Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

Abstract

Purpose: Familial exudative vitreoretinopathy (FEVR) is a group of inherited blinding eye diseases characterized by defects in the development of the retinal vessels. Recent studies have identified genetic variants in tetraspanin 12 (TSPAN12) as a cause of FEVR. The purpose of this study was to identify novel TSPAN12 mutations in Chinese patients with FEVR and to describe the associated phenotypes.

Methods: Mutation screening was performed by directly sequencing PCR products of genomic DNA with primers designed to amplify the seven coding exons and adjacent intronic regions of the FEVR-causing gene TSPAN12. Clinical phenotypes of the patients with TSPAN12 mutations were documented. Wild-type and mutant TSPAN12 proteins were assayed for the Norrin-β-catenin signaling pathway with luciferase reporter assays.

Results: Three novel heterozygous mutations in TSPAN12 were identified: c.566G>A (p.C189Y), c.177delC (p.Y59fsX67), and c.C254T (p.T85M). All three mutations involved highly conserved residues and were not present in 200 normal individuals. Ocular phenotypes included increased ramification of the peripheral retinal vessels, a peripheral avascular zone, inferotemporal dragging of the optic disc and macula, and retinal folds. The probands showed relatively severe retinopathy, whereas the other family members were often asymptomatic. In SuperTopFlash (STF) cell line transfection studies, C189Y, Y59fsX67, and T85M mutants failed to induce luciferase reporter activity in response to Norrin.

Conclusions: We found three novel TSPAN12 mutations in Chinese patients with autosomal dominant FEVR, and suggest that TSPAN12 mutations cause FEVR. The phenotypes associated with the TSPAN12 mutations showed extensive variation in disease severity among members of the same family, which implied the complexity of FEVR mutations and phenotypes.

Figure 1

Chromatograms and pedigrees of three families with familial exudative vitreoretinopathy. Three novel mutations were identified in TSPAN12. A: In Family A, the affected mother and son had the c.566G>A (p.C189Y) mutation. C: In Family B, the patient and her affected mother had the c.177delC (p.Y59fsX67) mutation. E: In Family C, the affected mother and son had the c.C254T (p.T85M) mutation. The columns from left to right display the pedigree and the sequence chromatograms of these patients (A, C, E) and the normal controls (B, D, F). Arrows indicate the positions of the altered nucleotides.

Figure 2

Protein sequence alignment of human TSPAN12 with its orthologs. The conserved amino acid residues are shaded. The orthologs are from the following species: Homo sapiens (NP_036470), Pan troglodytes (XP_001142754), Musmusculus (NP_766595), Rattus norvegicus (NP_001015026), Bos taurus (NP_001039977), Equus caballus (XP_001502093), Canis lupus familiaris (XP_855095), Monodelphis domestica (XP_001364876), Gallus gallus (NP_001007850), Taeniopygia guttata (XP_002192381), Ornithorhynchus anatinus (XP_001516347), and Danio rerio (NP_957446). A: The residue of the missense mutation p.C189Y is highly conserved. B: The residue of the missense mutation p.T85M is also highly conserved.

Figure 3

Fundus photographs of Family A with familial exudative vitreoretinopathy. A and B: Fundus photographs of the proband from Family A (individual II:1 in Figure 1), showing a retinal fold and a dragged macula. C and D: The unaffected father without the mutation has normal fundi. E and F: Fundus photographs of the asymptomatic mother with the c.566G>A mutation show normal posterior fundi. G and H: The mother has areas of avascularity and abnormal vessels in the peripheral retina.

Figure 4

Fundus photographs of Family B with familial exudative vitreoretinopathy. A and B: Fundus photographs of the proband from Family B (individual II:1 in Figure 1), showing the retinal vessels drawn up in a retinal fold that is obscuring the macula. C and D: The unaffected father has normal fundi. E and F: Fundus photographs of the asymptomatic mother with the c.177delC mutation show normal posterior fundi. G and H: The mother has increased vessel branching in the equatorial area and an avascular zone on the peripheral retina.

Figure 5

Fundus photographs of Family C with familial exudative vitreoretinopathy. A and B: Fundus photographs of the proband from Family C (individual II:1 in Figure 1), showing a retinal fold and a dragged macula. C and D: The mother has areas of avascularity and abnormal vessels in the peripheral retina.

Figure 6

Luciferase assays with the SuperTopFlash cell line transfected with the indicated plasmids. SuperTopFlash (STF) cells/well were transfected with 800 ng DNA (200 ng of Norrin, 200 ng of FZD4, 200 ng of LRP5, 100 ng of pSV-β-galactosidase control vector, and 100 ng of TSPAN12 plasmid [wild-type or mutation]) and 1.5 µl Lipofectamine 2000 transfection reagent. Forty-eight hours after transfection, the cells were harvested and washed twice with PBS. Luciferase activities were measured with a dual-luciferase assay kit. Reporter activity was normalized to the coexpressed β-galactosidase activity in each well. Each test was performed in triplicate. The reporter assay was repeated three times, and a representative result was obtained.

Figure 7

Western blot analysis by SDS-PAGE of the TSPAN12 mutants. Total protein (10 μg) isolated from cell lysates from luciferase assays was mixed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer and subjected to SDS-PAGE and western blot analysis using anti-Flag antibody to detect TSPAN12 and FZD4 expression. Beta-actin was used as the loading control. The expression level of TSPAN12 C189Y was compatible with that of the wild-type. However, the T85M and Y59fs mutant proteins were not stable.

Figure 8

Immunofluorescence staining of the TSPAN12 C189Y mutant. Cos 7 cells were transfected either with human wild-type or mutant TSPAN12 cloned into the pCMV6-entry vector, or empty vector. Cells were washed with PBS after 48 h and fixed with 4% PFA for 15 min. Mouse monoclonal anti-Flag antibody and Alexa Fluor 594 goat anti-mouse immunoglobulin (IgG) secondary antibody were used to detect TSPAN12 expression with the standard immunostaining method. Red channel, TSPAN12; blue channel, 4',6-diamidino-2-phenylindole (DAPI) for nuclei staining.

Figure 9

Diagram showing 9 known TSPAN12 mutations and three novel mutations identified in this study. The novel mutations are red.