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Patients with the very rare eye pathology Wagner disease (OMIM #143200) present with an abnormal (empty) vitreous, retinal detachment and altered electroretinogram (ERG). The disease is progressive and can eventually lead to blindness. No therapy can be offered to date. The genetic basis is the presence of mutations in the VCAN gene, encoding the large extracellular matrix molecule versican, which is a component of the vitreous. All identified mutations map to the canonical splice sites flanking exon 8, resulting in low number of aberrant splice products and a severe increase in two (V2, V3) of the four naturally occurring splice variants. The pathomechanism of Wagner's disease is poorly understood and a mouse model may afford further insight. The hdf-/- mice, named for their initial phenotype of heart defects, carry a null allele for Vcan that leads to embryonic lethality when homozygous, but heterozygote animals are viable. Here we investigated a possible eye phenotype in the heterozygous animals. While the overall morphology of retina and ciliary body appears to be normal, older (17 months) mutant animals show a decrease in ERG signalling profiles affecting the a-, b- and c-waves. This aspect of altered ERG profile demonstrates similarities to the human disease manifestation and underlines the suitability of heterozygous hdf+/- mice as a model for Wagner disease.
Wagner disease (OMIM# 143200) is a rare autosomal dominant progressive disorder belonging to the group of hereditary vitreoretinal degenerations (http://www.ncbi.nlm.nih.gov/books/NBK3821/). Vision impairment causes a major handicap for the affected. Clinically, the disease presents typically with the hallmark features of an empty vitreous cavity with fibrillary condensations, avascular strands and veils. Additionally, chorio-retinal atrophy, which may be reflected in altered ERG, and peripheral tractional retinal detachment are frequently observed. The syndrome shows full penetrance with manifestation at early adolescence. The disease is furthermore characterised by more heterogeneous clinical aspects, that is, cataracts and myopia. As VCAN has been shown to be mutated in the majority of patients with Wagner syndrome, the potential pathomechanism likely involves the large core protein isoforms V0 and/or V1 of the extracellular matrix chondroitin sulphate proteoglycan versican with a length of 3396 and 2409 amino acids, respectively. Furthermore, versican has been found as a component of the vitreous. To date all identified mutations in patients with Wagner disease map to the canonical splice sites bordering exon 8. Under normal conditions, 4 alternative splice isoforms are produced, which differ in the presence of exons 7 and 8 (variant V0 contains exon 7 and 8, V1 lacks exon 7, V2 lacks exon 8 and V3 lacks exon 7 and 8). It has been suggested that the splice site mutations lead to aberrant splice products and most strikingly to abnormal quantity of the normal transcript isoforms, in particular a dramatic increase in V2. The protein domains encoded by the large exons 7 and 8 carry 23 potential glucosaminoglycan attachment sites, suggesting that the larger isoforms contribute to a gel-maintenance function within the vitreous. In order to shed more light on the disease mechanism, we explored the phenotype of the mouse mutant model hdf which carries a non-functional Vcan gene. Mouse and human versican share a similar genomic organisation of the gene with 77% identity at the DNA sequence level and 64% at the amino acid level, with variations spread throughout the protein rather than being clustered (http://www.ncbi.nlm.nih.gov/gene/, Jan 2016). Furthermore, alternative splicing of the two largest exons is conserved in both species, yielding the 4 isoforms. These facts led us to investigate the suitability of Vcan knockout mice as a model for Wagner disease. Homozygous hdf mice (Vcan null) have previously been described; they are embryonic lethal due to severe heart and limb developmental defects. In contrast, heterozygous animals are viable and appear at first sight normal, but eye phenotypes have not been investigated. Since the Wagner disease phenotype is dominant and mutations are heterozygous, the occurrence of an eye phenotype in the heterozygous hdf+/- mouse mutant seemed plausible.
In humans heterozygous mutations in the VCAN gene cause the dominantly inherited erosive vitreoretinopathy of Wagner disease. To understand the underlying pathophysiology we investigated the effects of the heterozygous vcan null mutation in hdf+/- mice on eyes.
At age 5.5 months and 12 months, the scotopic and photopic ERGs did not differ between mutant and wild type siblings (data not shown). Furthermore, ophthalmoscopic analysis of wild type and mutant mice at 5.5 months did not reveal a difference (data not shown). At age 17 months, in the mutant animal eyes, the b-wave amplitude of the scotopic ERG was significantly reduced by 22% on average (Fig. 1B; 10 [mcds]: t(10) = 3.9, P = 0.00296; 100 [mcds]: t(10) = 3.97, P = 0.00264; 1000 [mcds]: t(10) = 3.64, P = 0.00454; 3000 [mcds]: t(10) = 4.06, P = 0.002287). The a-wave amplitude showed, for some light intensities, statistically significant differences (10 [mcds]: t(10)=0.91, P = 0.3824; 100 [mcds]: t(10)=2.99, P = 0.0136; 1000 [mcds]: t(10) = 3.07, P = 0.0118; 3000 [mcds]: t(10) = 2.18, P = 0.0544; 30000 [mcds]: t(10) = 2.69, P = 0.0228; 60000 [mcds]: t(10) = 2.02, P = 0.0710; 90000 [mcds]: t(10) = 2.3, P = 0.0439; 300000 [mcds]: t(10) = 1.95, P = 0.0797). The c-wave was also decreased (Figure 1c, t(10) = 4.99, P = 0.00054), indicating that in addition to photoreceptors and inner layer of retina, the retinal pigment epithelium (RPE) is also involved.
