Through the Cat-Map Gateway: A Brief History of Cataract Genetics.

Clouding of the transparent eye lens, or cataract(s), is a leading cause of visual impairment that requires surgical replacement with a synthetic intraocular lens to effectively restore clear vision. Most frequently, cataract is acquired with aging as a multifactorial or complex trait. Cataract may also be inherited as a classic Mendelian trait-often with an early or pediatric onset-with or without other ocular and/or systemic features. Since the early 1990s, over 85 genes and loci have been genetically associated with inherited and/or age-related forms of cataract. While many of these underlying genes-including those for lens crystallins, connexins, and transcription factors-recapitulate signature features of lens development and differentiation, an increasing cohort of unpredicted genes, including those involved in cell-signaling, membrane remodeling, and autophagy, has emerged-providing new insights regarding lens homeostasis and aging. This review provides a brief history of gene discovery for inherited and age-related forms of cataract compiled in the Cat-Map database and highlights potential gene-based therapeutic approaches to delay, reverse, or even prevent cataract formation that may help to reduce the increasing demand for cataract surgery.

A Cataract-Causing Mutation in the TRPM3 Cation Channel Disrupts Calcium Dynamics in the Lens.

TRPM3 belongs to the melastatin sub-family of transient receptor potential (TRPM) cation channels and has been shown to function as a steroid-activated, heat-sensitive calcium ion (Ca2+) channel. A missense substitution (p.I65M) in the TRPM3 gene of humans (TRPM3) and mice (Trpm3) has been shown to underlie an inherited form of early-onset, progressive cataract. Here, we model the pathogenetic effects of this cataract-causing mutation using 'knock-in' mutant mice and human cell lines. Trpm3 and its intron-hosted micro-RNA gene (Mir204) were strongly co-expressed in the lens epithelium and other non-pigmented and pigmented ocular epithelia. Homozygous Trpm3-mutant lenses displayed elevated cytosolic Ca2+ levels and an imbalance of sodium (Na+) and potassium (K+) ions coupled with increased water content. Homozygous TRPM3-mutant human lens epithelial (HLE-B3) cell lines and Trpm3-mutant lenses exhibited increased levels of phosphorylated mitogen-activated protein kinase 1/extracellular signal-regulated kinase 2 (MAPK1/ERK2/p42) and MAPK3/ERK1/p44. Mutant TRPM3-M65 channels displayed an increased sensitivity to external Ca2+ concentration and an altered dose response to pregnenolone sulfate (PS) activation. Trpm3-mutant lenses shared the downregulation of genes involved in insulin/peptide secretion and the upregulation of genes involved in Ca2+ dynamics. By contrast, Trpm3-deficient lenses did not replicate the pathophysiological changes observed in Trpm3-mutant lenses. Collectively, our data suggest that a cataract-causing substitution in the TRPM3 cation channel elicits a deleterious gain-of-function rather than a loss-of-function mechanism in the lens.

Charged multivesicular body protein 4b forms complexes with gap junction proteins during lens fiber cell differentiation.

Charged multivesicular body protein 4b (CHMP4B) is a core sub-unit of the endosomal sorting complex required for transport III (ESCRT-III) machinery that serves myriad remodeling and scission processes of biological membranes. Mutation of the human CHMP4B gene underlies rare forms of early-onset lens opacities or cataracts, and CHMP4B is required for lens growth and differentiation in mice. Here, we determine the sub-cellular distribution of CHMP4B in the lens and uncover a novel association with gap junction alpha-3 protein (GJA3) or connexin 46 (Cx46) and GJA8 or Cx50. Immunofluorescence confocal microscopy revealed that CHMP4B localized to cell membranes of elongated fiber cells in the outer cortex of the lens-where large gap junction plaques begin to form-particularly, on the broad faces of these flattened hexagon-like cells in cross-section. Dual immunofluorescence imaging showed that CHMP4B co-localized with gap junction plaques containing Cx46 and/or Cx50. When combined with the in situ proximity ligation assay, immunofluorescence confocal imaging indicated that CHMP4B lay in close physical proximity to Cx46 and Cx50. In Cx46-knockout (Cx46-KO) lenses, CHMP4B-membrane distribution was similar to that of wild-type, whereas, in Cx50-KO lenses, CHMP4B localization to fiber cell membranes was lost. Immunoprecipitation and immunoblotting analyses revealed that CHMP4B formed complexes with Cx46 and Cx50 in vitro. Collectively, our data suggest that CHMP4B forms plasma membrane complexes, either directly and/or indirectly, with gap junction proteins Cx46 and Cx50 that are often associated with "ball-and-socket" double-membrane junctions during lens fiber cell differentiation.

