A novel cardiomyopathy phenotype linked to missense mutation of CHD7

Loss-of-function of the chromatin remodeler CHD7 causes CHARGE syndrome, characterized by variable penetrance and diverse abnormalities. However, establishing genotype-phenotype correlations has been challenging, as most CHD7 inactivating mutations are null alleles. Through CHD7 missense variant analysis at potential phosphorylation sites, we identified T730 (T720 in mice) as a critical residue associated with pathogenesis. Using a CHD7 T730 missense mutation (Chd7T720A) and a frameshift null allele (Chd7fs) in a mouse model, we found that Chd7fs/fs mice were non-viable, while Chd7fs/+ mice exhibited haploinsufficiency-related circling behavior. Notably, Chd7fs/T720A mice died before postnatal day 2, indicating the Chd7T720A allele is hypomorphic. Micro-CT analysis at E18.5 revealed heterozygous mice primarily exhibited hypertrophic cardiomyopathy (HCM), while homozygous mice developed both HCM and dilated cardiomyopathy (DCM). RNA-seq analysis of neonatal Chd7T720A/T720A hearts revealed a disrupted transcriptome, which in males and females was characterized by downregulation of mitochondrial energy metabolism genes and enrichment of ETS family transcription factor targets. We further identified GSK3, GSK3, HIPK1, and DYRK2 as candidate kinases for this site, suggesting a regulatory role in CHD7. This missense mutation causing developmental heart abnormalities establishes the first genotype-phenotype correlation for CHD7, and offers new insights into CHARGE syndrome pathogenesis. Mouse hearts from neonatal mice at postnatal day 0 (P0) were immediately processed for total RNA extraction. The study included three biological replicates for each experimental group, encompassing male and female hearts of Chd7+/+, Chd7T720A/+, and Chd7T720A/T720A mice.染色质重塑因子（chromatin remodeler）CHD7的功能丧失可引发CHARGE综合征（CHARGE syndrome），该疾病以可变外显率与多样化表型异常为特征。然而，由于绝大多数CHD7失活突变均为无效等位基因，建立基因型-表型关联始终颇具挑战。本研究通过对潜在磷酸化位点处的CHD7错义变异进行分析，鉴定出T730（小鼠中对应位点为T720）作为与发病机制密切相关的关键残基。我们利用携带CHD7 T730错义突变（Chd7T720A）与移码无效等位基因（Chd7fs）的小鼠模型开展实验，结果显示：Chd7fs/fs纯合子小鼠无法存活，而Chd7fs/+杂合子小鼠表现出单倍体剂量不足相关的转圈行为。值得注意的是，Chd7fs/T720A复合杂合子小鼠在出生后第2天前死亡，这表明Chd7T720A等位基因属于低功能等位基因。对胚胎第18.5天（E18.5）的小鼠进行显微CT（micro-CT）分析发现，杂合子小鼠主要表现为肥厚型心肌病（hypertrophic cardiomyopathy, HCM），而纯合子小鼠则同时出现肥厚型心肌病与扩张型心肌病（dilated cardiomyopathy, DCM）。对新生Chd7T720A/T720A纯合子小鼠的心脏进行RNA测序（RNA-seq）分析显示，其转录组发生显著紊乱，无论雌雄个体，均呈现出线粒体能量代谢相关基因的下调，以及ETS家族转录因子靶基因的富集。我们进一步鉴定出GSK3、GSK3、HIPK1及DYRK2为该位点的候选激酶，提示这些激酶对CHD7存在调控功能。这一导致发育性心脏异常的错义突变，首次建立了CHD7的基因型-表型关联，为CHARGE综合征的发病机制提供了全新的研究视角。本研究于出生后第0天（P0）立即采集新生小鼠心脏样本以开展总RNA提取，每个实验组均设置3次生物学重复，涵盖Chd7+/+野生型、Chd7T720A/+杂合型及Chd7T720A/T720A纯合型小鼠的雌雄心脏组织。