Novel non-sense mutation in RP2 (c.843_844insT/p.Arg282fs) is associated with a severe phenotype of retinitis pigmentosa without evidence of primary retinal pigment epithelium involvement

Abstract

Retinitis pigmentosa (RP) relates to a heterogeneous group of rod-cone dystrophies of varying genetic aetiology. There is currently great interest in gene replacement therapy. Phenotyping is of particular importance because some RP genes are expressed ubiquitously and it is critically important to understand which retinal layer is primarily affected. RP2 is increasingly diagnosed in patients suffering from X-linked RP, which causes outer retinal degeneration. We present a case of a previously unreported null mutation in RP2 associated with severe RP. Loss of the retinal pigment epithelium (RPE) was noted in the central macula but not around the disc or peripherally. There was therefore no evidence of independent degeneration of the RPE. Hence despite expression in all retinal cells, RP2 deficiency does not appear to be pathogenic to the RPE. This observation may be helpful in considering the promoter and route of delivery of adeno-associated viral gene therapy vectors encoding RP2.

Background

Retinitis pigmentosa (RP) may relate to any one of a number of rod-cone dystrophies of varying genetic aetiology.¹ With a prevalence of 1:4000, it is among the most common causes of inherited blindness in humans and has recently overtaken diabetic retinopathy as the most common cause of blindness in the working age population.² Of the many causative genes involved in RP, 10%–20% of all cases are X-linked recessive.³ There are at least three genes on the X chromosome known to be mutated in RP.³ The two most commonly implicated are RP3, encoding retinitis pigmentosa GTPase regulator and RP2, coding for the RP2 protein.⁴ More rarely, mutations in OFD1 which causes a syndromic ciliopathy have recently been reported.⁵

Previous mutations have been described largely in exon 2 and to a lesser extent in exon 1 of RP2, while deletions of exons 4 and 5 have also been reported.⁶ A previous report of a null mutation in exon 3 showed an unusual phenotype⁷ with focal areas of atrophy in the retinal pigment epithelium (RPE) in addition to photoreceptor loss. Since RP2 is expressed in all retinal layers,⁸ we were particularly interested to see if this observation could be confirmed by more advanced loss of the RPE late in the disease.

Case presentation

A 54-year-old man with a history of RP was referred to our centre. He gave a medical history of rapid loss of visual acuity during childhood. His visual acuity at 9 years of age was 6/12, declining to 6/24 right eye and 6/36 left eye by age of 10 years. When he reached 18 years, his best corrected visual acuity had reduced to 6/60 in both eyes. He also had high myopia as a child, with a refraction of −8.50/–1.00 dioptre (D) x 45,–8.50/−1.00 D x 135 at the age of 6 years. By the age of 28 years, his visual acuity was Hand Movement, 1/60, and by the age of 42 years, he had perception of light only in both eyes. At his first visit to our centre, his visual acuity remained localisation of light only in both eyes.

He reported no known family history of RP although his maternal grandfather was known to have very poor vision from the age of 20 years. He had two female siblings with no inherited ocular pathology, each of whom had a son and a daughter, also with no known inherited ocular pathology (figure 1).

Figure 1.

Pedigree of the affected individual’s family. There are no other confirmed cases of retinitis pigmentosa within the family, however maternal grandfather (I:1) is reported to have had poor sight from an early age although he was not examined.

Clinical examination revealed pendular nystagmus, bilateral pseudophakia and normal intraocular pressures. There was atrophy and ‘bone spicule’ pigmentary changes of the retina bilaterally and the optic discs were pale.

Spectral domain optical coherence tomography revealed significant atrophy of the photoreceptor layer including at the fovea and also of the nerve fibre layer extending to the disc (figure 2). Autofluorescence photographs showed central thinning of the RPE and unmasking of the underlying scleral reflectivity, but the RPE appeared well preserved around the optic nerve head and anterior to the vascular arcades (figure 3).

Figure 2.

(A, B) Optical coherence tomography through the central macula/fovea of the right (A) and left (B) eyes showing the end-stage nature of the condition with complete loss of the photoreceptor outer nuclear layer (arrows). (C) Autofluorescence image showing the fundus of the right and left eyes. There is some mild central atrophy but the retinal pigment epithelium around the optic discs (solid arrow heads) and anterior to the vascular arcades (open arrow heads) appears to be intact, even at this very late stage of the disease.

Figure 3.

Schematic diagram represents on the top, the RP2 genomic sequence and on the bottom, how this relates to the RP2 protein amino acid sequence. TOP: the exon sequence is represented by black boxes and intronic sequence is represented by lines. Introns 1 and 3 contain dashed lines as their sequence is greater than 10 000 base pairs. The dashed lines highlight the borders of the exons. BOTTOM: The orange portion of the amino acid sequence represents the hydrophilic N terminus, the blue represents the cofactor C homologous portion and the green highlights the c-terminus domain. Within this domain and the third exon is the mutation identified in this case c. 843_844insT (p.Arg282fs).