Whole-exome sequencing prioritizes candidate genes for hereditary cataract in the Emory mouse mutant.

The Emory cataract (Em) mouse mutant has long been proposed as an animal model for age-related or senile cataract in humans-a leading cause of visual impairment. However, the genetic defect(s) underlying the autosomal dominant Em phenotype remains elusive. Here, we confirmed development of the cataract phenotype in commercially available Em/J mice [but not ancestral Carworth Farms White (CFW) mice] at 6-8 months of age and undertook whole-exome sequencing of candidate genes for Em. Analysis of coding and splice-site variants did not identify any disease-causing/associated mutations in over 450 genes known to underlie inherited and age-related forms of cataract and other lens disorders in humans and mice, including genes for lens crystallins, membrane/cytoskeleton proteins, DNA/RNA-binding proteins, and those associated with syndromic/systemic forms of cataract. However, we identified three cataract/lens-associated genes each with one novel homozygous variant including predicted missense substitutions in Prx (p.R167C) and Adamts10 (p.P761L) and a disruptive in-frame deletion variant (predicted missense) in Abhd12 (p.L30_A32delinsS) that were absent in CFW and over 35 other mouse strains. In silico analysis predicted that the missense substitutions in Prx and Adamts10 were borderline neutral/damaging and neutral, respectively, at the protein function level, whereas, that in Abhd12 was functionally damaging. Both the human counterparts of Adamts10 and Abhd12 are clinically associated with syndromic forms of cataract known as Weil-Marchesani syndrome 1 and polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract syndrome, respectively. Overall, while we cannot exclude Prx and Adamts10, our data suggest that Abhd12 is a promising candidate gene for cataract in the Em/J mouse.

Autophagy Requirements for Eye Lens Differentiation and Transparency.

Recent evidence points to autophagy as an essential cellular requirement for achieving the mature structure, homeostasis, and transparency of the lens. Collective evidence from multiple laboratories using chick, mouse, primate, and human model systems provides evidence that classic autophagy structures, ranging from double-membrane autophagosomes to single-membrane autolysosomes, are found throughout the lens in both undifferentiated lens epithelial cells and maturing lens fiber cells. Recently, key autophagy signaling pathways have been identified to initiate critical steps in the lens differentiation program, including the elimination of organelles to form the core lens organelle-free zone. Other recent studies using ex vivo lens culture demonstrate that the low oxygen environment of the lens drives HIF1a-induced autophagy via upregulation of essential mitophagy components to direct the specific elimination of the mitochondria, endoplasmic reticulum, and Golgi apparatus during lens fiber cell differentiation. Pioneering studies on the structural requirements for the elimination of nuclei during lens differentiation reveal the presence of an entirely novel structure associated with degrading lens nuclei termed the nuclear excisosome. Considerable evidence also indicates that autophagy is a requirement for lens homeostasis, differentiation, and transparency, since the mutation of key autophagy proteins results in human cataract formation.

Mutation of the EPHA2 Tyrosine-Kinase Domain Dysregulates Cell Pattern Formation and Cytoskeletal Gene Expression in the Lens.

