Genome-wide association identifies novel ROP risk loci in a multiethnic cohort.

We conducted a genome-wide association study (GWAS) in a multiethnic cohort of 920 at-risk infants for retinopathy of prematurity (ROP), a major cause of childhood blindness, identifying 1 locus at genome-wide significance level (p < 5×10-8) and 9 with significance of p < 5×10-6 for ROP ≥ stage 3. The most significant locus, rs2058019, reached genome-wide significance within the full multiethnic cohort (p = 4.96×10-9); Hispanic and European Ancestry infants driving the association. The lead single nucleotide polymorphism (SNP) falls in an intronic region within the Glioma-associated oncogene family zinc finger 3 (GLI3) gene. Relevance for GLI3 and other top-associated genes to human ocular disease was substantiated through in-silico extension analyses, genetic risk score analysis and expression profiling in human donor eye tissues. Thus, we identify a novel locus at GLI3 with relevance to retinal biology, supporting genetic susceptibilities for ROP risk with possible variability by race and ethnicity.

Patterns of Gene Expression, Splicing, and Allele-Specific Expression Vary among Macular Tissues and Clinical Stages of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness, and elucidating its underlying disease mechanisms is vital to the development of appropriate therapeutics. We identified differentially expressed genes (DEGs) and differentially spliced genes (DSGs) across the clinical stages of AMD in disease-affected tissue, the macular retina pigment epithelium (RPE)/choroid and the macular neural retina within the same eye. We utilized 27 deeply phenotyped donor eyes (recovered within a 6 h postmortem interval time) from Caucasian donors (60-94 years) using a standardized published protocol. Significant findings were then validated in an independent set of well-characterized donor eyes (n = 85). There was limited overlap between DEGs and DSGs, suggesting distinct mechanisms at play in AMD pathophysiology. A greater number of previously reported AMD loci overlapped with DSGs compared to DEGs between disease states, and no DEG overlap with previously reported loci was found in the macular retina between disease states. Additionally, we explored allele-specific expression (ASE) in coding regions of previously reported AMD risk loci, uncovering a significant imbalance in C3 rs2230199 and CFH rs1061170 in the macular RPE/choroid for normal eyes and intermediate AMD (iAMD), and for CFH rs1061147 in the macular RPE/choroid for normal eyes and iAMD, and separately neovascular AMD (NEO). Only significant DEGs/DSGs from the macular RPE/choroid were found to overlap between disease states. STAT1, validated between the iAMD vs. normal comparison, and AGTPBP1, BBS5, CERKL, FGFBP2, KIFC3, RORα, and ZNF292, validated between the NEO vs. normal comparison, revealed an intricate regulatory network with transcription factors and miRNAs identifying potential upstream and downstream regulators. Findings regarding the complement genes C3 and CFH suggest that coding variants at these loci may influence AMD development via an imbalance of gene expression in a tissue-specific manner. Our study provides crucial insights into the multifaceted genomic underpinnings of AMD (i.e., tissue-specific gene expression changes, potential splice variation, and allelic imbalance), which may open new avenues for AMD diagnostics and therapies specific to iAMD and NEO.

DeMixSC: a deconvolution framework that uses single-cell sequencing plus a small benchmark dataset for improved analysis of cell-type ratios in complex tissue samples.

Bulk deconvolution with single-cell/nucleus RNA-seq data is critical for understanding heterogeneity in complex biological samples, yet the technological discrepancy across sequencing platforms limits deconvolution accuracy. To address this, we introduce an experimental design to match inter-platform biological signals, hence revealing the technological discrepancy, and then develop a deconvolution framework called DeMixSC using the better-matched, i.e., benchmark, data. Built upon a novel weighted nonnegative least-squares framework, DeMixSC identifies and adjusts genes with high technological discrepancy and aligns the benchmark data with large patient cohorts of matched-tissue-type for large-scale deconvolution. Our results using a benchmark dataset of healthy retinas suggest much-improved deconvolution accuracy. Further analysis of a cohort of 453 patients with age-related macular degeneration supports the broad applicability of DeMixSC. Our findings reveal the impact of technological discrepancy on deconvolution performance and underscore the importance of a well-matched dataset to resolve this challenge. The developed DeMixSC framework is generally applicable for deconvolving large cohorts of disease tissues, and potentially cancer.

