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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.

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.

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.

Congenital cataracts cause approximately one-third of blindness in infants worldwide. If untreated they can cause permanent blindness by interfering with the sharp focus of light onto the retina and thus fail to establish appropriate synaptic connections between the retina and the visual cortex. Between 8 and 30% (see later) of congenital cataracts are inherited, and our understanding of their genetic architecture is increasing. Delineating the relationship between the genes and mutations causing cataracts and their phenotypic presentation can help us to understand the biology of the lens and provide a framework for the clinical approach to diagnosis and treatment.

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. By 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.

Lens opacities or cataracts in children are usually congenital and/or hereditary and less often are secondary to trauma, infections, or systemic disorders such as diabetes or galactosemia. Congenital cataracts cause approximately one-third of blindness in infants worldwide. Between 8% and 25% of congenital cataracts are inherited, and knowledge of their genetic architecture is increasing. Delineating the relationship between the genes and mutations causing cataracts and their phenotypic presentation can help us to understand the biology of the lens and provide a framework for the clinical approach to diagnosis and therapy.

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.

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.

First discovered in a light-sensitive retinal mutant ofDrosophila, 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.

Age-related cataract is a leading cause of vision loss and the most common reason for eye surgery in the elderly. Epidemiological studies of affected siblings and twins indicate that genetic factors may account for as much as 50% of the risk for age-related cataract. Family-based linkage studies have identified a number of genetic susceptibility loci for age-related cataract, and case–control association studies have implicated a number of diverse candidate genes involved in: antioxidant metabolism, galactose metabolism, the heat-shock stress response, and the ephrin signaling pathway. Rigorous characterization of the underlying genetic causes may lead to nonsurgical treatments for age-related cataract.

The pathogenesis of inherited cataracts of all kinds recapitulates the developmental and cell biology of the lens. Just as each novel mutation provides additional information about the structural or functional biology of the affected gene, each newly identified gene provides insight into the developmental and cellular biology of the lens. The set of genes currently known to be associated with cataract is far from complete, especially for age-related cataract, and there is much additional information to be discovered through further genetic studies.

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=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.

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.

The fusion of cells to generate syncytial tissues is a crucial event in the development of many organisms. In the lens of the vertebrate eye, proteins and other macromolecules diffuse from cell to cell via the large molecule diffusion pathway (LMDP). We used the tamoxifen-induced expression of GFP to investigate the nature and role of the LMDP in living, intact lenses. Our data indicate that the LMPD preferentially connects cells lying within a stratum of the lens cortex and that formation of the LMPD depends on the expression of Lim2, a claudin-like molecule. The conduits for intercellular protein exchange are most likely regions of partial cellular fusion, which are commonly observed in wild-type lenses but rare or absent in Lim2-deficient lenses. The observation that lens tissue constitutes a stratified syncytium has implications for the transparency, refractive function and pathophysiology of the tissue.

Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent exvivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.

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.

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.

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 βγ-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.

Hereditary cataract is a clinically and genetically heterogeneous lens disease that accounts for a significant proportion of visual impairment and blindness in childhood. The alphaA-crystallin (CRYAA) gene (CRYAA) encodes a member of the small-heat-shock protein (sHSP) family of molecular chaperones and is primarily and abundantly expressed in the ocular lens. Here, we have used linkage analysis to identify a novel missense mutation in CRYAA that underlies an autosomal dominant form of 'nuclear' cataract segregating in a four-generation Caucasian family. A maximum two-point LOD score (Z(max)) of 2.19 (maximum recombination fraction, theta(max)=0) and multipoint Z(max) of 3.3 (theta(max)=0) was obtained at marker D21S1885. Haplotype analysis indicated that the disease gene lay in the approximately 2.7 Mb physical interval between D21S1912 and D21S1260 flanking CRYAA on 21q22.3. Sequence analysis identified a C --> T transition in exon 1 of CRYAA from affected individuals that was predicted to result in the nonconservative substitution of cysteine for arginine at codon 49 (R49C). Transfection studies of lens epithelial cells revealed that, unlike wild-type CRYAA, the R49C mutant protein was abnormally localized to the nucleus and failed to protect from staurosporine-induced apoptotic cell death. This study has identified the first dominant cataract mutation in CRYAA located outside the phylogenetically conserved 'alpha-crystallin core domain' of the sHSP family.

