Human iPSC-Derived Retinal Pigment Epithelium: A Model System for Prioritizing and Functionally Characterizing Causal Variants at AMD Risk Loci
Highlights
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iPSC-RPE have morphological and molecular profiles similar to human fetal RPE
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RPE molecular data integration can efficiently prioritize variants at AMD risk loci
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rs943080 is the probable causal AMD risk variant in the VEGFA locus
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rs943080 is a regulatory variant associated with lower gene expression of VEGFA
Introduction
Age-related macular degeneration (AMD) is a leading cause of vision loss that affects 1.6 million people over the age of 50 years in the United States (CDC, 2018) and has limited therapeutic options (Al-Zamil and Yassin, 2017). Disease development manifests in progressive degeneration in response to oxidative stress and inflammation of the retinal pigment epithelium (RPE) (Kinnunen et al., 2012), a monolayer consisting of a few million cells most densely located in the macula of the eye (Panda-Jonas et al., 1996). AMD has a strong genetic component (Seddon et al., 2005), and through a large international study of 16,144 AMD cases and 17,832 controls, 52 independent AMD risk variants mapping to 34 AMD-associated loci have been identified (Fritsche et al., 2016). As in many common diseases (Gallagher and Chen-Plotkin, 2018, Gusev et al., 2014, Maurano et al., 2012, Visscher et al., 2017), the majority of these loci have strongly associated variants in non-coding regions of the genome, suggesting that they may act through gene regulation. Regulatory variants that affect human disease, such as the variant that affects IRX3 expression in obesity (Smemo et al., 2014), can have strong effects, but it can be challenging to identify causal distal regulatory variants and link them with their target genes. Indeed, for AMD, while some candidate target genes have been identified (Fritsche et al., 2016), the causal variants and the downstream processes by which they mediate their effects are generally unknown.
Regulatory genetic variation is often cell-type specific and can be studied through genetic analysis of molecular traits such as gene regulation and expression (Albert and Kruglyak, 2015). However, characterizing genetic variation in human RPE is challenging because the number of RPE cells in the human eye is limited (Panda-Jonas et al., 1996), can be affected by lifetime environmental exposures, and requires invasive procedures to collect samples. Induced pluripotent stem cell-derived RPE (iPSC-RPE) is a promising alternative to human RPE for genetic studies as a virtually unlimited number of cells can be obtained with the same genetic background non-invasively. iPSC-RPE has been shown to display characteristics of mature human RPE including polygonal and pigmented morphology, polarity of protein expression and secretion, phagocytosis of photoreceptor outer segments, and maintenance of RPE phenotypes after transplantation into mouse retina (Maruotti et al., 2015). Additionally, stem cell-derived RPE have been effectively transplanted into rodent and primate models, supporting their relevance in vivo (Davis et al., 2017, Kamao et al., 2014, Stanzel et al., 2014). Thus, iPSC-RPE could be an effective model system for functionally characterizing regulatory variation associated with AMD.
The identification and functional characterization of causal genetic variants has been improved through fine-mapping algorithms that can incorporate diverse epigenetic annotations. For example, accessible chromatin and active regulatory regions such as promoters and enhancers marked by histone 3 lysine-27 acetylation (H3K27ac) have been shown to be enriched for genetic variants associated with human diseases in cell types relevant for disease and can improve the prioritization of genome-wide association study (GWAS) causal variants through fine-mapping strategies (Pickrell, 2014). Additionally, while many GWAS loci harbor genes that have been implicated in AMD, such as VEGFA, which encodes the vascular endothelial growth factor (VEGF) protein that is targeted by three current treatments for AMD (Gragoudas et al., 2004, Heier et al., 2012, Rosenfeld et al., 2006), the causal risk variant and the mechanism of increased disease risk is not known. Thus, the molecular characterization of gene expression and regulatory regions in iPSC-RPE could improve fine mapping of AMD and lead to insights into mechanisms underlying genetic risk variants.
To investigate the utility of iPSC-RPE as a model system to characterize AMD risk variants, we generated iPSC-RPE from six human subjects and integrated gene expression, chromatin accessibility, and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) data with complementary published data from human adult subjects to identify potential causal variants at AMD risk loci. We show that the iPSC-RPE shows morphological and molecular characteristics that are similar to those of native RPE including a characteristic polygonal shape, strong melanin pigmentation and expression, and strong zonula occludens 1 (ZO-1), bestrofin 1 (BEST1), and microphthalmia-associated transcription factor (MITF) immunostaining. We show that iPSC-RPE gene expression profiles are highly similar to that of human fetal RPE, and that their ATAC-seq (assay for transposase-accessible chromatin using sequencing) peaks are enriched for relevant transcription factor motifs. We performed fine mapping of AMD risk loci integrating the molecular data from iPSC-RPE, human fetal RPE, and published human retina and RPE samples. At one locus, VEGFA, we show that the rs943080 risk allele is associated with regulatory protein binding in iPSC-RPE in a potentially disease-dependent manner, and that the risk allele results in decreased overall VEGFA expression, potentially through regulation by a non-coding transcript. These results establish a molecular hypothesis for the VEGFA genetic risk locus on AMD and illustrate the potential of iPSC-RPE as a model system to study the molecular function of genetic variation associated with AMD.
