3.2.3. Prph2Y141C/+ and Prph2Y141C/Y141C

Like the C213Y mutation, the Y141C mutation of Prph2 is also located in the D2 loop. The OSs in the Y141C knockin mouse model (*Prph2Y141C*/+ and *Prph2Y141C*/*Y141C* for heterozygous and homozygous animals, respectively) displayed structural anomalies. In *Prph2Y141C*/+ mice, the OS was shortened

with some structural alterations of the discs, including lengthening and vesicular structures [82]. When compared to the *Prph2*+/− mice, the structure was better conserved in the *Prph2Y141C*/+ retina (Figure 1B). In the *Prph2Y141C*/*Y141C* mice, OS formation is initialized but neither mature discs nor lamellae could be observed. Instead, the OS contained flattened whorl shaped membranous structures with vesicular arrangements lining up adjacent to them. Scotopic ERG revealed a significant decrease in rod function displayed by the *Prph2Y141C*/+ mice at P30 (Figure 2). Interestingly, the scotopic ERG did not deteriorate further at P180 [82].

Non-reducing SDS-PAGE/immunoblots of *Prph2Y141C*/+ retinal lysates showed abnormal high molecular weight aggregates at 250kDA. This was further evaluated by sucrose gradient velocity sedimentation experiments which revealed the formation of the expected intermediate and higher order complexes with an aberrant high molecular weight band indicating that the Y141C mutation results in the formation of abnormal large aggregates [82]. In the *Prph2Y141C*/*Y141C* retinas, the observed phenotype was even more pronounced with an increase in the aberrant high molecular weight aggregates at the expense of the formation of the normal intermediate and higher order complexes. While in WT mice Rom1 is normally excluded from higher order complexes, in the mutant retina, a portion of Rom1 was incorporated in the aberrant high molecular weight aggregates. Surprisingly though, mutant Prph2 was transported correctly to the OS which is shown by IF staining of Prph2 (green) and IS marker STX3B (red) (Figure 3). However, the formation of these high molecular weight aggregates likely interfered with the normal function of Prph2 and likely Rom1 in the OS. This o ffers an explanation for the structural and functional phenotypes observed in the knockin mice.

Patients carrying the Y141C mutation in *PRPH2* display primarily defects in the macula with some reported cases of more rod-associated phenotypes such as night blindness and RP [30,82,83]. The *Prph2Y141C*/+ mice, which represent patients' genotype, exhibit a decline in rods' function, mimicking the observed rod phenotype in some patients. Again, the di fference between the knockin model and the patient's phenotypes can be seen in the cone function. The most prominent phenotype found in patients with the Y141C mutation is the functional and structural decline in cones. The *Prph2Y141C*/+ mice on the other hand displayed only a slight functional decline in the photopic ERG which was not found to be significant [82], indicating that the cone function in these mice is not severely a ffected. While the heterozygous mice do not show significant decline in cone function, they do display a flecking in funduscopic analyses [82], which mimics findings in the fundus of patients. Recently, a study was undertaken to identify a potential explanation for the huge variation in phenotypes observed in patients by determining the role Rom1 plays in the observed phenotype [88]. Mice heterozygous or homozygous for the Y141C mutation were crossed with Rom1 knockout mice to produce mice that express mutant Prph2 in absence of Rom1 (*Prph2Y141C*/+/*Rom1*−/− and *Prph2Y141C*/*Y141C*/*Rom1*−/−). Absence of Rom1 abolished the formation of the abnormal high molecular weight aggregates and accumulation of mutant Prph2 in the IS and ONL in *Prph2Y141C*/*Y141C*/*Rom1*−/− retinas [88]. The depletion of Rom1 also changed the symptoms seen in the Y141C model. While the photopic ERG amplitudes in *Prph2Y141C*/+/*Rom1*−/− mice were comparable to that of the WT, a significant reduction in the scotopic ERG responses were observed [88].

When compared to the cone-rod functional defects noted for the *Prph2Y141C*/+ mice, *Prph2Y141C*/+/*Rom1*−/− mice mainly displayed a rod-dominant functional defect. In addition, the funduscopic anomalies were almost completely abolished in the *Prph2Y141C*/+/*Rom1*−/− mice. The results obtained in this study prove that alteration in the level of Rom1 can change the phenotype caused by a pathogenic *Prph2* mutation, and thus potentially provide an explanation to the variable phenotypes seen in patients carrying the same *PRPH2* mutation. Further studies combining *Prph2* mutations and mutations in *Rom1* should provide further insights into the complexity of phenotypes seen in the various *PRPH2* related diseases.
