First Author: M.Hassall UK
Co Author(s): A. Barnard P. Charbel Issa S. Aslam R. MacLaren
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We identified a colony of inbred mice with complete achromatopsia and hypothesised that a novel genetic mutation was responsible. A local colony of inbred mice (129S6/SvEvTac origin), that had been bred in isolation for over a decade, were found to have completely absent cone electroretinogram (ERG) responses. We investigated the inheritance and genetic basis of this phenotype of cone photoreceptor function loss (cpfl).
Translational laboratory research performed at the University of Oxford Nuffield Laboratory of Ophthalmology.
We performed dark- and light-adapted electroretinography (ERG) to assess retinal function. An affected 129S6/SvEvTac colony animal was outcrossed to a wild-type C57BL/6J mouse to produce first filial generation hybrid offspring (F1). The F1 animals were intercrossed to investigate inheritance in the F2 generation. Chi-squared test with Yates’s continuity correction was used for statistical analysis of phenotypic outcomes. Genomic DNA was extracted from several mice and fluorescent dideoxynucleotide sequencing was performed on particular genes known to cause a similar cpfl phenotype and/or identified as causing achromatopsia in humans (Gnat2, Cnga3, Cngb3, Pde6c Hcn1, Syne2). The eyes of a subset of animals underwent histological sectioning and cone arrestin immunostaining.
All founder colony animals tested lacked cone pathway function by ERG testing (n= 9), although rod pathway based ERG responses remained unaffected. None of the C57BL/6 outcross F1 progeny displayed a deficit in cone ERG function (n = 15). However, several animals in the F2 generation showed the original cpfl phenotype, indicating a recessive inheritance pattern. Targeted re-sequencing revealed that the colony did not carry any of the spontaneous mutations previous found in other inbred lines. Instead a novel missense mutation was identified in exon 6 of the Cngb3 gene, which causes an amino acid substitution at a highly conserved residue. Genotyping of the F2 generation found that, out of 59 animals tested: 15 were wildtype, 29 were heterozygous and 15 were homozygous for the novel Cngb3 mutation. These numbers were not significantly different from the expected genotypic ratio (X = 0.9745, df = 2, p = 0.6143). All mice homozygous for the mutation demonstrated the achromatopsia phenotype. Ocular imaging (cSLO and OCT) showed no retinal degeneration or other anatomical problems. Histological sectioning and IHC for Cone Arrestin showed normal retinal architecture and cone cell morphology.
The existing mouse model of Cngb3 achromatopsia is a knock-out and functionally null. Our model demonstrates a novel homozygous missense mutation and complete achromatopsia, similar to the human condition. The absence of cone function in the presence of mutant Cngb3 protein makes this mouse an optimal model for future pre-clinical development of gene therapy.