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

Patients and clinical examinations

This study adhered to the ARVO statement on human subjects and was approved by the Institutional Review Board of the Xinhua Hospital affiliated with the Shanghai Jiao Tong University School of Medicine and the Institutional Review Board of the Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital, China. The study complied with the guidelines of the Declaration of Helsinki and the Guidance of Sample Collection of Human Genetic Disease of the Ministry of Public Health of China. Informed consent was obtained from all participants in the study. 85 outpatients diagnosed as FEVR were recruited: 47 were male and 38 were female. The age of all participants ranged from 2 months to 14 years old. Their parents were also recruited in this study. All participants were in good health when they were recruited, except for the eye. For minors, written consent was obtained from their parents. All participants underwent careful ophthalmology examinations, and were diagnosed by a clinical ophthalmologist and a geneticist based mainly on the presence of retinal abnormalities in fundus photographs deemed typical of FEVR, which included an avascular peripheral retina together with exudative and/or sequelae of retinal traction, such as macular ectopia, retinal folds, and retinal detachment. Fundus fluorescein angiography (FFA) was performed in selected cases to confirm further the diagnosis. In the 200 normal matched controls, all individuals underwent an eye examination and no signs of eye diseases were observed. Venous blood samples were obtained from all subjects in EDTA Vacutainers.

Detection of TSPAN12 mutations

Genomic DNA samples were extracted from the peripheral blood of the subject patients using a whole blood DNA extraction kit (BioTeke, Beijing, China). Venous blood in EDTA vautainers was stored in 4 ºC and processed within 24 h after blood drawn. Genomic DNA samples were extracted blood DNA extraction kit following manufacture’s instruction (Bioteke). All patients had been confirmed beforehand as carrying no mutations in FZD4, LRP5, or NDP. To identify mutations in the coding exons of the TSPAN12 gene, oligonucleotide primers flanking the coding regions and neighboring intronic sequences were designed (Table 1). The exons of the TSPAN12 gene were analyzed via direct sequencing of the PCR products. PCR working conditions are as following: initial denature temperature 95 ºC for 3 min, followed by 33 cycles of reaction: template denature at 95 ºC for 15 s, annealing for 15 s at 59 ºC and extension at 72 ºC for 20 s. A final step of 7 min reaction extension at 72 ºC was applied to fill in the gaps of PCR product. Amplified products were purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), and were sequenced with forward and reverse primers by the BigDye Terminator v3.1 Cycle Sequencing Kit (ABI Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. After sequence changes were detected in the probands, samples from the other family members were analyzed with direct sequencing.

Construction of expression plasmids

The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites. All mutations were introduced into the wild-type TSPAN12 cDNA by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). LRP5, FZD4, and Norrin expression vectors (generously provided by Dr. Jeremy Nathans of Johns Hopkins University) have been described previously [12]. The recombinant plasmids containing TSPAN12-Myc-Flag fusion constructs were first verified with DNA sequencing, and then prepared for transfection using Qiagen plasmid Maxi preparation kit (Qiagen).

Luciferase assays

The SuperTopFlash (STF) reporter, in which firefly luciferase was driven by seven lymphoid enhancer factor/T-cell factor (LEF/TCF) consensus binding sites, was a kind gift from Dr. Jeremy Nathans (John Hopkins University, Baltimore, MD). This reporter plasmid was stably transfected into HEK 293 cells as reported previously to generate the STF cell line. In 24-well plates, 160 K STF cells/well were transfected with 800 ng DNA and 1.5 µl Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA). The DNA mix contained 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). Forty-eight hours after transfection, the cells were harvested and washed twice with PBS (1X; 120 mM NaCl, 20 mM KCl 10 mM NaPO₄, 5 mM KPO₄, pH 7.4), and the luciferase activities were measured with a Dual-Luciferase Assay Kit (Promega) according to the manufacturer’s instructions. 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.

