National Eye Institute Workshop to Identify Gaps, Needs, and Opportunities in Ophthalmic Genetics [NEI Strategic Planning]

National Eye Institute Workshop to Identify Gaps, Needs, and Opportunities in Ophthalmic Genetics

June 4-5, 2009

Diseases, Biological Systems, Approaches and Methodologies

White Papers

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Janey Wiggs


Corneal dystrophies are a group of heterogenous conditions that are characterized by the progressive loss of corneal transparency that results from the accumulation of deposits within the different corneal layers. Most corneal dystrophies develop during the 2nd to 4th decades of life, and are treated surgically by corneal transplantation. Currently there are no known medical treatments for these conditions. The majority of corneal dystrophies are rare conditions that follow mendelian inheritance. Two significant exceptions are Fuchs endothelial dystrophy and keratoconus, both age-related conditions with complex inheritance patterns.

Genetics of mendelian corneal dystrophies: The majority of the mendelian corneal dystrophies are inherited as autosomal dominant traits. There is considerable genetic heterogeneity and also phenotypic variability (even within the same family). Corneal dystrophies may affect all layers of the corneal tissue: epithelium, stroma and Descemet’s membrane. A number of genes responsible for these conditions have been identified (see Table) and one gene, TGFB1 is the cause of a group of corneal dystrophies (granular, Avellino, lattice and Reis Bucklers). Macular dystrophy (caused by mutations in CHST6 (carbohydrate sulfotransferase 6 gene) is inherited as an autosomal recessive trait.

Genetics of Fuchs endothelial dystrophy and keratoconus: Mutations in COL8A2 have been associated with some early-onset Fuchs endothelial dystrophy families, but most patients with adult onset Fuchs dystrophy do not have mutations in this gene. Like other age-related conditions, it is likely that multiple factors, both genetic and environmental may contribute to this condition. A genome wide linkage scan recently has identified several chromosome regions that may contain susceptibility genes. UV light exposure may also be an environmental risk factor for Fuchs. Keratoconus is also likely to result from multiple risk factors. A recent genome scan has been completed and has identified several candidate regions. Recently a gene responsible for Leber’s congenital amaurosis, CRB1, has also been implicated in keratoconus. Patients with Down’s syndrome are more likely to develop keratoconus, a finding that has suggested a locus on chromosome 21, and eye rubbing may be an environmental risk factor.

Consensus on phenotyping: A recent publication, IC3D Classification of the Corneal Dystrophies (see reviews below) has developed a consensus nomenclature for the corneal dystrophies based on genetic defects.

Genetic resources for cornea: Population-based studies, clinic-based studies, some animal models, affected tissue from penetrating keratoplasty.

Major cornea genetics studies

Mendelian corneal dystrophies: Many studies of linkage and gene identification have been published (see reviews and table below).

Fuchs and keratoconus: Recent genome scans for both conditions have been published (references below). These studies have identified relatively large chromosome regions with multiple possible candidate genes. A large multicenter study collecting Fuchs cases and controls lead by Dr. S. Iygengar (Case Western Ohio) is ongoing, as well as other studies in Fuchs lead by J. Gottsch (Hopkins) and G. Klintworth (Duke).

Aldave, A. J. et al. Arch Ophthalmol 2007;125:177-186.

Next Steps

Short-term goals to advance the field:


  1. Gene testing panel, sequencing array to establish screening/diagnosis/prognosis
  2. Animal models for testing potential therapeutics (gene replacement therapy could be considered for macular dystrophy)


  1. Sufficiently powered studies for Fuchs and keratoconus such that genes of modest effect could be identified. Gene-gene and gene-environment studies should also be considered.

Long-term goals to advance the field:

  1. Functional studies to inform potential therapeutics
  2. Translation of modifiable risk factors (for Fuchs and keratoconus) to clinical practice


Weiss JS, Møller HU, Lisch W, Kinoshita S, Aldave AJ, Belin MW, Kivelä T, Busin M, Munier FL, Seitz B, Sutphin J, Bredrup C, Mannis MJ, Rapuano CJ, Van Rij G, Kim EK, Klintworth GK. The IC3D classification of the corneal dystrophies. Cornea. 2008 Dec;27 Suppl 2:S1-83.

