Mutations in PRPH2 are a relatively common cause of sight-robbing inherited retinal degenerations (IRDs). Peripherin-2 (PRPH2) is a photoreceptor-specific tetraspanin protein that structures the disk rim membranes of rod and cone outer segment (OS) organelles, and is required for OS morphogenesis. PRPH2 is noteworthy for its broad spectrum of disease phenotypes; both inter- and intra-familial heterogeneity have been widely observed and this variability in disease expression and penetrance confounds efforts to understand genotype-phenotype correlations and pathophysiology. Here we report the generation and initial characterization of a gene-edited animal model for PRPH2 disease associated with a nonsense mutation (c.1095:C>A, p.Y285X), which is predicted to truncate the peripherin-2 C-terminal domain. Young (P21) Prph2Y285X/WT mice developed near-normal photoreceptor numbers; however, OS membrane architecture was disrupted, OS protein levels were reduced, and in vivo and ex vivo electroretinography (ERG) analyses found that rod and cone photoreceptor function were each severely reduced. Interestingly, ERG studies also revealed that rod-mediated downstream signaling (b-waves) were functionally compensated in the young animals. This resiliency in retinal function was retained at P90, by which time substantial IRD-related photoreceptor loss had occurred. Altogether, the current studies validate a new mouse model for investigating PRPH2 disease pathophysiology, and demonstrate that rod and cone photoreceptor function and structure are each directly and substantially impaired by the Y285X mutation. They also reveal that Prph2 mutations can induce a functional compensation that resembles homeostatic plasticity, which can stabilize rod-derived signaling, and potentially dampen retinal dysfunction during some PRPH2-associated IRDs.

Mutations in the photoreceptor-specific tetraspanin gene peripherin-2 (PRPH2) lead to widely varying forms of retinal degeneration ranging from retinitis pigmentosa to macular dystrophy. Both inter- and intra-familial phenotypic heterogeneity has led to much interest in uncovering the complex pathogenic mechanisms of PRPH2-associated disease. Majority of disease-causing mutations in PRPH2 reside in the second intradiscal loop, wherein seven cysteines control protein folding and oligomerization. Here, we utilize knockin models to evaluate the role of three D2 loop cysteine mutants (Y141C, C213Y and C150S), alone or in combination. We elucidated how these mutations affect PRPH2 properties, including oligomerization and subcellular localization, and contribute to disease processes. Results from our structural, functional and molecular studies revealed that, in contrast to our understanding from prior investigations, rods are highly affected by PRPH2 mutations interfering with oligomerization and not merely by the haploinsufficiency associated with these mutations. On the other hand, cones are less affected by the toxicity of the mutant protein and significantly reduced protein levels, suggesting that knockdown therapeutic strategies may sustain cone functionality for a longer period. This observation provides useful data to guide and simplify the current development of effective therapeutic approaches for PRPH2-associated diseases that combine knockdown with high levels of gene supplementation needed to generate prolonged rod improvement.

The light-sensitive outer segment organelle of photoreceptor cells contains a stack of hundreds of flat, disc-shaped membranes called discs. The rims of these discs contain a photoreceptor-specific tetraspanin protein peripherin-2 (also known as rds or PRPH2). Mutations in the PRPH2 gene lead to a wide variety of inherited retinal degenerations in humans. The vast majority of these mutations occur within a large, intradiscal loop of peripherin-2, known as the D2 loop. The D2 loop mediates well-established intermolecular interactions of peripherin-2 molecules among themselves and a homologous protein ROM1. These interactions lead to the formation of large, highly ordered oligomers. In this chapter, we discuss the supramolecular organization of peripherin-2/ROM1 complexes and their contribution to the process of outer segment disc morphogenesis and enclosure.

