A novel CFH mutation affects FHL-1 expression and metabolism in EOMD patient derived iPSC-RPE.

Abstract

Purpose : Early onset macular drusen (EOMD) is a rare inherited retinal degeneration with similar clinical features to AMD. EOMD patients express high penetrance genetic variants in the complement factor H (CFH) gene that affect expression of the truncated isoform, factor H-like protein 1 (FHL-1). FHL-1 is highly localized to Bruch’s membrane where it functions as an alternative pathway inhibitor of the complement system. However, little is known about the role of FHL-1 in the retinal pigmented epithelium (RPE). In this study, we generated and characterized EOMD-patient derived iPSC-RPE cells, and hypothesized that reduced FHL-1 expression results in the formation of basal deposits and reprogramming of RPE metabolism.

Methods : PBMCs isolated from EOMD patients were reprogrammed into iPSCs and differentiated into RPE using standard protocols. Sanger sequencing was performed to identify the novel CFH gene mutation. Minigene assays in HEK293T transfected cells were used to evaluate mutant FHL-1 expression. iPSC-RPE cultured on Matrigel-coated filters for 4-weeks were examined for ultrastructural differences using TEM. RPE cells cultured on flat bottom dishes were also harvested for WB, qPCR and immunocytochemistry of RPE markers, and targeted metabolomics was performed using LCMS/GCMS. iPSC-RPE from healthy age-matched donors were used as controls.

Results : EOMD patients expressed a novel c.351-2A>G mutation in the conserved AG dinucleotide at the 3’ splice site of exon 4 in the CFH gene, resulting in exon 4 skipping and no expression of mutant FHL-1 protein in the minigene assay. iPSC-RPE generated from EOMD patients heterozygous for the mutation exhibited typical RPE morphology, pigmentation, and RPE marker expression, but had a 50% reduction in FHL-1 secretion. EOMD iPSC RPE cells formed large multilaminar crystalline-like deposits, consistent with the appearance of calcium oxalate. In addition, numerous electron lucent vesicles were noted in the basal deposits, and Oil Red O staining confirmed the presence of basal lipid deposits. Metabolite analysis revealed increased glucose consumption and lipid accumulation in the iPSC-RPE derived from EOMD patients.

Conclusions : EOMD iPSC-RPE have decreased FHL-1 expression, form basal laminar calcium and lipid deposits, and have increased glucose consumption and lipid accumulation, suggesting a crucial role for FHL-1 in the maintenance of RPE homeostasis.

This is a 2021 ARVO Annual Meeting abstract.