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Molecular Genetics of Inherited Eye Diseases

Dr. Shomi S Bhattacharya
Image Dr. Shomi S Bhattacharya

Molecular Genetics defines a field of study where in the absence of a detectable biochemical defect (true for a majority of inherited diseases) a reverse genetics approach allows the chromosomal assignment and isolation of the disease causing genes. Advent of recombinant DNA technology led to the cloning of chromosome specific DNA markers in the early 1980s. The ability to detect restriction fragment length polymorphisms (RFLPs) in the Human Genome, permitted the use of these markers as genetic tools for localizing the disease trait in the family to a particular chromosomal region thereby facilitating the eventual identification of the disease gene.

Identification / characterization of genes involved in inherited eye diseases and investigating the functional consequences of mutations have constituted the major focus of my research programme to date. The genetic register of patients available at the laboratory provides a unique resource and the opportunity to undertake molecular genetic investigations for this purpose. The close interaction between my research group and clinical colleagues has been crucial to our success and underpins our contribution to translational research. The procedures for gene mapping and identification of disease genes via a conventional positional cloning approach or the more novel bioinformatics based approach utilizing the information available in the various Human Genome database, are proving invaluable in this endeavor. Along with clinical colleagues, assessment of large pedigrees suitable for gene mapping for the various types of inherited eye diseases (retinal degenerative diseases, retinitis pigmentosa, cone, cone-rod dystrophies and macular dystrophies, corneal dystrophies, congenital cataracts, dominant optic atrophy and inherited glaucomas) can be undertaken to facilitate identification of these genes.

Based on total genome linkage analysis using hypervariable microsatellite markers for the human genome, some 30 novel ophthalmic loci have so far been identified by my group. A positional cloning/candidate gene approach have been pursued for most of these loci.  Notable achievements have been in the identification of genes for retinitis pigmentosa (and Leber congenital amaurosis (), cone () and cone-rod dystrophies (and), cataract (andand) and dominant optic atrophy (). Functional studies of the mutant in frog oocyte expression system indicated impairment of water transport across cell membrane.  Mutations in (Keratoepithelin) in a variety of clinically distinct corneal dystrophy, have also been identified and a new locus for congenital hereditary corneal dystrophy (CHED) on chromosome 20p mapped by our group. A large family segregating glaucoma and iridogoniodysplasia was localised to the terminal short arm of chromosome 6. We are expanding activity in glaucoma genetic research and recently we demonstrated the duplication of the genomic interval containing the gene as causative of glaucoma in one of our families. We have undertaken clinical characterization and genetic mapping of over 50 families with the dominant form of optic atrophy. Almost all families genetically linked to the tip of long arm of chromosome 3 (q28-ter). It led to the identification of the gene for this condition which plays an important role in mitochondrial integrity. With the near completion of the Human Genome Sequencing project, vast amount of sequence information has been made available to the genetics community through public databases. We have access to and expertise in a variety of bioinformatics software programmes that allow gene prediction, protein and nucleotide sequence alignment, identification of gene families and functional domain in proteins.  Using this expertise, we have already identified and fully characterized a number of novel genes that are candidates for eye disease.  Extensive mutation screening in these and other disease genes has been carried out in our clinically well-characterized panel of patients.

We have initiated studies on the functional characterization of mutations in eye diseases and the following two examples define this approach. and have shown to be implicated in retinal degeneration by our group. The concentration of cGMP in photoreceptors is central to the process of phototransduction. In the mammalian retina, two membrane guanylate cyclases, retGC-1 and retGC-2, synthesize cGMP in mammalian photoreceptors. cGMP gates cation channels which control the membrane potential and signaling states of rods and cones. Light stimulates the degradation of cGMP through the activation of a cGMP-phosphodiesterase, causing the cGMP-gated channels to close. This reduces intracellular Na+ and Ca2+ concentrations, hyperpolarizes the cell, and slows the release of neurotransmitter. Lowered Ca2+ levels allow the Ca2+-binding proteins guanylate cyclase activating protein 1 (GCAP-1) and GCAP-2 to stimulate retGCs. The Y99C substitution mutation that we identified in GCAP-1 in a cone dystrophy family is expected to disrupt the EF3 Ca2+-binding hand. Therefore, even in the presence of high levels of Ca, GCAP1 continues to activate RetGC1 leading to increased levels of cGMP synthesis. The constitutive activation remained in the presence of wild-type enzyme, as expected for a dominant disease. High levels of cGMP have been shown to be harmful to photoreceptor survival in animal models of retinal degeneration.