Genetic variations in ephrin type-A receptor 2 (EPHA2) have been associated with inherited and age-related forms of cataract in humans. Here, we have characterized the eye lens phenotype and transcript profile of germline Epha2 knock-in mutant mice homozygous for either a missense variant associated with age-related cataract in humans (Epha2-Q722) or a novel insertion-deletion mutation (Epha2-indel722) that were both located within the tyrosine-kinase domain of EPHA2. Confocal imaging of ex vivo lenses from Epha2-indel722 mice on a fluorescent reporter background revealed misalignment of epithelial-to-fiber cell meridional-rows at the lens equator and severe disturbance of Y-suture formation at the lens poles, whereas Epha2-Q722 lenses displayed mild disturbance of posterior sutures. Immunofluorescent labeling showed that EPHA2 was localized to radial columns of hexagonal fiber cell membranes in Epha2-Q722 lenses, whereas Epha2-indel722 lenses displayed disorganized radial cell columns and cytoplasmic retention of EPHA2. Immunoprecipitation/blotting studies indicated that EPHA2 formed strong complexes with Src kinase and was mostly serine phosphorylated in the lens. RNA sequencing analysis revealed differential expression of several cytoskeleton-associated genes in Epha2-mutant and Epha2-null lenses including shared downregulation of Lgsn and Clic5. Collectively, our data suggest that mutations within the tyrosine-kinase domain of EPHA2 result in lens cell patterning defects and dysregulated expression of several cytoskeleton-associated proteins.

Inherited cataracts: Genetic mechanisms and pathways new and old.

Cataract(s) is the clinical equivalent of lens opacity and is caused by light scattering either by high molecular weight protein aggregates in lens cells or disruption of the lens microarchitecture itself. Genetic mutations underlying inherited cataract can provide insight into the biological processes and pathways critical for lens homeostasis and transparency, classically including the lens crystallins, connexins, membrane proteins or components, and intermediate filament proteins. More recently, cataract genes have been expanded to include newly identified biological processes such as chaperone or protein degradation components, transcription or growth factors, channels active in the lens circulation, and collagen and extracellular matrix components. Cataracts can be classified by age, and in general congenital cataracts are caused by severe mutations resulting in major damage to lens proteins, while age related cataracts are associated with variants that merely destabilize proteins thereby increasing susceptibility to environmental insults over time. Thus there might be separate pathways to opacity for congenital and age-related cataracts whereby congenital cataracts induce the unfolded protein response (UPR) and apoptosis to destroy the lens microarchitecture, while in age related cataract high molecular weight (HMW) aggregates formed by denatured crystallins bound by α-crystallin result in light scattering without severe damage to the lens microarchitecture.

Mutation of the TRPM3 cation channel underlies progressive cataract development and lens calcification associated with pro-fibrotic and immune cell responses.

Transient-receptor-potential cation channel, subfamily M, member 3 (TRPM3) serves as a polymodal calcium sensor in diverse mammalian cell-types. Mutation of the human TRPM3 gene (TRPM3) has been linked with inherited forms of early-onset cataract with or without other eye abnormalities. Here, we have characterized the ocular phenotypes of germline "knock-in" mice that harbor a human cataract-associated isoleucine-to-methionine mutation (p.I65M) in TRPM3 (Trpm3-mutant) compared with germline "knock-out" mice that functionally lack TRPM3 (Trpm3-null). Despite strong expression of Trpm3 in lens epithelial cells, neither heterozygous (Trpm3+/- ) nor homozygous (Trpm3-/- ) Trpm3-null mice developed cataract; however, the latter exhibited a mild impairment of lens growth. In contrast, homozygous Trpm3-M/M mutants developed severe, progressive, anterior pyramid-like cataract with microphthalmia, whereas heterozygous Trpm3-I/M and hemizygous Trpm3-M/- mutants developed anterior pyramidal cataract with delayed onset and progression-consistent with a semi-dominant lens phenotype. Histochemical staining revealed abnormal accumulation of calcium phosphate-like deposits and collagen fibrils in Trpm3-mutant lenses and immunoblotting detected increased αII-spectrin cleavage products consistent with calpain hyper-activation. Immunofluorescent confocal microscopy of Trpm3-M/M mutant lenses revealed fiber cell membrane degeneration that was accompanied by accumulation of alpha-smooth muscle actin positive (α-SMA+ve) myofibroblast-like cells and macrosialin positive (CD68+ve) macrophage-like cells. Collectively, our mouse model data support an ocular disease association for TRPM3 in humans and suggest that (1) Trpm3 deficiency impaired lens growth but not lens transparency and (2) Trpm3 dysfunction resulted in progressive lens degeneration and calcification coupled with pro-fibrotic (α-SMA+ve) and immune (CD68+ve) cell responses.

