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Dominant, Gain-of-Function Mutant Produced by Truncation of RPGR

From the Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts.

	Abstract

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PURPOSE. The retinitis pigmentosa GTPase regulator (RPGR) is essential in the maintenance of photoreceptor viability. Mutations in the X-linked RPGR gene have generally been assumed to be recessive. This study was undertaken to investigate whether certain mutant RPGR alleles may act dominantly.

METHODS. An RPGR transgene representing the RPGR ORF15 variant was placed under a non–tissue-specific promoter and introduced into transgenic mice. The transgene was crossed into both a wild type (WT) and an RPGR null background. Its expression was analyzed by RT-PCR, immunoblot analysis, and immunofluorescence. Photoreceptor survival was assessed by electroretinography and histology.

RESULTS. The RPGR transgene transcript underwent photoreceptor-specific, alternative splicing involving the purine-rich region of the ORF15 exon, generating a shortened mRNA and a premature stop codon. This truncation mutant caused more rapid photoreceptor degeneration than that in the RPGR null (knockout) mutant. The disease course was similar, whether the transgene was coexpressed with WT RPGR or expressed alone in the RPGR null background.

CONCLUSIONS. Certain truncated forms of RPGR can behave as a dominant, gain-of-function mutant. These data suggest that human RPGR mutations are not necessarily null and some may also act as dominant alleles, leading to a more severe phenotype than a null mutant.

The in vivo function of RPGR is not fully understood. RPGR is localized in rod and cone photoreceptor connecting cilia,^{6 12} which link the biosynthetic inner segments and the light-sensing outer segments. RPGR localizes to the connecting cilia through binding, via its N-terminal domain, to an RPGR-interacting protein (RPGRIP).^{13 14 15 16} RPGRIP itself is essential for photoreceptor function, as loss of RPGRIP causes Leber congenital amaurosis^{17 18} in humans and a severe form of photoreceptor degeneration in RPGRIP knockout mice.¹⁶

RPGR presents an unusual challenge to cell biological and biochemical studies. One difficulty lies in the complexity of RPGR transcript splicing. Earlier studies identified a major transcript consisting of 19 exons, known as the constitutive or default transcript, expressed in multiple tissues.^{1 19 20} A retina-enriched transcript, referred to as the ORF15 transcript, shares the same exons 1 through 13, but utilizes exon 14 through part of intron 15 as a large terminal exon (ORF15 exon).² The ORF15 exon has a highly repetitive, purine-rich internal region. Additional alternative splicing is found in both the constitutive and the ORF15 transcripts.^{19 20 21} In mice, the repetitive purine-rich region in the ORF15 exon appears to act as a splicing enhancer and promote alternative splicing leading to partial removal of the repetitive sequence.²¹ The physiological significance of this complex splicing remains unclear. In human genetic studies, a large number of disease-causing mutations have been identified in the ORF15 exon, but none has been found in those exons specific for the constitutive transcript. Furthermore, at the protein level, the ORF15 variant is found only in photoreceptors, whereas the constitutive variant is expressed at higher levels outside of photoreceptors.^{12 21} These observations suggest that the ORF15 variant may be the functionally significant isoform in photoreceptors.

Disease-causing mutations in the ORF15 exon cause a shift of the reading frame, whereas in-frame deletions and insertions are thought to be nonpathogenic. The repetitive purine-rich region codes for alternating glycine and glutamic acid residues that are several hundred residues in length. Such a sequence is not expected to fold into a compact structure but probably exists as an extended "linker" connecting the globular N- and C-terminal domains. The C-terminal sequence is highly conserved among vertebrate species and is abolished by frame-shift mutations, suggesting that the C-terminal tail is necessary for function.² In contrast, the length of the linker region does not appear to be under a rigid functional restraint. Indeed, the lengths of this region vary considerably among species² and even among different strains of mice.

RPGR mutations are generally considered loss-of-function alleles. Although phenotype variations have been noted, X-linkage of the RPGR gene dictates that only a wild-type (WT) or a mutant allele is expressed in any given photoreceptor because of random X-inactivation. This precludes a definitive determination by clinical studies as to whether dominance or variable residual function may underlie allelic differences. In this study, we used the transgenic approach to demonstrate that a truncated RPGR acts as a dominant gain-of-function mutant, causing rapid photoreceptor loss, regardless of whether normal RPGR is present.

