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A. McMahon, I. A. Butovich, S. N. Jackson, A. S. Woods, W. Kedzierski; Lipid Analysis of Stargardt-3 Mice Supports a Simple Loss of Function Pathogenic Mechanism. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3416.
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Stargardt disease-3 (STGD3) is caused by mutations in elongase of very long chain fatty acids-4 (ELOVL4). The enzyme catalyzes an extension of a fatty acid chain, a rate-limiting step of C28-C36 fatty acid synthesis which takes place in the endoplasmic reticulum. Studies of cultured cells expressing an ELOVL4 transgene have demonstrated that the mutated ELOVL4 protein leaves the endoplasmic reticulum and carries away the wild-type ELOVL4 protein. If this dominant negative mechanism plays a major role in STGD3 pathogenesis, it should result in about a 75% reduction of C28-C36 fatty acid synthesis in Stargardt-3 patients and mouse genetic models of the human pathology. To test this, we analyzed C28-C36 acyl lipids in Elovl4-expressing tissues of our Stgd3 mice which carry a 5-bp STGD3-pathogenic mutation in the mouse Elovl4 gene.
Stgd3-homozygous, Stgd3-heterozygous and wt littermate mice were euthanized and their tissues collected. Lipids were isolated and analyzed using thin layer chromatography and high performance liquid chromatography-mass spectrometry.
Analysis of mouse tissues detected C28-C36 acyl lipids in the skin and retina, but not in the brain. Mouse epidermis was found to contain two abundant lipid species, acylceramides and diacylglycerols, which have residues of C28-C36 fatty acids. Both lipid species were completely missing from the skin of neonatal Stgd3-homozygous mice. In Stgd3-heterozygous mice, their levels were reduced by approximately 50% in the epidermis. Similarly, the level of C28-C36 acyl phosphatidylcholines was lower by a half in the retina of adult Stgd3-heterozygous mice.
Lipid analysis in Stgd3-homozygous mice demonstrates that the Stargardt-3 mutation completely abolishes the synthetic activity of Elovl4. Furthermore, studies of Stgd3-heterozygous mice show that the mutation reduces the levels of C28-C36 acyl lipids by a half, not by 75% as the dominant negative mechanism predicts. These findings suggest that in Stgd3-heterozygous mice the pathogenesis involves a more complex mechanism or is a simple loss of function mechanism.
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