TRPM3_miR-204: a complex locus for eye development and disease.

First discovered in a light-sensitive retinal mutant of Drosophila, the transient receptor potential (TRP) superfamily of non-selective cation channels serve as polymodal cellular sensors that participate in diverse physiological processes across the animal kingdom including the perception of light, temperature, pressure, and pain. TRPM3 belongs to the melastatin sub-family of TRP channels and has been shown to function as a spontaneous calcium channel, with permeability to other cations influenced by alternative splicing and/or non-canonical channel activity. Activators of TRPM3 channels include the neurosteroid pregnenolone sulfate, calmodulin, phosphoinositides, and heat, whereas inhibitors include certain drugs, plant-derived metabolites, and G-protein subunits. Activation of TRPM3 channels at the cell membrane elicits a signal transduction cascade of mitogen-activated kinases and stimulus response transcription factors. The mammalian TRPM3 gene hosts a non-coding microRNA gene specifying miR-204 that serves as both a tumor suppressor and a negative regulator of post-transcriptional gene expression during eye development in vertebrates. Ocular co-expression of TRPM3 and miR-204 is upregulated by the paired box 6 transcription factor (PAX6) and mutations in all three corresponding genes underlie inherited forms of eye disease in humans including early-onset cataract, retinal dystrophy, and coloboma. This review outlines the genomic and functional complexity of the TRPM3_miR-204 locus in mammalian eye development and disease.

Biology of Inherited Cataracts and Opportunities for Treatment.

Cataract, the clinical correlate of opacity or light scattering in the eye lens, is usually caused by the presence of high-molecular-weight (HMW) protein aggregates or disruption of the lens microarchitecture. In general, genes involved in inherited cataracts reflect important processes and pathways in the lens including lens crystallins, connexins, growth factors, membrane proteins, intermediate filament proteins, and chaperones. Usually, mutations causing severe damage to proteins cause congenital cataracts, while milder variants increasing susceptibility to environmental insults are associated with age-related cataracts. These may have different pathogenic mechanisms: Congenital cataracts induce the unfolded protein response and apoptosis. By contrast, denatured crystallins in age-related cataracts are bound by α-crystallin and form light-scattering HMW aggregates. New therapeutic approaches to age-related cataracts use chemical chaperones to solubilize HMW aggregates, while attempts are being made to regenerate lenses using endogenous stem cells to treat congenital cataracts.

A charged multivesicular body protein (CHMP4B) is required for lens growth and differentiation.

