New retinal genes have been identified and studied in the investigator's laboratory in order to identify etiologies for hereditary blinding retinal diseases, to study how these genes cause disease, and to use this information to find the best treatment or cure for them. To date, it has been demonstrated that: (1) a patient with late-onset cone-rod dystrophy has a nonsense codon mutation in HRG4, a photoreceptor gene identified in the investigator's laboratory; (2) a transgenic mouse model carrying the same mutation as the patient showed an ERG abnormality, consistent with a problem in neurotransmission at the photoreceptor synapse and late-onset retinal degeneration; (3) HRG4 is present in the rod and cone photoreceptor synapses, making it the first synaptic protein implicated in retinal degeneration; and (4) HRG4 is homologous to UNC119, a mutated protein in C. elegans with problems of coordination, feeding, and chemosensation. For continuation of the HRG4 study, 4 aims are proposed.
Specific aim 1 will test the hypothesis that HRG4 plays a significant role in the synaptic vesicle cycle which forms the basis of synaptic neurotransmission. The status of synaptic vesicle cycle proteins and their interactions will be compared in the transgenic model versus normal mice.
Aim 2 is to determine the identity of the partner or target protein with which HRG4 interacts in order to elucidate the function of HRG4. Direct binding assays, co-immunoprecipitation experiments using the recombinant HRG4, and the yeast two-hybrid strategy will be used.
Aim 3 is to construct an HRG4 gene-targeted (knock-out) mouse model and to study the effect of null expression of this gene on the phenotype and mechanism of genesis of the phenotype. The goals of this aim are to (i) investigate the function and pathogenic mechanism of HRG4 and (ii) to obtain an animal model in which structure-function studies of HRG4 can be carried out by transgene expression against a clean background, and which ultimately can be used to investigate the mechanism and the treatment of retinal degeneration.
Aim 4 is to screen the HRG4 gene for mutations in patients with retinopathies by (a) searching for patients whose retinopathy has been mapped to 17q11 and screening them, if found; (b) searching for patients with neurofibromatosis mapping to 17q11.2 and retinopathy and screening them in order to determine the presence of a contiguous chromosome syndrome and the involvement of HRG4 in the retinopathy; (c) conducting rapid random mutational screening using denaturing gradient gel electrophoresis or heteroduplex analysis on unmapped cases of retinopathy; (d) investigating the possible interaction of mutations in HRG4 and other retinal degeneration genes by screening both; and (e) performing co-segregation analysis and screening of normal DNA for any mutations found in order to confirm the pathogenic relationship and establish the significance of HRG4 for retinal diseases.