Background Inherited cataract is a clinically important and genetically heterogeneous cause of visual impairment. Typically, it presents at an early age with or without other ocular/systemic signs and lacks clear phenotype-genotype correlation rendering both clinical classification and molecular diagnosis challenging. Here we have utilized trio-based whole exome sequencing to discover mutations in candidate genes underlying autosomal dominant cataract segregating in three nuclear families. Results In family A, we identified a recurrent heterozygous mutation in exon-2 of the gene encoding γD-crystallin (CRYGD; c.70C > A, p.Pro24Thr) that co-segregated with ‘coralliform’ lens opacities. Families B and C were found to harbor different novel variants in exon-2 of the gene coding for gap-junction protein α8 (GJA8; c.20T > C, p.Leu7Pro and c.293A > C, p.His98Pro). Each novel variant co-segregated with disease and was predicted in silico to have damaging effects on protein function. Conclusions Exome sequencing facilitates concurrent mutation-profiling of the burgeoning list of candidate genes for inherited cataract, and the results can provide enhanced clinical diagnosis and genetic counseling for affected families. Electronic supplementary material The online version of this article (doi:10.1186/s40246-014-0019-6) contains supplementary material, which is available to authorized users.

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.

Inherited forms of cataract are a clinically important and genetically heterogeneous cause of visual impairment that usually present at an early age with or without systemic and/or other ocular abnormalities. Here we have identified a new locus for inherited cataract and high-tension glaucoma with variable anterior segment defects, and characterized an underlying mutation in the gene coding for transient receptor potential cation channel, subfamily M, member-3 (TRPM3, melastatin-2). Genome-wide linkage analysis mapped the ocular disease locus to the pericentric region of human chromosome 9. Whole exome and custom-target next-generation sequencing detected a heterozygous A-to-G transition in exon-3 of TRPM3 that co-segregated with disease. As a consequence of alternative splicing this missense mutation was predicted to result in the substitution of isoleucine-to-methionine at codon 65 (c.195A>G; p.I65 M) of TRPM3 transcript variant 9, and at codon 8 (c.24A>G; p.I8 M) of a novel TRPM3 transcript variant expressed in human lens. In both transcript variants the I-to-M substitution was predicted in silico to exert damaging effects on protein function. Furthermore, transient expression studies of a recombinant TRPM3-GFP reporter product predicted that the I-to-M substitution introduced an alternative translation start-site located 89 codons upstream from the native initiator methionine found in eight other TRPM3 transcript variants (1-8). Collectively, these studies have provided the first evidence that TRPM3 is associated with inherited ocular disease in humans, and further provide support for the important role of this cation channel in normal eye development.

Purpose Lens fiber cell membranes contain aquaporin-0 (AQP0), which constitutes approximately 50% of the total fiber cell membrane proteins and has a dual function as a water channel protein and an adhesion molecule. Fiber cell membranes also develop an elaborate interlocking system that is required for maintaining structural order, stability, and lens transparency. Herein, we used an AQP0-deficient mouse model to investigate an unconventional adhesion role of AQP0 in maintaining a normal structure of lens interlocking protrusions. Methods The loss of AQP0 in AQP0(-/-) lens fibers was verified by Western blot and immunofluorescence analyses. Changes in membrane surface structures of wild-type and AQP0(-/-) lenses at age 3 to 12 weeks were examined with scanning electron microscopy. Preferential distribution of AQP0 in wild-type fiber cell membranes was analyzed with immunofluorescence and immunogold labeling using freeze-fracturing transmission electron microscopy. Results Interlocking protrusions in young differentiating fiber cells developed normally but showed minor abnormalities at approximately 50 μm deep in the absence of AQP0 in all ages studied. Strikingly, protrusions in maturing fiber cells specifically underwent uncontrolled elongation, deformation, and fragmentation, while cells still retained their overall shape. Later in the process, these changes eventually resulted in fiber cell separation, breakdown, and cataract formation in the lens core. Immunolabeling at the light microscopy and transmission electron microscopy levels demonstrated that AQP0 was particularly enriched in interlocking protrusions in wild-type lenses. Conclusions This study suggests that AQP0 exerts its primary adhesion or suppression role specifically to maintain the normal structure of interlocking protrusions that is critical to the integrity and transparency of the lens.

Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because hCx46 mutations have been linked to congenital cataracts. We studied two hCx46 mutants, N63S, a missense mutation in the first extracellular domain, and fs380, a frame-shift mutation that shifts the translational reading frame at amino acid residue 380. We expressed wild-type Cx46 and the two mutants in Xenopus oocytes. Production of the expressed proteins was verified by SDS-PAGE after metabolic labeling with [35S]methionine or by immunoblotting. Dual two-microelectrode voltage-clamp studies showed that hCx46 formed both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants could form intercellular channels in paired Xenopus oocytes. The hCx46 mutants were also impaired in their ability to form hemi-gap-junctional channels. When N63S or fs380 was coexpressed with wild-type connexins, both mutations acted like “loss of function” rather than “dominant negative” mutations, because they did not affect the gap junctional conductance induced by either wild-type hCx46 or wild-type hCx50.

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in CatFr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (pH2O) on the order of 1 micron/sec whereas normal fiber cell membrane pH2O was 17 micron/sec frog, 32 micron/sec rabbit and 43 micron/sec mouse. CatFr mouse lens fiber cell pH2O was reduced by 13 micron/sec for heterozygous and 30 micron/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the pH2O conferred by MIP is not sensitive to Hg2+ whereas that of CHIP28 (AQP1) is blocked by Hg2+. The fiber cell membrane pH2O was also not sensitive to Hg2+ whereas lens epithelial cell pH2O (136 micron/sec in rabbit) was blocked by Hg2+. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous CatFr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses.

7 páginas, 1 figura, 2 tablas.-- Licence Creative Commons, attribution, Non-commercial licence.-- et al., [AIMS]: To determine the different morphologies of autosomal dominant cataract (ADC), assess the intra- and interfamilial variation in cataract morphology, and undertake a genetic linkage study to identify loci for genes causing ADC and detect the underlying mutation. [METHODS]: Patients were recruited from the ocular genetic database at Moorfields Eye Hospital. All individuals underwent an eye examination with particular attention to the lens including anterior segment photography where possible. Blood samples were taken for DNA extraction and genetic linkage analysis was carried out using polymorphic microsatellite markers. [RESULTS]: 292 individuals from 16 large pedigrees with ADC were examined, of whom 161 were found to be affected. The cataract phenotypes could all be described as one of the eight following morphologies—anterior polar, posterior polar, nuclear, lamellar, coralliform, blue dot (cerulean), cortical, and pulverulent. The phenotypes varied in severity but the morphology was consistent within each pedigree, except in those affected by the pulverulent cataract, which showed considerable intrafamilial variation. Positive linkage was obtained in five families; in two families linkage was demonstrated to new loci and in three families linkage was demonstrated to previously described loci. In one of the families the underlying mutation was isolated. Exclusion data were obtained on five families. [CONCLUSIONS]: Although there is considerable clinical heterogeneity in ADC, the phenotype is usually consistent within families. There is extensive genetic heterogeneity and specific cataract phenotypes appear to be associated with mutations at more than one chromosome locus. In cases where the genetic mutation has been identified the molecular biology and clinical phenotype are closely associated., The work was supported by a grants from theWellcome Trust (043073 to AS and SSB; 053416 to ATM and SSB). ACWI was supported by a grant from The Friends of Moorfields Eye Hospital and DSM is a Wellcome Prize student.

Mutations in gap junctional channels have been linked to certain forms of inherited congenital cataract (D. Mackay, A. Ionides, V. Berry, A. Moore, S. Bhattacharya, and A. Shiels. Am. J. Hum. Genet. 60: 1474-1478, 1997; A. Shiels, D. Mackay, A. Ionides, V. Berry, A. Moore, and S. Bhattacharya. Am. J. Hum. Genet. 62: 526-532, 1998). We used the Xenopus oocyte pair system to investigate the functional properties of a missense mutation in the human connexin 50 gene (P88S) associated with zonular pulverulent cataract. The associated phenotype for the mutation is transmitted in an autosomal dominant fashion. Xenopus oocytes injected with wild-type connexin 50 cRNA developed gap junctional conductances of approximately 5 microS 4-7 h after pairing. In contrast, the P88S mutant connexin failed to form functional gap junctional channels when paired homotypically. Moreover, the P88S mutant functioned in a dominant negative manner as an inhibitor of human connexin 50 gap junctional channels when coinjected with wild-type connexin 50 cRNA. Cells injected with 1:5 and 1:11 ratios of P88S mutant to wild-type cRNA exhibited gap junctional coupling of approximately 8% and 39% of wild-type coupling, respectively. Based on these findings, we conclude that only one P88S mutant subunit is necessary per gap junctional channel to abolish channel function.

