Mutation discovered in a feline model of human congenital retinal blinding disease

Abstract

Purpose: To elucidate the gene defect in a pedigree of cats segregating for autosomal dominant rod-cone dysplasia (Rdy), a retinopathy characterized extensively from a clinical perspective. Disease expression in Rdy cats is comparable to that in young patients with congenital blindness (Leber congenital amaurosis [LCA] or retinitis pigmentosa [RP]).

Methods: A pedigree segregating for Rdy was generated and phenotyped by clinical ophthalmic examination methods including ophthalmoscopy and full-field flash electroretinography. Short tandem repeat loci tightly linked to candidate genes for autosomal dominant retinitis pigmentosa in humans were genotyped in the pedigree.

Results: Significant linkage was established to the candidate gene CRX (LOD = 5.56, = 0) on cat chromosome E2. A single base pair deletion was identified in exon 4 (n.546delC) in affected individuals but not in unaffected littermates. This mutation generates a frame shift in the transcript, introducing a premature stop codon truncating the putative CRX peptide, which would eliminate the critical transcriptional activation region. Clinical observations corroborate previously reported clinical reports about Rdy. Results show that the cone photoreceptor system was more severely affected than the rods in the early disease process.

Conclusions: A putative mutation causative of the Rdy phenotype has been described as a single base pair deletion in exon 4 of the CRX gene, thus identifying the first animal model for CRX-linked disease that closely resembles the human disease. As such, it will provide valuable insights into the mechanisms underlying these diseases and their variable presentation, as well as providing a suitable model for testing therapies for these diseases.

Figure 1.

Cat pedigree derived at MU, segregating for autosomal dominant rod-cone dysplasia (Rdy). White symbols: unaffected individuals; partially filled symbols: Rdy-affected individuals.

Figure 2.

Fundus photographs of a 12-week-old normal Abyssinian cat (A) and an affected mixed breed (Rdy) cat (B). The generalized grayish discoloration of the fundus in the affected cat is most notable in the area centralis, as is the generalized vascular attenuation. Arrows: marked changes in the area centralis of the affected cat.

Figure 3.

Dark-adapted (scotopic) ERGs from a 7-week-old normal mixed-breed kitten showing responses to three different light intensities: 300, 1000, and 3000 mcd · s/m². Similar recordings are shown for an Rdy-affected littermate at ages 7 and 11 weeks, obtained with identical procedures as for the normal kitten. Note the initial recordable ERG in the affected kitten at 7 weeks, with clear a- and b-wave recordings, both of low amplitude and increased implicit time. Within 4 weeks a- and b-waves from the same kitten were nonrecordable and were replaced by a late-onset negative waveform. Amplitude and implicit time calibrations are shown on the ordinate in microvolts and on the abscissa in milliseconds. Note that the amplitude calibration varies in the ordinate for most of the recordings.

Figure 4.

(A) Genomic nucleotide sequence of the feline CRX gene, nucleotides 539-559 of the coding sequence, Rdy unaffected cat; (B) genomic sequence of homologous region in an Rdy-affected cat carrying one wild-type and one affected allele. Note the polymorphism (C/G) at the marked (*) position where the affected allele demonstrates a 1-bp deletion. Downstream of the deletion, the wild-type and affected alleles are 1 bp out of frame with one another, which can be clearly seen in the remaining sequence.

Figure 5.

CRX protein structure in Felis catus. Comparison between the wild-type feline CRX protein (A) to the putative truncated CRX protein (B). Y, exon splice junctions; *start codon; X stop codons. Shaded boxes: protein domains, defined as the homeobox, the WSP domain, the transcriptional transactivation domains 1 and 2 (TTD1 and TTD2), and the OTX tail. Domains are drawn to scale.

Figure 6.

CLUSTAL W (1.83) multiple-sequence amino acid alignment of human CRX protein compared with the wild-type and Rdy feline CRX protein. Underscore: the conserved homeobox (DNA binding) domain; italic: the reported WSP domain; bold: the putative transcriptional activation domains. The amino acids after the second transcription domain demarcate the conserved OTX tail. X, the premature termination codon.