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Mika Asai-Coakwell, Xiao Hua Dai, Lindsey March, Elfride De Baere, Peter J. Francis, Yves Sauve, W Ted Allison, Robert K. Koenekoop, Andrew Waskiewicz, Ordan J. Lehmann; Examining the contribution of Growth differentiation factor 6 (GDF6) to retinal dystrophies. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4530.
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GDF6, a TGF-β superfamily member, is essential for specifying the dorso-ventral retinal axis and we therefore investigated its contribution to early onset retinal dystrophies.
249 Lebers Congenital Amaurosis (LCA) and Juvenile RP (JRP) DNA samples were screened for GDF6 mutations by direct sequencing and identified variants characterized by immunoblot and luciferase reporter assays. Eight additional BMP/GDFs were sequenced in samples heterozygous for a GDF6 variant, with whole exome sequencing of 2 samples. Murine (E18) and zebrafish Gdf6 mutants were characterized with retinal histology and TUNEL assay, with ERGs performed in Gdf6+/- mice.
Four GDF6 variants (A249E, D57H, E292D, A199T) were identified in 4 LCA/JRP samples. One proband was a compound heterozygote (A249E and D57H), and the heterozygous parents displayed subtle ERG changes (paternal: reduced b-wave amplitude; maternal: delayed b-wave implicit time). Of the three probands with a single GDF6 variant, one carried a deletion of 3 alanine residues in GDF11 (also present in 2 of 1450 control chromosomes). Immunoblot analysis demonstrated altered levels of mature ligand compared to wild type (WT) [decreases: A249E, 56%; D57H,99%; increases: A199T,137%; E292D,4%] with luciferase analysis revealing significant decreases in transactivation of the reporter for each variant (p<0.001, t-test). A subset of Gdf6+/-mice exhibited anomalous ERGs with up to 50% decreases in a-wave and b-wave amplitudes compared to WT. The number of apoptotic signals was substantially increased in both murine and zebrafish homozygous mutants [mean/section: Gdf6-/- 30.4 (WT 8.3), gdf6-/- 76.4 (WT 3.3), (p<0.001, t-test)].
These data provide evidence from human genetics and two model organisms that perturbed GDF6 function contributes to early onset retinal dystrophies, extending the range of phenotypes associated with GDF6 mutation. The identification of multiple LCA probands with heterozygous GDF6 mutations is compatible with multi-allelic inheritance of a second TGF-β variant. Notably, recapitulation of key aspects of the human phenotypes in multiple animal models provides opportunities for developing novel therapies.
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