Poulaki V, Colby K. Semin Ophthalmol. Genetics of anterior and stromal corneal dystrophies.2008 Jan-Feb;23(1):9-17.

Aldave AJ, Sonmez B. Elucidating the molecular genetic basis of the corneal dystrophies: are we there yet? Arch Ophthalmol. 2007 Feb;125(2):177-86.

Afshari NA, Li YJ, Pericak-Vance MA, Gregory S, Klintworth GK. Genome-wide linkage scan in fuchs endothelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2009 Mar;50(3):1093-7.

Sundin OH, Broman KW, Chang HH, Vito EC, Stark WJ, Gottsch JD. A common locus for late-onset Fuchs corneal dystrophy maps to 18q21.2-q21.32. Invest Ophthalmol Vis Sci. 2006 Sep;47(9):3919-26.

Gajecka M, Radhakrishna U, Winters D, Nath SK, Rydzanicz M, Ratnamala U, Ewing K, Molinari A, Pitarque JA, Lee K, Leal SM, Bejjani BA. Localization of a gene for keratoconus to a 5.6-Mb interval on 13q32. Invest Ophthalmol Vis Sci. 2009 Apr;50(4):1531-9.

McMahon TT, Kim LS, Fishman GA, Stone EM, Zhao XC, Yee R, Malicki J. CRB1 Gene Mutations Are Associated With Keratoconus In Patients With Leber Congenital Amaurosis. Invest Ophthalmol Vis Sci. 2009 Apr 30. [Epub ahead of print]

Lens and Cataract

Janey Wiggs


Cataract can be defined as any opacity of the crystalline lens. Cataracts typically develop with age, but may also affect children. Early-onset or congenital cataracts are particularly serious because they have the potential for inhibiting visual development and cause blindness from amblyopia, or other problems related to their surgical removal. Most inherited cataract is caused by mutations in genes encoding for proteins involved in the maintenance of lens transparency and homeostasis. Information about these proteins and their functions has lead to an increased understanding about lens biology and cataract formation.

Genetics of early-onset or congenital cataracts: About 70% of early-onset or congenital cataracts involve the lens only, while the remaining cases are associated with other ocular abnormalities or syndromes. Nonsyndromic cataracts are most frequently inherited as autosomal dominant traits, but also can be inherited in an autosomal recessive, or X-linked fashion. Phenotypically identical cataracts can result from mutations at different genetic loci and may have different inheritance patterns, while phenotypically variable cataracts can be found in a single large family. There are at least 39 mapped loci for early-onset or congenital cataracts, and mutations in 26 genes have been associated with these conditions. Of these, approximately 50% have mutations in crystallins, approximately 25% have mutations in connexins, and the remainder divided among the genes for heat shock transcription factor-4 (HSF4), aquaporin-0 (AQP0, MIP), and beaded filament structural protein-2 (BFSP2) (see review by Hejtmancik for a detailed mutation list).

Genetics of age-related cataracts: Studies have suggested that multiple risk factors, both genetic and environmental, are associated with age-related cataracts. Family aggregations have been reported and segregation analyses suggest modest family correlations. A genomewide scan, using model-free linkage analysis of affected sib pairs, several potential susceptiblity loci for age-related cortical cataract in white individuals. These linkage regions are large and contain many potential disease associated genes. Some genes causing early-onset cataracts or syndromic cataracts may also contribute to age-related cataracts. For example, a novel variant of galactokinase, causing an early-onset cataract was also shown to be associated with an increase in bilateral cataracts in adults.

Consensus on phenotyping: Cataracts, which can be defined as lens opacities, have multiple causes, but are often associated with breakdown of the lens microarchitecture, possibly including vacuole formation and disarray of lens cells, which can cause large fluctuations in density resulting in light scattering. Early-onset forms include: polar opacities, zonular, pulverulent, sutural, and cerulean (blue dot). Age-related cataracts are classified as nuclear sclerosis, anterior or posterior capsular, or cortical.

Genetic resources for cataract: Population-based studies, clinic-based studies, some animal models, some ‘special populations’ with high incidence (India).

Major cataract genetics studies

Early-onset cataract: Many studies of linkage and gene identification have been published (see Hetmanjik review). Of the 39 known loci, 26 causative genes have been identified.

