Advances in cell therapies using stem cells/progenitors as a novel approach for neurovascular repair of the diabetic retina.

BACKGROUND: Diabetic retinopathy, a major complication of diabetes mellitus, is a leading cause of sigh-loss in working age adults. Progressive loss of integrity of the retinal neurovascular unit is a central element in the disease pathogenesis. Retinal ischemia and inflammatory processes drive interrelated pathologies such as blood retinal barrier disruption, fluid accumulation, gliosis, neuronal loss and/or aberrant neovascularisation. Current treatment options are somewhat limited to late-stages of the disease where there is already significant damage to the retinal architecture arising from degenerative, edematous and proliferative pathology. New preventive and interventional treatments to target early vasodegenerative and neurodegenerative stages of the disease are needed to ensure avoidance of sight-loss. MAIN BODY: Historically, diabetic retinopathy has been considered a primarily microvascular disease of the retina and clinically it is classified based on the presence and severity of vascular lesions. It is now known that neurodegeneration plays a significant role during the pathogenesis. Loss of neurons has been documented at early stages in pre-clinical models as well as in individuals with diabetes and, in some, even prior to the onset of clinically overt diabetic retinopathy. Recent studies suggest that some patients have a primarily neurodegenerative phenotype. Retinal pigment epithelial cells and the choroid are also affected during the disease pathogenesis and these tissues may also need to be addressed by new regenerative treatments. Most stem cell research for diabetic retinopathy to date has focused on addressing vasculopathy. Pre-clinical and clinical studies aiming to restore damaged vasculature using vasoactive progenitors including mesenchymal stromal/stem cells, adipose stem cells, CD34+ cells, endothelial colony forming cells and induced pluripotent stem cell derived endothelial cells are discussed in this review. Stem cells that could replace dying neurons such as retinal progenitor cells, pluripotent stem cell derived photoreceptors and ganglion cells as well as Müller stem cells are also discussed. Finally, challenges of stem cell therapies relevant to diabetic retinopathy are considered. CONCLUSION: Stem cell therapies hold great potential to replace dying cells during early and even late stages of diabetic retinopathy. However, due to the presence of different phenotypes, selecting the most suitable stem cell product for individual patients will be crucial for successful treatment.

Generation and characterization of iPSC-derived renal proximal tubule-like cells with extended stability.

The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.

miR-130a activates the VEGFR2/STAT3/HIF1α axis to potentiate the vasoregenerative capacity of endothelial colony-forming cells in hypoxia.

Hypoxia modulates reparative angiogenesis, which is a tightly regulated pathophysiological process. MicroRNAs (miRNAs) are important regulators of gene expression in hypoxia and angiogenesis. However, we do not yet have a clear understanding of how hypoxia-induced miRNAs fine-tune vasoreparative processes. Here, we identify miR-130a as a mediator of the hypoxic response in human primary endothelial colony-forming cells (ECFCs), a well-characterized subtype of endothelial progenitors. Under hypoxic conditions of 1% O2, miR-130a gain-of-function enhances ECFC pro-angiogenic capacity in vitro and potentiates their vasoreparative properties in vivo. Mechanistically, miR-130a orchestrates upregulation of VEGFR2, activation of STAT3, and accumulation of HIF1α via translational inhibition of Ddx6. These findings unveil a new role for miR-130a in hypoxia, whereby it activates the VEGFR2/STAT3/HIF1α axis to enhance the vasoregenerative capacity of ECFCs.

Plasma level of lipocalin 2 is increased in neovascular age-related macular degeneration patients, particularly those with macular fibrosis.