Genome-wide association identifies novel ROP risk loci in a multi-ethnic cohort.

We conducted a genome-wide association study (GWAS) in a multiethnic cohort of 920 at-risk infants for retinopathy of prematurity (ROP), a major cause of childhood blindness, identifying 2 loci at genome-wide significance level (p<5×10-8) and 7 at suggestive significance (p<5×10-6) for ROP ≥ stage 3. The most significant locus, rs2058019, reached genome-wide significance within the full multiethnic cohort (p=4.96×10-9); Hispanic and Caucasian infants driving the association. The lead single nucleotide polymorphism (SNP) falls in an intronic region within the Glioma-associated oncogene family zinc finger 3 (GLI3) gene. Relevance for GLI3 and other top-associated genes to human ocular disease was substantiated through in-silico extension analyses, genetic risk score analysis and expression profiling in human donor eye tissues. Thus, we report the largest ROP GWAS to date, identifying a novel locus at GLI3 with relevance to retinal biology supporting genetic susceptibilities for ROP risk with possible variability by race and ethnicity.

Placental Inflammation Significantly Correlates with Reduced Risk for Retinopathy of Prematurity.

Retinopathy of prematurity (ROP), a blinding condition affecting preterm infants, is an interruption of retinal vascular maturation that is incomplete when born preterm. Although ROP demonstrates delayed onset following preterm birth, representing a window for therapeutic intervention, there are no curative or preventative measures available for this condition. The in utero environment, including placental function, is increasingly recognized for contributions to preterm infant disease risk. The current study identified a protective association between acute placental inflammation and preterm infant ROP development using logistic regression, with the most significant association found for infants without gestational exposure to maternal preeclampsia and those with earlier preterm birth. Expression analysis of proteins with described ROP risk associations demonstrated significantly decreased placental high temperature requirement A serine peptidase-1 (HTRA-1) and fatty acid binding protein 4 protein expression in infants with acute placental inflammation compared with those without. Within the postnatal peripheral circulation, HTRA-1 and vascular endothelial growth factor-A demonstrated inverse longitudinal trends for infants born in the presence of, compared with absence of, acute placental inflammation. An agnostic approach, including whole transcriptome and differential methylation placental analysis, further identify novel mediators and pathways that may underly protection. Taken together, these data build on emerging literature showing a protective association between acute placental inflammation and ROP development and identify novel mechanisms that may inform postnatal risk associations in preterm infants.

Implication of N-Methyl-d-Aspartate Receptor in Homocysteine-Induced Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of vision loss. Elevated homocysteine (Hcy) (Hyperhomocysteinemia) (HHcy) has been reported in AMD. We previously reported that HHcy induces AMD-like features. This study suggests that N-Methyl-d-aspartate receptor (NMDAR) activation in the retinal pigment epithelium (RPE) is a mechanism for HHcy-induced AMD. Serum Hcy and cystathionine-ß-synthase (CBS) were assessed by ELISA. The involvement of NMDAR in Hcy-induced AMD features was evaluated (1) in vitro using ARPE-19 cells, primary RPE isolated from HHcy mice (CBS), and mouse choroidal endothelial cells (MCEC); (2) in vivo using wild-type mice and mice deficient in RPE NMDAR (NMDARR-/-) with/without Hcy injection. Isolectin-B4, Ki67, HIF-1α, VEGF, NMDAR1, and albumin were assessed by immunofluorescence (IF), Western blot (WB), Optical coherence tomography (OCT), and fluorescein angiography (FA) to evaluate retinal structure, fluorescein leakage, and choroidal neovascularization (CNV). A neovascular AMD patient's serum showed a significant increase in Hcy and a decrease in CBS. Hcy significantly increased HIF-1α, VEGF, and NMDAR in RPE cells, and Ki67 in MCEC. Hcy-injected WT mice showed disrupted retina and CNV. Knocking down RPE NMDAR improved retinal structure and CNV. Our findings underscore the role of RPE NMDAR in Hcy-induced AMD features; thus, NMDAR inhibition could serve as a promising therapeutic target for AMD.

Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids.

Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium-retinal organoid-that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.

Long-term outcomes for pediatric patients having transscleral fixation of the capsular bag with intraocular lens for ectopia lentis.

