Human Genetics, Faculty of Medicine and Health Sciences, University of Oldenburg, 26129 Oldenburg, Germany.
Institute of Developmental Biology and Neurobiology, Johannes Gutenberg-University of Mainz, 55099 Mainz, Germany.
Int J Mol Sci. 2020 Nov 10;21(22):8418. doi: 10.3390/ijms21228418.
X-chromosomal retinitis pigmentosa (RP) frequently is caused by mutations in the retinitis pigmentosa GTPase regulator () gene. We evaluated the potential of PTC124 (Ataluren, Translama) treatment to promote ribosomal read-through of premature termination codons (PTC) in . Expression constructs in HEK293T cells showed that the efficacy of read-through reagents is higher for UGA than UAA PTCs. We identified the novel hemizygous nonsense mutation c.1154T > A, p.Leu385* (NM_000328.3) causing a UAA PTC in and generated patient-derived fibroblasts. Immunocytochemistry of serum-starved control fibroblasts showed the RPGR protein in a dot-like expression pattern along the primary cilium. In contrast, RPGR was no longer detectable at the primary cilium in patient-derived cells. Applying PTC124 restored RPGR at the cilium in approximately 8% of patient-derived cells. RT-PCR and Western blot assays verified the pathogenic mechanisms underlying the nonsense variant. Immunofluorescence stainings confirmed the successful PTC124 treatment. Our results showed for the first time that PTC124 induces read-through of PTCs in and restores the localization of the RPGR protein at the primary cilium in patient-derived cells. These results may provide a promising new treatment option for patients suffering from nonsense mutations in or other genetic diseases.
X 连锁性视网膜炎色素变性（RP）通常由视网膜炎 GTP 酶调节因子（RPGR）基因突变引起。我们评估了 PTC124（Ataluren，Translama）治疗促进提前终止密码子（PTC）核糖体通读的潜力。在 HEK293T 细胞中的表达构建体表明，读通试剂对 UGA 的功效高于 UAA PTC。我们确定了新的半合子无义突变 c.1154T > A，p.Leu385*（NM_000328.3）导致 RPGR 中的 UAA PTC，并生成了患者来源的成纤维细胞。血清饥饿对照成纤维细胞的免疫细胞化学显示 RPGR 蛋白沿着初级纤毛呈点状表达模式。相比之下，在患者来源的细胞中，RPGR 不再可检测到初级纤毛。应用 PTC124 可使约 8%的患者来源细胞中的 RPGR 恢复到纤毛上。RT-PCR 和 Western blot 检测证实了该无义变异的致病机制。免疫荧光染色证实了 PTC124 治疗的成功。我们的研究结果首次表明，PTC124 可诱导 RPGR 中的 PTC 通读，并恢复患者来源细胞中 RPGR 蛋白在初级纤毛上的定位。这些结果可能为 RPGR 或其他遗传疾病中存在无义突变的患者提供一种有前途的新治疗选择。
