Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a Novel BEST1 Mutation
Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a Novel BEST1 Mutation
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DOI：
10.1167/iovs.15-16910
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发表时间：
2015-07-01
影响因子：
4.4
通讯作者：
Marmorstein, Alan D.
中科院分区：
文献类型：
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作者：
Johnson, Adiv A.;Bachman, Lori A.;Marmorstein, Alan D.
目的. BEST 1基因突变编码雌激素样蛋白-1（Best1），可导致常染色体隐性雌激素样蛋白病（ARB）。编码bestrophin-1是一个五聚体阴离子通道，定位于RPE的基底外侧质膜。在这里，我们描述了在我们的实践中在患者中鉴定的突变R141H（CGC> CAC）和I366fsX18（c.1098_1100 + 7del）对Best1运输、寡聚化和通道活性的影响。采用全细胞膜片钳技术检测转染HEK293细胞的Cl-电流。通过共聚焦显微镜在分化的人诱导多能干细胞衍生的RPE（iPSC-RPE）细胞中评估Best1定位，随后通过腺病毒介导的基因转移表达突变体。在iPSC-RPE和MDCK细胞中通过共免疫沉淀评价寡聚化。与Best1相比，Best1（I366fsX18）电流增加，而Best1（R141H）Cl-电流减少。Best1（R141H）与Best1或Best1（I366fsX18）的共表达导致了挽救的通道活性。Best1、Best1（R141H）和Best1（I366fsX18）均在iPSC-RPE细胞中正确定位; Best1（R141H）和Best1（I366fsX18）在iPSC-RPE细胞中与内源性Best1共免疫沉淀，在MDCK细胞中相互共免疫沉淀。Best1的前366个氨基酸足以介导通道活性和同源寡聚化。Best1和Best1（R141H）的组合不会引起疾病，而Best1（R141H）与Best1（I366fsX18）一起引起ARB。由于两种组合产生的Cl-电流相当，这表明该患者的ARB不是由通道活性丧失引起的。此外，Best1（I366fsX18）与Best1的不同之处在于，它缺乏大部分的胞质C-末端结构域，这表明该区域的丢失对该患者ARB的发病机制有重要作用。
PURPOSE. Mutations in BEST1, encoding bestrophin-1 (Best1), cause autosomal recessive bestrophinopathy (ARB). Encoding bestrophin-1 is a pentameric anion channel localized to the basolateral plasma membrane of the RPE. Here, we characterize the effects of the mutations R141H (CGC > CAC) and I366fsX18 (c.1098_1100+7del), identified in a patient in our practice, on Best1 trafficking, oligomerization, and channel activity.METHODS. Currents of Cl- were assessed in transfected HEK293 cells using whole-cell patch clamp. Best1 localization was assessed by confocal microscopy in differentiated, human-induced pluripotent stem cell-derived RPE (iPSC-RPE) cells following expression of mutants via adenovirus-mediated gene transfer. Oligomerization was evaluated by coimmunoprecipitation in iPSC-RPE and MDCK cells.RESULTS. Compared to Best1, Best1(I366fsX18) currents were increased while Best1(R141H) Cl- currents were diminished. Coexpression of Best1(R141H) with Best1 or Best1(I366fsX18) resulted in rescued channel activity. Overexpressed Best1, Best1(R141H), and Best1(I366fsX18) were all properly localized in iPSC-RPE cells; Best1(R141H) and Best1(I366fsX18) coimmunoprecipitated with endogenous Best1 in iPSC-RPE cells and with each other in MDCK cells.CONCLUSIONS. The first 366 amino acids of Best1 are sufficient to mediate channel activity and homo-oligomerization. The combination of Best1 and Best1(R141H) does not cause disease, while Best1(R141H) together with Best1(I366fsX18) causes ARB. Since both combinations generate comparable Cl- currents, this indicates that ARB in this patient is not caused by a loss of channel activity. Moreover, Best1(I366fsX18) differs from Best1 in that it lacks most of the cytosolic C-terminal domain, suggesting that the loss of this region contributes significantly to the pathogenesis of ARB in this patient.