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.