Differential Lysyl oxidase like 1 expression in pseudoexfoliation glaucoma is orchestrated via DNA methylation.

Pseudoexfoliation syndrome (PXF) is the most common cause of secondary open angle glaucoma worldwide. Single nucleotide polymorphisms (SNPs) in the gene Lysyl oxidase like 1 (LOXL1) are strongly associated with the development of pseudoexfoliation glaucoma (PXFG). However, these SNPs are also present in 50-80% of the general population, suggestive of other factors being involved in the pathogenesis of PXFG. In this study, we aimed to investigate the influence of epigenetic regulation, specifically DNA methylation, on LOXL1 expression in PXFG using human tenons fibroblasts (HTFs), aqueous humour and serum samples from donors with and without PXFG. LOXL1 expression in HTFs was measured by qPCR and Western Blotting and LOXL1 concentration in aqueous humour was determined by ELISA. Global DNA methylation levels were quantified using an ELISA for 5-methylcytosine. MeDIP assays assessed the methylation status of the LOXL1 promoter region. Expression of methylation-associated enzymes (DNMT1, DNMT3a and MeCP2) were determined by qPCR and inhibited by 0.3 µM 5-azacytidine (5-aza). Results showed that LOXL1 expression was significantly decreased in PXFG HTFs compared with Control HTFs at gene (Fold change 0.37 ± 0.05, P < 0.01) level and showed a decrease, when measured at the protein level (Fold change 0.65 ± 0.42, P = 0.22), however this was not found to be significant. LOXL1 concentration was increased in the aqueous of PXFG patients compared with Controls (2.76 ± 0.78 vs. 1.79 ± 0.33 ng/ml, P < 0.01). Increased global methylation (56.07% ± 4.87% vs. 32.39% ± 4.29%, P < 0.01) was observed in PXFG HTFs compared with Control HTFs, as was expression of methylation-associated enzymes (DNMT1 1.58 ± 0.30, P < 0.05, DNMT3a 1.89 ± 0.24, P < 0.05, MeCP2 1.63 ± 0.30, P < 0.01). Methylation-associated enzymes were also increased when measured at protein level (DNMT1 5.70 ± 2.64, P = 0.04, DNMT3a 1.79 ± 1.55, P = 0.42, MeCP2 1.64 ± 1.33, P = 0.45). LOXL1 promoter methylation was increased in patients with PXFG compared to Control patients in both blood (3.98 ± 2.24, 2.10 ± 1.29, P < 0.05) and HTF cells (37.31 ± 22.0, 8.66 ± 10.40, P < 0.01). Treatment of PXFG HTFs with in 5-azacytidine increased LOXL1 expression when compared with untreated PXFG HTFs (Fold change 2.26 ± 0.67, P < 0.05). These data demonstrate that LOXL1 expression is altered in PXFG via DNA methylation and that reversal of these epigenetic changes may represent future potential therapeutic targets in the management of PXFG.

Prevalence of Pseudoexfoliation Glaucoma Risk-associated Variants Within Lysyl Oxidase-like 1 in an Irish Population.

PRECIS: High-risk alleles of risk-associated single-nucleotide polymorphisms (SNPs) within the lysyl oxidase-like 1 (LOXL1) gene are associated with pseudoexfoliation in patients recruited from an Irish population. PURPOSE: SNPs within the LOXL1 gene have been identified as a major risk factor for pseudoexfoliation syndrome (PXF) and pseudoexfoliation glaucoma (PXFG), specifically SNPs within exon 1 and intron 1 regions of the gene. The common haplotype (G-G) of 2 SNPs within exon 1, rs1048661, and rs3825942, is the strongest associated risk factor for PXF in white populations, but is switched in some populations to act as protective or low risk. Herein, a study was undertaken to genotype an Irish population for PXF/PXFG risk-associated SNPs within LOXL1. MATERIALS AND METHODS: Patient cohorts of PXFG, PXF, and controls were recruited and genotyped for risk-associated SNPs within exon 1 (rs1048661 and rs3825942), along with 3 SNPs within intron 1 (rs1550437, rs6495085, and rs6495086) of LOXL1. RESULTS: The risk G alleles of rs1048661 and rs3825942 were most prevalent in PXFG patients, and a significant association was found between rs3825942 and pseudoexfoliation (P=0.04). Genotypes of several intron 1 SNPs were found to be present at higher frequencies within the pseudoexfoliation patient cohort (PXF/PXFG) compared with control patients, wherein rs6495085 showed statistical association (P=0.04). The G-G-G haplotype of rs1048661, rs3825942, and rs6495085 was the most prevalent in PXFG patients compared with control patients or patients with PXF alone. Patients with the G-G-G haplotype were more likely to need surgery, suggestive of a more severe form of disease. CONCLUSION: Collectively, these results represent the first study to assess the association of LOXL1 SNPs with PXFG in an Irish population.