	Experimental Procedures

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Transgenic Mice
A cDNA representing the mouse RPGR ORF15 variant was obtained by reverse transcription and PCR (RT-PCR) and has been described elsewhere (clone T12).²¹ Clone T12 was among the larger cDNAs that could be amplified from the C57BL mouse retina. It is shortened by 654 bp in the purine-rich region of the ORF15 exon, compared with the published sequence determined from the 129/Sv mouse strain.² We have found that the genomic purine-rich region of the C57BL/6 strain is shorter by approximately 90 bp than the 129/Sv strain. Hence, approximately 534 bp of the purine-rich sequence appears lost in clone T12 cDNA from the corresponding genomic region of the C57BL/6 mice. Clone T12 cDNA was subcloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA) between the cytomegalovirus (CMV) enhancer-promoter and a bovine growth hormone (BGH) polyadenylation sequence. The transgene construct was used to generate transgenic mice by pronuclear injections. Founder animals were crossed into either a WT C57BL/6 background or into an RPGR knockout background.⁶ Homozygosity of the transgene was first screened by semiquantitative PCR, and candidate homozygous animals were mated with C57BL/6 mice. They were considered homozygotes if 100% of their offspring was transgene positive in two litters or more. Histology and electroretinography were performed as previously described.²² All data were collected on three animals or more (one eye from each animal) for each time point. All procedures involving animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Reverse Transcription–Polymerase Chain Reaction
RT-PCR was performed as previously described.²¹ Photoreceptor-enriched RNA was harvested by gently shaking dissected retinas in phosphate-buffered saline (PBS) and taking the supernatant, which contained RNA primarily from the broken photoreceptor inner segments. T7 (5'-ACGACTCACTATAGGGAGACCCAAGC) and ORF11R (5'-GGACTCCATTGGCATTTTAGACGGC) primers were used to amplify the RPGR ORF15 transgene transcript. R50 (5'-CGAGTCGCCCCTGCTATCTTTCCGA) and D9R (5'-CCAGTTGACCTTCATTATTTCCACCAGCTGC) primers were used to amplify an N-terminal region, shared by the endogenous WT RPGR and the transgene transcripts. GPDHP1 (5'-TGAAGGTCGGTGTGAACGGATTTGGC) and GPDHP2R (5'-CATGTAGGCCATGAGGTCCACCAC) primers were used to amplify the mouse GPDH cDNAs as a control.

Antibodies, Immunoblot Analysis, and Immunofluorescence
Anti-RPGR S1 and NT antibodies, used for the detection of RPGR proteins, have been described.¹² For immunoblot analysis, an axoneme-enriched preparation from retinas, which also enriches RPGR, was prepared as described.²¹ Immunofluorescence staining was performed as described.¹²

Transient Transfection
Transfection of COS-7 cells was performed using transfection reagent (Geneshuttle 40; Quantum Biotechnologies, Surrey, British Columbia, Canada) according to the manufacturer’s instructions. cDNA fragments encoding RPGR variants and mutants were inserted into a vector under the control of a CMV enhancer/ß-actin hybrid (CBA) promoter.²⁴ The CBA promoter contained an artificial intron and in our pilot studies CBA promoter-expression constructs appeared to reduce aberrant splicing compared with the conventional CMV promoter, allowing more faithful expression of the ORF15 sequence. The following variants-mutants were studied: RPGR constitutive variant, RPGR ORF15 clone T12, the 2.9-kb truncation mutant, and the 1.5-kb truncation mutant. At 48 hours after transfection, cells were washed with PBS, fixed in 4% paraformaldehyde/PBS, blocked in 5% goat serum and PBS, and incubated with primary antibodies overnight at 4°C. After they were washed, cells were incubated with the secondary antibody conjugated to Alexa 488 (Molecular Probes, Eugene, OR) for 1 hour at room temperature. Cell nuclei were counterstained with Hoechst dye 33342.

