discordant subjects provides a unique platform to functionally assess the effect of genetic modifiers.
摘要：
背景：肥厚型心肌病（HCM）是最常见的遗传性心脏病，也是心力衰竭和心源性猝死的常见原因。最近，我们对HCM的遗传基础和致病机制的理解有了显著提高，但是对各种致病基因变异的综合作用和遗传修饰在疾病表现中的影响知之甚少。这里,我们着手调查2名具有广泛HCM家族史的兄弟姐妹的基因型-表型关系，两者都在MYBPC3基因中携带致病性截断变体（p。Lys600Asnfs*2)，但表现出高度分歧的临床表现。
结果：我们使用了基于诱导多能干细胞（iPSC）的疾病建模和CRISPR/Cas9介导的基因组编辑的组合，以产生患者特异性心肌细胞（iPSC-CM）和缺乏致病性MYBPC3变体的等基因对照。突变iPSC-CM发展受损的线粒体生物能学，这取决于突变的存在。此外，我们可以从严重受影响的个体中检测到iPSC-CM中激发-收缩耦合的改变。发现致病性MYBPC3变体是必要的，但还不够,诱导iPSC-CM过度兴奋，表明存在额外的遗传修饰剂。突变携带者的全外显子组测序鉴定出MYH7基因中具有未知意义的变体（p。Ile1927Phe）独特地存在于患有严重HCM的个体中。我们最终通过在编辑变体后对iPSC-CM进行功能性评估来评估这种意义未知的变体的致病性。
结论：我们的结果表明，当与MYBPC3中的截短变体组合发现时，MYH7中未知意义的p.Ile1927Phe变体可被视为HCM表达的修饰物。总的来说,我们的研究表明,基于iPSC的临床不一致受试者建模提供了一个独特的平台,从功能上评估遗传修饰剂的作用.
结果：我们使用了基于诱导多能干细胞（iPSC）的疾病建模和CRISPR/Cas9介导的基因组编辑的组合，以产生患者特异性心肌细胞（iPSC-CM）和缺乏致病性MYBPC3变体的等基因对照。突变iPSC-CM发展受损的线粒体生物能学，这取决于突变的存在。此外，我们可以从严重受影响的个体中检测到iPSC-CM中激发-收缩耦合的改变。发现致病性MYBPC3变体是必要的，但还不够,诱导iPSC-CM过度兴奋，表明存在额外的遗传修饰剂。突变携带者的全外显子组测序鉴定出MYH7基因中具有未知意义的变体（p。Ile1927Phe）独特地存在于患有严重HCM的个体中。我们最终通过在编辑变体后对iPSC-CM进行功能性评估来评估这种意义未知的变体的致病性。
结论：我们的结果表明，当与MYBPC3中的截短变体组合发现时，MYH7中未知意义的p.Ile1927Phe变体可被视为HCM表达的修饰物。总的来说,我们的研究表明,基于iPSC的临床不一致受试者建模提供了一个独特的平台,从功能上评估遗传修饰剂的作用.
