There is considerable clinical heterogeneity associated with retinal degenerative diseases due to mutations in ciliary proteins. Investigations on the impact of disease-causing mutations on the function of the causative protein have been difficult due to the lack of a platform to analyze protein function. In this study, we have utilized zebrafish development as a paradigm to elucidate the pathogenic potential of selected disease-associated mutations in RPGR. We show that RPGR mutations exhibit variable effects on its ability to rescue the rpgr-knockdown phenotype in zebrafish.
All patients carrying RPGR
mutations exhibit photoreceptor degeneration; however, we did not detect any defects in photoreceptor development or opsin trafficking in rpgr
-morphants by 4–5 dpf (Supplementary Material, Fig. S2
). These results likely suggest that RPGR is not essential for retinal differentiation and development. Notably, RPGR patients and animal models exhibit normal photoreceptor development but undergo relatively late-onset photoreceptor degeneration and blindness (15
). Hence, we cannot rule out an effect on photoreceptor survival in adult zebrafish, as also proposed with bbs
). A lack of retinal phenotype in rpgr
-knockdown zebrafish embryos is not unexpected. Because of the duplication of the zebrafish genome (51
), a functional redundancy of RPGR is highly likely. Tissue- and cell-type specific alternative isoforms of RPGR have also been reported in mice, bovine and human tissues. Hence, it is possible that the specific isoform of RPGR, investigated in the present study, may not play a role in retinal development. While this study was going on, we identified another potential RPGR isoform by in silico
analysis of the updated genome of zebrafish. Further investigations are necessary to delineate the function of this isoform in retinal development in zebrafish.
Our studies provide in vivo
evidence for the functional significance of the N-terminal domain of RPGR (encoded by exons 1–15) in ciliary functions (23
). The T99 residue is highly conserved during evolution as well as in the RCC1 protein (10
); hence, the T99N mutation may result in altered conformation, a detrimental effect on an as yet uncharacterized activity of RPGR, ciliary localization and/or interaction with other proteins. Truncation mutations in RPGR-RLD that are associated with a syndromic phenotype in patients exhibit an expected detrimental effect on protein function whereas the G173R mutation (associated with hearing loss, sinusitis and recurrent respiratory infections) (17
) seems to retain partial function in zebrafish development.
Mutations in exon ORF15 exhibit a hypomorphic effect in our assays. These data are consistent with previous observations that ORF15-variants in patients are associated with a relatively mild phenotype (12
). Moreover, the RPGR1-ORF15
variants encode the RPGR-RLD as part of exons 1–15, which are sufficient to rescue the rpgr
-MO phenotype. Intriguingly, animal models of exon ORF15 mutations exhibit discordant phenotype (slow or rapid photoreceptor degeneration and a dominant gain of function effect) based upon the length of the Glu–Gly repeats encoded by exon ORF15 (36
). We predict that relative dosage of the truncated ORF15-mutant proteins may alter the functional conformation of the RPGR protein, resulting in a mild versus severe phenotype in the cognate model system. Further studies are necessary to piece-together the complex association of mutations in exon ORF15 of RPGR with its function.
PCP proteins, which localize to cilia, control CE movements during axis elongation in vertebrates (44
). Our data suggest that RPGR is essential for vertebrate embryonic development and complete loss of RPGR function during development likely results in lethality. Support of this hypothesis also comes from previous observations that RPGR is expressed in mouse embryonic stem cells (unpublished data), and that attempts to generate a complete loss of function mouse mutant of Rpgr
have so far been unsuccessful. Moreover, the reported Rpgr
-ko mouse (35
) is not complete null because expression of alternative RPGR isoforms can be detected in the photoreceptors (23
). However, the precise function of RPGR during vertebrate development remains to be established.
Why do RPGR mutations not exhibit lethality in humans? We suggest that a majority of RPGR mutations detected in RP patients are hypomorphs with reduced function. This functional retention seems to be sufficient to rescue the developmental phenotype. Only selected mutations in RPGR may severely compromise the protein function. Given high protein trafficking demands due to periodic outer segment disc shedding and renewal (55
), even hypomorphic RPGR mutations have deleterious impact on the integrity of ciliary protein complexes and intracellular transport in photoreceptors. We predict that multiple isoforms of RPGR (generated by alternate splicing or with alternate promoter) may complement the detrimental effects of such mutations in extra-retinal tissues in higher vertebrates during development.
Although previous studies have reported a predominantly retinal phenotype associated with RPGR-T99N and RPGR-E589X mutations, we propose that detailed clinical analysis of patients predicted to have deleterious mutations based on the zebrafish assays reported here should be performed to analyze potential heterogenic phenotype(s), such as olfactory dysfunction, which can assist in early diagnosis of the disease (58
). We and others have shown that hypomorphic mutations in the ciliary protein CEP290 are associated with predominantly LCA and olfactory defects (58
) whereas predicted null mutations cause Joubert Syndrome, Bardet–Biedl Syndrome and Meckel–Gruber Syndrome (38
). Although olfactory dysfunction was not significantly observed in the RPGR patients tested (58
), additional studies using patients with other mutations should be performed for such analyses.
Taken together, our work analyzes the effect of RPGR mutations on its function using a zebrafish model system. This system offers a platform to understand the behavior of mutant RPGR in vivo so that future therapeutic strategies can be appropriately designed to target a complete or partial loss of function of the mutant RPGR protein in patients with ciliary disorders.