Age-related cataract: Several population based studies have examined a variety of environmental exposures for cataract associated risk. At least one genome-wide linkage study has been performed (Beaver Dam) and several candidate gene association studies have been published.

Next Steps

Short-term goals to advance the field:


  1. Gene testing panel, sequencing array to establish screening/diagnosis/prognosis
  2. Animal models for testing potential therapeutics


  1. Analysis of risk factors in population-based studies (environmental and modifiable)

Long-term goals to advance the field:

  1. Functional studies to inform potential therapeutics
  2. Translation of modifiable risk factors to clinical practice


Hejtmancik JF, Congenital Cataracts and their Molecular Genetics. Semin Cell Dev Biol. 2008 April; 19(2): 134–149.

Shiels A, Hejtmancik JF, Genetic Origins of Cataract. Arch Ophthalmol. 2007;125(2):165-173.

Aquaporins in Ocular Disease

Alan S. Verkman


The aquaporins (AQPs) are small integral membrane proteins (30 kDa/monomer) expressed widely in the animal and plant kingdoms, with 13 members in mammals. AQPs are expressed in epithelia and endothelia involved in fluid transport. Structural analysis by x-ray and electron crystallography indicate that the AQPs assemble in tetramers in which each monomer consists of six tilted alpha-helical domains enclose an aqueous pore. Molecular dynamics simulations suggest tortuous, single-file passage of water through a narrow pore of less than 0.3-nm diameter, in which steric and electrostatic factors prevent the transport of protons and other small molecules. AQP1, AQP2, AQP4, AQP5 and AQP8 are primarily water-selective, whereas AQP3, AQP7 and AQP9 (called ‘aquaglyceroporins’) also transport glycerol and possibly other small solutes.

Several AQPs are expressed in the eye. At the ocular surface, AQP1 is expressed in corneal endothelium, AQP3 and AQP5 in corneal epithelium, and AQP3 in conjunctival epithelium. AQPs are also expressed in lens fiber cells (AQP0), lens epithelium (AQP1), ciliary epithelium (AQP1, AQP4) and retinal Müller cells (AQP4). Mutations in AQP0 produce congenital cataracts in humans. Analysis of knockout mice lacking individual AQPs suggests their involvement in maintenance of corneal and lens transparency, corneal epithelial repair, intraocular pressure regulation, retinal signal transduction, and retinal swelling following injury. The neuroinflammatory demyelinating disease neuromyelitis optica, which causes optic neuritis and transverse myelitis, is associated with circulating AQP4 autoantibodies.

Major Studies

AQP0 mutations produce hereditary cataracts in mice and humans. Cataract-producing AQP0 mutations are thought to produce endoplasmic reticulum-retained and non-functional AQP0; however, the mechanism linking AQP0 loss-of-function and cataracts remains unclear. Proposed mechanisms include loss of AQP0-facilitated fiber-fiber adherence, and impaired fiber cell dehydration.

Though disease-producing loss-of-function mutations in other AQPs have not been identified, studies from knockout mice have revealed various AQP functions in ocular tissues. The roles of AQPs in the eye can in large part be attributed to their water and/or glycerol transporting functions. Examples include: AQP1/AQP4-dependent active fluid secretion by ciliary epithelium; AQP5-dependent osmosis across cornea, and AQP3-dependent corneal epithelial proliferation. The molecular roles of AQPs in maintaining corneal and lens transparency are less clear, as is the role of AQP4 in retina. Whether and how AQP4 autoantibodies cause optic neuritis in neuromyelitis optica is not known.

Known Genes

AQP0 (MIP; major intrinsic protein in lens fiber): Cataracts
Other AQPs expressed in eye: AQP1, AQP3, AQP4, AQP5

Next Steps

Much basic research remains in defining cell-level mechanisms for the ocular AQP functions, in establishing the relevance to human eye disease of conclusions from knockout mice, and in developing AQP-modulating drugs. Specific areas of basic research in the biology of ocular AQPs include elucidation of the precise cellular role of endothelial AQP1 in corneal fluid balance, of lens epithelial AQP1 in cataractogenesis, of corneal epithelial AQP3 in cell regeneration, and of retinal AQP4 in light-neural signal transduction and retinal fluid balance. The role of AQP4 autoantibodies in optic neuritis in NMO warrants investigation.

