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The severity of retinal pathology in homozygous Crb1rd8/rd8 mice is dependent on additional genetic factors.

Luhmann UF, Carvalho LS, Holthaus SM, Cowing JA, Greenaway S, Chu CJ, Herrmann P, Smith AJ, Munro PM, Potter P, Bainbridge JW, Ali RR - Hum. Mol. Genet. (2014)

Bottom Line: Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines.By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified.This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and u.luhmann@ucl.ac.uk.

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The Crb1rd8/rd8 mutation leads to reduced Crb1 mRNA levels and the absence of the Crb1 protein at the OLM. mRNA expression analysis for Crb1 (A) and Crb2 (B) by quantitative real-time RT-PCR and protein localization of Crb1 by immunohistochemistry (C–F) in retinas of wild-type (Crb1+/+) and homozygous Crb1rd8/rd8 mice from the different lines at 8 weeks of age. (A) Quantitative real-time PCR detecting the exon 8 to mutant exon 9 boundary of Crb1 revealed similar reduction of mutant Crb1 transcripts in all homozygous Crb1rd8/rd8 mice compared with wild type. Asterisks indicate a statistically significant difference at P < 0.001 using one-way ANOVA with Tukey's multiple comparison test. (B) No significant compensatory increase of Crb2 mRNA levels in either of the Crb1rd8/rd8 lines (P = 0.1693; one-way ANOVA with Tukey's multiple comparison test). (C–F) Immunohistochemistry for Crb1 on sagittal retinal sections shows a specific signal (red) for Crb1 at the level of the OLM (white arrow head) in the wild type (C), which was not detected in all other homozygous Crb1rd8/rd8 mouse lines (D–F). ONL, outer nuclear layer. Scale bar: 25 µm.
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DDU424F3: The Crb1rd8/rd8 mutation leads to reduced Crb1 mRNA levels and the absence of the Crb1 protein at the OLM. mRNA expression analysis for Crb1 (A) and Crb2 (B) by quantitative real-time RT-PCR and protein localization of Crb1 by immunohistochemistry (C–F) in retinas of wild-type (Crb1+/+) and homozygous Crb1rd8/rd8 mice from the different lines at 8 weeks of age. (A) Quantitative real-time PCR detecting the exon 8 to mutant exon 9 boundary of Crb1 revealed similar reduction of mutant Crb1 transcripts in all homozygous Crb1rd8/rd8 mice compared with wild type. Asterisks indicate a statistically significant difference at P < 0.001 using one-way ANOVA with Tukey's multiple comparison test. (B) No significant compensatory increase of Crb2 mRNA levels in either of the Crb1rd8/rd8 lines (P = 0.1693; one-way ANOVA with Tukey's multiple comparison test). (C–F) Immunohistochemistry for Crb1 on sagittal retinal sections shows a specific signal (red) for Crb1 at the level of the OLM (white arrow head) in the wild type (C), which was not detected in all other homozygous Crb1rd8/rd8 mouse lines (D–F). ONL, outer nuclear layer. Scale bar: 25 µm.

Mentions: To further understand whether the Crb1rd8 allele, possibly by altered expression of the predicted truncated CRB1 protein (16), contributes to the phenotypic variability in different strains and whether expression of Crb2 may be altered in a compensatory way, we quantified Crb1 and Crb2 transcript levels relative to wild type in weakly and severely affected Crb1rd8/rd8 mice (Fig. 3). We also evaluated whether the genetic background of different inbred mouse strains influences the expression level of Crb1 (Supplementary Material, Fig. S1). Crb1 transcripts were significantly reduced to ∼20% of wild type in all Crb1rd8/rd8 mice, independent of the severity of their phenotypes (Fig. 3A). No significant changes in Crb2 transcript levels were observed, although subtle effects cannot be excluded (Fig. 3B). Although Crb1 transcript levels were significantly influenced by different genetic backgrounds, the influence of the homozygous Crb1rd8/rd8 mutation on Crb1 transcript levels was always much more pronounced (Supplementary Material, Fig. S1). Consistent with these data, immunohistochemistry using a C-terminal antibody for Crb1 revealed a weak specific signal across the whole superior and inferior OLM in wild-type retina (OLM; Fig. 3C, white arrow head), but not in any retina from Crb1rd8/rd8 mice (Fig. 3D–F).Figure 3.