Immunocytochemistry fails to detect expression of the lens major intrinsic protein (MIP) in 16-day embryos of the congenitally cataractous mouse, CAT, which inherits a dominant mutation assigned to the distal end of mouse chromosome 10. In situ hybridisation, however, detects MIP mRNA in CAT embryo lens fibre cells at a level approximating 60% of that detected in embryonic lens fibres of the non-cataractous MF1 mouse. Northern blot hybridisation reveals that the most abundant MIP mRNA transcript in the adult CAT lens is truncated when compared to that in the adult MF1 lens. The results are consistent with a cataractogenic mutation in the mouse gene for MIP (Mip) which has also been mapped to the distal end of mouse chromosome 10.

Lens opacities, or cataract(s), may be inherited as a classic Mendelian disorder usually with early-onset or, more commonly, acquired with age as a multi-factorial or complex trait. Many genetic forms of cataract have been described in mice and other animal models. Considerable progress has been made in mapping and identifying the genes and mutations responsible for inherited forms of cataract, and genetic determinants of age-related cataract are beginning to be discovered. To provide a convenient and accurate summary of current information focused on the increasing genetic complexity of Mendelian and age-related cataract we have created an online chromosome map and reference database for cataract in humans and mice (Cat-Map).

Cloned cDNAs coding for eye-lens fibre cell-membrane proteins, MIP and MP70, were used to detect restriction fragment length polymorphisms (RFLPs) in genomic DNA from inbred mice with autosomally inherited cataracts. Whereas distinct RFLPs associated with the MIP gene were identified in the Cba Cat and Nct mutants, no such genetic variation was associated with the MP70 gene. RFLPs associated with the mouse MIP gene may provide informative DNA markers in gene linkage studies of murine hereditary cataracts.

Aims: To determine whether complement factor H ( CFH ) genotypes have a pharmacogenetic effect on the treatment of exudative age-related macular degeneration (AMD) with ranibizumab. Methods: A retrospective study of 156 patients with exudative AMD treated with intravitreal ranibizumab monotherapy was conducted. AMD phenotypes were characterised by clinical examination, visual acuity, fundus photography, fluorescein angiography and injection timing. Patients received intravitreal ranibizumab injections as part of routine ophthalmological care and were followed for a minimum of 9 months. Each patient was genotyped for the single nucleotide polymorphism rs1061170 (Y402H) in the CFH gene. Results: Baseline lesion size and angiographic type, as well as mean visual acuities at baseline, 6 months, and 9 months were similar among the three CFH genotypes. Over 9 months, patients with both risk alleles received approximately one more injection (p = 0.09). In a recurrent event analysis, patients homozygous for the CFH Y402H risk allele had a 37% significantly higher risk of requiring additional ranibizumab injections (p = 0.04). Conclusions: In this study cohort, the response to treatment of AMD with ranibizumab differed according to CFH genotype, suggesting that determining patients’ CFH genotype may be helpful in the future in tailoring treatment for exudative AMD with intravitreal ranibizumab.

To determine whether there is an association between complement factor H (CFH) or LOC387715 genotypes and response to treatment with photodynamic therapy (PDT) for exudative age-related macular degeneration (AMD). Sixty-nine patients being treated for neovascular AMD with PDT were genotyped for the CFH Y402H and LOC387715 A69S polymorphisms by allele-specific digestion of PCR products. AMD phenotypes were characterized by clinical examination, fundus photography, and fluorescein angiography. Adjusting for age, pre-PDT visual acuity (VA), and lesion type, mean VA after PDT was significantly worse for the CFH TT genotype than for the TC or CC genotypes (P=0.05). Post-PDT VA was significantly worse for the CFH TT genotype in the subgroup of patients with predominantly classic choroidal neovascular lesions (P=0.04), but not for the patients with occult lesions (P=0.22). For the LOC387715 A69S variant, there was no significant difference among the genotypes in response to PDT therapy. The CFH Y402H variant was associated with a response to PDT treatment in this study. Patients with the CFH TT genotype fared significantly worse with PDT than did those with the CFH TC and CC genotypes, suggesting a potential relationship between CFH genotype and response to PDT.

