Human LCA16 iPSC-RPE recapitulates the phenotype associated with the nonsense mutation c.158 G>A (p.W53X)

Abstract

Purpose: Mutations in the KCNJ13 gene lead to a dysfunctional inwardly-rectifying potassium channel, Kir7.1, and result in one variant of Lebers Congenital Amaurosis (LCA16). Kir7.1 acts in the RPE apical processes to control the subretinal space environment which affects vision. We sought to determine the ability of using human iPSC-RPE to recapitulate aspects of the LCA16 phenotype associated with the nonsense mutation c.158 G>A (p.W53X) in vitro.

Methods: Skin biopsy (5mm punch) was obtained from a male LCA16 patient with a confirmed W53X mutation (proband) and from a healthy male after informed consent. The study was approved by UW-Madison Institutional Review Board. Fibroblast cells were used to generate iPSCs and were differentiated to RPE cells in culture according to described methods. Pigmented and hexagonal colonies were purified and used for propagating iPSC-RPE cells maintained in 6 mm inserts for 6-8 weeks with media replacement twice weekly. We used sequencing to confirm the presence of the LCA16 mutation. Immunocytochemistry was performed to determine the expression of RPE-specific proteins. We performed whole-cell patch clamp to register Kir7.1 current from iPSC-RPE cell lines. Results were compared with current recordings using heterologous expression of wild-type and mutant proteins in CHO-K1 cells.

Results: iPSC-RPE cells appeared pigmented with characteristic cobblestone morphology. Both the proband and the sibling control cell lines expressed the RPE-specific markers Mitf and ZO-1 protein and developed a resistance of more than 800 MOhm. A truncated Kir7.1 protein was detected in LCA16 cells by Western blot analysis. Control cells measured Kir7.1 current that reversed at -53 mV compared to a depolarized membrane potential of -30 mV for LCA16 cells. Rb⁺ generated about 9-fold increase in inward current measured at -150 mV potential in control cells, but had no effect on the LCA16 cells.

Conclusions: The Kir7.1 channel controls both potassium recycling around the resting membrane potential as well as a hyperpolarized membrane potential, and both were evident in control cells. TheLCA16 mutation W53X resulted in a dysfunctional truncated protein that depolarized the membrane potential. Using patient-derived iPSC-RPE cells, we have successfully reproduced a measurable aspect of the Kir7.1 phenotype which gives us the potential to safely test novel therapies for LCA16 in vitro.