An intriguing possibility, which remains speculative at this time, is the clinical development of modulators of AQP function or expression. At the ocular surface, AQP3 and AQP5 up-regulation are predicted to accelerate epithelial resurfacing and reduce corneal edema, respectively. Inducers of AQP1 expression in corneal endothelium might reduce corneal edema and associated opacity following injury. Induction of lens AQPs might slow cataractogenesis. AQP1/AQP4 inhibition represents a possible strategy for reducing intraocular pressure associated with glaucoma. In the retina, AQP4 inhibitors might offer neuroprotection in ischemic and other retinopathies.

Suggested areas for investment of NEI resources: Repository for distribution of AQP knockout / transgenic mice; Small-molecule discovery of AQP modulators; Basic research on cellular mechanisms of AQP functions in ocular tissues, AQP4 autoantibodies in neuromyelitis optica, and AQPs in human eye diseases.


Verkman, A.S., J. Ruiz-Ederra and M. Levin (2008). Functions of aquaporins in the eye. Prog. Ret. Eye Res. 27:420-433.

Connexins in Ocular Disease

Daniel A. Goodenough


Gap junctions are clusters of intercellular channels that directly connect the cytoplasms of adjacent cells. A long evolutionary history has permitted the adaptation of gap junctional intercellular channels for a wide variety of uses. This diversity of function is reflected in a 21-member family of structural proteins, the connexins (Cx), each with multiple channel conductance states, phosphorylation states, and with the ability to assemble in heterotypic and heteromeric configurations, diversifying their functional complexity. Cellular activities facilitated by gap junctions fall into two general classes: synchrony/coordination and stimulus/suppression. Among excitable cells, gap junctions, also known as electrical synapses, are common in circuits requiring speed or synchronous firing. In other tissues, gap junctions allow intercellular transfer of small molecules and ions. For example, in the ocular lens, exchange of nutrients and signals required for the prevention of cataract and proper regulation of postnatal lens growth require gap junctions. Indeed, mutations in lens connexin genes are a common cause of hereditary cataract.

Knockout of Cx50 results in both a pulverulent cataract and a smaller lens with concomitant microphthalmia due to a slower mitotic rate in the lens epithelium. Replacement of the Cx50 coding sequences with the Cx46 sequences (Cx50KI46) results in a rescue of the cataract but not of the mitotic rate defect, indicating that there is a quality inherent in Cx50 that is required. Back-crossing these animals with Cx46–/– and Cx50–/– animals has revealed that different forms of dominant cataract result from incongruous mixing of connexins, and that both connexin identity and the locus of gene expression can dramatically affect junctional coupling in the lens.

The ciliary epithelium consists of a double layer of epithelial cells. The pigmented epithelium (PE) rests on the connective tissue stroma and the non-pigmented epithelium (NPE) is polarized with its basal lamina facing the posterior chamber of the eye. The PE and NPE interact via their apical membranes with gap junctions containing Cx43. The NPE forms tight junctions delineating a boundary between the blood and the aqueous humor as part of the blood-aqueous barrier. The combined ion transporters and pumps located in the epithelia of the ciliary epithelium provide the source of the aqueous humor. The gap junctions between the PE and NPE are critical to coordinating the ionic pumping of the two epithelial cell types. The water following this NaCl movement can pass from PE to NPE cells via gap junctions. Targeted disruption of Cx43 in the ciliary epithelium results in a decrease or loss of intraocular pressure.

In the retina, gap junctions are involved in interneuronal electrical signaling. Targeted disruption of Cx36 results in a loss of scotopic vision due to the ablation of gap junctions between AII Amacrine and cone ON bipolar cells and possibly gap junctions between rods and cones. Deletion of Cx57 significantly reduces horizontal cell receptive field size and this connexin’s distribution is modulated by light. It is speculated that the transjunctional voltage dependence of Cx45 channels could support the transmission of direction selectivity. Connexins are found in other ocular locations. Multiple connexin proteins are found between corneal epithelial cells; many of these may have redundant functions since knockout of connexin genes does not result in a corneal phenotype. In humans, oculodentodigital dysplasia, a pleomorphic, syndromic condition affecting a large number of cell types, results from mutations in Cx43. Some patients show abnormalities in eye development and in the development of glaucoma. In families carrying the Cx43 L113P mutation, the ophthalmic features include epicanthus, microcornea, and the presence of glaucoma. The retinal pigment epithelium (RPE) expresses Cx43. It has been reported that RPE regulates proliferation in the underlying neural retina by ATP efflux through Cx43 hemichannels, although targeted deletion of Cx43 from the RPE does not result in retinal abnormalities.