	Results

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We introduced into transgenic mice two expression constructs representing different RPGR splice variants, initially designed to explore which might be relevant for in vivo function. One transgene, representing an RPGR ORF15 variant, contained an in-frame ORF15 exon with a shortened purine-rich repetitive region (Fig. 1A) . Nine lines of transgenic mice carrying this construct were established and analyzed. Eight were found to express the transgene at very low levels detectable only by RT-PCR, but one line expressed sufficient transgene product to allow detection by RT-PCR (Fig. 1B) , immunoblot analysis (Fig. 1D) , and immunofluorescence. The transgene protein accumulated to a much lower level than endogenous WT RPGR, as indicated by immunoblot and by immunofluorescence (data not show). The transgene protein migrated at approximately 140 kDa, smaller than the native RPGR ORF15 variant²¹ or the same transgene cDNA expressed in COS cells (clone T12; 200 kDa).²¹ By immunofluorescence, the transgene protein was found in the connecting cilium, the same location as the endogenous WT protein (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Expression of RPGR ORF15 transgene and aberrant splicing. (A) Schematic diagrams of the gene structure of murine RPGR (top; drawn from published sequences2 ) and RPGR ORF15 transgene construct (lower). Open boxes: exons. CMV, CMV enhancer-promotor; BGH polyA, BGH polyadenylation signal. Arrows: PCR primers. Shaded box: purine-rich region in the ORF15 exon. (B) RT-PCR analysis of retinal RNAs from WT, knockout (RPGR-), and Tg/knockout (Tg+;RPGR-) mice. PCR primers are shown at the top. Two smaller-than-expected transcripts were amplified (top). Primers R50 and D9R amplified both endogenous and transgenic RPGR transcripts to determine relative expression levels (middle). GPDH (glyceraldehyde phosphate dehydrogenase; bottom) was included (B, C) as an internal standard. (C) RT-PCR analysis indicates that the 2.9-kb splice variant is present only in the retina (Ret). Analysis of photoreceptor-enriched mRNA suggests the 2.9-kb variant is photoreceptor specific. Br, brain; Li, liver. (D) Immunoblot analysis found a transgene product migrating at 140 kDa. The NT antibody recognizes an N-terminal epitope, and the S1 antibody recognizes an epitope in exon 13 of RPGR. (E) Two alternatively spliced, out-of-frame mutant transcripts were found in the transgenic retina. Splicing in the 2.9-kb variant joined nucleotides 2579-3391 and terminated the open reading frame after 15 nonnative amino acid residues. Splice sites in the 2.9-kb variant conform to the canonical consensus, but nonconsensus sites23 appear to be used in the 1.5-kb variant. Nucleotide numbering is based on the published sequence.2

The transgenic mice showed photoreceptor degeneration (Fig. 2) but were otherwise indistinguishable from WT mice in general health and growth characteristics. The rate of degeneration was faster than that of mice without RPGR (RPGR knockout mice), suggesting the mutant confers an additional deleterious effect. We therefore performed genetic analyses to explore how the truncation mutant might interact with the WT RPGR. Breeding experiments indicated that the transgene was integrated into an autosome, making it possible to cross it into the RPGR knockout background. Three genotype combinations were generated and analyzed: mice hemizygous for the transgene in the WT (Tg⁺;RPGR⁺) background, mice hemizygous for the transgene in the RPGR null (Tg⁺;RPGR^-) background, and mice homozygous for the transgene in the RPGR null (Tg^+/+;RPGR^-) background.

View larger version (109K): [in this window] [in a new window]   FIGURE 2. Photoreceptor degeneration caused by the truncated RPGR mutant. (A) Light micrographs of retinal sections from WT, RPGR-, and Tg+;RPGR- mice at different ages showing more severe disease in mice expressing the transgene. (B) Increasing gene dosage by crossing the transgene to homozygosity (Tg+/+;RPGR-) further accelerated the disease course. (C) Dark-adapted (rod-dominant) ERGs recorded in mice 40 days of age showed reduction of both a- and b-wave amplitudes consistent with loss of photoreceptors (right). At this age, mice carrying a null RPGR mutation maintained normal ERG responses (left).

These results show that the truncation mutant produced a deleterious effect beyond what could be accounted for by the loss of RPGR function alone. Because this effect persisted whether on an RPGR null (Tg⁺;RPGR^-) or WT (Tg⁺;RPGR⁺) background, the truncated RPGR was best described as a gain-of-function rather than a dominant-negative mutant. The truncation mutant might be generally cytotoxic or it might mediate a photoreceptor-specific cell death. To distinguish between these possibilities we expressed this transgene product in cultured cells. The truncated RPGR (2.9K) could be expressed transiently in COS cells (Fig. 3A) and stably in mouse embryonic stem cells (data not shown) without overt cytotoxicity, nor did we see aberrant accumulation of the mutant protein in the endoplasmic reticulum (ER). These observations suggest that its deleterious effect may be expressed only in a photoreceptor milieu.

