| BackgroundSevere myopic anisometropia is a condition in which the degree of myopia of both eyes differs. It is closely associated with binocular defects, amblyopia, and strabismus, which can affect the patient’s normal life. Visual function damage may be in the eye with higher diopter. Severe myopic anisometropia is characterized by over and asymmetry development of axial lengths between two eyes, and it is associated with genetic propensity, but there is no relative gene reported. Our group paid close attention to the anisometropic patients in our clinical settings and we have collected 2 families with severe myopic anisometropia. And the clinical report of one family had been published on Opotometry and Vision Science in 2012. We have identified the gene UNC5D which was associated with myopic anisometropia by pathogenic gene location for candidates genealogy study. A missense mutation of gene UNC5D which lead to the substitution of Arg for Cys of UNC5D protein was found in all five patients. Herein, to verify the mechanism of mutant UNC5D in pathogenesis of changes in relative genes and extracellular matrix, UNC5D cDNA sequence was subcloned into vectors and a series of studies were performed in vitro.PurposeTo speculate the pathogenesis of the sclera thinning and axial lengths over and asymmetry expanding which lead to anisometropia, the mechanism of mutant UNC5D in changes of relative genes and extracellular matrix was studied.Methods1 To predict the effect of this missense mutation on the protein, Kyte Doolittle Hydropathy Plots was used to predict the hydrophobicity of the wild-type and mutant protein. And the online PolyPhen-2, Align-GVGD and SIFT tool were used to distinguish between functionally neutral and deleterious mutations in both the mutant and wild-type version of UNC5D protein.2 The human wild type UNC5D cDNA sequence was constructed by gene sythesis and sub-cloned into pLVX-Tight-Puro. Mutant UNC5D plasmid was constructed after PCR induced mutagenesis. HEK293T cells were co-transduced with lentiviral vectors expressing UNC5D (wild type or mutant type) and pLVX-Tet-On-Advanced. Gene UNC5D expression was induced by culturing cells in the presence of doxycycline. Total RNA was extracted for Affymetrix 3’ IVT gene chip analysis, and qPCRs were performed to verify the chip results.3 pLVX-IRES-ZsGreenl lentiviral vectors expressing wild and mutant UNC5D contained HA tag were constructed and HDFs cells were infected. (1) QPCRs were used to conform the difference of relative expression between wild group, mutant group and control group; (2) Difference of sub-cellular localization of wild UNC5D and mutant UNC5D in HDFs by fluorescence Microscope; (3) The interaction between HSP70B’ and Smad2/3 was tested by Co-IP. (4) The relative expressions of COL1A1, MMP-1 and MMP-2 between wild group, mutant group and control group were tested by QPCR after treated by TGF-β1 and COL1A1 protein expression levels in different groups were checked by Western Blot; (5) P-Smad2/3 expressions between wild group, mutant group and control group were tested by Western Blot after treated by TGF-β1.Results1 Bioinformatics analysis:(1) The identified missense mutation in UNC5D gene resulted in the substitution of a highly conserved arginine by cysteine (R433C). Arginine has strong hydrophily while cysteine belongs to hydrophobic amino. The missense mutation led to the change of hydrophobicity of UNC5D protein. (2) The mutation was predicted to be Probably Damaging on protein function by Polyphen2 (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://sift.jcvi.org/), and most likely interfere with the function by Align-GVGD (http://agvgd.iarc.fr).2 Lentiviral vectors pLVX-Tight-UNC5D-WT and pLVX-Tight-UNC5D-MU were successfully constructed and HEK293T cells were co-transiently transfected with pLVX-Tet-On-Advanced by each plasmid respectively and UNC5D was in response to Dox treatment. Affymetrix 3" IVT gene chip showed that 14 genes significantly (p< 0.05) up-regulated (more than twofold up/down) in mutant group as compared to wild group, with 2 of these deregulated more than five fold up and 2 genes significantly (p< 0.05) deregulated. Strongly up-regulated genes in mutant group was HSPA6,22.0835 fold compared with wild group and 27.58 fold compared with control group. The expression level for HSPA6 was similar between wild group and control group. The results of qPCR were similar with the chip results. HSPA6 was upregulated in mutant group,7.82 fold compared with wild group and 25.16 fold compared with control group. The expression level of HSPA6 in wild group was 3.22 fold as it was in control group.3 pLVX-IRES-ZsGreenl lentiviral vectors expressing wild and mutant UNC5D contained HA tag were constructed successfully and HDFs cells were infected. (1) HSPA6 was up-regulated in mutant group,6.02 fold compared with wild group and 15.59 fold compared with control group by qPCR tests. The expression level of HSPA6 in wild group was 2.59 fold as it was in control group by qPCR tests. (2) No obviously difference of sub-cellular localization of wild UNC5D and mutant UNC5D in HDFs cells by fluorescence Microscope. (3) Results of Co-IP indicated there was interaction between HSP70B’and Smad2/3. (4) After treated by TGF-β1 the relative expression of COL1A1 which was tested by qPCR was down-regulated, MMP-2 was up-regulated in mutant group compared with wild group and control group, and the difference of expression of MMP-1 was not found among three groups. And COL1A1 protein expression was less than that of other two groups checked by Western Blot; (5) P-Smad2/3 expression of mutant group was lowest between three groups tested by Western Blot after treated by TGF--β1.ConclusionsHSPA6 up-regulation induced by mutation in UNC5D might decrease the phosphorylation level of Smad2/3 and impede the TGF-β signaling which may be result of the changes in sclera extracellular matrix which can cause the sclera rebuild. Our study firstly provides a relative gene research of pathogenesis of severe myopic anisometropia. |
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