BACKGROUND: Previously, we and others have reported higher populations of circulating neutrophils in patients with neovascular age-related macular degeneration (nAMD). Neutrophil gelatinase-associated lipocalin (NGAL, also known as lipocalin-2, LCN2), an important innate immune mediator, is known to be critically involved in sterile inflammation-mediated organ failure, fibrosis, cancer progression and retinal degeneration. This study investigated the plasma levels of LCN2, matrix metalloproteinase 9 (MMP9) and LCN2/MMP9 complex in different types of nAMD and examined whether the levels were related to patients' responsiveness to anti-VEGF therapy. RESULTS: One hundred and seventy-four nAMD patients, including 108 with choroidal neovascularisation (CNV), 32 with retinal angiomatous proliferation (RAP), 23 with polypoidal choroidal vasculopathy (PCV) and 11 unclassified patients, and 43 healthy controls were recruited to this case-control study. Fifty-eight nAMD patients had macular fibrosis and 110 patients did not. Out of the 174 nAMD patients, 80 patients responded completely, 90 responded partially, and 4 did not respond to the anti-VEGF therapy. The plasma levels of LCN2 in nAMD patients (181.46 ± 73.62 ng/ml) was significantly higher than that in healthy controls (152.24 ± 49.55 ng/ml, P = 0.047). However, the difference disappeared after adjusting for age. A positive correlation between plasma level of LCN2 and age was observed in nAMD patients (r = 0.29, P = 0.0002) but not in healthy controls. The plasma level of LCN2 was also positively correlated with circulating neutrophils in nAMD patients (r = 0.34, p = 0.0007) but not in healthy controls (r = 0.057, p = 0.77). No correlation was observed between age and circulating neutrophils. Further analysis of nAMD subtypes uncovered a significantly higher level of LCN2 in patients with macular fibrosis even after adjusting for age. No relationship was observed between plasma levels of LCN2 and patients' responsiveness to anti-VEGF therapy. The plasma levels of MMP9 and LCN2/MMP9 complex were comparable between nAMD and controls. CONCLUSIONS: Our results suggest that higher plasma levels of LCN2 in nAMD are related to ageing and increased population of circulating neutrophils. Our results also suggest that higher levels of LCN2 may increase the risk of macular fibrosis in nAMD.

VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions.

Purpose: Müller glia are important in retinal health and disease and are a major source of retinal VEGF-A. Of the different VEGF family members, the role of VEGF-A in retinal health and disease has been studied extensively. The potential contribution of other VEGF family members to retinal pathophysiology, however, remains poorly defined. This study aimed to understand the role of VEGF-B in Müller cell pathophysiology. Methods: The expression of different VEGFs and their receptors in human MIO-M1 and mouse QMMuC-1 Müller cell lines and primary murine Müller cells was examined by RT-PCR, ELISA, and Western blot. The effect of recombinant VEGF-B or VEGF-B neutralization on Müller cell viability and survival under normal, hypoxic, and oxidative (4-hydroxynonenal [4-HNE]) conditions was evaluated by Alamar Blue, Yo-Pro uptake, and immunocytochemistry. The expression of glial fibrillary acidic protein, aquaporin-4, inward rectifying K+ channel subtype 4.1, glutamine synthetase, and transient receptor potential vanilloid 4 under different treatment conditions was examined by RT-PCR, immunocytochemistry, and Western blot. Transient receptor potential vanilloid 4 channel activity was assessed using a Fura-2-based calcium assay. Results: VEGF-B was expressed in Müller cells at the highest levels compared with other members of the VEGF family. VEGF-B neutralization did not affect Müller cell viability or functionality under normal conditions, but enhanced hypoxia- or 4-HNE-induced Müller cell death and decreased inward rectifying K+ channel subtype 4.1 and aquaporin-4 expression. Recombinant VEGF-B restored Müller cell glutamine synthetase expression under hypoxic conditions and protected Müller cells from 4-HNE-induced damage by normalizing transient receptor potential vanilloid 4 channel expression and activity. Conclusions: Autocrine production of VEGF-B protects Müller cells under pathologic conditions.

Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors.

Preclinical research using different rodent model systems has largely contributed to the scientific progress in the pain field, however, it suffers from interspecies differences, limited access to human models, and ethical concerns. Human induced pluripotent stem cells (iPSCs) offer major advantages over animal models, i.e., they retain the genome of the donor (patient), and thus allow donor-specific and cell-type specific research. Consequently, human iPSC-derived nociceptors (iDNs) offer intriguingly new possibilities for patient-specific, animal-free research. In the present study, we characterized iDNs based on the expression of well described nociceptive markers and ion channels, and we conducted a side-by-side comparison of iDNs with mouse sensory neurons. Specifically, immunofluorescence (IF) analyses with selected markers including early somatosensory transcription factors (BRN3A/ISL1/RUNX1), the low-affinity nerve growth factor receptor (p75), hyperpolarization-activated cyclic nucleotide-gated channels (HCN), as well as high voltage-gated calcium channels (VGCC) of the CaV2 type, calcium permeable TRPV1 channels, and ionotropic GABAA receptors, were used to address the characteristics of the iDN phenotype. We further combined IF analyses with microfluorimetric Ca2+ measurements to address the functionality of these ion channels in iDNs. Thus, we provide a detailed morphological and functional characterization of iDNs, thereby, underpinning their enormous potential as an animal-free alternative for human specific research in the pain field for unveiling pathophysiological mechanisms and for unbiased, disease-specific personalized drug development.

