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

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.

• Authors: Greg D Appleyard, George W Forsyth, Laura M Kiehlbauch, Kristen N Sigfrid, Heather L J Hanik, Anita Quon, Matthew E Loewen, Bruce H Grahn
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 47 Issue 5 Pg. 1810-6 (May 2006) ISSN: 0146-0404 [Print] United States
• PMID: 16638985 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

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

Topics

• Animals
• DNA, Complementary (genetics)
• DNA, Mitochondrial (genetics)
• Dog Diseases (genetics, pathology)
• Dogs
• Electron Transport Complex IV (genetics)
• Female
• Gene Expression Regulation
• Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) (genetics)
• Male
• Mitochondria, Muscle (genetics, 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, pathology, veterinary)
• Reverse Transcriptase Polymerase Chain Reaction (veterinary)