PURPOSE: To report the long-term outcomes of transscleral fixation of capsular tension rings (CTR) with intraocular lens (IOL) placement in pediatric patients with ectopia lentis. SETTING: John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. DESIGN: Retrospective case series. METHODS: Pediatric patients requiring lens extraction with transscleral capsular bag fixation with a CTR and IOL for ectopia lentis between January 2006 and January 2016 were analyzed. RESULTS: Thirty-seven patients (67 eyes) who had transscleral fixation of the capsular bag using a CTR fixated with 9-0 or 10-0 polypropylene (Prolene), 8-0 polytetrafluoroethylene (Gore-Tex), or 9-0 nylon were identified. The mean age at time of surgery was 7.25 years (2 to 18 years) and the mean follow-up was 35.3 months (0.25 to 120 months). The proportion of eyes showing improvement in corrected distance visual acuity (CDVA) postoperatively was 78.5%, which demonstrated significance with a 95% confidence interval. In the immediate postoperative period, 1 eye developed a hyphema and 1 eye required IOL repositioning. Long-term complications included posterior capsule opacification in 35 eyes (52%) and uveitis-glaucoma-hyphema syndrome in 1 eye (1.5%). Three eyes (4.4 %) required IOL repositioning for spontaneous delayed IOL dislocation, 2 sutured with 8-0 polytetrafluoroethylene at postoperative month 8 and postoperative year 3 and 1 sutured with 9-0 polypropylene at postoperative year 7. CONCLUSION: Transscleral fixation of the capsular bag using a CTR improved CDVA and provided IOL stability in pediatric patients with ectopia lentis.

Genetics of age-related macular degeneration (AMD).

Age-related macular degeneration (AMD) is a progressive blinding disease and represents the leading cause of visual impairment in the aging population. AMD affects central vision which impairs one's ability to drive, read and recognize faces. There is no cure for this disease and current treatment modalities for the exudative form of the disease require repeated intravitreal injections which may be painful, are incompletely efficacious, and represent a significant treatment burden for both the patient and physician. As such, AMD represents a significant and important clinical problem.It is anticipated that in three years' time, 196 million individuals will be affected with AMD. Over 250 billion dollars per year are spent on care for AMD patients in the US. Over half of the heritability is explained by two major loci, thus AMD is considered the most well genetically defined of the complex disorders. A recent GWAS on 43,566 subjects identified novel loci and pathways associated with AMD risk, which has provided an excellent platform for additional functional studies. Genetic variants have been investigated, particularly with respect to anti-VEGF treatment, however to date, no pharmacogenomic associations have been consistently identified across these studies. It may be that if the goal of personalized medicine is to be realized and biomarkers are to have predictive value for determining the magnitude of risk for AMD at the genetic level, one will need to examine the relationships between these pathways across disease state and relative to modifiable risk factors such as hypertension, smoking, body mass index, and hypercholesterolemia. Further studies investigating protective alleles in populations with low AMD prevalence may lead to this goal.

Retinopathy of prematurity: A comprehensive risk analysis for prevention and prediction of disease.

BACKGROUND: Retinopathy of prematurity (ROP) is a blinding morbidity of preterm infants. Our current screening criteria have remained unchanged since their inception and lack the ability to identify those at greatest risk. OBJECTIVES: We sought to comprehensively analyze numerous proposed maternal, infant, and environmental ROP risk variables in a robustly phenotyped population using logistic regression to determine the most predictive model for ROP development and severity. We further sought to determine the statistical interaction between significant ROP risk variables, which has not previously been done in the field of ROP. We hypothesize that our comprehensive analysis will allow for better identification of risk variables that independently correlate with ROP disease. Going forward, this may allow for improved infant risk stratification along a time continuum from prenatal to postnatal development, making prevention more feasible. METHODS: We performed a retrospective cohort analysis of preterm infants referred for ROP screening in one neonatal intensive care unit from 2010-2015. The primary outcome measure was presence of ROP. Secondary outcome measures were ROP requiring treatment and severe ROP not clearly meeting current treatment criteria. Univariate, stepwise regression and statistical interaction analyses of 57 proposed ROP risk variables was performed to identify variables which were significantly associated with each outcome measure. RESULTS: We identified 457 infants meeting our inclusion criteria. Within this cohort, numerous factors showed a significant individual association with our ROP outcome measures; however, stepwise regression analysis found the most predictive model for overall ROP risk included estimated gestational age, birth weight, the need for any surgery, and maternal magnesium prophylaxis. The corresponding Area Under the Curve (AUC) for this model was 0.8641, while the traditional model of gestational age and birth weight predicted ROP disease less well with an AUC of 0.8489. Development of severe ROP was best predicted by estimated gestational age (week), the need for any surgery and increased probability of death or moderate-severe BPD at 7 days. Finally, the model most predictive for type 1 ROP included estimated gestational age (week) and the presence of severe chronic lung disease. No significant statistical interaction was found between variables. CONCLUSIONS: Our work is unique as we report comprehensive analysis of the greatest number of proposed ROP risk variables to date in a robustly phenotyped population. We describe novel risk models for our ROP outcome measures and demonstrate independence of these variables using statistical modeling not previously applied to ROP. This may better allow for individual infant risk stratification and importantly mitigation of future risk.