Interdigitated aluminium and titanium sensors for assessing epithelial barrier functionality by electric cell-substrate impedance spectroscopy (ECIS).

Electric cell-substrate impedance spectroscopy (ECIS) enables non-invasive and continuous read-out of electrical parameters of living tissue. The aim of the current study was to investigate the performance of interdigitated sensors with 50 µm electrode width and 50 µm inter-electrode distance made of gold, aluminium, and titanium for monitoring the barrier properties of epithelial cells in tissue culture. At first, the measurement performance of the photolithographic fabricated sensors was characterized by defined reference electrolytes. The sensors were used to monitor the electrical properties of two adherent epithelial barrier tissue models: renal proximal tubular LLC-PK1 cells, representing a normal functional transporting epithelium, and human cervical cancer-derived HeLa cells, forming non-transporting cancerous epithelial tissue. Then, the impedance spectra obtained were analysed by numerically fitting the parameters of the two different models to the measured impedance spectrum. Aluminium sensors proved to be as sensitive and consistent in repeated online-recordings for continuous cell growth and differentiation monitoring as sensors made of gold, the standard electrode material. Titanium electrodes exhibited an elevated intrinsic ohmic resistance in comparison to gold reflecting its lower electric conductivity. Analysis of impedance spectra through applying models and numerical data fitting enabled the detailed investigation of the development and properties of a functional transporting epithelial tissue using either gold or aluminium sensors. The result of the data obtained, supports the consideration of aluminium and titanium sensor materials as potential alternatives to gold sensors for advanced application of ECIS spectroscopy.

Sustained intraocular vascular endothelial growth factor neutralisation does not affect retinal and choroidal vasculature in Ins2Akita diabetic mice.

The purpose of this study was to understand the influence of sustained intravitreal vascular endothelial growth factor neutralisation on the retinal and choroidal vasculature in diabetic eyes. Ins2Akita diabetic mice received five intravitreal injections of anti-mouse vascular endothelial growth factor antibody or goat immunoglobulin G (0.2 µg/µL/eye) over a 4-month period. Retinal and choroidal vascular changes were analysed by confocal microscopy of tissue flat-mounts. Retinal gene expression of vascular endothelial growth factor family members (vascular endothelial growth factors A, B, C and D), vascular endothelial growth factor receptors (sVEGFR-1 and VEGFR-2) and tight junctions (claudin 1, 2, 5; occludin and zonula occludens-1) were analysed by quantitative reverse transcription polymerase chain reaction. Vascular endothelial growth factor A and claudin 5 were significantly increased in diabetic retinae. Gene expression was unaffected by anti-vascular endothelial growth factor treatment. The number of acellular vessels was increased in diabetic retinae and reduced following anti-vascular endothelial growth factor treatment. Retinal and choroidal vascular density and area were unaffected by sustained vascular endothelial growth factor neutralisation. Our results suggest that five consecutive intravitreal anti-vascular endothelial growth factor injections do not cause significant vascular changes in the retina and choroid in diabetic and non-diabetic mice.

Topical Delivery of Anti-VEGF Drugs to the Ocular Posterior Segment Using Cell-Penetrating Peptides.

Purpose: To evaluate the efficacy of anti-VEGF agents for treating choroidal neovascularization (CNV) when delivered topically using novel cell-penetrating peptides (CPPs) compared with delivery by intravitreal (ivit) injection. Methods: CPP toxicity was investigated in cell cultures. Ivit concentrations of ranibizumab and bevacizumab after topical administration were measured using ELISA. The biological efficacy of topical anti-VEGF + CPP complexes was compared with ivit anti-VEGF injections using an established model of CNV. Results: CPPs were nontoxic in vitro. In vivo, after topical eye drop delivery, CPPs were present in the rat anterior chamber within 6 minutes. A single application of CPP + bevacizumab eye drop delivered clinically relevant concentrations of bevacizumab to the posterior chamber of the rat eye in vivo. Similarly, clinically relevant levels of CPP + ranibizumab and CPP + bevacizumab were detected in the porcine vitreous and retina ex vivo. In an established model of CNV, mice treated with either a single ivit injection of anti-VEGF, twice daily CPP + anti-VEGF eye drops or daily dexamethasone gavage for 10 days all had significantly reduced areas of CNV when compared with lasered eyes without treatment. Conclusions: CPPs are nontoxic to ocular cells and can be used to deliver therapeutically relevant doses of ranibizumab and bevacizumab by eye drop to the posterior segment of mouse, rat, and pig eyes. The CPP + anti-VEGF drug complexes were cleared from the retina within 24 hours, suggesting a daily eye drop dosing regimen. Daily, topically delivered anti-VEGF with CPP was as efficacious as a single ivit injection of anti-VEGF in reducing areas of CNV in vivo.

