Insights into autosomal recessive bestrophinopathy from a disease in a dish model.

Abstract

Purpose : To test the hypothesis that autosomal recessive bestrophinopathy (ARB) is the human “null “ condition for Bestrophin 1.

Methods : We have recruited a 13 year old female Caucasian subject with ARB due to compound heterozygous mutations (R141H & I366fsX18) and her parents into a clinical study (NCT02162953, Stem Cell Models of Best Disease and Other Retinal Degenerative Diseases) which aims to collect skin biopsies from 100 subjects with bestrophinopathies and their unaffected parents and siblings. Skin fibroblasts from these subjects were reprogramed to induced pluripotent stem cells (iPSCs). Each subject’s iPSCs were differentiated into retinal pigment epithelial (RPE) cells. Successful differentiation of iPSCs to RPE is determined by a rigorous set of morphologic and molecular criteria.

Results : Western blot of iPSC-RPE lysates for Best1 from the subject and her father (Best1^+/R141H) were compared to lysates from 3 unrelated BEST1^+/+ controls. Best1 expression was reduced, relative to the control in lysates from the father and absent from the subject with ARB. All anti-Best1 antibodies only recognize epitopes in the region deleted from the I366fsX18. However, we have previously shown that this mutant is stable, active as a Ca²⁺ activated anion channel, and that it oligomerizes with wild type Best1 when heterologously expressed in HEK293 and MDCK cells. We used this knowledge to probe for the Best1 ^I366fsX18 mutant in RPE lysates from the subject and the Mother (Best1^{+/ I366fsX18}) of the subject. Following radiolabeling of iPSC-RPE cells with [³⁵S] cysteine & methionine Best1 was immunoprecipitated from lysates. No Best1 was detected in lysates from the ARB subject. We next performed phagocytosis assays using FITC-labelled bovine photoreceptor outer segments (OS). Both binding and internalization of OS were significantly impaired in iPSC-RPE from the subject versus iPSC-RPE from her father. Mother, and unrelated controls.

Conclusions : Based on these data, we conclude that ARB in this patient results at least in part from absent or severely diminished expression of Best1 leading to a defect in OS phagocytosis.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.