Major Studies

Positional cloning has been the principal method used for identifying mutations in humans with hereditary cataract and with ODDD.

Known Genes

A partial list of coding mutations causing cataract in human lens connexins:

Cx50: S50P S276F, W45S, P88Q, D47N, V79L.
Cx46: L11S, R76G, V28M.

There have been no reported regulatory mutations.

Next Steps

Given that the eye is a privileged compartment, it is a location where there is a potential future for targeted gene therapy. Current advances in viral gene transfer offer the possibility of introducing wild-type genes to replace mutants and to selectively express siRNA to downregulate overexpressed or improperly regulated gene activity. Given the high costs of primate research, a central facility permitting exploration of these methods would facilitate the transfer of technologies to humans.


Gong,X., C.Cheng, and C.H.Xia. 2007. Connexins in lens development and cataractogenesis. J.Membr.Biol. 218:9-12.

Mese,G., G.Richard, and T.W.White. 2007. Gap junctions: basic structure and function. J.Invest Dermatol. 127:2516-2524.

Mouse Models in Ocular Disease

Simon John


Review mouse models currently available for study of ocular disease: Many models are available for a wide variety ocular diseases. There are more mouse models than for other mammalian species. Despite this important models exist in other mammalian species (eg spontaneous and induced glaucomas in rat and RCS rat) and other systems are used to study heritable ocular disease or test interventions (dog, zebrafish, flies). Inherited and or induced models exist for various diseases and conditions but not all of these models are well characterized in a uniform fashion. Although experimentally induced models do not allow us to study the initial causes of inherited disease, they can be useful for assessing genetic susceptibility factors and testing treatments. There are genetic models for cataracts, retinal degenerations, glaucoma, abnormal ocular development, night blindness, mucoploysaccharidoses, choridemia and others. Since they are easier to study and detect, developmental conditions are over-represented. One review (Budzynski, 2006) indicated that 36% of ocular disease models in mice involve ocular development. There are also many models of retinal degeneration.

There is a deficiency of later onset models and models of common complex human conditions such as age-related macular degeneration, open-angle glaucoma and diabetic retinopathy. A larger focus on generating and studying such models would be useful.

Major Studies

Provide an overview of the approaches used to create and characterize mouse models that contribute to ocular disease: Genetic mouse models are provided in a variety of ways (spontaneous mutations, induced point mutations, transgenic and knockout technologies etc, detailed in Adams review). Methods of analyses are tailored to the specific condition and study but include clinical approaches (slit-lamp, ophthalmoscope, fluorescein angiography, fundus photography, SLO, gonioscopy etc) and the full range of histological, molecular, genetic genomic, proteomic, biochemical and cell biological approaches. Physiological analyses are also important including electroretinography, IOP assessment and aqueous humor outflow. Genetic experiments identify and characterize causative genes, identify and characterize genes that modify susceptibility to or severity of disease, and manipulate specific pathways or processes to test hypothesis. Viral vectors are also used to express genes in areas of interest or to knockdown the function of specific genes using small inhibitory RNA approaches.

Known Genes

Describe genes that are responsible for ocular disease in the mouse models and note if mutations are regulatory or coding: Many genes contribute to ocular disease in mice. A search of the Mouse Genome Informatics database (<a href=’);> identified 334 genes associated with the search terms vision or ocular disease. Gene targeting that typically abrogates gene function was used to induce mutations in the great majority of these genes. A significant proportion of these genes were identified from mice with spontaneous mutation or chemically induced point mutations. The point mutations are often coding or splice junction mutations but other types exist. Some of the mutations involve gene traps, which decrease or abrogate the production of normal transcripts.