The pathology associated with diabetic retinopathy.

This review summarizes the pathological features of diabetic retinopathy. The lesions occurring in the diabetic retina have been described over many decades using descriptive and experimental approaches based on clinical studies on patients, human post-mortem material, animal models and various in vitro systems. We have also accumulated a wealth of knowledge about basic molecular mechanisms and key pathogenic processes that drive these abnormalities in diabetic retina. Despite these advances, there are still limited therapeutic options for diabetic retinopathy with those currently available only addressing late-stage disease. With a particular focus on the earlier stages of diabetes, there is growing appreciation the complex neuronal, glial and microvascular abnormalities which progressively disrupt retinal function. This is especially true from the perspective of the neurovascular unit during health and disease. Based on a strong appreciation of cellular and molecular pathology that underpins diabetic retinopathy, further advances are anticipated as we drive towards development of efficacious therapeutic options that can address all stages of disease.

Peripheral blood mononuclear cells from neovascular age-related macular degeneration patients produce higher levels of chemokines CCL2 (MCP-1) and CXCL8 (IL-8).

BACKGROUND: Infiltrating immune cells including monocytes/macrophages have been implicated in the pathogenesis of neovascular age-related macular degeneration (nAMD). The aim of this study was to investigate the cytokine and chemokine expression and secretion profile of peripheral blood mononuclear cells (PBMCs) from nAMD patients and the relationship between the cytokine/chemokine expression profile and clinical phenotype of nAMD, including macular fibrosis, macular atrophy or the responsiveness to anti-VEGF therapy. METHODS: One hundred sixty-one nAMD patients and 43 controls were enrolled in this study. nAMD patients were divided into subgroups based on the presence/absence of (1) macular atrophy, (2) macular fibrosis and (3) responsiveness to anti-VEGF therapy; 25-30 ml of peripheral blood were obtained from all participants and 5 ml were used for serum collection, and the remaining were used for PBMC isolation using density gradient centrifugation. Intracellular cytokine expressions by PBMCs following phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation were examined using flow cytometry. Cytokine productions in lipopolysaccharides (LPS)-or 1% oxygen -treated PBMC were measured using cytometric bead array (CBA) assay. In addition, cytokine and chemokine levels in the serum were also measured by CBA assay. RESULTS: PBMCs from nAMD patients secreted higher levels of IL-8, CCL2 and VEGF, especially following LPS and 1% oxygen stimulation, than those from controls. 60~80% of IL-8 producing cells were CD11b+CD3- monocytes. The percentage of CD11b+CD3- IL-8+ was significantly increased in nAMD patients compared to controls. PBMCs from nAMD patients without macular fibrosis produced the highest levels of IL-8 and CCL2, whilst PBMCs from nAMD patients with macular atrophy produced highest levels of VEGF. In addition, PBMCs from patients who partially responded to anti-VEGF produced higher levels of IL-8 compared to the cells from complete responders. Interestingly, serum level of CCL2 was not increased in nAMD patients although there was a trend of increased IL-8 in nAMD patients. CONCLUSIONS: PBMCs, in particular monocytes, may contribute to CNV development in nAMD through secreting elevated levels of IL-8, CCL2 and VEGF after they are recruited to the macula. Apart from VEGF, IL-8 and CCL2 may be additional targets for nAMD management.

Sex Differences in Renal Proximal Tubular Cell Homeostasis.

