The GGLEAM Study: Understanding Glaucoma in the Ohio Amish.

Glaucoma leads to millions of cases of visual impairment and blindness around the world. Its susceptibility is shaped by both environmental and genetic risk factors. Although over 120 risk loci have been identified for glaucoma, a large portion of its heritability is still unexplained. Here we describe the foundation of the Genetics of GLaucoma Evaluation in the AMish (GGLEAM) study to investigate the genetic architecture of glaucoma in the Ohio Amish, which exhibits lower genetic and environmental heterogeneity compared to the general population. To date, we have enrolled 81 Amish individuals in our study from Holmes County, Ohio. As a part of our enrollment process, 62 GGLEAM study participants (42 glaucoma-affected and 20 unaffected individuals) received comprehensive eye examinations and glaucoma evaluations. Using the data from the Anabaptist Genealogy Database, we found that 80 of the GGLEAM study participants were related to one another through a large, multigenerational pedigree containing 1586 people. We plan to integrate the health and kinship data obtained for the GGLEAM study to interrogate glaucoma genetics and pathophysiology in this unique population.

Mutation of FOXC1 and PITX2 induces cerebral small-vessel disease.

Patients with cerebral small-vessel disease (CSVD) exhibit perturbed end-artery function and have an increased risk for stroke and age-related cognitive decline. Here, we used targeted genome-wide association (GWA) analysis and defined a CSVD locus adjacent to the forkhead transcription factor FOXC1. Moreover, we determined that the linked SNPs influence FOXC1 transcript levels and demonstrated that patients as young as 1 year of age with altered FOXC1 function exhibit CSVD. MRI analysis of patients with missense and nonsense mutations as well as FOXC1-encompassing segmental duplication and deletion revealed white matter hyperintensities, dilated perivascular spaces, and lacunar infarction. In a zebrafish model, overexpression or morpholino-induced suppression of foxc1 induced cerebral hemorrhage. Inhibition of foxc1 perturbed platelet-derived growth factor (Pdgf) signaling, impairing neural crest migration and the recruitment of mural cells, which are essential for vascular stability. GWA analysis also linked the FOXC1-interacting transcription factor PITX2 to CSVD, and both patients with PITX2 mutations and murine Pitx2-/- mutants displayed brain vascular phenotypes. Together, these results extend the genetic etiology of stroke and demonstrate an increasing developmental basis for human cerebrovascular disease.

FoxC1 is essential for vascular basement membrane integrity and hyaloid vessel morphogenesis.

PURPOSE: Alterations in FOXC1 dosage lead to a spectrum of highly penetrant, ocular anterior segment dysgenesis phenotypes. The most serious outcome is the development of glaucoma, which occurs in 50% to 75% of patients. Therefore, the need to identify specific pathways and genes that interact with FOXC1 to promote glaucoma is great. In this study, the authors investigated the loss of foxC1 in the zebrafish to characterize phenotypes and gene interactions that may impact glaucoma pathogenesis. METHODS: Morpholino knockdown in zebrafish, RNA and protein marker analyses, transgenic reporter lines, and angiography, along with histology and transmission electron microscopy, were used to study foxC1 function and gene interactions. RESULTS: Zebrafish foxC1 genes were expressed dynamically in the developing vasculature and periocular mesenchyme during development. Multiple ocular and vascular defects were found after the knockdown of foxC1. Defects in the hyaloid vasculature, arteriovenous malformations, and coarctation of the aorta were observed with maximal depletion of foxC1. Partial loss of foxC1 resulted in CNS and ocular hemorrhages, defects in intersegmental vessel patterning, and increased vascular permeability. To investigate the basis for these disruptions, the ultrastructure of foxC1-depleted hyaloid vascular cells was studied. These experiments, along with laminin-111 immunoreactivity, revealed disruptions in basement membrane integrity. Finally, codepletion of laminin alpha-1 and foxC1 uncovered a genetic interaction between these genes during development. CONCLUSIONS: Genetic interactions between FOXC1 and basement membrane components influence vascular stability and may impact glaucoma development and increase stroke risk in FOXC1 patients.