Electrophysiological testing might have been helpful in the early stages of disease, however, we have moved away from routine electrophysiological testing and only perform this if the genetic testing is equivocal. We however now refer to electrooculography as a possible test in cases where RPE involvement is suspected in early disease stages.

Investigations

Next generation sequencing was undertaken to identify the mutation responsible. Rather than carrying out whole exome or whole genome sequencing, the first step in identifying a genetic mutation is often sequencing a relatively smaller number of genes, which are already known to cause a particular disease. The patient’s blood was sent for analysis using a customised system (HaloPlex enrichment system kit (Agilent technologies)) to capture the coding regions of 55 retinal genes common genes associated with rod-cone dystrophy, including RP2. Libraries were then pooled and sequenced using next generation sequencing on an Illumina MiSeq instrument (Illumina) using a MiSeq v2 kit as per manufacturer’s instructions. Reads were aligned using Burrows-Wheeler Aligner software and variants called using platypus.^{9 10}

A putative pathogenic variant was discovered within the third exon of the RP2 gene (on the X chromosome at Xp11.3), which was confirmed with Sanger sequencing. Analysis of the mutation found him to be hemizygous for an insertion of a single thymine nucleotide at c. 843_844, corresponding to arginine 282 and a frameshift (p.Arg282fs6X)—a predicted null mutation.

Although there were no other affected family members available for segregation analysis, such a significant null mutation in RP2 is highly likely to be the causative mutation, as described further below.

Outcome and follow-up

The patient continues to be monitored in clinic, however without a treatment option, vision has continued to deteriorate to perception of light in both eyes.

Discussion

We present a case of a previously unreported frameshift mutation in exon 3 of the RP2 gene as a putative cause of X-linked RP. In contrast to a previous report, the null mutation we observed did not have an obvious RPE phenotype even in the very late stages of the disease. In time, loss of the retinal pigment cells leads to atrophy of the underlying choroid and exposure of the underlying sclera, as seen in choroideremia¹¹ and dominantly inherited RPE65 mutations.¹² This does not appear to be a feature seen in RP2 mutations, at least in this case.

Mutation of the RP2 gene was first identified as a cause of X-linked RP in 1998 and is implicated in 10% of familial X-linked RP.¹³ The RP2 gene is found at Xp11.3 and encodes 5 exons of 1050 base pairs resulting in a 350 amino acid membrane localising protein.¹⁴ The relatively small size of the cDNA makes RP2 encodable with adeno-associated viral (AAV) vectors and hence there is great interest in potentially treating patients with RP2 mutations through a clinical trial. Successful rescue of photoreceptors by gene therapy has recently been demonstrated in an RP2 knockout mouse model of cone-rod dystrophy using an AAV 8 vector.¹⁵

The RP2 protein is hypothesised to be involved in trafficking membrane bound proteins from the inner segment to the outer segment of photoreceptors.¹⁶ It has been found to localise to the plasma membrane of both rod and cone photoreceptors.⁸ On a molecular level, RP2 functions a GTPase activating protein for the GTPase ADP-ribosylation factor-like 3, which forms part of the putative molecular machinery in accomplishing this task.¹⁷ Although the RP2 protein is ubiquitously expressed, mutations typically affect only the photoreceptors.¹⁸ This is likely to be because photoreceptors are among the most metabolically active cells in the body and require continuous trafficking of proteins from the inner segment (where protein synthesis occurs) to the outer segment (where phototransduction occurs).¹⁹

It is notable that two-thirds of all RP2 mutations that result in RP are caused by premature termination of translation due to frame shift or introduction of a premature stop codon.¹⁴ Hence, we propose that the RP2 mutation identified is the cause of RP in this patient. In this case, the mechanism of non-sense-mediated decay would most likely result in no RP2 protein being translated. In cases where premature stop codons are in the terminal exon, truncation of the c-terminus of RP2 results in a misfolded and non-functional protein. Rather than localising to the plasma membrane as is the case for the wild type protein, it localises to the cytoplasm and is susceptible to enhanced lysosomal degradation.^{14 20}

We hypothesise that delivery of the RP2 gene to the subretinal space of human patients with early stage RP secondary to RP2 mutations might be an effective disease modifying treatment. Although a ubiquitous promoter would be a safe choice, there is no evidence yet that the RPE is the primary driver of the RP2 disease. Hence, AAV vectors should be designed with photoreceptor (including cone) targeting properties.

Learning points.

• RP2 is a ubiquitously expressed gene and has a small coding sequence (1 kb) causing a severe, X-linked phenotype of retinitis pigmentosa (RP).

• We present a novel frameshift mutation of RP2 with no evidence of independent degeneration of the RPE.

• Therefore, the development of gene therapy using an adeno-associated viral vector may be an effective modifying treatment for patients with early stage RP secondary to RP2 mutations.