Eyes from the individual mutants were analysed for ERG beforehand and were subjected to histological analysis of the retina. All retinas displayed a proper morphological distribution of the various cell layers positioned at the central or peripheral retina or at the ciliary body when comparing heterozygous mutant and wild type siblings (Fig. 1D). Furthermore, quantitative analysis of the thickness of the retina or the outer nuclear layer also did not reveal a difference between mutant and wild type siblings (Fig. 1E, central and peripheral retina: ratio retina [µm] /ONL [µm] nuclei (t(4) = 0.03285, P = 0.9754 and t(4) = 0.3736, P = 0.7277, respectively) and for ratio ONL [µm] /ONL nuclei (t(4) = 1.303, P = 0.2624 and t(4) = 2.724, P = 0.0528, respectively).
Taking the morphological and ERG studies together, a functional defect is likely due to reduced signalling from the retinal cells. Of note, a collagen (Col2a1) mouse model for Stickler Syndrome, a connective tissue disease with syndromic features affecting limbs and an eye phenotype very similar to that observed in Wagner disease, had previously been shown to display morphological alterations in the ciliary body only in older mice (18 months). Therefore, we also examined potential changes in the ciliary body in the hdf mouse strain. Based on the image observation, no obvious difference between wild type and mutant animals could be detected (Fig. 1D).
The dominantly inherited Wagner disease is associated with heterozygous mutations in the VCAN gene, leading to abnormal splicing. While the hallmark clinical phenotype is affecting the vitreous, retinal complications are common, also reflected by abnormal ERG. The hdf mouse model is characterised by a null mutation in the Vcan gene. We sought to explore an eye phenotype in heterozygous hdf+/- mice, possibly pheno-copying the human disease. In younger mice, no difference was seen between the wild type and mutant animals but the ERG showed abnormalities in older animals. A slight reduction of a-wave suggested that the photoreceptor cells are involved. A reduction of the b-wave implicated synaptic or post-synaptic effects on the bipolar cells. Generally, the b-wave may also be affected when the blood flow through the central retinal artery is blocked or the membrane potential of Müller cells is changed as result of an altered extracellular potassium concentration. Which of these conditions apply here needs to be further investigated. In addition to the b-wave, the c-wave has also been reduced in the older hdf+/- mice, suggesting an involvement of the RPE or the RPE-photoreceptor complex. However, as the a-wave is normal a potential damage of the photoreceptors is at least not detectable. The c-wave is not a typical feature of the human ERG, but is found in several animals, including mice. Interestingly, the age-related progressive nature of the phenotype in Wagner patients is mimicked in the hdf+/- mouse model, showing an altered retinal function at increased age. Whether the patho-mechanism is also comparable remains to be investigated because the mouse strain carries an expression null allele, while human patients still may express the two smallest splice variants V2 and V3 from the affected allele. It has previously been suggested that the splice defect might result in an imbalanced ratio of transcript isoforms that may lead to an imbalance of versican protein isoforms. The observation that retinal morphology was not altered in hdf+/- mice suggests that the abnormal ERG response may be caused by cellular signalling defects rather than cell death. Furthermore, the intact ciliary body suggests that the heterozygous null allele of Vcan does not exert a major structural function with respect to the ciliary body, unlike the Col2a1 null gene.
Taken together, a functional similarity of the hdf+/- mouse to the human Wagner disease has been demonstrated. However, further experiments are needed to establish the hdf-mouse strain as a valid animal model for this hereditary vitreoretinal degeneration.
The hdf+/- mouse strain was generated at the Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, USA. The animals were bred to yield a segregating, viable population of heterozygous and wild type siblings. Mice were kept at the Institute of Pathology, University of Zurich.
At age 5.5 months, animals were transferred to the Department of Ophthalmology, Charité Universitätsmedizin, Berlin, Germany, where Ganzfeld ERG (scotopic and photopic electroretinogram) measurements were performed as described at age 5.5 months, 12 months and 17 months. After the last time point, 12 animals (6 wild type and 6 hdf+/- mice) were sacrificed and eyes were removed for morphological analysis at the Institute of Medical and Molecular Genetics, University of Zurich.
Eyes were fixed and embedded in paraffin, and 2–3 µm sections were cut and were stained with haematoxylin-eosin as described. Analysis was performed using Zeiss Axioplan2 imaging light microscopy. Sections were evaluated at the ciliary body and also at the central and peripheral retina. Thickness (µm) of the entire retina and ONL (outer nuclear layer) as well as the nuclei in the ONL was measured (Fig. 1A). To correct for normal variation in eye sizes, statistical analysis was done after calculating the ratio of retinal thickness and OLN thickness and the ratio of ONL thickness and cell number in the ONL. For each of the 6 animals, (2 wild types and 4 hdf+/- mutants) 3 slides were evaluated to yield a total of 15 measurements per animal. For statistical evaluation GraphPad v5 was used, first to assess normal distribution of the data using D’Agostino & Pearson test and second to assess significance using one-way ANOVA with Bartlett’s test.
ProRetina Deutschland e.V. to KR.
We would like to thank Susanne Hubman for excellent technical work on retinal histology.
Animals were kept in compliance with the National Institute of Health guidelines as approved by the Swiss Cantonal Veterinary Office.