Congenital complex corneal choristoma associated with unilateral bony calvarial defects, subcutaneous nodules, and alopecia.

Complex corneal choristoma is a rare finding and its relationship with systemic disease is poorly understood. We present a 3-day-old boy with the constellation of left-sided congenital complex corneal choristoma, limbal dermoid, ipsilateral bony skull defects, and bilateral subcutaneous vascularized nodules with overlying alopecia and scalp nevi. To our knowledge this is the first case of complex choristoma involving the presence of associated bony calvarial defects. This presentation may contribute to a greater understanding of the systemic findings associated with the condition. Furthermore, it may shed light on the possible syndromic spectrum associated with complex choristoma and its underlying pathophysiology.

FLT1 genetic variation predisposes to neovascular AMD in ethnically diverse populations and alters systemic FLT1 expression.

PURPOSE: Current understanding of the genetic risk factors for age-related macular degeneration (AMD) is not sufficiently predictive of the clinical course. The VEGF pathway is a key therapeutic target for treatment of neovascular AMD; however, risk attributable to genetic variation within pathway genes is unclear. We sought to identify single nucleotide polymorphisms (SNPs) associated with AMD within the VEGF pathway. METHODS: Using a tagSNP, direct sequencing and meta-analysis approach within four ethnically diverse cohorts, we identified genetic risk present in FLT1, though not within other VEGF pathway genes KDR, VEGFA, or VASH1. We used ChIP and ELISA in functional analysis. RESULTS: The FLT1 SNPs rs9943922, rs9508034, rs2281827, rs7324510, and rs9513115 were significantly associated with increased risk of neovascular AMD. Each association was more significant after meta-analysis than in any one of the four cohorts. All associations were novel, within noncoding regions of FLT1 that do not tag for coding variants in linkage disequilibrium. Analysis of soluble FLT1 demonstrated higher expression in unaffected individuals homozygous for the FLT1 risk alleles rs9943922 (P = 0.0086) and rs7324510 (P = 0.0057). In silico analysis suggests that these variants change predicted splice sites and RNA secondary structure, and have been identified in other neovascular pathologies. These data were supported further by murine chromatin immunoprecipitation demonstrating that FLT1 is a target of Nr2e3, a nuclear receptor gene implicated in regulating an AMD pathway. CONCLUSIONS: Although exact variant functions are not known, these data demonstrate relevancy across ethnically diverse genetic backgrounds within our study and, therefore, hold potential for global efficacy.

Soluble mediators of diabetic macular edema: the diagnostic role of aqueous VEGF and cytokine levels in diabetic macular edema.

Diabetic macular edema (DME) is a significant cause of vision loss and represents an important clinical and public health problem. It is characterized by breakdown of the blood retinal barrier with fluid accumulation in the sub-retinal and intra-retinal spaces. Although several hypotheses exist for the causes of diabetic macular edema, specific molecular mechanisms remain unclear. Current thinking includes the role of vascular endothelial growth factor (VEGF) and inflammatory cytokines in vascular permeability. We review studies showing a relationship between elevated aqueous VEGF, monocyte chemoattractant protein -1, interleukin 6, or interleukin 8 in association with DME and as predictors of DME. The presence of mediators in both the angiogenesis and inflammatory pathways data suggest a multifactorial model for the development of DME. Further studies targeting individual cytokine activity will be important to our understanding of the pathogenesis and treatment.

