A mutation in the start codon of γ-crystallin D leads to nuclear cataracts in the Dahl SS/Jr-Ctr strain

Abstract

Cataracts are a major cause of blindness. The most common forms of cataracts are age- and UV-related and develop mostly in the elderly, while congenital cataracts appear at birth or in early childhood. The Dahl salt-sensitive (SS/Jr) rat is an extensively used model of salt-sensitive hypertension that exhibits concomitant renal disease. In the mid-1980s, cataracts appeared in a few animals in the Dahl S colony, presumably the result of a spontaneous mutation. The mutation was fixed and bred to establish the SS/Jr-Ctr substrain. The SS/Jr-Ctr substrain has been used exclusively by a single investigator to study the role of steroids and hypertension. Using a classical positional cloning approach, we localized the cataract gene with high resolution to a less than 1-Mbp region on chromosome 9 using an F1(SS/Jr-Ctr × SHR) × SHR backcross population. The 1-Mbp region contained only 13 genes, including 4 genes from the γ-crystallins (Cryg) gene family, which are known to play a role in cataract formation. All of the γ-crystallins were sequenced and a novel point mutation in the start codon (ATG → GTG) of the Crygd gene was identified. This led to the complete absence of the CRYGD protein in the eyes of the SS/Jr-Ctr strain. In summary, the identification of the genetic cause in this novel cataract model may provide an opportunity to better understand the development of cataracts, particularly in the context of hypertension.

Figure 1. Gross appearance of nuclear cataract at 21 days of age

(A) Normal appearance of eye in wild-type SS/Jr strain. (B) Appearance of cataract in eye of SS/Jr-Ctr strain which exhibits spontaneous cataracts. (C) Gross dissection of eye from wild-type and (D) cataract animals. The lens of the SS/Jr eye is clear and transparent, whereas the lens from the SS/Jr-Ctr eye is opaque and cloudy.

Figure 2. Measurement of eye weight in SS/Jr and SS/Jr-Ctr animals

Total eye weight (left + right eye) is shown for each strain and sex. For either sex, eyes from SS/Jr-Ctr were significantly smaller than eyes from SS/Jr (n=6).

Figure 3. Representative whole-eye and high resolution images of lens from SS/Jr and SS/Jr-Ctr at 21 days of age

(A–B) Longitudinal histology section through whole-eye (1X, H&E) which demonstrates the normal appearance of key anatomical structures in each strain, aside from obvious lens fiber changes observed in eye from SS/Jr-Ctr. (C–D) Higher magnification images (10X, H&E) showing normal lens appearance in eye from SS/Jr and disruption of lens fibers in eye from SS/Jr-Ctr. (E–F) Lens from SS/Jr-Ctr also demonstrates distinct calcification (40X, Von Kossa stain), whereas wild-type SS/Jr have normal appearance.

Figure 4. Genome-wide linkage analysis for cataract status in F₁(SS/Jr-Ctr X SHR) X SHR population at 21 days of age

(A) LOD plot of cataract status (category trait analysis-presence or absence). (B) LOD plot of continuous traits, including body weight, eye weight (left, right, and total), eye to eye or eye to nose distance. The chromosome location is shown on the x-axis and LOD score is shown on the y-axis. The significance threshold for significant quantitative trait locus (QTL) was determined by permutation testing and is denoted by the line around LOD~3.

Figure 5. Haplotype analysis and refinement of cataract mutation to rat chromosome 9

(A) Location of the 95% confidence interval (CI) of the genomic interval linked to the cataract mutation (solid black bar) next to the physical map (left) and haplotype analysis of the F₁(SS/Jr-Ctr X SHR) X SHR population (right). The haplotype analysis provides a refinement of the QTL based on genotype and absence of cataract (haplotype 1–2) or cataract status (haplotype 3–5). Haplotype 3–5 exhibit cataracts and share the SS/Jr-Ctr/SHR genotype at 09_0059708371 (denoted by the arrow). (B) Refinement of the cataract QTL using key recombinant animals from BC. All three animals exhibited cataracts and shared the SS/Jr-Ctr allele at only microsatellite markers D9Rat85 (63,689,889 Mb) to D9Mit2 (63,839,862 Mb). The genomic interval containing the cataract mutation is denoted by the boxed area.

Figure 6. Sequence and western blot analysis of whole eye and other organs from SS/Jr and SS/Jr-Ctr

(A) DNA sequencing chromatographs shows a point mutation in the start codon (ATG → GTG) in SS/Jr-Ctr and F₁ DNA compared to wild-type SS/Jr. The start codon (ATG) which encodes a methionine is predicated to be a valine in the SS/Jr-Ctr and F₁. (B) Western blot analysis of eye. No CRYGD protein is evident in the SS/Jr-Ctr eye, but is observed in wild-type SS/Jr animals. In eyes from F₁ animals, there is approximately 50% reduction in the amount of CRYGD protein observed. (C) Western blot analysis of homogenates from various organs and eye. No CRYGD protein is evident in tissues related to cardiovascular health and disease (heart or kidney).