The gene for transcription factor localises to chromosome 14q11. In photoreceptors, binds to two regions of the rod opsin promoter; deletion and mutation studies of these promoter regions clearly show that is important in regulating rod opsin production. A mutation screen of patients in one of our autosomal dominant RP family that maps to this region, identified a Ser50Thr substitution in the transactivation domain of the protein. The substitution was in a highly conserved region of the protein and alters a consensus sequence for phosphorylation/dephosphorylation by proline-directed serine/threonine protein kinases and protein phosphatases. Transactivation experiments with the rod opsin promoter demonstrate that, at subsaturation concentrations, the mutant (Ser50Thr) NRL results in a significant increase in reporter gene (luciferase) expression over wild-type when NRL is co-expressed with CRX, another transcription factor regulating rod opsin gene expression. Over expression of wild-type rhodopsin may result in photoreceptor degeneration which may explain the molecular basis of disease in our family.

A close working relationship with clinical colleagues acknowledges the critical contribution of clinical research in genotype / phenotype correlation studies and the important information this is likely to provide in our understanding of the aetiology of these diseases. Furthermore, based on the exclusion of the known loci for the different types of inherited eye diseases, we can confidently predict that many new genetic eye disease loci remain to be identified. We anticipate that future work will be greatly aided by DNA chip technology and microarray systems.  The ability for rapid analysis of gene expression between normal and mutant tissues will facilitate future research direction. Additionally, our genetic studies are expanding into the mapping of complex traits, such as, age related macular disease (AMD), glaucoma and age related cataract. Clinically, polygenic diseases account for the majority of patients with inherited eye diseases and by targeting this area of research we hope to address the commoner problems afflicting the human population.

My research group is committed to basic research and programmes of work in the areas of novel disease gene identification, functional genomics, elucidation of disease mechanism and development of gene targeted animal models that may underpin translational research. Site directed mutagenesis and protein expression is used extensively for our research.  Currently a mouse knock-in model for a dominant form of photoreceptor dystrophy is complete which should allow a proper and systematic evaluation of the cellular basis of the disease and disease progression during the lifetime of the animal. In earlier work we demonstrated a major breakthrough in gene therapy by the rescue of photoreceptor ultrastructure and electrophysiological function in a well-known mouse model (rds mouse, lacks peripherin) of retinal degeneration. Subretinal injection of a functional gene in adeno-associated viral vector led to these interesting results, firmly establishing “proof of principle” for the success of gene therapy. The remarkable results obtained by Professor Robin Ali in the recent gene therapy treatment of LCA patients, further emphasizes ongoing work in this area. Using an integrated approach, characterization of the cellular and biochemical basis of the disease should ultimately lead to better protocols for pharmacogenomics and treatment by gene and/or gene based therapies. Working in close association with clinical colleagues, careful genotype / phenotype studies are likely to identify patients for clinical trials who will benefit most from this technological revolution.

In summary my future research programme includes:

  • Accurate genotype-phenotype correlation
  • Identification and characterization of novel monogenic ophthalmic disease genes
  • Identifying susceptibility loci for genetically complex diseases such as AMD and Glaucoma
  • Basic molecular biology of genes in the visual system
  • Functional / biochemical characterization of ophthalmic disease genes
  • Development of animal models to investigate disease mechanism

Shomi S Bhattacharya

University College London
Institute of Ophthalmology, Molecular Genetics Group

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