Novel TSPAN12 mutations in patients with familial exudative vitreoretinopathy and their associated phenotypes

• Introduction
• Methods
• Results
• Discussion

Abstract

Purpose: Mutations in tetraspanin 12 (TSPAN12) have recently been identified as a cause of autosomal dominant familial exudative vitreoretinopathy (FEVR). The purpose of this study was to detect TSPAN12 mutations in Chinese patients with FEVR and to describe the associated phenotypes.

Methods: Sanger sequencing was used to analyze the seven coding exons and their adjacent regions of TSPAN12 in 49 unrelated FEVR patients. Clinical phenotypes of the patients with TSPAN12 mutations were documented.

Results: Three novel heterozygous mutations in TSPAN12 were identified in three patients from unrelated families: c.146C>T (p.Thr49Met), c.313T>C (p.Cys105Arg), and c.601delC (p.Leu201PhefsX14). All three mutations involved highly conserved residues and were not present in 180 normal individuals. Ocular phenotypes included retinal folds, inferotemporal dragging of the optic disc and macula, increased vessels in the equatorial region, and a peripheral avascular zone. A father and his daughter had the same mutation but the father only had mild peripheral fundus changes while his daughter had obvious dragged disc and macular ectopia.

Conclusions: Our results suggest that TSPAN12 mutations are responsible for FEVR. Similar to patients with mutations in NDP, LRP5, or FZD4, the phenotypes associated with TSPAN12 mutations showed great variations between different individuals within a family and between the two eyes in individual patients.

Familial exudative vitreoretinopathy (FEVR, OMIM 133780) is a hereditary vitreoretinal disorder characterized by developmental anomalies of the retinal vessels [1]. The primary anomaly is a premature arrest of the vascularization in the peripheral retina, which may lead to retinal neovascularization, vitreoretinal traction, exudates, fibrovascular masses, vitreous hemorrhages, retinal folds, and tractional retinal detachment. FEVR exhibits strikingly variable phenotypes among patients, even for patients from the same family or between the two eyes of an individual patient. The most severe form results in complete blindness, whereas minimally affected individuals can be totally asymptomatic, in which nonperfusion or avascular zones in the peripheral retina may only be identified by fluorescein angiography [2].

FEVR can be inherited as an autosomal dominant (OMIM 133780) [1,3-5], autosomal recessive (OMIM 601813) [6,7], or X-linked (OMIM 305390) [8,9] trait, and the autosomal dominant form is the most common [10,11]. Mutations in NDP (OMIM 300658) [8,12], FZD4 (OMIM 604579) [13], and LRP5 (OMIM 603506) [14,15] have been reported to be responsible for FEVR. The proteins encoded by these genes have all been shown to participate in the Wnt/Norrin signaling pathway [16-19]. Mutations in NDP, FZD4, and LRP5 account for approximately half of all FEVR cases, which indicates that additional causative genes remain to be identified [20-26].

Recently, heterozygous mutations in tetraspanin 12 (TSPAN12; OMIM 613138), which is a component of the Norrin-FZD4-LRP5 signaling complex [16,19], have been found to be responsible for autosomal dominant FEVR [27,28]. However, the frequency of TSPAN12 mutations and their associated phenotypes need further study.

In this study, Sanger sequencing was used to analyze the coding and adjacent intronic regions of TSPAN12 in 49 unrelated Chinese FEVR patients. Three novel heterozygous mutations were identified, and the associated phenotypes were described.

After sequencing the coding and adjacent regions of TSPAN12 in 49 unrelated FEVR patients, three novel heterozygous mutations were detected in three patients: c.146C>T, c.313T>C, and c.601delC (Figure 1). These three mutations were not present in the 180 normal individuals.

The c.146C>T mutation in exon 3 changed the encoded residue from a hydrophilic threonine to a sulfur-containing hydrophobic methionine at codon 49 (p.Thr49Met), which involves a highly evolutionarily conserved residue (Figure 2). This mutation was predicted to probably be damaging by PolyPhen-2. The c.313T>C mutation in exon 5 changed the encoded residue from a thiol-containing cysteine to a positively charged basic arginine at codon 105 (p.Cys105Arg), which is highly conserved (Figure 2). This mutation was also predicted to probably be damaging by PolyPhen-2. The c.601delC mutation in exon 7 not only changed the residue at codon 201, but also created a frame shift with 14 additional new residues before a premature termination at codon 215 (p.Leu201PhefsX14). This mutation also involved an evolutionarily conserved region.