Infantile nystagmus is a clinically and genetically heterogeneous eye movement disorder. Here we map and identify the genetic mutation underlying X-linked idiopathic infantile nystagmus (XL-IIN) segregating in two Caucasian-American families.Eye movements were recorded using binocular infrared digital video-oculography. Genomic DNA was prepared from blood or buccal-cells, and linkage analysis was performed using short tandem repeat (STR) and single nucleotide polymorphism (SNP) markers. Pedigree and haplotype data were managed using Cyrillic, and LOD scores calculated using MLINK. Mutation profiling of PCR-amplified exons was performed by dye-terminator cycle-sequencing and analyzed by automated capillary electrophoresis.Video-oculography of affected males recorded conjugate, horizontal, pendular nystagmus with increasing-velocity waveforms in primary gaze converting to jerk nystagmus in eccentric gaze. Linkage analysis detected significantly positive two-point LOD scores (Z) at markers DXS8078 (Z=4.82, recombination fraction [theta]=0) and DXS1047 (Z=3.87, theta=0). Haplotyping indicated that the IIN locus mapped to the physical interval DXS8057-(11.59 Mb)-rs6528335 on Xq25-q26. Sequencing of positional-candidate genes detected a c.425TG transversion in exon-6 of the gene for FERM domain containing-7 (FRMD7) that cosegregated with affected and carrier status. In addition, the same change was found to cosegregate with IIN in a genetically unrelated family but was not detected in 192 control individuals.The c.425TG change is predicted to result in the missense substitution of the phylogenetically conserved leucine at codon 142 for an arginine (p.L142R), and supports a causative role for FRMD7 mutations in the pathogenesis of XL-IIN.

Purpose To determine whether the complement factor H (CFH) Y402H variant is associated with specific age-related macular degeneration (AMD) clinical phenotypes. Design Retrospective, case-control study. Methods One hundred and eighty-eight white subjects with AMD and 189 control subjects were genotyped for the T-to-C polymorphism in exon 9 of the CFH gene by restriction-fragment length analysis and deoxyribonucleic acid (DNA) sequencing using genomic DNA from mouthwash samples. AMD phenotypes were characterized by clinical examination, fundus photography, and fluorescein angiography. Results Heterozygosity for the at-risk genotype (TC) increased the likelihood for AMD 2.1-fold (95% confidence interval [CI], 1.3 to 3.3), whereas homozygosity for the genotype (CC) increased the likelihood for AMD 6.5-fold (95% CI, 3.4 to 12.5) in our population. The C allele was associated significantly with predominantly classic choroidal neovascularization (odds ratio [OR], 2.01; 95% CI, 1.34 to 3.30). Neovascular lesion size was similar among the three genotypes ( P = .67). Conclusions The Y402H CFH variant carried a significantly increased risk for developing AMD in our population. Genotype and phenotype correlations regarding choroidal neovascular lesion type were observed.

Purpose To investigate whether there is an association between complement factor H (CFH) or LOC387715 genotypes with response to treatment with intravitreal bevacizumab for exudative age-related macular degeneration (AMD). Design Retrospective cohort study. Participants The study cohort consisted of 86 patients being treated for neovascular AMD with bevacizumab alone. Methods Genotype determination for the CFH Y402H and LOC387715 A69S polymorphisms was performed by allele-specific digestion of polymerase chain reaction products. All patients were treated with 1.25 mg intravitreal bevacizumab at 6-week intervals until choroidal neovascularization was no longer active. Main Outcome Measures CFH Y402H and LOC387715 A69S polymorphisms. Choroidal neovascular lesion characteristics were ascertained by fluorescein angiography. Snellen visual acuity (VA) was measured before and after treatment. Results For the CFH Y402H polymorphism, patients with the CFH TT genotype had the largest choroidal neovascular lesions ( P = 0.02). With treatment, VA improved from 20/248 to 20/166 for the CFH TT genotype and from 20/206 to 20/170 for the TC genotype, but fell from 20/206 to 20/341 for the CFH CC genotype ( P = 0.016). Only 10.5% of patients with the CFH CC genotype demonstrated improved VA with treatment, compared with 53.7% of CFH TT and TC genotypes ( P = 0.004). For the LOC387715 A69S variant, patients with the TT genotype had the largest choroidal neovascular lesions ( P = 0.012). There was no significant difference in response to bevacizumab treatment according to LOC387715 genotype. Conclusions The AMD-associated CFH Y402H and LOC387715 A69S variants were associated with differences in choroidal neovascular lesion size in this study. Patients with the CFH CC genotype fared significantly worse with intravitreal bevacizumab than did those with the CFH TC and TT genotypes, suggesting a potential pharmacogenetic relationship. Prospective studies to confirm or refute this observation should be considered.