Studies in human patients and animals have revealed sex-specific differences in susceptibility to renal diseases. Because actions of female sex hormones on normal renal tissue might protect against damage, we searched for potential influences of the female hormone cycle on basic renal functions by studying excretion of urinary marker proteins in healthy human probands. We collected second morning spot urine samples of unmedicated naturally ovulating women, postmenopausal women, and men daily and determined urinary excretion of the renal tubular enzymes fructose-1,6-bisphosphatase and glutathione-S-transferase-α Additionally, we quantified urinary excretion of blood plasma proteins α1-microglobulin, albumin, and IgG. Naturally cycling women showed prominent peaks in the temporal pattern of urinary fructose-1,6-bisphosphatase and glutathione-S-transferase-α release exclusively within 7 days after ovulation or onset of menses. In contrast, postmenopausal women and men showed consistently low levels of urinary fructose-1,6-bisphosphatase excretion over comparable periods. We did not detect changes in urinary α1-microglobulin, albumin, or IgG excretion. Results of this study indicate that proximal tubular tissue architecture, representing a nonreproductive organ-derived epithelium, undergoes periodical adaptations phased by the female reproductive hormone cycle. The temporally delimited higher rate of enzymuria in ovulating women might be a sign of recurring increases of tubular cell turnover that potentially provide enhanced repair capacity and thus, higher resistance to renal damage.

Higher plasma levels of complement C3a, C4a and C5a increase the risk of subretinal fibrosis in neovascular age-related macular degeneration: Complement activation in AMD.

BACKGROUND: The aim of this study was to investigate the plasma levels of complement C3a, C4a, and C5a in different types of neovascular age-related macular degeneration (nAMD) and whether the levels were related to patients' responsiveness to anti-VEGF therapy. RESULTS: Ninety-six nAMD patients (including 61 with choroidal neovascularisation (CNV), 17 with retinal angiomatous proliferation (RAP), 14 with polypoidal choroidal vasculopathy (PCV) and 4 unclassified patients) and 43 controls were recruited to this case-control study. Subretinal fibrosis was observed in 45 nAMD patients and was absent in 51 nAMD patients. In addition, the responsiveness to anti-VEGF (Lucentis) therapy was also evaluated in nAMD patients. Forty-four patients were complete responders, 48 were partially responders, and only 4 patients did not respond to the therapy. The plasma levels of C3a, C4a and C5a were significantly higher in nAMD patients compared to controls. Further analysis of nAMD subgroups showed that the levels of C3a, C4a and C5a were significantly increased in patients with CNV but not RAP and PCV. Significantly increased levels of C3a, C4a and C5a were also observed in nAMD patients with subretinal fibrosis but not in those without subretinal fibrosis. Higher levels of C3a were observed in nAMD patients who responded partially to anti-VEGF therapy. CONCLUSIONS: Our results suggest increased systemic complement activation in nAMD patients with CNV but not RAP and PCV. Our results also suggest that higher levels of systemic complement activation may increase the risk of subretinal fibrosis in nAMD patients.

Alterations in Circulating Immune Cells in Neovascular Age-Related Macular Degeneration.

Neovascular age-related macular degeneration (nAMD) is the leading cause of irreversible blindness in developed countries. Recent advances have highlighted the essential role of inflammation in the development of the disease. In addition to local retinal chronic inflammatory response, systemic immune alterations have also been observed in AMD patients. In this study we investigated the association between the frequency of circulating leukocyte populations and the prevalence as well as clinical presentations of nAMD. Leukocyte subsets of 103 nAMD patients (most of them were receiving anti-VEGF therapy prior to enrolment) and 26 controls were analysed by flow cytometry by relative cell size, granularity and surface markers. Circulating CD11b(+) cells and CD16(hi)HLA-DR(-) neutrophils were significantly increased (P = 0.015 and 0.009 respectively) in nAMD when compared to controls. The percentage of circulating CD4(+) T-cells was reduced in nAMD patients without subretinal fibrosis (P = 0.026) compared to patients with subretinal fibrosis. There was no correlation between the percentage of circulating leukocytes and the responsiveness to anti-VEGF therapy in nAMD patients. Our results suggest that higher levels of circulating CD11b(+) cells and neutrophils are associated with nAMD and that reduced levels of CD4(+) T-cells are associated with the absence of subretinal fibrosis in nAMD.

Whole-mitochondrial genome sequencing in primary open-angle glaucoma using massively parallel sequencing identifies novel and known pathogenic variants.

