Role of SETD5 in Moyamoya Disease Pathogenesis

摘要 烟雾病（MMD）发生时，远端颈内动脉进行性狭窄， 最终闭塞，并且是儿科中风的常见原因。许多致病性遗传变异具有 已被确定为引起MMD，但尚未确定共同的发病机制。病理学 受影响的血管显示闭塞性病变由纤维增生细胞组成， 平滑肌细胞（SMC）特异性α-肌动蛋白（SMA）;因此，我们提出SMC迁移和增殖 可能是疾病的驱动因素。多个编码蛋白质的基因参与染色质重塑， 已被鉴定为引起MMD，包括基因SETD5中的杂合功能丧失（LOF）变体。设置5 与核受体-辅阻遏物（Ncor）复合物相互作用以调节组蛋白乙酰化。我们以前的 对ACTA 2中引起MMD的致病性变体的研究表明，这些变体损害SMC分化， 未完全分化的细胞具有增加的增殖和迁移， 能源生产。重要的是，促进氧化磷酸化的治疗恢复了分化， 在具有导致MMD的Acta2变体的小鼠SMC中，迁移减少，表明潜在的治疗方法 战略基于这些结果和MMD的已确定遗传触发因素列表，我们提出了一个共同的 致病机制：SMC特化过程中异常的染色质重塑导致细胞增殖 并移动以阻塞血管。在这里，我们将在两个实验中在具有SETD 5中的LOF变体的细胞中测试这一假设。 具体目标。1)我们将评估SETD5中的LOF变体是否影响SMC分化和表型。我们 将使用Crispr/Cas9基因编辑将SETD5 LOF等位基因导入人类诱导多能干细胞 （iPSC）。我们将这些iPSC与同基因对照一起分化为神经嵴祖细胞，然后分化为神经嵴祖细胞。 SMC，并将表征所得细胞的分化、增殖、迁移和代谢。（二） 我们将评估SETD5是否影响SMC分化关键位点的染色质重塑。我们将 使用靶向Crispr/Cas9基因编辑在人中的SETD5蛋白的C末端引入3xFlag标签 并将使用这些细胞来鉴定其中SETD5在iPSC、神经嵴祖细胞 和SMC染色质免疫沉淀测序。我们将评估SETD 5中的LOF变体是否影响 组蛋白乙酰化和基因转录在所确定的基因座。这些目标的完成将把SETD 5- 依赖性染色质重塑与SMC表型，并阐明LOF的分子机制， SETD5中的变体导致MMD。这些结果有可能确定治疗或预防的治疗策略 具有SETD5 LOF变体的患者中的MMD。最后，这些数据将极大地促进我们对一个 MMD发病机制的潜在共同途径。

ABSTRACT Moyamoya disease (MMD) occurs when the distal internal carotid arteries are progressively narrowed and eventually occluded, and is a common cause of pediatric stroke. Numerous pathogenic genetic variants have been identified to cause MMD, but a common mechanism of pathogenesis has yet to be defined. Pathology from affected vessels shows the occlusive lesions are comprised of fibroproliferative cells that stain positive for smooth muscle cell (SMC)-specific α-actin (SMA); we therefore propose that SMC migration and proliferation may be drivers of the disease. Multiple genes encoding proteins that participate in chromatin remodeling have been identified to cause MMD, including heterozygous loss of function (LOF) variants in the gene SETD5. SETD5 interacts with the nuclear receptor-corepressor (Ncor) complex to regulate histone acetylation. Our previous work on MMD-causing pathogenic variants in ACTA2 showed that these variants impair SMC differentiation, and the incompletely differentiated cells have increased proliferation and migration and rely on glycolysis for cellular energy production. Importantly, treatments that boost oxidative phosphorylation restored differentiation and reduced migration in mouse SMCs with MMD-causing Acta2 variants, suggesting a potential therapeutic strategy. Based on these results and the list of identified genetic triggers for MMD, we propose a common pathogenic mechanism: aberrant chromatin remodeling during SMC specification leads to cells that proliferate and migrate to occlude the vessels. Here, we will test this hypothesis in cells with LOF variants in SETD5 in two specific aims. 1) We will assess whether LOF variants in SETD5 impact SMC differentiation and phenotype. We will use Crispr/Cas9 gene editing to introduce SETD5 LOF alleles into human induced pluripotent stem cells (iPSCs). We will differentiate these iPSCs alongside isogenic controls into neural crest progenitors and then into SMCs, and will characterize the differentiation, proliferation, migration, and metabolism of the resulting cells. 2) We will assess whether SETD5 impacts chromatin remodeling at loci critical for SMC differentiation. We will introduce a 3xFlag tag at the C-terminus of the SETD5 protein using targeted Crispr/Cas9 gene editing in human iPSCs and will use these cells to identify genomic loci where SETD5 is acting in iPSCs, neural crest progenitors, and SMCs by chromatin immunoprecipitation sequencing. We will assess whether LOF variants in SETD5 affect histone acetylation and gene transcription at the identified loci. Completion of these aims will link SETD5- dependent chromatin remodeling with SMC phenotype and elucidate the molecular mechanisms by which LOF variants in SETD5 cause MMD. The results have the potential to identify therapeutic strategies to treat or prevent MMD in patients with SETD5 LOF variants. Finally, these data will dramatically advance our understanding of a potential common pathway for MMD pathogenesis.