A broad spectrum of autosomal-dominant inherited retinal diseases (IRDs), ranging from mild macular pattern dystrophy to severe cone-rod degeneration, is associated with PRPH2 variants (peripherinopathies). We present detailed clinical and molecular characterization of patients affected by peripherinopathies, aiming to expand the mutational spectrum, and propose novel genotype-phenotype correlations.
Observational, retrospective case series.
Patients with an IRD related to a molecularly proven PRPH2 variant.
Data from ophthalmic examinations and retinal imaging were collected for each follow-up visit. The standard imaging protocol included OCT, blue-light autofluorescence, near-infrared autofluorescence, and ultra-widefield fundus imaging. Genetic analysis was performed with a genomic approach by next-generation sequencing.
Results of ophthalmic examination, retinal imaging, and molecular genetic analysis.
Overall, a total of 19 patients with an IRD and a (likely) pathogenic PRPH2 variant were identified. Their age at presentation had a median of 48 years, whereas the symptomatic disease onset was in their 30s or 40s in 74% of cases. The median follow-up time was 4 years. Clinically, 6 patients were diagnosed with cone-rod dystrophy and 13 with pattern dystrophy. Among the 13 PRPH2 pathogenic variants identified in our cohort, 7 were missense, 3 nonsense, 2 frame shifting, and 1 splice site. Missense variants in the D2 loop were associated with cone-rod dystrophies and poor visual prognosis, whereas predicted loss-of-function alleles with pattern dystrophies and retention of a good visual function into adulthood. Overall, the following 7 variants were novel and never associated to a clinical phenotype: c.68delT, c.290G>A, c.413T>G, c.642C>G, c.702_706dupCAGTT, c.771_772delinsGA, and c.850C>G.
Here, we report the findings of a retrospective case series that provided a detailed clinical and molecular characterization of 19 patients harboring 13 different PRPH2 pathogenic variants, 7 of which were previously unreported, expanding the mutational spectrum of the PRPH2 gene. Loss-of-function variants might be preferentially associated with mild-pattern dystrophies, whereas missense dominant-negative variants might be preferentially associated with severely blinding cone-rod degenerations. Further studies are needed to better define the pathogenetic mechanisms and the functional effects of most variants to allow the development of successful gene therapy.
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The photoreceptor-specific glycoprotein retinal degeneration slow (RDS, also called PRPH2) is necessary for the formation of rod and cone outer segments. Mutations in RDS cause rod and cone-dominant retinal disease, and it is well established that both cell types have different requirements for RDS. However, the molecular mechanisms for this difference remain unclear. Although RDS glycosylation is highly conserved, previous studies have revealed no apparent function for the glycan in rods. In light of the highly conserved nature of RDS glycosylation, we hypothesized that it is important for RDS function in cones and could underlie part of the differential requirement for RDS in the two photoreceptor subtypes. We generated a knockin mouse expressing RDS without the N-glycosylation site (N229S). Normal levels of RDS and the unglycosylated RDS binding partner rod outer segment membrane protein 1 (ROM-1) were found in N229S retinas. However, cone electroretinogram responses were decreased by 40% at 6 months of age. Because cones make up only 3-5% of photoreceptors in the wild-type background, N229S mice were crossed into the nrl(-/-) background (in which all rods are converted to cone-like cells) for biochemical analysis. In N229S/nrl(-/-) retinas, RDS and ROM-1 levels were decreased by ~60% each. These data suggest that glycosylation of RDS is required for RDS function or stability in cones, a difference that may be due to extracellular versus intradiscal localization of the RDS glycan in cones versus rods.

Intrinsically disordered proteins contain some residual structures, which may fold further upon binding to the partner protein for function. The residual structures observed in two intrinsically disordered proteins, including the C-terminal segment of peripherin-2 (63 residues) and measles virus nucleocapsid protein Ntail (125 residues), were compared using NMR. Differences in the chemical shifts of alpha-, beta- and carbonyl carbons between the observed structure and calculated random coil revealed the existence of a helix and some possible beta-structures in both proteins. The intensity of signals in the C-terminal segment of peripherin-2 in NMR spectra was informative and locally low, particularly in the middle and N-terminal parts: this suggested the broadening of the signals caused by the formation of residual structures in those areas. Furthermore, the protection of exchange of amide protons was significantly observed at the N-terminus. Conversely, the intensities of signals for Ntail were random beyond the overall areas of protein, and indicated no characteristic pattern. Only a faint protection of amide-proton exchange in Ntail was observed in the C-terminus. It was concluded that Ntail was more intrinsically disordered than the C-terminal segment of peripherin-2. The combination of chemical shifts with the amide-proton exchanges and signal intensities was useful for the analyses of the remaining secondary structures. The beta-structure might be more detectable by the protection of amide-proton exchange than the helical structure, although the changes in chemical shifts were sensitive for the detection of elements of both secondary structures.