Charged multivesicular body protein 4B (CHMP4B) functions as a core component of the endosome sorting complex required for transport-III (ESCRT-III) machinery that facilitates diverse membrane remodeling and scission processes in eukaryotes. Mutations in the human CHMP4B gene underlie rare, inherited forms of early-onset lens opacities or cataract. Here we have characterized the lens phenotypes of mutant (knock-in) mice harboring a human cataract-associated mutation (p.D129V) in CHMP4B (Chmp4b-mutant) and conditional knockdown mice deficient in lens CHMP4B (Chmp4b-CKD). In situ hybridization localized Chmp4b transcripts to lens epithelial cells and elongating fiber cells at the lens equator. Heterozygous Chmp4b-mutant (D/V) mice were viable and fertile with lenses grossly similar to those of wild-type. However, homozygous Chmp4b-mutant (V/V) mice died by embryonic day 15.5 (E15.5) with grossly abnormal eye and brain histology. Chmp4b-CKD mice displayed variable degrees of lens dysmorphology including lens ablation. Immuno-localization of aquaporin-0 (AQP0) revealed lens fiber cell degeneration in homozygous Chmp4b-mutant (V/V) mouse embryos and in embryonic and postnatal Chmp4b-CKD mice. DNA fragmentation (TUNEL) analysis revealed global cell death in homozygous Chmp4b-mutant (V/V) embryos, whereas, cell death was confined to the lens of Chmp4b-CKD mice. Immuno-localization of the monocyte/macrophage marker macrosialin (CD68) suggested that severe lens degeneration in Chmp4b-CKD mice resulted in an ocular immune cell response. Collectively, these mouse data suggest that (1) heterozygous, germ-line mutations in Chmp4b may not manifest as cataract, (2) homozygous, germ-line mutations in Chmp4b are embryonic lethal, and (3) conditional loss of Chmp4b results in arrest of lens growth and differentiation.

Epha2 and Efna5 participate in lens cell pattern-formation.

Ephrin type-A receptor 2 (EPHA2) and one of its ligands, ephrin-A5 (EFNA5), have been associated with loss of eye lens transparency, or cataract, - an important cause of visual impairment. Here we show that mice functionally lacking EPHA2 (Epha2-null), EFNA5 (Efna5-null), or both receptor and ligand (Epha2/Efna5-null) consistently develop mostly transparent lenses with an internal refractive disturbance and a grossly disturbed cellular architecture. In situ hybridization localized Epha2 and Efna5 transcripts to lens epithelial cells and nascent fiber cells at the lens equator. In vivo labeling of Epha2-null lenses with a thymidine analog detected a significant decrease in lens epithelial cell proliferation within the germinative zone resulting in impaired early lens growth. Ex vivo imaging of Epha2-null, Efna5-null, and Epha2/Efna5-null lenses labelled in vivo with a membrane-targeted red fluorescent protein revealed misalignment of elongating fiber cells at the lens equator and loss of Y-suture pattern formation near the anterior and posterior poles of the lens. Immuno-fluorescent labeling of lens major intrinsic protein or aquaporin-0 (MIP/AQP0) showed that the precise, radial column patterning of hexagonal fiber cells throughout the cortex region was disrupted in Epha2-null, Efna5-null and Epha2/Efna5-null lenses. Collectively, these data suggest that Epha2 and Efna5 participate in the complex, global patterning of lens fiber cells that is necessary for maximal optical quality.

Germ-line and somatic EPHA2 coding variants in lens aging and cataract.

Rare germ-line mutations in the coding regions of the human EPHA2 gene (EPHA2) have been associated with inherited forms of pediatric cataract, whereas, frequent, non-coding, single nucleotide variants (SNVs) have been associated with age-related cataract. Here we sought to determine if germ-line EPHA2 coding SNVs were associated with age-related cataract in a case-control DNA panel (> 50 years) and if somatic EPHA2 coding SNVs were associated with lens aging and/or cataract in a post-mortem lens DNA panel (> 48 years). Micro-fluidic PCR amplification followed by targeted amplicon (exon) next-generation (deep) sequencing of EPHA2 (17-exons) afforded high read-depth coverage (1000x) for > 82% of reads in the cataract case-control panel (161 cases, 64 controls) and > 70% of reads in the post-mortem lens panel (35 clear lens pairs, 22 cataract lens pairs). Novel and reference (known) missense SNVs in EPHA2 that were predicted in silico to be functionally damaging were found in both cases and controls from the age-related cataract panel at variant allele frequencies (VAFs) consistent with germ-line transmission (VAF > 20%). Similarly, both novel and reference missense SNVs in EPHA2 were found in the post-mortem lens panel at VAFs consistent with a somatic origin (VAF > 3%). The majority of SNVs found in the cataract case-control panel and post-mortem lens panel were transitions and many occurred at di-pyrimidine sites that are susceptible to ultraviolet (UV) radiation induced mutation. These data suggest that novel germ-line (blood) and somatic (lens) coding SNVs in EPHA2 that are predicted to be functionally deleterious occur in adults over 50 years of age. However, both types of EPHA2 coding variants were present at comparable levels in individuals with or without age-related cataract making simple genotype-phenotype correlations inconclusive.

