Complement dysregulation in an iPSC-RPE model with CFH/FHL-1 haploinsufficiency.

Abstract

Purpose : Early onset macular drusen (EOMD) is an inherited retinal degeneration where patients develop clinical features similar to AMD early in life. This rare condition is associated with high penetrance genetic variants in the complement factor H (CFH) gene which result in loss of CFH and factor H-like protein 1 (FHL-1) expression. We identified an EOMD family with a novel c.351-2A>G variant in the conserved 3’ splice site of exon 4. Induced pluripotent stem cells (iPSC)-derived retinal pigment epithelium (RPE) generated from two EOMD patients heterozygous for the mutation showed significantly reduced (~ 50%) CFH and FHL-1 expression, consistent with haploinsufficiency. This study will examine the effects of CFH/FHL-1 haploinsufficiency on complement inhibition and regulators of complement activators (RCA) in RPE.

Methods : iPSC-RPE derived from healthy controls and EOMD subjects were seeded on Matrigel coated dishes and transwell filters. Media CFH/FHL-1 levels were quantified by WB and ELISA, and an in situ C3b breakdown was performed to assay CFH/FHL-1 function. Expression of soluble complement components were evaluated via real-time PCR and WB. Expression of RCA members were also assessed with real-time PCR and immunostaining. RPE cultured on chamber slides were treated with 10% NHS and stained for C5b-9 to visualize MAC deposition.

Results : EOMD RPE cells had a significantly decreased CFH protein secretion per cell and reduced in situ C3b cleavage activity. In naïve conditions, EOMD RPE had elevated levels of soluble C3bα, C3bβ, and C3bα fragments, consistent with higher C3 turnover. No differences in C5 and CFI protein expression was found. Transcript levels of CD46 (membrane cofactor protein; MCP), CD55 (decay accelerating factor; DAF) and clusterin (CLU) were unchanged, while CD59 and vitronectin (VTN) were significantly increased. On immunostaining, VTN and CLU-positive deposits were found only in EOMD RPE and increased basal localization of CD55 was observed. In response to treatment with 10% NHS, MAC deposition was significantly increased in EOMD RPE compared to controls.

Conclusions : These findings indicate that reduction in CFH and FHL-1 expression contributes to complement dysregulation, suggesting that the RPE cells derived from EOMD patients could be more susceptible to complement-mediated stress.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.