背景:遗传性视网膜营养不良(IRD)是全球范围内无法治愈的失明的主要原因之一。IRD是由编码视网膜必需蛋白的基因突变引起的,导致光感受器退化和视觉功能丧失。由于缺乏对其病理生理学的重要部分的了解,IRD产生了巨大的全球财务负担,分子诊断,以及几乎没有非姑息治疗方案。用于IRD的患者来源的诱导多能干细胞(iPSC)似乎是解决这些问题的绝佳选择,作为IRD病理生理学深入研究和测试新治疗方法的特殊工具。
方法:从8名与PROM1相关的IRD患者的队列中,我们确定了3名患者携带相同的变体(c.1354dupT),但表达三种不同的IRD表型:锥形和杆状营养不良(CORD),色素性视网膜炎(RP),和Stargardt病4型(STGD4)。这三个目标患者,每个人都有一个健康的亲戚,接受了全面的眼科检查,并通过临床外显子组测序(CES)扩展了他们的遗传小组研究。随后,产生非整合性患者来源的iPSC,并对其进行充分表征.使用CRISPR/Cas9进行c.1354dupT突变的校正,并且在患者来源的iPSC系中通过流式细胞术和蛋白质印迹确认PROM1基因的遗传恢复。
结果:CES显示,2名具有c.1354dupT突变的目标患者在与补体系统或光感受器分化和过氧化物酶体生物发生障碍相关的基因中呈现单等位基因变异,分别。证实了患者来源的iPSC细胞系的多能性和功能性,目标突变的校正完全恢复了基因修复的患者来源的iPSC系中编码Prominin-1(CD133)的能力。
结论:PROM1基因的c.1354dupT突变与IRD的三种不同的AR表型相关。这种多向效应可能与视网膜营养不良相关的其他基因中单等位基因变体的影响有关。然而,需要提供进一步的证据。未来的实验应该包括基因编辑的患者来源的iPSC,因为它有潜力作为疾病建模工具来阐明这一问题。
BACKGROUND: Inherited retinal dystrophies (IRD) are one of the main causes of incurable blindness worldwide. IRD are caused by mutations in genes that encode essential proteins for the retina, leading to photoreceptor degeneration and loss of visual function. IRD generates an enormous global financial burden due to the lack of understanding of a significant part of its pathophysiology, molecular diagnosis, and the near absence of non-palliative treatment options. Patient-derived induced pluripotent stem cells (iPSC) for IRD seem to be an excellent option for addressing these questions, serving as exceptional tools for in-depth studies of IRD pathophysiology and testing new therapeutic approaches.
METHODS: From a cohort of 8 patients with PROM1-related IRD, we identified 3 patients carrying the same variant (c.1354dupT) but expressing three different IRD phenotypes: Cone and rod dystrophy (CORD), Retinitis pigmentosa (RP), and Stargardt disease type 4 (STGD4). These three target patients, along with one healthy relative from each, underwent comprehensive ophthalmic examinations and their genetic panel study was expanded through clinical exome sequencing (CES). Subsequently, non-integrative patient-derived iPSC were generated and fully characterized. Correction of the c.1354dupT mutation was performed using CRISPR/Cas9, and the genetic restoration of the PROM1 gene was confirmed through flow cytometry and western blotting in the patient-derived iPSC lines.
RESULTS: CES revealed that 2 target patients with the c.1354dupT mutation presented monoallelic variants in genes associated with the complement system or photoreceptor differentiation and peroxisome biogenesis disorders, respectively. The pluripotency and functionality of the patient-derived iPSC lines were confirmed, and the correction of the target mutation fully restored the capability of encoding Prominin-1 (CD133) in the genetically repaired patient-derived iPSC lines.
CONCLUSIONS: The c.1354dupT mutation in the PROM1 gene is associated to three distinct AR phenotypes of IRD. This pleotropic effect might be related to the influence of monoallelic variants in other genes associated with retinal dystrophies. However, further evidence needs to be provided. Future experiments should include gene-edited patient-derived iPSC due to its potential as disease modelling tools to elucidate this matter in question.