Impaired glutamylation of RPGR ^ORF15 underlies the cone-dominated phenotype associated with truncating distal ORF15 variants

Abstract

Pathogenic variants in the Retinitis pigmentosa GTPase regulator (RPGR) gene lead to a clinically severe form of X-linked retinal dystrophy. However, it remains unclear why some variants cause a predominant rod, while others result in a cone-dominated phenotype. Post-translational glutamylation of the photoreceptor-specific RPGR ORF15 isoform by the TTLL5 enzyme is essential for its optimal function in photoreceptors, and loss of TTLL5 leads to retinal dystrophy with a cone phenotype. Here we show that RPGR retinal disease, studied in a single cohort of 116 male patients, leads to a clear progressive shift from rod- to cone-dominating phenotype as the RPGR ORF15 variant location approaches the distal part of the Open Reading Frame 15 (ORF15) region. The rod photoreceptor involvement on the contrary diminishes along the RGPR sequence, and the variants associated with the cone only phenotype are located predominantly in the very distal part, including the C-terminal basic domain. Moreover, these distal truncating RPGR ORF15 variants disrupt the interaction with TTLL5 and lead to a significant impairment of RPGR glutamylation. Thus, consistent with the phenotype of TTLL5 pathogenic variants, our study shows that RPGR ORF15 variants, which disrupt its basic domain and the interaction with TTLL5, also impair RPGR glutamylation and lead to the cone phenotype. This has implications for ongoing gene therapy clinical trials where the application of RPGR with impaired glutamylation may be less effective in treating RGPR dystrophies and may even convert a rod–cone dystrophy into a cone dystrophy phenotype.

Identification of the RPGR Gene Pathogenic Variants in a Cohort of Polish Male Patients with Retinitis Pigmentosa Phenotype

“…Moreover, studies around the world on RPGR retinal diseases have shown marked genotypic and phenotypic heterogeneity. In a large study by Cehajic-Kapetanovic and colleagues [21] where they analyzed the data of a single cohort of 116 male patients from the genetic databases at two clinical centers (Oxford, United Kingdom, and Bonn, Germany), a total of 60 different pathogenic variants were identified in the ORF15 region of the RPGR gene. They found the commonest form of RPGR-related disease to be the rod-cone phenotype, also known as the "classic" X-linked RP phenotype, which was present in 56% of 60 patients (60/108) compared to 94% [22], 85% [23], or 70% [24] of the total number of patients observed in studies from Germany, China, and a European multicenter study, respectively.…”

Retinal Ciliopathies and Potential Gene Therapies: A Focus on Human iPSC-Derived Organoid Models

“…This alternative open reading frame, known as ORF15, comprises a highly repetitive region encoding a domain rich in glutamic acid-glycine (Glu-Gly) amino acid residues. Due to its repetitive nature, the terminal exon is prone to mutations, with the majority of reported RPGR mutations occurring in this region [ 61 , 62 ], although mutations throughout the entire RPGR ORF15 sequence can cause retinal disease [ 63 , 64 ]. Post-translational modification of the RPGR ORF15 isoform occurs through glutamylation within the Glu-Gly motifs of the ORF15 region.…”

“…Whether this directly affects cytoskeletal regulation by interfering with interactions involving RPGR and gelsolin, or if it constitutes a distinct disease mechanism, requires further investigation. The absence of glutamylation may give rise to a unique pathological condition, such as mutations resulting in distal truncations in the C-terminal domain, which is essential for TTLL5 interactions and leads to a cone-dominant phenotype—in contrast to the N-terminal mutations associated with a rod-dominant phenotype [ 64 ].…”

Retinitis pigmentosa GTPase regulator-related retinopathy and gene therapy

“…[ 36 , 37 ] Several studies identified that variants in exons 1–14 and at the 5’end of ORF15 are associated with rod-cone dystrophies, whereas variants located towards the 3‘end of ORF15 are more often associated with cone/cone-rod dystrophies. [ 3 , 38 39 40 41 42 ] However, conflicting reports of genotype–phenotype correlation exists. Some reported variants in exon 1–14 were associated with more severe disease phenotypes than ORF15 variants,[ 5 , 43 , 44 ] while others report the opposite.…”