Expression of TSPAN12 in Cos 7 cells

Cos 7 cells (American Type Culture Collection [ATCC], Manassas, VA) were cultured in DMEM (ATCC, Manassas, VA) with high glucose (HyClone) supplemented with 10% fetal bovine serum and 1% (vol/vol) penicillin/streptomycin at 37 °C in a 5% CO₂ atmosphere. Cells were seeded in six-well plates (Corning Inc., Corning, NY) and transfected at 50% confluency with 1 μg of wild-type and mutant pCMV6-entry-TSPAN12 or empty vector using Lipofectimine 2000 (Invitrogen) according to the manufacturer’s instructions. After 48 h, the cells were washed with PBS, and fixed with 4% paraformaldehyde (PFA) for 15 min. Anti-Flag antibody was used to detect TSPAN12 expression using standard immunostaining method.

Identification of novel TSPAN12 mutations and phenotypes

We identified three proband patients with FEVR in our clinic. To identify causative genes for FEVR, we sequenced all the exons and the flanking intonic sequences for four known FEVR genes: FZD4, LRP5, Norrin, and TSPAN12. Three novel mutations in the coding sequence of the TSPAN12 gene were found in these families, including c.566G>A (p.C189Y), c.177delC (Y59fsX67), and c.C254T (p.T85M; Figure 1).

In Family A, we identified a missense mutation in exon 7, c.566G>A, segregating with the disease. This mutation led to the replacement of a cysteine by a tyrosine at codon 189 (p.C189Y; Figure 1), which involved a highly evolutionarily conserved residue (Figure 2A) from Homo sapiens to Danio rerio. The proband of the family was a 1-year-old boy with bilateral retinal folds affecting the macula (Figure 3). His mutation-carrying mother had normal vision but with areas of avascular zone and abnormal vessels in the peripheral retina (Figure 3).

We identified one base pair deletion in exon 4, c.177delC, in the proband of Family B and her asymptomatic mother. This mutation resulted in a frameshift followed by premature termination at codon 67 (p.Tyr59fsX67; Figure 1), which also involved highly evolutionarily conserved residues (Figure 2C) from Homo sapiens to Danio rerio. The proband was a 2-year-old girl with a complaint of esotropia. Fundus examination showed typical falciform retinal folds across the fovea in both eyes (Figure 4). Her father had normal fundi, but her mother had typical fundus changes in FFA examination, including increased ramification of the peripheral retinal vessels and an avascular zone in the peripheral retina, though she had normal visual acuity of 1.0 in both eyes and had no vision complaints (Figure 4).

In Family C, a missense mutation c.C254T (p.T85M) segregating with the disease was identified (Figure 1). This mutation led to the change in a threonine residue to a methionine at codon 85 (p.T85M), which is extremely conserved from Homo sapiens to Danio rerio. (Figure 1, Figure 2B). The proband was a 7-year-old boy with dragged disc in his left eye (Figure 5). There was pigment deposit in his right retina. His mutation-carrying mother had normal vision but had areas of avascular peripheral retina. All three mutations cosegregated with the disease phenotype of the respective families, and were not detected in 200 ethnically matched control individuals.

Defective Norrin signaling mediated by mutant TSPAN12 proteins in vitro

To determine the effect of the TSPAN12 mutants on its biologic activity in Norrin/β-catenin signaling, we performed luciferase assays in STF cells using wild-type and three mutant TSPAN12 cDNAs. c.177delC, c.566G>A, and c.C254T were introduced into TSPAN12 with the site-direct mutagenesis method. Our in vitro analyses suggested that compared to wild-type TSPAN12, all three TSPAN12 mutants failed to induce luciferase reporter activity in STF cells in response to Norrin (Figure 6). Western blot analysis indicated that the T85M and Y59fsX67 mutant proteins were not stable (Figure 7). The C189Y mutant expression level was compatible to that of the wild-type (Figure 7 and Figure 8).The results suggest that the three TSPAN12 mutations identified in our study are pathogenic, and are consistent with the notion that defective Norrin/TSPAN12 signaling underlies FEVR.