Coulson Zachary, Kolb Justin, Sabha Nesrin, Karimi Esmat, Hourani Zaynab, Ottenheijm Coen, Granzier Henk, Dowling James J
Program for Genetics and Genome Biology, Hospital for Sick Children, Toronto, ON, Canada.
Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
Skelet Muscle. 2025 Mar 20;15(1):8. doi: 10.1186/s13395-025-00378-2.
Biallelic pathogenic variants in the nebulin (NEB) gene lead to the congenital muscle disease nemaline myopathy. In-frame deletion of exon 55 (ΔExon55) is the most common disease-causing variant in NEB. Previously, a mouse model of Neb was developed; however, it presented an uncharacteristically severe phenotype with a near complete reduction in Neb transcript expression that is not observed in NEB exon 55 patients. We identified by RNA sequencing that the cause of this unexpectedly severe presentation in mice is the generation of a pseudoexon containing two premature termination codons (and promoting nonsense mediated decay) at the Neb exon 55 deletion site. To prove that this is the cause of the loss of Neb transcript, and to generate a more faithful model of the human disease, we used CRISPR gene editing to remove the pseudoexon sequence and replace it with human intron 54 sequence containing a validated cas9 gRNA protospacer. The resulting "hmz" mice have a significant reduction in pseudoexon formation (93.6% reduction), and a re-introduction of stable Neb transcript expression. This new model has the characteristic features of nemaline myopathy at the physiological, histological, and molecular levels. Importantly, unlike the existing exon 55 deletion mice (which die by age 7 days), it survives beyond the first months and exhibits obvious signs of neuromuscular dysfunction. It thus provides a new, robust model for studying pathomechanisms and developing therapies for NEB related nemaline myopathy.
肌动蛋白（NEB）基因的双等位基因致病变异会导致先天性肌肉疾病杆状体肌病。外显子55的框内缺失（ΔExon55）是NEB中最常见的致病变异。此前，已构建了Neb的小鼠模型；然而，该模型呈现出异常严重的表型，Neb转录本表达几乎完全降低，而这在NEB外显子55患者中并未观察到。我们通过RNA测序确定，小鼠中这种意外严重表型的原因是在Neb外显子55缺失位点产生了一个包含两个提前终止密码子（并促进无义介导的衰变）的假外显子。为了证明这是Neb转录本缺失的原因，并生成一个更忠实反映人类疾病的模型，我们使用CRISPR基因编辑去除假外显子序列，并用包含经过验证的cas9 gRNA原间隔序列的人类内含子54序列进行替换。由此产生的“hmz”小鼠假外显子形成显著减少（减少93.6%），并重新引入了稳定的Neb转录本表达。这个新模型在生理、组织学和分子水平上具有杆状体肌病的特征。重要的是，与现有的外显子55缺失小鼠（7天龄时死亡）不同，它能存活超过最初几个月，并表现出明显的神经肌肉功能障碍迹象。因此，它为研究NEB相关杆状体肌病的发病机制和开发治疗方法提供了一个新的、可靠的模型。