The severity of retinal pathology in homozygous Crb1rd8/rd8 mice is dependent on additional genetic factors.

Luhmann UF, Carvalho LS, Holthaus SM, Cowing JA, Greenaway S, Chu CJ, Herrmann P, Smith AJ, Munro PM, Potter P, Bainbridge JW, Ali RR - Hum. Mol. Genet. (2014)

The Crb1rd8/rd8 mutation leads to reduced Crb1 mRNA levels and the absence of the Crb1 protein at the OLM. mRNA expression analysis for Crb1 (A) and Crb2 (B) by quantitative real-time RT-PCR and protein localization of Crb1 by immunohistochemistry (C–F) in retinas of wild-type (Crb1+/+) and homozygous Crb1rd8/rd8 mice from the different lines at 8 weeks of age. (A) Quantitative real-time PCR detecting the exon 8 to mutant exon 9 boundary of Crb1 revealed similar reduction of mutant Crb1 transcripts in all homozygous Crb1rd8/rd8 mice compared with wild type. Asterisks indicate a statistically significant difference at P < 0.001 using one-way ANOVA with Tukey's multiple comparison test. (B) No significant compensatory increase of Crb2 mRNA levels in either of the Crb1rd8/rd8 lines (P = 0.1693; one-way ANOVA with Tukey's multiple comparison test). (C–F) Immunohistochemistry for Crb1 on sagittal retinal sections shows a specific signal (red) for Crb1 at the level of the OLM (white arrow head) in the wild type (C), which was not detected in all other homozygous Crb1rd8/rd8 mouse lines (D–F). ONL, outer nuclear layer. Scale bar: 25 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4262495&req=5

DDU424F3: The Crb1rd8/rd8 mutation leads to reduced Crb1 mRNA levels and the absence of the Crb1 protein at the OLM. mRNA expression analysis for Crb1 (A) and Crb2 (B) by quantitative real-time RT-PCR and protein localization of Crb1 by immunohistochemistry (C–F) in retinas of wild-type (Crb1+/+) and homozygous Crb1rd8/rd8 mice from the different lines at 8 weeks of age. (A) Quantitative real-time PCR detecting the exon 8 to mutant exon 9 boundary of Crb1 revealed similar reduction of mutant Crb1 transcripts in all homozygous Crb1rd8/rd8 mice compared with wild type. Asterisks indicate a statistically significant difference at P < 0.001 using one-way ANOVA with Tukey's multiple comparison test. (B) No significant compensatory increase of Crb2 mRNA levels in either of the Crb1rd8/rd8 lines (P = 0.1693; one-way ANOVA with Tukey's multiple comparison test). (C–F) Immunohistochemistry for Crb1 on sagittal retinal sections shows a specific signal (red) for Crb1 at the level of the OLM (white arrow head) in the wild type (C), which was not detected in all other homozygous Crb1rd8/rd8 mouse lines (D–F). ONL, outer nuclear layer. Scale bar: 25 µm.
Mentions: To further understand whether the Crb1rd8 allele, possibly by altered expression of the predicted truncated CRB1 protein (16), contributes to the phenotypic variability in different strains and whether expression of Crb2 may be altered in a compensatory way, we quantified Crb1 and Crb2 transcript levels relative to wild type in weakly and severely affected Crb1rd8/rd8 mice (Fig. 3). We also evaluated whether the genetic background of different inbred mouse strains influences the expression level of Crb1 (Supplementary Material, Fig. S1). Crb1 transcripts were significantly reduced to ∼20% of wild type in all Crb1rd8/rd8 mice, independent of the severity of their phenotypes (Fig. 3A). No significant changes in Crb2 transcript levels were observed, although subtle effects cannot be excluded (Fig. 3B). Although Crb1 transcript levels were significantly influenced by different genetic backgrounds, the influence of the homozygous Crb1rd8/rd8 mutation on Crb1 transcript levels was always much more pronounced (Supplementary Material, Fig. S1). Consistent with these data, immunohistochemistry using a C-terminal antibody for Crb1 revealed a weak specific signal across the whole superior and inferior OLM in wild-type retina (OLM; Fig. 3C, white arrow head), but not in any retina from Crb1rd8/rd8 mice (Fig. 3D–F).Figure 3.

Bottom Line: Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines.By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified.This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and u.luhmann@ucl.ac.uk.

Show MeSH
Related in: MedlinePlus