Morpholino-mediated increase in soluble Flt-1 expression results in decreased ocular and tumor neovascularization.

BACKGROUND: Angiogenesis is a key process in several ocular disorders and cancers. Soluble Flt-1 is an alternatively spliced form of the Flt-1 gene that retains the ligand-binding domain, but lacks the membrane-spanning and intracellular kinase domains of the full-length membrane bound Flt-1 (mbFlt-1) protein. Thus, sFlt-1 is an endogenous inhibitor of VEGF-A mediated angiogenesis. Synthetic mopholino oligomers directed against splice site targets can modulate splice variant expression. We hypothesize that morpholino-induced upregulation of sFlt-1 will suppress angiogenesis in clinically relevant models of macular degeneration and breast cancer. METHODS AND FINDINGS: In vivo morpholino constructs were designed to target murine exon/intron 13 junction of the Flt-1 transcript denoted VEGFR1_MOe13; standard nonspecific morpholino was used as control. After nucleofection of endothelial and breast adenocarcinoma cell lines, total RNA was extracted and real-time RT-PCR performed for sFlt-1 and mbFlt-1. Intravitreal injections of VEGFR1_MOe13 or control were done in a model of laser-induced choroidal neovascularization and intratumoral injections were performed in MBA-MD-231 xenografts in nude mice. VEGFR1_MOe13 elevated sFlt-1 mRNA expression and suppressed mbFlt-1 mRNA expression in vitro in multiple cellular backgrounds (p<0.001). VEGFR1_MOe13 also elevated sFlt/mbFlt-1 ratio in vivo after laser choroidal injury 5.5 fold (p<0.001) and suppressed laser-induced CNV by 50% (p = 0.0179). This latter effect was reversed by RNAi of sFlt-1, confirming specificity of morpholino activity through up-regulation of sFlt-1. In the xenograft model, VEGFR1_MOe13 regressed tumor volume by 88.9%, increased sFlt-1 mRNA expression, and reduced vascular density by 50% relative to control morpholino treatment (p<0.05). CONCLUSIONS: Morpholino oligomers targeting the VEGFR1 mRNA exon/intron 13 junction promote production of soluble FLT-1 over membrane bound FLT-1, resulting in suppression of lesional volume in laser induced CNV and breast adenocarcinoma. Thus, morpholino manipulation of alternative splicing offers translational potential for therapy of angiogenic disorders.

EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma.

EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip) revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease.

Identification of target genes in their native cellular context: an analysis of EWS/FLI in Ewing's sarcoma.

Ewing's sarcoma is the second most common tumor of bone in children and young adults, and requires highly intensive chemotherapy along with surgery and/or radiation for successful treatment. Because these therapies are associated with significant short- and long-term side effects, new therapeutic approaches are needed. Most cases of Ewing's sarcoma contain somatic translocations between chromosomes 11 and 22 that result in the t(11;22)(q24;q12). This translocation encodes the EWS/FLI fusion protein. EWS/FLI formation appears to be the critical oncogenic event in the development of Ewing's sarcoma. It is hoped that an in-depth understanding of the mechanism that EWS/FLI uses to cause oncogenic transformation will result in new therapies for this disease. Unfortunately, this hope has not been realized. One difficulty has been the lack of an appropriate model system in which to study the fusion oncoprotein. We recently described and validated the use of retroviral RNA interference approaches to study EWS/FLI in Ewing's sarcoma cell lines. We now put this model into a historical context, and describe the benefits (both perceived and observed) of this model over previous approaches using heterologous cell types.

Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma.

Our understanding of Ewing's sarcoma development mediated by the EWS/FLI fusion protein has been limited by a lack of knowledge regarding the tumor cell of origin. To circumvent this, we analyzed the function of EWS/FLI in Ewing's sarcoma itself. By combining retroviral-mediated RNA interference with reexpression studies, we show that ongoing EWS/FLI expression is required for the tumorigenic phenotype of Ewing's sarcoma. We used this system to define the full complement of EWS/FLI-regulated genes in Ewing's sarcoma. Functional analysis revealed that NKX2.2 is an EWS/FLI-regulated gene that is necessary for oncogenic transformation in this tumor. Thus, we developed a highly validated transcriptional profile for the EWS/FLI fusion protein and identified a critical target gene in Ewing's sarcoma development.