PURPOSE: The aim of this study was to determine whether mutations in mitochondrial DNA play a role in high-pressure primary open-angle glaucoma (OMIM 137760) by analyzing new data from massively parallel sequencing of mitochondrial DNA. METHODS: Glaucoma patients with high-tension primary open-angle glaucoma and ethnically matched and age-matched control subjects without glaucoma were recruited. The entire human mitochondrial genome was amplified in two overlapping fragments by long-range polymerase chain reaction and used as a template for massively parallel sequencing on an Ion Torrent Personal Genome Machine. All variants were confirmed by conventional Sanger sequencing. RESULTS: Whole-mitochondrial genome sequencing was performed in 32 patients with primary open-angle glaucoma from India (n = 16) and Ireland (n = 16). In 16 of the 32 patients with primary open-angle glaucoma (50% of cases), there were 22 mitochondrial DNA mutations consisting of 7 novel mutations and 8 previously reported disease-associated sequence variants. Eight of 22 (36.4%) of the mitochondrial DNA mutations were in complex I mitochondrial genes. CONCLUSION: Massively parallel sequencing using the Ion Torrent Personal Genome Machine with confirmation by Sanger sequencing detected a pathogenic mitochondrial DNA mutation in 50% of the primary open-angle glaucoma cohort. Our findings support the emerging concept that mitochondrial dysfunction results in the development of glaucoma and, more specifically, that complex I defects play a significant role in primary open-angle glaucoma pathogenesis.

Enrichment of pathogenic alleles in the brittle cornea gene, ZNF469, in keratoconus.

Keratoconus, a common inherited ocular disorder resulting in progressive corneal thinning, is the leading indication for corneal transplantation in the developed world. Genome-wide association studies have identified common SNPs 100 kb upstream of ZNF469 strongly associated with corneal thickness. Homozygous mutations in ZNF469 and PR domain-containing protein 5 (PRDM5) genes result in brittle cornea syndrome (BCS) Types 1 and 2, respectively. BCS is an autosomal recessive generalized connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Some individuals with heterozygous PRDM5 mutations demonstrate a carrier ocular phenotype, which includes a mildly reduced corneal thickness, keratoconus and blue sclera. We hypothesized that heterozygous variants in PRDM5 and ZNF469 predispose to the development of isolated keratoconus. We found a significant enrichment of potentially pathologic heterozygous alleles in ZNF469 associated with the development of keratoconus (P = 0.00102) resulting in a relative risk of 12.0. This enrichment of rare potentially pathogenic alleles in ZNF469 in 12.5% of keratoconus patients represents a significant mutational load and highlights ZNF469 as the most significant genetic factor responsible for keratoconus identified to date.

Mutational analysis of MIR184 in sporadic keratoconus and myopia.

PURPOSE: A mutation miR-184(+57C>T) in the seed region of miR-184 (encoded by MIR184 [MIM*613146]) results in familial severe keratoconus combined with early-onset anterior polar cataract and endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning (EDICT) syndrome (MIM#614303). In order to investigate the phenotypic spectrum resulting from MIR184 mutation, MIR184 was sequenced in a keratoconus cohort of mixed ethnicity and a Chinese axial myopia cohort. METHODS: Sequencing of MIR184 was performed in 780 unrelated keratoconus patients and 96 unrelated Han southern Chinese subjects with axial myopia. Effects of identified mutations on RNA secondary structure were predicted computationally using mFold and RNAFold algorithms. MIR184 amplicons from patients harboring mutations were cloned and transfected into human embryonic kidney 293T (HEK293T) cells, and mature mutant miR-184 expression was analyzed by stem-loop real-time quantitative PCR (RT-qPCR). RESULTS: Two novel heterozygous substitution mutations in MIR184 were identified in the two patients with isolated keratoconus: miR-184(+8C>A) and miR-184(+3A>G). Computational modeling predicted that these mutations would alter the miR-184 stem-loop stability and secondary structure. Ex vivo miR-184 expression analysis demonstrated that miR-184(+8C>A) almost completely repressed the expression of miR-184 (P = 0.022), and miR-184(+3A>G) reduced the expression of miR-184 by approximately 40% (P = 0.002). There was no significant association of rs41280052, which lies within the stem-loop of miR-184, with keratoconus. No MIR184 mutations were detected in the axial myopia cohort. CONCLUSIONS: Two novel heterozygous substitution mutations in MIR184 were identified in two patients with isolated keratoconus: miR-184(+8C>A) and miR-184(+3A>G). Mutations in MIR184 are a rare cause of keratoconus and were found in 2 of 780 (0.25%) cases.

Mutational spectrum of the ZEB1 gene in corneal dystrophies supports a genotype-phenotype correlation.