Cataracts are a clinically diverse and genetically heterogeneous disorder of the crystalline lens and a leading cause of visual impairment. Here we report linkage of autosomal dominant "progressive childhood posterior subcapsular" cataracts segregating in a white family to short tandem repeat (STR) markers D20S847 (LOD score [Z] 5.50 at recombination fraction [theta] 0.0) and D20S195 (Z=3.65 at theta =0.0) on 20q, and identify a refined disease interval (rs2057262-(3.8 Mb)-rs1291139) by use of single-nucleotide polymorphism (SNP) markers. Mutation profiling of positional-candidate genes detected a heterozygous transversion (c.386A--T) in exon 3 of the gene for chromatin modifying protein-4B (CHMP4B) that was predicted to result in the nonconservative substitution of a valine residue for a phylogenetically conserved aspartic acid residue at codon 129 (p.D129V). In addition, we have detected a heterozygous transition (c.481G--A) in exon 3 of CHMP4B cosegregating with autosomal dominant posterior polar cataracts in a Japanese family that was predicted to result in the missense substitution of lysine for a conserved glutamic acid residue at codon 161 (p.E161K). Transfection studies of cultured cells revealed that a truncated form of recombinant D129V-CHMP4B had a different subcellular distribution than wild type and an increased capacity to inhibit release of virus-like particles from the cell surface, consistent with deleterious gain-of-function effects. These data provide the first evidence that CHMP4B, which encodes a key component of the endosome sorting complex required for the transport-III (ESCRT-III) system of mammalian cells, plays a vital role in the maintenance of lens transparency.

PURPOSE. To characterize the optical properties of lenses from mice deficient in the gene for lens intrinsic membrane protein-2 (Lim2), which encodes the second most abundant integral protein (Lim2) of lens fiber cell plasma membranes. METHODS. Lim2-deficient mice were derived from a library of gene-trap embryo stem cells. Genotyping was performed by polymerase chain reaction (PCR) amplification of tail genomic DNA and resequencing. Lim2 expression was analyzed by reverse transcription (RT)-PCR and Northern blotting of lens total RNA, immunoblotting of lens membrane extracts, and immunofluorescence confocal microscopy of lens sections. Lens morphology was assessed by light microscopy, and lens refractive properties were quantified with a laser imaging system. RESULTS. Genomic PCR amplification and resequencing indicated that the gene-trap vector had disrupted intron 3 of Lim2, effectively resulting in a null allele (Lim2 Gt ), as verified by RT-PCR amplification and sequencing, RNA blotting, immunoblotting, and immunofluorescence confocal microscopy. Heterozygous Lim2 gene-trap lenses (Lim2 Gt/+ ) were morphologically indistinguishable from wild type, whereas homozygous Lim2 gene-trap lenses (Lim2 Gt/Gt ) consistently developed faint, central pulverulent cataracts. Laser imaging analysis indicated that rays passing through the peripheral cortex of the Lim2 Gt/Gt lens were more strongly refracted than normal, suggesting that the internal gradient refractive index of the lens was disturbed. CONCLUSIONS. These data show that heterozygous loss of Lim2 is insufficient to trigger cataracts in mice, and they provide the first direct evidence that Lim2 plays a critical role in establishing the correct internal refractive properties of the crystalline lens.

