Purpose: The Rd4/+ mouse inherits an autosomal dominant retinal degeneration that cosegregates with a large inversion spanning nearly all of mouse chromosome 4 (Chr 4). This inversion is homozygous lethal. The hypothesis for the study was that disruption of a gene at one of the two breakpoints in the Rd4 chromosome is responsible for the retinal degeneration. The purpose was to identify the disrupted gene.
Methods: Genotyping was performed by PCR and gel electrophoresis. The Rd4/+ phenotype was confirmed by ERG. Fluorescence in situ hybridization (FISH) analysis was performed with bacterial artificial chromosome (BAC) probes. Northern and quantitative PCR procedures were used to evaluate Gnb1 mRNA expression. Protein expression was measured by Western blot.
Results: To identify the Rd4 gene defect, the breakpoints were first localized with a testcross and the locus refined by using FISH. Genetic testcross data revealed that the inversion breakpoints are located within a few centimorgans of both the telomeric and centromeric ends of Chr 4. Initial FISH analysis showed the proximal breakpoint of the inversion to be in the centromere itself. Therefore, we focused on the distal breakpoint and found that it lies in the second intron of the gene Gnb1, coding for the transducin beta1-subunit (Tbeta1) protein that is directly involved in the response to light of rod photoreceptors. Before the beginning of retinal degeneration in Rd4/+ retina, the levels of Gnb1 mRNA and Tbeta1 protein are 50% of those in wild-type retina.
Conclusions: The results suggest that disruption of the Gnb1 gene is responsible for Rd4 retinal disease.