All patients with the three novel mutations showed typical signs of FEVR. The patient with the c.146C>T mutation in Family A was a six-year-old boy with strabismus. Falciform retinal folds in the left fundus were seen upon ophthalmoscopic observation. His parents were recorded to have normal visual acuity, but they were not available for additional examination. The patient with the c.313T>C mutation in Family B was a nine-year-old boy with a complaint of poor visual acuity in the left eye. He had normal visual acuity in the right eye but only recognized hand movement with the left eye. His right fundus was normal, but his left fundus showed the following changes: macular degeneration with pigment proliferation, mild temporal dragging of the optic disc, pigmentary deposits on the midperipheral retina, increased ramification of the peripheral retinal vessels, and an avascular zone on the peripheral retina (Figure 3). His parents were recorded to have normal visual acuity, but they were not available for additional examination. The heterozygous c.601delC mutation in Family C was detected in the proband and in her asymptomatic father. The proband was a three-year-old girl with a complaint of esotropia. Fundus examination showed typical inferotemporal dragging of the optic disc and macula in both eyes (Figure 4). Her mother had normal fundi (Figure 5A,B); but her asymptomatic father had typical fundus changes, although he had normal visual acuity of 1.0 in both eyes. The asymptomatic father had straightening and increased branching of his peripheral retinal vessels (Figure 5C-F) but showed normal macula on optical coherence tomography scan (Figure 5G,H).

In this study, three novel TSPAN12 mutations were detected in three patients with FEVR, but not in 180 normal individuals. Segregation analysis in one family indicated a dominant role for the mutation. The two missense mutations occurred in evolutionarily conserved regions of TSPAN12 and were predicted to be pathogenic. These lines of evidence provide support for the conclusion that these mutations are the cause of FEVR in these patients.

Previously, nine TSPAN12 mutations in patients with FEVR were reported. All 12 mutations, including the three reported in this study, are distributed throughout all coding exons, except for exon 2 (Figure 6). These 12 mutations can be classified into missense mutations (six), nonsense mutations (two), insertion or deletion mutations (two), and splice-site mutations (two). Of the 12 mutations, each has only been found in a single family, except for the p.Ala237Pro mutations that have been detected in four families. For the 15 families with the 12 TSPAN12 mutations, ten had a family history of FEVR, which suggests that this is an autosomal dominant trait with incomplete penetrance, whereas the other five families were isolated cases without a family history. The approximately 6.1% (3/49) mutation frequency detected in our patients is comparable to the 10% (7/70) frequency detected in one previous study [28]. The patients in that study were determined to have no mutations in the other known FEVR genes (NDP, FZD4, or LRP5) that might account for approximately half of FEVR cases [30]. In another study, two TSPAN12 mutations were present in 5 of 11 probands (45.5%), but these patients were previously excluded from having mutations in NDP, FZD4 or LRP5. Four of the five probands with the same mutation shared an at-risk haplotype of at least 5.8 Mb, suggesting a founder effect [27].

For the patients with the previously reported TSPAN12 mutations, clinical phenotypes include the following: retinal folds, temporal dragging of the optic disc and/or macula, traction of the posterior retinal vessels, increased vascular ramifications in the equatorial area, a peripheral retinal avascular zone, retinal pigmentary disturbances that mimic retinitis pigmentosa, exudates, and retinal detachments. Retinal folds were the most common finding. FEVR in asymptomatic individuals can be detected using fluorescein angiography (Figure 5), but mild changes in the peripheral retina may be neglected, which might partly contribute to the nonpenetrance in a few families. Large phenotypic variations have been observed between different individuals within a family and between different eyes in individual patients. There is no particular genotype–phenotype correlation between particular mutations and certain clinical signs, which suggests haploinsufficiency as the underlying mechanism that induces the manifestation of the disease. Overall, the ocular manifestations caused by TSPAN12 mutations are similar to those due to mutations in FZD4, LRP5, or NDP [21,23,24].

The proteins encoded by TSPAN12, NDP, FZD4, and LRP5 are important components of the Norrin/Fz4 signaling pathway (Norrin/Fz4/Lrp5/Tspan12 signaling pathway), which controls retinal vascular development [16,19]. Recognizing additional mutations in these genes may not only provide useful information for clinical diagnosis and genetic counseling, but may also enrich our understanding of the functional domains of these proteins. Mutations in these four genes all lead to FEVR, which suggests that additional components that participate in this signaling pathway may be reasonable candidates in those FEVR patients without mutations in these four genes.

The authors thank the family members for their participation. This study was supported by the National Science Fund for Distinguished Young Scholars (30725044 to Q.Z.) National 973 Plan of China (2010CB529904 to Q.Z.).