Differential mitochondrial DNA and gene expression in inherited retinal dysplasia in miniature Schnauzer dogs

Invest Ophthalmol Vis Sci. 2006 May;47(5):1810-6. doi: 10.1167/iovs.05-0819.

Abstract

Purpose: To investigate the molecular basis of inherited retinal dysplasia in miniature Schnauzers.

Methods: Retina and retinal pigment epithelial tissues were collected from canine subjects at the age of 3 weeks. Total RNA isolated from these tissues was reverse transcribed to make representative cDNA pools that were compared for differences in gene expression by using a subtractive hybridization technique referred to as representational difference analysis (RDA). Expression differences identified by RDA were confirmed and quantified by real-time reverse-transcription PCR. Mitochondrial morphology from leukocytes and skeletal muscle of normal and affected miniature Schnauzers was examined by transmission electron microscopy.

Results: RDA screening of retinal pigment epithelial cDNA identified differences in mRNA transcript coding for two mitochondrial (mt) proteins--cytochrome oxidase subunit 1 and NADH dehydrogenase subunit 6--in affected dogs. Contrary to expectations, these identified sequences did not contain mutations. Based on the implication of mt-DNA-encoded proteins by the RDA experiments we used real-time PCR to compare the relative amounts of mt-DNA template in white blood cells from normal and affected dogs. White blood cells of affected dogs contained less than 30% of the normal amount of two specific mtDNA sequences, compared with the content of the nuclear-encoded glyceraldehyde-3-phosphate dehydrogenase (GA-3-PDH) reference gene. Retina and RPE tissue from affected dogs had reduced mRNA transcript levels for the two mitochondrial genes detected in the RDA experiment. Transcript levels for another mtDNA-encoded gene as well as the nuclear-encoded mitochondrial Tfam transcription factor were reduced in these tissues in affected dogs. Mitochondria from affected dogs were reduced in number and size and were unusually electron dense.

Conclusions: Reduced levels of nuclear and mitochondrial transcripts in the retina and RPE of miniature Schnauzers affected with retinal dysplasia suggest that the pathogenesis of the disorder may arise from a lowered energy supply to the retina and RPE.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA, Complementary / genetics
  • DNA, Mitochondrial / genetics*
  • Dog Diseases / genetics*
  • Dog Diseases / pathology
  • Dogs
  • Electron Transport Complex IV / genetics
  • Female
  • Gene Expression Regulation*
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / genetics
  • Male
  • Mitochondria, Muscle / genetics*
  • Mitochondria, Muscle / ultrastructure
  • NADH Dehydrogenase / genetics
  • Oligonucleotide Array Sequence Analysis / veterinary
  • Pigment Epithelium of Eye / metabolism
  • RNA / isolation & purification
  • RNA, Messenger / analysis
  • Retina / metabolism
  • Retinal Dysplasia / genetics
  • Retinal Dysplasia / pathology
  • Retinal Dysplasia / veterinary*
  • Reverse Transcriptase Polymerase Chain Reaction / veterinary

Substances

  • DNA, Complementary
  • DNA, Mitochondrial
  • RNA, Messenger
  • RNA
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)
  • NADH Dehydrogenase
  • Electron Transport Complex IV