Mutations and mechanisms in congenital and age-related cataracts.

The crystalline lens plays an important role in the refractive vision of vertebrates by facilitating variable fine focusing of light onto the retina. Loss of lens transparency, or cataract, is a frequently acquired cause of visual impairment in adults and may also present during childhood. Genetic studies have identified mutations in over 30 causative genes for congenital or other early-onset forms of cataract as well as several gene variants associated with age-related cataract. However, the pathogenic mechanisms resulting from genetic determinants of cataract are only just beginning to be understood. Here, we briefly summarize current concepts pointing to differences in the molecular mechanisms underlying congenital and age-related forms of cataract.

Lens transcriptome profile during cataract development in Mip-null mice.

Major intrinsic protein or aquaporin-0 (MIP/AQP0) functions as a water channel and a cell-junction molecule in the vertebrate eye lens. Loss of MIP function in the lens leads to degraded optical quality and cataract formation by pathogenic mechanisms that are unclear. Here we have used microarray-hybridization analysis to detect lens transcriptome changes during cataract formation in mice that are functionally null for MIP (Mip-/-). In newborn Mip-/- lenses (P1) 11 genes were up-regulated and 18 were down-regulated (>2-fold, p=<0.05) and a similar number of genes was differentially regulated at P7. The most up-regulated genes (>6-fold) in the Mip-/- lens at P1 included those coding for a mitochondrial translocase (Timmdc1), a matrix metallopeptidase (Mmp2), a Rho GTPase-interacting protein (Ubxn11) and a transcription factor (Twist2). Apart from Mip, the most down-regulated genes (>4-fold) in the Mip-/- lens at P1 included those coding for a proteasome sub-unit (Psmd8), a ribonuclease (Pop4), and a heat-shock protein (Hspb1). Lens fiber cell degeneration in the Mip-/- lens was associated with increased numbers of TUNEL-positive cell nuclei and dramatically elevated levels of calpain-mediated proteolysis of αII-spectrin. However red-ox status, measured by glutathione and free-radical levels, was similar to that of wild-type. These data suggest that while relatively few genes (∼1.5% of the transcriptome) were differentially regulated >2-fold in the Mip-/- lens, calpain hyper-activation acts as a terminal pathogenic event during lens fiber cell death and cataract formation.

Lens ER-stress response during cataract development in Mip-mutant mice.

Major intrinsic protein (MIP) is a functional water-channel (AQP0) that also plays a key role in establishing lens fiber cell architecture. Genetic variants of MIP have been associated with inherited and age-related forms of cataract; however, the underlying pathogenic mechanisms are unclear. Here we have used lens transcriptome profiling by microarray-hybridization and qPCR to identify pathogenic changes during cataract development in Mip-mutant (Lop/+) mice. In postnatal Lop/+ lenses (P7) 99 genes were up-regulated and 75 were down-regulated (>2-fold, p=<0.05) when compared with wild-type. A pathway analysis of up-regulated genes in the Lop/+ lens (P7) was consistent with endoplasmic reticulum (ER)-stress and activation of the unfolded protein response (UPR). The most up-regulated UPR genes (>4-fold) in the Lop/+ lens included Chac1>Ddit3>Atf3>Trib3>Xbp1 and the most down-regulated genes (>5-fold) included two anti-oxidant genes, Hspb1 and Hmox1. Lop/+ lenses were further characterized by abundant TUNEL-positive nuclei within central degenerating fiber cells, glutathione depletion, free-radical overproduction, and calpain hyper-activation. These data suggest that Lop/+ lenses undergo proteotoxic ER-stress induced cell-death resulting from prolonged activation of the Eif2ak3/Perk-Atf4-Ddit3-Chac1 branch of the UPR coupled with severe oxidative-stress.

