The Increased Burden of Rare Variants in Four Matrix Metalloproteinase-Related Genes in Childhood Glaucoma Suggests a Complex Genetic Inheritance of the Disease.

Childhood glaucoma encompasses congenital and juvenile primary glaucoma, which are heterogeneous, uncommon, and irreversible optic neuropathies leading to visual impairment with a poorly understood genetic basis. Our goal was to identify gene variants associated with these glaucoma types by assessing the mutational burden in 76 matrix metalloproteinase-related genes. We studied 101 childhood glaucoma patients with no identified monogenic alterations using next-generation sequencing.

Candidate Glaucoma Biomarkers: From Proteins to Metabolites, and the Pitfalls to Clinical Applications.

Glaucoma is an insidious group of eye diseases causing degeneration of the optic nerve, progressive loss of vision, and irreversible blindness. The number of people affected by glaucoma is estimated at 80 million in 2021, with 3.5% prevalence in people aged 40-80.

Systemic Alterations of Immune Response-Related Proteins during Glaucoma Development in the Murine Model DBA/2J.

Animal models of glaucoma, a neurodegenerative disease affecting the retina, offer the opportunity to study candidate molecular biomarkers throughout the disease. In this work, the DBA/2J glaucomatous mouse has been used to study the systemic levels of several proteins previously identified as potential biomarkers of glaucoma, along the pre- to post-glaucomatous transition. Serum samples obtained from glaucomatous and control mice at 4, 10, and 14 months, were classified into different experimental groups according to the optic nerve damage at 14 months old.

CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix.

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG.

The association study of lipid metabolism gene polymorphisms with AMD identifies a protective role for APOE-E2 allele in the wet form in a Northern Spanish population.

Purpose: To elucidate the potential role of eleven single nucleotide polymorphisms (SNPs) in the most relevant lipid metabolism genes in Northern Spanish patients with age-related macular degeneration (AMD).

Methods: A case-control study of 228 unrelated native Northern Spanish patients diagnosed with AMD (73 dry and 155 wet) and 95 healthy controls was performed. DNA was isolated from peripheral blood and genotyped for the SNPs APOE rs429358 and rs7412; CTEP rs3764261; LIPC rs10468017 and rs493258; LPL rs12678919; ABCA1 rs1883025; ABCA4 rs76157638, rs3112831 and rs1800555; and SCARB1 rs5888, using TaqMan probes.

Identification of myocilin as a blood plasma protein and analysis of its role in leukocyte adhesion to endothelial cell monolayers.

Myocilin is an extracellular glycoprotein with a poorly understood biological function and typically known because of its association with glaucoma. In this study, we analyzed the expression and biological activity of human myocilin in some non-ocular tissues. Western immunoblot showed the presence of myocilin in blood plasma as well as in liver and lymphoid tissues (thymus and lymph node).

The Zinc-Metallothionein Redox System Reduces Oxidative Stress in Retinal Pigment Epithelial Cells.

Oxidative stress affects all the structures of the human eye, particularly the retina and its retinal pigment epithelium (RPE). The RPE limits oxidative damage by several protective mechanisms, including the non-enzymatic antioxidant system zinc-metallothionein (Zn-MT). This work aimed to investigate the role of Zn-MT in the protection of RPE from the oxidative damage of reactive oxygen intermediates by analytical and biochemical-based techniques.

Quantitative study of zinc and metallothioneins in the human retina and RPE cells by mass spectrometry-based methodologies.

The retina contains the highest concentration of zinc in the human eye and it is primarily associated with the retinal pigment epithelium (RPE). Metallothioneins (MTs) are the main cytosolic zinc-ion-binding proteins, exerting a tight control in the number of atoms of Zn-bound to the MTs related with their antioxidant and neuroprotective functions. In order to study the Zn-MT system in retina and RPE, we have implemented mass spectrometry (MS)-based technologies: two complementary element detection methodologies (HPLC- and laser ablation (LA)-ICP-MS) have been successfully employed to study metal content in the human eye as well as to perform speciation studies of Zn-MTs.

Metallothionein polymorphisms in a Northern Spanish population with neovascular and dry forms of age-related macular degeneration.

Background: To elucidate the potential role of single nucleotide polymorphisms (SNPs) in the metallothionein (MT) genes in Northern Spanish patients with age-related macular degeneration (AMD).

Methods: A total of 130 unrelated Northern Spanish natives diagnosed with AMD (46 dry, 35 neovascular, and 49 mixed) and 96 healthy controls, matched by age and ethnicity, were enrolled in a case-control study. DNA was isolated from peripheral blood and genotyped for 14 SNPs located at 5 MT genes (MT1A: rs11076161, rs 11640851, rs8052394, and rs7196890; MT1B: rs8052334, rs964372, and rs7191779; MT1M: rs2270836 and rs9936741; MT2A: rs28366003, rs1610216, rs10636, and rs1580833; MT3: rs45570941) using TaqMan probes.

LOXL1 gene variants and their association with pseudoexfoliation glaucoma (XFG) in Spanish patients.

Background: LOXL1 gene is the most important genetic risk factor known so far for pseudoexfoliation glaucoma (XFG). Our purpose was to evaluate the potential association of individual genetic variants of the lysyl oxidase-like 1 (LOXL1) gene and haplotypes with XFG in Spanish patients.