PURPOSE. To examine Ca 2+ - and pH-mediated regulation of water permeability of endogenously expressed aquaporin (AQP)0 in lens fiber cells and AQP1 in lens epithelial cells. METHODS. Large, right-side-out membrane vesicles were formed from freshly isolated groups of lens fiber cells. Osmotic shrinking or swelling of these vesicles was used to determine the water permeability of endogenously expressed AQP0. The results were compared with those in similar studies of freshly isolated lens epithelial cells, which endogenously expressed AQP1, and of oocytes, which exogenously expressed AQP0. RESULTS. In the lens or in oocytes, decreasing external pH from 7.5 to 6.5 caused a two- to fourfold increase in the water permeability of mammalian AQP0. Several lines of evidence suggest that this effect is mediated by the binding of H + to a histidine in the first extracellular loop (His40). Lens AQP1 lacks His40 and also lacks pH sensitivity. Increasing Ca 2+ caused a two- to fourfold increase in the water permeability of endogenous AQP0. The Ca 2+ effect on mouse AQP0 was a 2.5-fold increase in the lens, whereas in oocytes, it was a 4-fold decrease. In either environment, the effect was mediated through calmodulin, most likely through its binding to the proximal domain of the C terminus. Lens AQP1 does not have a similar domain and does not have calcium sensitivity. CONCLUSIONS. In either the lens or oocytes, Ca 2+ and H + appear to affect the same mechanism, probably either the open probability of the water channel, or open-channel permeability. The difference between calcium's effects in lens versus oocytes was remarkable and is not understood. However, in the lens, Ca 2+ and H + are both increased in inner fiber cells, and so in the physiologically relevant environment, both may act to increase the water permeability of AQP0.

Autosomal dominant cataracts are a clinically and genetically heterogeneous eye-lens disorder that usually present in childhood with symptoms of impaired vision. The purpose of this study was to map and identify the mutation underlying autosomal dominant nuclear punctate cataracts segregating in a six generation Caucasian pedigree.Genomic DNA was prepared from blood leucocytes, genotyping was performed using microsatellite markers, and LOD scores were calculated using the LINKAGE programs. Mutation detection was performed using direct sequencing and restriction fragment length analysis.Significant evidence of linkage was obtained at marker D13S175 (LOD score [Z]=4.11, recombination fraction [theta]=0.0) and haplotyping indicated that the disease gene lay in the about 2 Mb physical interval between D13S1316 and D13S1236, which contained the gene for gap-junction protein a3 (GJA3) or connexin46. Sequencing of GJA3 detected a C-T transition in exon 2 that resulted in the gain of an Alu 1 restriction site and was predicted to cause a conservative substitution of proline to leucine at codon 59 (P59L). Restriction analysis confirmed that the novel Alu 1 site co-segregated with cataracts in the family but was not detected in a control panel of 170 normal unrelated individuals.The present study has identified a fifth mutation in GJA3, rendering this connexin gene one of the most common non-crystallin genes associated with autosomal dominant cataracts in humans.

Hereditary cataract is a clinically and genetically heterogeneous lens disorder that usually presents as a sight-threatening trait in childhood. The purpose of this study was to map and identify the mutation underlying an autosomal dominant form of coral-shaped cataract segregating in a three generation Caucasian pedigree.Genomic DNA was prepared from blood leucocytes, genotyping was performed using microsatellite markers, and LOD scores were calculated using the LINKAGE programs. Mutation detection was performed using direct sequencing and primer extension analysis. Following site-directed mutagenesis, mutant and wild type expression constructs were transfected into a human lens epithelial cell line (HLE B-3) and recombinant protein was detected by immunoblotting, imunofluorescence, and immunogold microscopy. Cell death was monitored by fluorescence activated cell sorting.Significant evidence of linkage was detected at markers D2S371 (LOD score [Z]=3.81, recombination fraction [theta]=0) and D2S369 (Z=3.64, theta=0). Haplotyping indicated that the disease gene lay in the approximate 10 Mb physical interval between D2S1384 and D2S128, containing the gamma-crystallin gene (CRYGA-CRYGD) cluster on chromosome 2q33.3-q34. Sequencing of the CRYGA-CRYGD cluster identified a C-A transversion in exon 2 of CRYGD that was predicted to result in the non-conservative substitution of threonine for proline at amino-acid residue 23 (P23T) in the processed CRYGD protein. Transfection studies suggested that the P23T mutant was less soluble than its wild type counterpart when expressed in HLE B-3 cells.This study has identified an eighth type of cataract morphology associated with CRYGD and suggests that a CRYGD mutation may underlie the historically important "coralliform" cataract first reported in 1895.