The primary open-angle glaucoma gene WDR36 functions in ribosomal RNA processing and interacts with the p53 stress-response pathway.

Primary open-angle glaucoma (POAG) is a genetically complex neuropathy that affects retinal ganglion cells and is a leading cause of blindness worldwide. WDR36, a gene of unknown function, was recently identified as causative for POAG at locus GLC1G. Subsequent studies found disease-associated variants in control populations, leaving the role of WDR36 in this disease unclear. To address this issue, we determined the function of WDR36. We studied Wdr36 in zebrafish and found it is the functional homolog of yeast Utp21. Utp21 is cell essential and functions in the nucleolar processing of 18S rRNA, which is required for ribosome biogenesis. Evidence for functional homology comes from sequence alignment, ubiquitous expression, sub-cellular localization to the nucleolus and loss-of-function phenotypes that include defects in 18S rRNA processing and abnormal nucleolar morphology. Additionally, we show that loss of Wdr36 function leads to an activation of the p53 stress-response pathway, suggesting that co-inheritance of defects in p53 pathway genes may influence the impact of WDR36 variants on POAG. Although these results overall do not provide evidence for or against a role of WDR36 in POAG, they do provide important baseline information for future studies.

FOXC1 is required for cell viability and resistance to oxidative stress in the eye through the transcriptional regulation of FOXO1A.

Mutations in the human FOXC1 transcription factor gene underlie Axenfeld-Rieger (AR) syndrome, a disorder characterized by anterior segment malformations in the eye and glaucoma. Through the use of an inducible FOXC1 protein, along with an intermediate protein synthesis blocker, we have determined direct targets of FOXC1 transcriptional regulation. FOXC1 regulates the expression of FOXO1A and binds to a conserved element in the FOXO1A promoter in vivo. The zebrafish foxO1a orthologs exhibit a robust expression pattern in the periocular mesenchyme. Furthermore, FOXO1A expression is reduced in cultured human trabecular meshwork (TM) cells and in the zebrafish developing eye when FOXC1 expression is knocked down by siRNAs and morpholino antisense oliognucleotides, respectively. We also demonstrate that reduced FOXC1 expression increases cell death in cultured TM cells in response to oxidative stress, and increases cell death in the developing zebrafish eye. These studies have uncovered a novel role for FOXC1 as an essential mediator of cellular homeostasis in the eye and indicate that a decreased resistance to oxidative stress may underlie AR-glaucoma pathogenesis. Given that FOXO1A influences cellular homeostasis when positively or negatively regulated; the dysregulation of FOXO1A activities in the eye through FOXC1 loss of function mutations and FOXC1 gene duplications provides an explanation into how seemingly similar human disorders can arise from both increases and decreases in FOXC1 gene dose.

FGF19 is a target for FOXC1 regulation in ciliary body-derived cells.

The forkhead C1 (FOXC1) transcription factor is involved in the development and regulation of several organs, including the eye, where FOXC1 alterations cause iris, trabecular meshwork and corneal anomalies. Using nickel agarose chromatin enrichment with human anterior segment cells, we previously identified the fibroblast growth factor 19 (FGF19) locus as a gene potentially regulated by FOXC1. Here, we demonstrate that FGF19 is a direct target of FOXC1 in the eye. FOXC1 positively regulates FGF19 expression in corneal and periocular mesenchymal cells in cell culture and in zebrafish embryos. Through the FGFR4 tyrosine kinase, FGF19 promotes MAPK phosphorylation in the developing and mature cornea. During development, loss of either FOXC1 or FGF19 results in complementary, but distinct, anterior segment dysgeneses. This study reveals an important role for FOXC1 in the direct regulation of the FGF19-FGFR4-MAPK pathway to promote both the development and maintenance of anterior segment structures within the eye.