Department of Ophthalmology, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA.
Sci Rep. 2018 Mar 14;8(1):4487. doi: 10.1038/s41598-018-21651-z.
Autosomal recessive bestrophinopathy (ARB) is caused by mutations in the gene BEST1 which encodes bestrophin 1 (Best1), an anion channel expressed in retinal pigment epithelial (RPE) cells. It has been hypothesized that ARB represents the human null phenotype for BEST1 and that this occurs due to nonsense mediated decay (NMD). To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with ARB and her parents. After differentiation to retinal pigment epithelial (iPSC-RPE) cells, both BEST1 mRNA and Best1 protein expression were compared to controls. BEST1 mRNA expression levels, determined by quantitative PCR, were similar in ARB iPSC-RPE, parental cells, and genetically unrelated controls. Western blotting revealed that CRALBP and RPE65 were expressed within the range delineated by unrelated controls in iPSC-RPE from the ARB donor and her parents. Best1 protein was detected in different clones of ARB iPSC-RPE, but at reduced levels compared to all controls. When tested for the ability to phagocytose photoreceptor outer segments, ARB iPSC-RPE exhibited impaired internalization. These data suggest that impaired phagocytosis is a trait common to the bestrophinopathies. Furthermore, ARB is not universally the result of NMD and ARB, in this patient, is not due to the absence of Best1.
常染色体隐性眼病(ARB)是由 BEST1 基因突变引起的,该基因编码的是在视网膜色素上皮(RPE)细胞中表达的阴离子通道蛋白——Bestrophin 1(Best1)。据推测,ARB 代表了 BEST1 的人类无效表型,这是由于无义介导的衰变(NMD)所致。为了验证这一假说,我们从 ARB 患者及其父母中生成诱导多能干细胞(iPSC)。在分化为视网膜色素上皮(iPSC-RPE)细胞后,我们将 Best1 基因的 mRNA 和蛋白表达与对照组进行了比较。通过定量 PCR 测定的 BEST1 mRNA 表达水平在 ARB iPSC-RPE、亲代细胞和遗传上无关的对照组中相似。Western blot 显示,CRALBP 和 RPE65 的表达水平在 ARB 供体及其父母的 iPSC-RPE 中与无关对照组的范围一致。在 ARB iPSC-RPE 的不同克隆中检测到了 Best1 蛋白,但与所有对照组相比水平降低。在对吞噬光感受器外节的能力进行测试时,ARB iPSC-RPE 的内化能力受损。这些数据表明,吞噬作用受损是 Bestrophin 病的共同特征。此外,ARB 并非普遍由 NMD 引起,并且在该患者中,ARB 并非由于 Best1 的缺失所致。