Judith Lechner

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.

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.

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

BackgroundThe 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.ResultsNinety-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.ConclusionsOur 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.

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.Genet Med 17 4, 279–284.

STAT3 Activation in Circulating Monocytes Contributes to Neovascular Age-Related Macular Degeneration.

Infiltrating macrophages are critically involved in pathogenic angiogenesis such as neovascular age-related macular degeneration (nAMD). Macrophages originate from circulating monocytes and three subtypes of monocyte exist in humans: classical (CD14+CD16-), non-classical (CD14-CD16+) and intermediate (CD14+CD16+) monocytes. The aim of this study was to investigate the role of circulating monocyte in neovascular age-related macular degeneration (nAMD). Flow cytometry analysis showed that the intermediate monocytes from nAMD patients expressed higher levels of CX3CR1 and HLA-DR compared to those from controls. Monocytes from nAMD patients expressed higher levels of phosphorylated Signal Transducer and Activator of Transcription 3 (pSTAT3), and produced higher amount of VEGF. In the mouse model of choroidal neovascularization (CNV), pSTAT3 expression was increased in the retina and RPE/choroid, and 49.24% of infiltrating macrophages express pSTAT3. Genetic deletion of the Suppressor of Cytokine Signalling 3 (SOCS3) in myeloid cells in the LysM-Cre+/-:SOCS3fl/fl mice resulted in spontaneous STAT3 activation and accelerated CNV formation. Inhibition of STAT3 activation using a small peptide LLL12 suppressed laser-induced CNV. Our results suggest that monocytes, in particular the intermediate subset of monocytes are activated in nAMD patients. STAT3 activation in circulating monocytes may contribute to the development of choroidal neovascularisation in AMD.

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

&NA; 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.

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 CD16hiHLA-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.

Heredity of Keratoconus

Epidemiological, family and twin studies support a role for heredity in the pathogenesis of keratoconus. Candidate gene analysis and genetic mapping have identified a limited number of genetic factors implicated in the development of keratoconus to date. Mutations in the visual system homeobox gene 1 (VSX1) are found in approximately 2–3% of keratoconus cases. As such, VSX1 represents the only major genetic defect identified to date causing keratoconus. The role of other candidate genes (SOD1, IL-1 and collagens) has not been firmly established. Most gene mapping studies have been performed on Caucasian families with autosomal dominant keratoconus or in isolated populations where genetic heterogeneity is minimised. Multiple loci have been mapped although no pathological mutations have been detected in linked disease intervals. Only the mapped locus on 5q21.2 has been convincingly replicated in two independent datasets. The clinical variability and evidence that keratoconus is linked to multiple chromosomal regions is consistent with an oligogenic or even polygenic model. A genetic model in which mutations in several different genes involved in common pathways cause keratoconus is most likely. Emerging data from genome wide association studies should bring further insights into the genetic basis of keratoconus.