Methods: Blood samples were collected from a total of 105 Spanish patients with XFG and 200 healthy controls.

CFH polymorphisms in a Northern Spanish population with neovascular and dry forms of age-related macular degeneration.

Purpose: To elucidate the potential role of single-nucleotide polymorphisms (SNPs) in complement factor H (CFH) gene in Northern Spanish patients with age-related macular degeneration (AMD).

Methods: A case-control study of 130 unrelated native Northern Spanish diagnosed with AMD (46 dry, 35 neovascular and 49 mixed) and 96 healthy controls matched by age and ethnicity were enrolled. DNA was isolated from peripheral blood and genotyped for AMD-associated SNPs (rs3753394, rs529825, rs800292, rs3766404, rs203674, rs10671170, rs3753396 and rs1065489) using TaqMan probes and restriction fragment length polymorphism (RFLP).

Sensitive targeted multiple protein quantification based on elemental detection of quantum dots.

A generic strategy based on the use of CdSe/ZnS Quantum Dots (QDs) as elemental labels for protein quantification, using immunoassays with elemental mass spectrometry (ICP-MS), detection is presented. In this strategy, streptavidin modified QDs (QDs-SA) are bioconjugated to a biotinylated secondary antibody (b-Ab2). After a multi-technique characterization of the synthesized generic platform (QDs-SA-b-Ab2) it was applied to the sequential quantification of five proteins (transferrin, complement C3, apolipoprotein A1, transthyretin and apolipoprotein A4) at different concentration levels in human serum samples.

A renal-like organic anion transport system in the ciliary epithelium of the bovine and human eye.

The purpose of this study was to determine the direction of organic anion (OA) transport across the ciliary body and the transport proteins that may contribute. Transport of several OAs across the bovine ciliary body was examined using ciliary body sections mounted in Ussing chambers and a perfused eye preparation. Microarray, reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry were used to examine OA transporter expression in human ocular tissues.

Quantitative selenium speciation by HPLC-ICP-MS(IDA) and simultaneous activity measurements in human vitreous humor.

A post-column isotope dilution analysis (IDA) methodology was applied to carry out quantitative speciation of selenium in human vitreous humor samples by size exclusion chromatography (SEC) coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS). Two main selenium species detected by SEC-ICP-MS were found to be associated to protein complexes. The expected molecular weights for both selenium-bound complexes were confirmed by MALDI-TOF(MS) and the results matched well with the theoretical mass of a GPx monomer (M, 22 kDa) and tetramer (T, 88 kDa).

The blood-aqueous barrier in health and disease.

The blood-aqueous barrier of the eye is composed by tight junctions in the ciliary process nonpigmented epithelium, the endothelial cells in the iris vasculature, and the inner wall endothelium of Schlemm's canal. Tight junctions are gatekeepers of the paracellular transport limiting the selective diffusion of ions and small solutes through the space between neighboring cells. Tight junctions (ie, junctional adhesion molecules, claudins, occludins, zonula occludens, cingulin) are part of the apical junctional complex that also includes the adherens junctions (ie, cadherin-catenin and nectin-afadin complexes) and the gap junctions (ie, connexins).

Quantitative bioimaging of trace elements in the human lens by LA-ICP-MS.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for the quantitative imaging of Fe, Cu and Zn in cryostat sections of human eye lenses and for depth profiling analysis in bovine lenses. To ensure a tight temperature control throughout the experiments, a new Peltier-cooled laser ablation cell was employed. For quantification purposes, matrix-matched laboratory standards were prepared from a pool of human lenses from eye donors and spiked with standard solutions containing different concentrations of natural abundance Fe, Cu and Zn.

Metallothioneins (MTs) in the human eye: a perspective article on the zinc-MT redox cycle.

Metallothioneins (MTs) are zinc-ion-binding proteins with a wide range of functions, among which are neuroprotection, maintenance of cellular zinc homeostasis, and defense against oxidative damage and inflammation. The human eye is enriched in MTs, and multiple isoforms may contribute to distinct antioxidant defense mechanisms in various ocular tissues. Zinc is a main regulator of MT gene and protein expression, and we recently applied bioanalytical techniques to address key questions on its relationship with MTs, including the stoichiometry of zinc-MT, the fate of zinc tracers ((nat)Zn and (68)Zn) in MTs during activation by exogenous zinc and cytokines, and the concentration of MTs in human ocular cells.

Gold internal standard correction for elemental imaging of soft tissue sections by LA-ICP-MS: element distribution in eye microstructures.

Laser ablation coupled to inductively coupled plasma mass spectrometry has been developed for the elemental imaging of Mg, Fe and Cu distribution in histological tissue sections of fixed eyes, embedded in paraffin, from human donors (cadavers). This work presents the development of a novel internal standard correction methodology based on the deposition of a homogeneous thin gold film on the tissue surface and the use of the (197)Au(+) signal as internal standard. Sample preparation (tissue section thickness) and laser conditions were carefully optimized, and internal normalisation using (197)Au(+) was compared with (13)C(+) correction for imaging applications.

Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin.

Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment.