Genetic Modifiers of rd3 Retinal Degeneration

Abstract

Purpose: : Previously, we identified several QTL that significantly modify the course of retinal degeneration in 431 10-week F2 progeny of an F1 intercross between B6(Cg)-Tyr^c-2J/J (B6a) and BALB/cByJ (BALB) albino mice that are both congenic and homozygous for the rd3 allele. The purpose of this work was to refine the loci of the two strongest QTL at Chrs 17 and 5, and to evaluate candidate genes.

Methods: : Utilizing genomic DNAs from the original 431 F2 mice, the Center for Inherited Disease Research genotyped a number of SNPs in each QTL in a follow-up study. The data was analyzed with the Map Manager QTX program. A yeast two-hybrid (Y2H) screen of 1 x 10⁶ transformed colonies from a human retina cDNA library was used to identify proteins interacting with RD3. Candidate genes were sequenced by standard methods and measured for expression by QPCR.

Results: : The Chr 17 QTL was reduced from ~40 Mb in the original map to a 1-LOD critical area of 16 Mb in the follow-up study utilizing 34 informative SNPs; the Chr 5 QTL from 23.5 to 9 Mb with 12 informative SNPs. The Y2H study revealed a non-redundant set of 41 cDNAs. Of these, two encoded genes that were orthologs of mouse genes in the Chr 17 critical area - Ppt2 and Hnrpm. Sequencing revealed no differences in the coding regions of either gene between the B6a and BALB strains. There was 1 sequence variant in the 3’ UTR of Hnrpm. The Crybb2 gene mapping to the reduced Chr 5 QTL was selected for study because of expression differences associated with retinal degeneration cited in the literature. There were no coding region differences in the cDNAs between the two strains. QPCR of the three candidate genes in B6a- and BALB-rd3/rd3 at 5 and 10 weeks showed a few differences but none were remarkable.

Conclusions: : Significant differences in expression or coding sequence between the B6a- and BALB-rd3/rd3 strains were not found in the three candidate genes studied so far. Other genes will be selected for future study based on function and implication in photoreceptor degeneration, but the critical areas of the QTL must be refined further by recombinant progeny testing to reduce the number of candidate genes.

Keywords: degenerations/dystrophies • gene modifiers • retina: distal (photoreceptors, horizontal cells, bipolar cells)