PURPOSE: Mutations in ZEB1 have been reported in posterior polymorphous corneal dystrophy (PPCD3; MIM #609141) and Fuchs' endothelial corneal dystrophy (FECD6; MIM #613270). Although PPCD and keratoconus are clinically and pathologically distinct, PPCD has been associated with keratoconus, suggesting a common genetic basis. The purpose of our study was to perform mutational screening of the ZEB1 gene in patients affected with keratoconus or PPCD. METHODS: Sanger sequencing of ZEB1 was performed in 70 unrelated patients with keratoconus and 18 unrelated patients with PPCD. Real-time quantitative PCR (RT-qPCR) was performed on RNA from cultured corneal keratocytes obtained from a keratoconic patient harboring a missense ZEB1 mutation (p.Gln640His) undergoing corneal transplantation. RESULTS: Mutational analysis of ZEB1 in PPCD identified a previously reported frameshift mutation (C.1578_1579INSG) and a novel nonsense mutation (C.2249C A) in exon 7 of ZEB1 causing the insertion of a stop codon: p.Ser750X. In the keratoconus cohort, a novel heterozygous pathogenic mutation in exon 7 (c.1920G > T; p.Gln640His) of ZEB1 was identified in a family affected with keratoconus and Fuchs' endothelial corneal dystrophy. RT-qPCR performed on cultured corneal keratocytes harboring the missense ZEB1 mutation (p.Gln640His) demonstrated that COL4A1 and COL4A2 were markedly downregulated, and COL4A3, COL4A4, and COL8A2 were moderately downregulated. CONCLUSIONS: Our data combined with the previously reported mutational spectrum of ZEB1 support a genotypephenotype correlation: missense substitutions in the ZEB1 protein are associated with FECD6 and keratoconus, whereas protein truncating ZEB1 mutations result in PPCD3. The dysregulation of α-type IV collagens represents a common link between ZEB1 mutation and the clinical phenotypes (PPCD3, FECD, and keratoconus).

Association of polymorphisms in the hepatocyte growth factor gene promoter with keratoconus.

PURPOSE: Keratoconus is a progressive disorder of the cornea that can lead to severe visual impairment or blindness. Although several genomic regions have been linked to rare familial forms of keratoconus, no genes have yet been definitively identified for common forms of the disease. METHODS: Two genome-wide association scans were undertaken in parallel. The first used pooled DNA from an Australian cohort, followed by typing of top-ranked single-nucleotide polymorphisms (SNPs) in individual DNA samples. The second was conducted in individually genotyped patients, and controls from the USA. Tag SNPs around the hepatocyte growth factor (HGF) gene were typed in three additional replication cohorts. Serum levels of HGF protein in normal individuals were assessed with ELISA and correlated with genotype. RESULTS: The only SNP observed to be associated in both the pooled discovery and primary replication cohort was rs1014091, located upstream of the HGF gene. The nearby SNP rs3735520 was found to be associated in the individually typed discovery cohort (P = 6.1 × 10(-7)). Genotyping of tag SNPs around HGF revealed association at rs3735520 and rs17501108/rs1014091 in four of the five cohorts. Meta-analysis of all five datasets together yielded suggestive P values for rs3735520 (P = 9.9 × 10(-7)) and rs17501108 (P = 9.9 × 10(-5)). In addition, SNP rs3735520 was found to be associated with serum HGF level in normal individuals (P = 0.036). CONCLUSIONS: Taken together, these results implicate genetic variation at the HGF locus with keratoconus susceptibility.

Mutation altering the miR-184 seed region causes familial keratoconus with cataract.

MicroRNAs (miRNAs) bind to complementary sequences within the 3' untranslated region (UTR) of mRNAs from hundreds of target genes, leading either to mRNA degradation or suppression of translation. We found that a mutation in the seed region of miR-184 (MIR184) is responsible for familial severe keratoconus combined with early-onset anterior polar cataract by deep sequencing of a linkage region known to contain the mutation. The mutant form fails to compete with miR-205 (MIR205) for overlapping target sites on the 3' UTRs of INPPL1 and ITGB4. Although these target genes and miR-205 are expressed widely, the phenotype is restricted to the cornea and lens because of the very high expression of miR-184 in these tissues. Our finding highlights the tissue specificity of a gene network regulated by a miRNA. Awareness of the important function of miRNAs could aid identification of susceptibility genes and new therapeutic targets for treatment of both rare and common diseases.