Gene targeting and /or gene trapping approaches are being used to mutate many of the genes in the mouse genome (see Adams et al.). Large scale efforts are producing impressive collections of knockout first alleles. These are null alleles that express a Lacz reporter gene. These alleles can be exposed to the site-specific recombinase Flp to generate a conditional allele, and then with the site-specific recombinae Cre to produce a null allele in a cell- or tissue- specific manner.

Next Steps

Describe next steps you recommend to advance the field. What communal research resources would represent a good investment for NEI? Many human conditions are caused by point mutations.

An efficient approach for identyfing new disease models and pathways would be to establish phenotyping centers to characterize the eyes of many of the new mutants that are being produced in specific genes.

Targeted mutations in some mouse genes do not cause phenotypes found in people with point mutations in the orthologous gene, while point mutations do. Point mutation resources are needed to complement the large gene knockout efforts. ENU mutagenesis is a valuable approach for producing these mutations and new generation sensitized screens will be more powerful and productive than previous versions for providing models and understanding of complex diseases. Sensitized mutagenesis screens using ENU, especially those sensitized by mutations in a gene that causes human disease will be valuable. These genetic screens will allow identification of other genes that interact with the first genes to modify disease and will identify new disease pathways. Genome wide collections of mouse mutations are likely to facilitate the identification of human disease genes, especially for diseases such as glaucoma, for which common large affect alleles do not appear to significantly contribute to disease.

A new twist on the ENU approach may emerge to produce banked point mutation resources. The mutations present in these banks can be identified using the new massively parallel sequencing techniques. As one approach, frozen sperm banks with associated databases would allow rapid and low cost mutation selection and recovery to live mice.

Mutation collections are needed on different strain backgrounds, as the commonly used B6 and 129 backgrounds are not optimal for studying various diseases. The production of mouse ES cells with different strain backgrounds will be important. Emerging oligonucleotide-based mutagenesis may prove an efficient strategy for producing subtle mutations in selected target genes in ES cells.

Emerging and sophisticated strategies that combine gene traps and transposons may provide efficient methods to produce genome wide collections of null and conditional alleles on these backgrounds. Additionally, they can be used to produce mutation collections on different backgrounds by breeding (no need for ES cells, much simpler and cost effective). These approaches can also be used in other species where it is possible to make transgenic animals.

Where human mutations become characterized it will often be valuable to humanize mice by replacing the mouse gene with normal and mutant versions of the human gene, or by adding a human transgene. BAC transgenics will often be a good approach to maintain endogenous expression patterns as they can assume human regulation in a mouse. Humanized models that are phenotypically well characterized using generally accepted/standardized approaches will be very valuable to the field.

Copy number variations are increasingly being implicated in disease and this will be true for ocular diseases. Assessing copy number variations in mouse genetic experiments will be important and the ability to manipulate the copy number of single or muliple mouse genes, small and larger genomic regions is important. Gene targeting and chromosome engineering techniques can produce desired duplications and deletions. BAC transgenics can increase copy number but this is not easily controllable. A powerful approach for producing genome wide deletions and duplications by breeding was recently reported (see Wu et al. 2007, cited in Adams review below). This method combines gene trapping and transposon technology and a centralized effort would produce genome wide collections of deletions, and duplications, in addition to insertional loss-of function and conditional rescue alleles.

Well-characterized collections of tissue and cell specific Cre and/or Flp recombinase (especially Cre) expressing mice will be valuable for determining how mutations cause specific diseases. Ideally these would include CreERT variants that require tamoxifen for activity, providing for spatial and temporal control of conditional mutations and fluorescent protein markers. Different diseases will require different sets of Cre mice that are tailored to manipulate the relevant cells and tissues for each disease (eg retinal ganglion cells, astrocytes, microglia endothelial cells and other cell types in glaucoma).

Similarly, well-characterized collections of fluorescent protein alleles that mark different cell types or organelles in different colors will be very valuable. Ideally different colors would be available for each cell type. These resources can also facilitate genetic screens.

Mice will be increasingly important in understanding complex genetic interactions and the genetic complexity of disease. Valuable new resources are being produced such as the community cross.Robert Williams and Gary Churchill are experts in this area. Emphasis on training in genetics and the value of these resources is important.

Review and Books

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