Overview of the Lens.

In order to accomplish its function of transmitting and focusing light, the crystalline lens of the vertebrate eye has evolved a unique cellular structure and protein complement. These distinct adaptations have provided a rich source of scientific discovery ranging from biochemistry and genetics to optics and physics. In addition, because of these adaptations, lens cells persist for the lifetime of an organism, providing an excellent model of the aging process. The chapters dealing with the lens will demonstrate how the different aspects of lens biology and biochemistry combine in this singular refractive organ to accomplish its critical role in the visual system.

Molecular Genetics of Cataract.

Lens opacities or cataract(s) represent a universally important cause of visual impairment and blindness. Typically, cataract is acquired with aging as a complex disorder involving environmental and genetic risk factors. Cataract may also be inherited with an early onset either in association with other ocular and/or systemic abnormalities or as an isolated lens phenotype. Here we briefly review recent advances in gene discovery for inherited and age-related forms of cataract that are providing new insights into lens development and aging.

Lens Biology and Biochemistry.

The primary function of the lens resides in its transparency and ability to focus light on the retina. These require both that the lens cells contain high concentrations of densely packed lens crystallins to maintain a refractive index constant over distances approximating the wavelength of the light to be transmitted, and a specific arrangement of anterior epithelial cells and arcuate fiber cells lacking organelles in the nucleus to avoid blocking transmission of light. Because cells in the lens nucleus have shed their organelles, lens crystallins have to last for the lifetime of the organism, and are specifically adapted to this function. The lens crystallins comprise two major families: the ßγ-crystallins are among the most stable proteins known and the α-crystallins, which have a chaperone-like function. Other proteins and metabolic activities of the lens are primarily organized to protect the crystallins from damage over time and to maintain homeostasis of the lens cells. Membrane protein channels maintain osmotic and ionic balance across the lens, while the lens cytoskeleton provides for the specific shape of the lens cells, especially the fiber cells of the nucleus. Perhaps most importantly, a large part of the metabolic activity in the lens is directed toward maintaining a reduced state, which shelters the lens crystallins and other cellular components from damage from UV light and oxidative stress. Finally, the energy requirements of the lens are met largely by glycolysis and the pentose phosphate pathway, perhaps in response to the avascular nature of the lens. Together, all these systems cooperate to maintain lens transparency over time.

Exome Sequencing Identifies a Missense Variant in EFEMP1 Co-Segregating in a Family with Autosomal Dominant Primary Open-Angle Glaucoma.

Primary open-angle glaucoma (POAG) is a clinically important and genetically heterogeneous cause of progressive vision loss as a result of retinal ganglion cell death. Here we have utilized trio-based, whole-exome sequencing to identify the genetic defect underlying an autosomal dominant form of adult-onset POAG segregating in an African-American family. Exome sequencing identified a novel missense variant (c.418C>T, p.Arg140Trp) in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1) that co-segregated with disease in the family. Linkage and haplotype analyses with microsatellite markers indicated that the disease interval overlapped a known POAG locus (GLC1H) on chromosome 2p. The p.Arg140Trp substitution was predicted in silico to have damaging effects on protein function and transient expression studies in cultured cells revealed that the Trp140-mutant protein exhibited increased intracellular accumulation compared with wild-type EFEMP1. In situ hybridization of the mouse eye with oligonucleotide probes detected the highest levels of EFEMP1 transcripts in the ciliary body, cornea, inner nuclear layer of the retina, and the optic nerve head. The recent finding that a common variant near EFEMP1 was associated with optic nerve-head morphology supports the possibility that the EFEMP1 variant identified in this POAG family may be pathogenic.