Lysyl Oxidase Like 1: Biological roles and regulation.

Lysyl Oxidase Like 1 (LOXL1) is a gene that encodes for the LOXL1 enzyme. This enzyme is required for elastin biogenesis and collagen cross-linking, polymerising tropoelastin monomers into elastin polymers. Its main role is in elastin homeostasis and matrix remodelling during injury, fibrosis and cancer development. Because of its vast range of biological functions, abnormalities in LOXL1 underlie many disease processes. Decreased LOXL1 expression is observed in disorders of elastin such as Cutis Laxa and increased expression is reported in fibrotic disease such as Idiopathic Pulmonary Fibrosis. LOXL1 is also downregulated in the lamina cribrosa in pseudoexfoliation glaucoma and genetic variants in the LOXL1 gene have been linked with an increased risk of developing pseudoexfoliation glaucoma and pseudoexfoliation syndrome. However the two major risk alleles are reversed in certain ethnic groups and are present in a large proportion of the normal population, implying complex genetic and environmental regulation is involved in disease pathogenesis. It also appears that the non-coding variants in intron 1 of LOXL1 may be involved in the regulation of LOXL1 expression. Gene alteration may occur via a number of epigenetic and post translational mechanisms such as DNA methylation, long non-coding RNAs and microRNAs. These may represent future therapeutic targets for disease. Environmental factors such as hypoxia, oxidative stress and ultraviolet radiation exposure alter LOXL1 expression, and it is likely a combination of these genetic and environmental factors that influence disease development and progression. In this review, we discuss LOXL1 properties, biological roles and regulation in detail with a focus on pseudoexfoliation syndrome and glaucoma.

Difference in intraocular pressure measurements between non-contact tonometry and Goldmann applanation tonometry and the role of central corneal thickness in affecting glaucoma referrals.

BACKGROUND: Patients at glaucoma risk are commonly identified by optometrists and subsequently referred to glaucoma specialists. Optometrists mainly use non-contact tonometry (NCT) for intraocular pressure (IOP) measurement. AIMS: To investigate the role of differences in IOP measurement between NCT and Goldmann applanation tonometry (GAT) and the effect of central corneal thickness (CCT) on these differences in optometrist referrals METHODS: Details of the initial clinical visit of patients referred with IOP > 21 mmHg in either eye as measured by NCT to a consultant glaucoma specialist were retrospectively reviewed. Demographic and referral data, IOP, CCT, and glaucoma diagnosis were obtained. The main outcome measure was the IOP measurement differences between NCT and GAT. RESULTS: Of the 98 patients referred, only 23% had IOP > 21 mmHg when measured by GAT. NCT (Nidek NT400, Reichert Puff, Pulsair Easy Eye) measured the IOP greater than GAT by a mean of 5.8 mmHg (NCT 24.1 ± 3.5, GAT 18.3 ± 3.0). The effect of CCT on IOP measurement was less for GAT (R2 0.034, p = 0.067) than for NCT (R2 0.088, p = 0.003). The NCT/GAT IOP differences increased with increasing CCT (R2 0.166, p < 0.0001). The NCT/GAT differences decreased with patient age (R2 0.048, p = 0.03). Patients were classified as normal 67% (66/98), ocular hypertension 11% (11/98), glaucoma suspect 14% (14/98), and glaucoma 7% (7/98). CONCLUSIONS: The difference in IOP measurement between NCT and GAT leads to a possible increase in glaucoma referrals, particularly in patients with thicker corneas. Repeat IOP using GAT and CCT measurement would help in triaging referrals.

Intermediate-term and long-term outcome of piggyback drainage: connecting glaucoma drainage device to a device in-situ for improved intraocular pressure control.

IMPORTANCE: This study provides results of a treatment option for patients with failed primary glaucoma drainage device. BACKGROUND: The study aimed to describe and evaluate the long-term intraocular pressure control and complications of a new technique joining a second glaucoma drainage device directly to an existing glaucoma drainage device termed 'piggyback drainage'. DESIGN: This is a retrospective, interventional cohort study. PARTICIPANTS: Eighteen eyes of 17 patients who underwent piggyback drainage between 2004 and 2013 inclusive have been studied. All patients had prior glaucoma drainage device with uncontrolled intraocular pressure. METHODS: The piggyback technique involved suturing a Baerveldt (250 or 350 mm) or Molteno3 glaucoma drainage device to an unused scleral quadrant and connecting the silicone tube to the primary plate bleb. MAIN OUTCOME MEASURES: Failure of intraocular pressure control defined as an intraocular pressure greater than 21 mmHg on maximal therapy on two separate occasions or further intervention to control intraocular pressure. RESULTS: The intraocular pressure was controlled in seven eyes (39%) at last follow-up with a mean follow-up time of 74.2 months. The mean preoperative intraocular pressure was 27.1 mmHg (95% confidence interval 23.8-30.3) compared with 18.4 mmHg (95% confidence interval 13.9-22.8) at last follow-up. The mean time to failure was 57.1 months (95% confidence interval 32.2-82), and the mean time to further surgery was 72.3 months (95% confidence interval 49.9-94.7). Lower preoperative intraocular pressure was associated with longer duration of intraocular pressure control (P = 0.048). If the intraocular pressure was controlled over 2 years, it continued to be controlled over the long term. Two eyes (11%) experienced corneal decompensation. CONCLUSIONS: Piggyback drainage represents a viable surgical alternative for the treatment of patients with severe glaucoma with failing primary glaucoma drainage device, particularly in those at high risk of corneal decompensation.

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.

Protein macroarray profiling of serum autoantibodies in pseudoexfoliation glaucoma.

PURPOSE: Complex repertoires of IgG autoantibodies have been detected against ocular antigens in patients with glaucoma. The goal was to identify and characterize the IgG autoantibody repertoires in sera of patients with pseudoexfoliation glaucoma (PXFG) with protein macroarrays. METHODS: Serum samples of 21 patients with PXFG and 19 age- and sex-matched control subjects were profiled on high-density colony protein macroarrays expressing His-tagged recombinant human proteins derived from a human fetal brain cDNA library. Statistically prevalent expression clones in the PXFG group were sequenced. mRNA expression of identified antigens was examined in the rat ganglion cell line RGC-5 and in human brain and optic nerve cDNA. The IgG immunoreactivity of the sera of 20 control and 26 PXFG patients to purified C6orf129 was analyzed in a reverse enzyme-linked immunosorbent assay. RESULTS: An increased prevalence was detected among the PXFG patients of serum antibodies to seven proteins: C6orf129; stathmin-like 4; transmembrane protein 9 domain family, member B; fibroblast growth factor receptor 3; cleft lip and palate transmembrane protein 1; EH-domain-containing protein 1; and eukaryotic translation elongation factor 2. All antigens were expressed in the RGC-5 cells and in cDNA from human brain and optic nerve, with the exception of stathmin-like 4, which was not expressed in the RGC-5 cells. The patients with PXFG had increased anti-C6orf129 IgG immunoreactivity compared with that in the control subjects (P < 0.05). CONCLUSIONS: Screening high-density protein arrays identifies unique antibody profiles that may discriminate between patients with and without PXFG. Characterization of the autoantibody repertoire may provide new insights into the pathophysiology of PXFG.