We took advantage of the robust “loss of a-wave” phenotype to determine the genetic basis of the observed inherited retinopathy. Our population of C57-outcrossed
rdx animals were screened by ERG to identify affected individuals and were then genotyped across a genomewide panel of 511 informative SNPs with an average spacing of 4.9 Mb.
17 This analysis positionally cloned the mutation to a 25-Mb interval on chromosome 9 between
rs4227585 and
rs4227682 (
Fig. 4A). Although many genes are present in this interval,
Mfrp and
Ctrp5 are located in tandem within this region and were investigated as strong candidates because they have been previously linked to inherited retinal diseases. They have been reported to be expressed as a bicistronic transcript,
2 and a splice site mutation in the
rd6 mouse that deletes exon 4 from the mature
Mfrp transcript results in flecked retina disease with progressive retinal degeneration.
2 Similarly, an S163R substitution in human CTRP5 causes late-onset macular degeneration in humans and mice.
10,21 Sequencing of the coding regions of these two genes from
rdx mice identified a single base pair deletion in exon 3 of
Mfrp, which we designated
Mfrp 174delG (
Fig. 4B). We then developed a PCR-RFLP genotyping assay to identify this allele (
Fig. 4C). Finally, to confirm that
rd6 and
Mfrp 174delG are indeed allelic, we performed a genetic complementation test using homozygous
rd6 mice obtained from The Jackson Laboratory (Bar Harbor, ME). Of 27
Mfrp rd6/174delG mice produced and examined by fundus photography at 2.5 to 3 months of age, all exhibited signs of flecked retina disease (
Fig. 4D). Phenotypic analysis of offspring from multiple homozygous
Mfrp 174delG crosses confirmed that the fundus fleck and retinal degeneration phenotypes described are 100% penetrant in
Mfrp 174delG/174delG animals. If translated to a stable protein, the
Mfrp 174delG allele is predicted to encode only the cytosolic N terminus, followed by 83 nonsense residues. In contrast, the
rd6 allele deletes a region between the transmembrane domain and the first CUB domain, without disrupting C-terminal structures (
Fig. 4E). After identification of the genetic basis of this phenotype,
Per3 −/− mice on the original 129/Sv background were bred to select against the
Mfrp 174delG allele, and the resultant
Per3 −/− Mfrp +/+ mice were deposited at The Jackson Laboratory (strain 010833).