Genetic modification of the schisis phenotype in a mouse model of X-linked retinoschisis

Abstract

X-linked retinoschisis (XLRS) is an inherited form of macular degeneration that is caused by mutations in the retinoschisin (RS1) gene. In addition to macular degeneration, other major characteristics of XLRS include splitting of the retina (schisis) and impaired synaptic transmission as indicated by a reduction in the electroretinogram b-wave. It has been known that patients carrying RS1 mutations show a broad range of phenotypic variability. Interestingly, phenotypic variation is observed even among family members with the same RS1 mutation, suggesting the existence of genetic or environmental factors that contribute to the severity of XLRS. However, in the human population, the cause of phenotypic variability and the contribution of genetic modifiers for this relatively rare disease are difficult to study and poorly understood. In this study, using a mouse model for XLRS, we show that genetic factors can contribute to the severity of the retinoschisis phenotype. We report evidence of a major genetic modifier of Rs1, which affects the disease severity in these animals. A quantitative trait locus (QTL), named modifier of Rs1 1 (Mor1), is mapped on chromosome (Chr) 7. When homozygous, the Mor1 allele from the inbred mouse strain AKR/J diminishes the severity of the schisis phenotype in Rs1(tmgc1)/Y male and Rs1(tmgc1)/Rs1(tmgc1) female mice. We also show that the penetrance of the disease phenotype is affected by additional genetic factor(s). Our study suggests that multiple genetic modifiers could potentially be responsible for the phenotypic variation in human XLRS.

Figure 1.—

Schisis progression and ERG b-wave response in Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y mice. (A) Schisis is most severe at 4 weeks of age and decreases over time. Note that adult (10-week-old) Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y mice do not have schisis. Images of H&E stained paraffin sections are shown. (B) ERG a-wave (top) and b-wave (bottom) responses in Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y mice at 4 and 9 weeks of age. The graphs show the average response at increasing light intensities for wild-type and Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y mice. Nine-week-old Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y mice have a reduced a-wave and slightly higher b-wave amplitude than 4-week-old mice. (C) The b/a-wave ratio in wild-type and Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y mice at 4 and 9 weeks of age. Nine-week-old Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y mice have an improved b/a-wave ratio compared to 4-week-old Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y mice. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; wt, wild type; bar, 20 μm; ***P < 0.0001.

Figure 2.—

Phenotypic variation in F₂ intercross (AKR × Rs1^tmgc1/Y) mice. Phenotypic variation observed in Rs1^tmgc1/Y F₂ mice at P30 with scoring used for the QTL analysis. The numbers at the top of the images are identification numbers of the F₂ mice. The numbers at the bottom represent the semiquantitative scores (schisis index). Images of H&E stained paraffin sections are shown. INL, inner nuclear layer; ONL, outer nuclear layer; bar, 20 μm.

Figure 3.—

QTL mapping of the modifier of Rs1. (A) Whole genome scan LOD score distribution for association of the schisis index in 45 Rs1^tmgc1/Y F₂ intercross mice. The QTL on Chr 7 was named Mor1 for modifier of Rs1 1. Three horizontal lines represent significant linkage as assessed by permutation testing (bottom, P < 0.05; middle, P < 0.01; top, P < 0.001). (B) LOD score distribution on Chr 7 for all Rs1^tmgc1/Y F₂ mice. After Mor1 was identified in the whole genome scan, 225 additional mice were genotyped and phenotyped for Chr 7. The LOD score distribution for the total 270 Rs1^tmgc1/Y F₂ intercross mice is shown. The genetic map was generated in R/qtl on the basis of our cross. D7Mit321 is the marker that is closest to the peak LOD score of 17.2 (P < 0.0001). Three horizontal lines represent significant linkage as noted in A. (C) Phenotypic distribution of F₂ mice at D7Mit321. Error bars represent ±1 standard error. 44TNJ includes both B6 and C3H alleles. (D) Mean schisis index of Rs1^tmgc1/Y F₂ mice with respect to AKR, B6, and C3H alleles. B6 and C3H alleles render the severe phenotype. Only mice that are AKR/AKR at D7Mit321 show significant reduction of the schisis index (ANOVA, P < 0.0001).

Figure 4.—

Mor1 allelic differences in B6 congenic mice. (A) Images are representative of the phenotypes in the B6 congenic (B6.Cg-Rs1^tmgc1) line at P30. All Mor1^AKR/Mor1^AKR mice do not exhibit the schisis phenotype. Mor1^AKR/Mor1^B6 (images not shown) and Mor1^B6/Mor1^B6 mice have a range of phenotypic severity from no schisis to severe schisis. Images of H&E stained paraffin sections are shown. (B) The scatter plot shows the genotypic effect of Mor1 on phenotypic severity in congenic mice. INL, inner nuclear layer; ONL, outer nuclear layer; bar, 20 μm; ***, ANOVA P < 0.0001.

Figure 5.—

Penetrance of the schisis phenotype in B6 and C3H congenic mice. Phenotypic distribution of B6 (B6.Cg-Rs1^tmgc1) and C3H (C3H.Cg-Rs1^tmgc1) congenic mice at P30. The penetrance of the schisis phenotype is significantly different between the two congenic strains (χ², P < 0.0006). Thirty-seven percent of the B6 congenic mice have the schisis phenotype, whereas 92% of the C3H congenic mice have schisis.

Figure 6.—

Population of transcripts in Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y and B6 congenic mice. (A) Representative electropherograms for Rs1 transcripts in the mouse retina. Wild-type mice show one major peak corresponding to the full-length wild-type transcript (738 bp), while male and female Rs1^tmgc1 mutant mice show a full-length transcript plus three alternative transcripts at 609 bp, 699 bp, and 787 bp. Control peaks are labeled with arrowheads. (B) Splicing variants of Rs1. The diagram illustrates the mouse Rs1 wild-type transcript and the three splice variants found in Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y retinas. The asterisks denote the formation of premature stop codons. (C) Relative amount of transcript variants found in Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y retinas. The amount of each transcript variant is shown as a percentage of total transcripts in Rs1^tmgc1/Rs1^tmgc1 and Rs1^tmgc1/Y mice and B6 congenic (B6.Cg-Rs1^tmgc1) mice that are Mor1^AKR/Mor1^AKR or Mor1^B6/Mor1^B6. Mice were tested at 4 weeks of age unless noted. The ratios for wild-type transcripts, transcripts that skip exon 2, and transcripts that skip both exons 2 and 3 are not significantly different among all groups tested. The ratio of wild-type transcripts with intronic sequence is significantly different (P < 0.05) between Mor1^B6/Mor1^B6 and 16-week-old Rs1^tmgc1/Rs1^tmgc1 mice.