View larger version (63K): [in this window] [in a new window]   FIGURE 3. (A) Immunofluorescence microscopy showing the distribution of transiently expressed RPGR variants. Top: COS7 cells were transfected with pCBA vector containing a constitutive and RPGR ORF15 variants and mutants and were stained with the RPGR NT antibody. The RPGR constitutive variant was centrally located next to the nuclei, consistent with a Golgi-enriched pattern, as has been reported.19 Note the cytoplasmic localization pattern of all three RPGR ORF15-derived variants and mutants. Bottom: nuclear dye counterstain. Arrowheads: location of transfected cell nuclei. RPGR ORF Tg, the 3.4 kb transgene as shown in Figure 1A ; Tg 2.9k, the 2.9-kb truncated RPGR mutant; Tg 1.5k, the 1.5-kb truncated RPGR mutant. (B). Electron micrographs of photoreceptor connecting cilia from WT and transgenic mice. Cross sections are shown.

	Discussion

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As a transgene representative of the RPGR ORF15 transcript, we chose a cDNA harboring an in-frame deletion within the repetitive purine-rich region. There were several rationales for analyzing this cDNA construct in transgenic mice. First, a cDNA containing the full-length ORF15 exon could not be obtained in mouse retinal RNA.²¹ Second, human genetic studies have indicated that short in-frame deletions in the repetitive region of the ORF15 exon has no adverse effect. Third, an RPGR cDNA containing the full-length ORF15 exon would be too large to be accommodated in commonly used gene therapy vectors for retinal cells. Thus, it would be interesting to see whether an RPGR cDNA with a larger in-frame deletion in the purine-rich region retained function.

Human RPGR mutations have generally been assumed to be loss-of-function alleles. Phenotype variations were attributed to different degrees of residual function. This study makes a conceptual advance by demonstrating that an RPGR mutant can lead to a gain of function. The transgenic approach was essential in making this determination. Because the RPGR gene is X-linked, a photoreceptor can express either a WT or a mutant protein, but not both. Therefore, clinical studies of genotype–phenotype correlation will not demonstrate definitively if a mutation leads to a severe phenotype, due to a gain-of-function effect or less residual function. The transgenic approach, with the transgene integrated into an autosome, is free of such constraint. Thus, we found that the truncation mutant expressed either alone or coexpressed with a WT RPGR caused a more severe form of photoreceptor disease than if RPGR was not expressed at all. The usual explanations of dominance, such as haploinsufficiency or a dominant–negative effect, clearly do not apply in this case. It thus appears that the truncated RPGR acts as a gain-of-function mutant. We could not formally rule out the possibility that insertional inactivation of a photoreceptor-essential gene may in fact underlie the photoreceptor phenotype. This possibility is exceedingly remote, considering that homozygosity of the transgene only moderately aggravates the disease. Had there been insertional inactivation, a gene dosage reduction by half in the heterozygotes would have become a complete ablation in the homozygotes. This would be expected to lead to a more drastic change in disease phenotype, pleiotropic involvement, and even lethality.

A second interesting observation of this work is the differential splicing of the purine-rich region of ORF15 exon in photoreceptors versus other cells. Our previous study of endogenous RPGR transcripts in mouse retinas suggests that the purine-rich region is partially removed by alternative splicing.²¹ In the present study, two smaller-than-expected transcripts were derived from the RPGR transgene, and the extent of deletion in the purine-rich region was tissue specific. The amplification of the smaller transcripts cannot be explained by a difficulty in reverse transcription through this region, but could be explained by a retina-specific alternative splicing. These observations support the notion that alternative splicing occurs in the purine-rich region of endogenous RPGR transcript.

Our conclusion that a truncated RPGR can act as a gain-of-function dominant mutant finds support in a recent study of canine RPGR mutations.⁷ Two disease-causing RPGR mutations, XLPRA1 and -2, were found in dogs. Both are frame-shift mutations within the ORF15 exon and are located less than 20 codons apart. The two mutants, however, lead to drastically different levels of disease severity. XLPRA1 produces a very mild phenotype, in that photoreceptor degeneration becomes apparent only after 13 months of age. XLPRA2 causes a much more severe phenotype, with loss of cells beginning at 1 month of age. Because the two mutants truncate the reading frame near each other and both have lost the highly conserved C-terminal domain, the degree of function loss should be comparable. Thus, the greater disease severity associated with XLPRA2 suggests a novel deleterious function. Indeed, XLPRA2 encodes 30 aberrant amino acid residues at its C terminus and accumulates in the endoplasmic reticulum on transfection into fibroblasts,⁷ implying a deleterious effect in addition to the loss of RPGR function. These observations suggest that XLPRA1 is a loss-of-function allele, whereas XLPRA2 acts as a dominant gain-of-function mutant. The truncated murine RPGR we have studied encodes 15 aberrant amino acids at its C terminus, although it does not accumulate abnormally in the ER. Because the endogenous RPGR expression level is low, sequestration in the ER may not be a strong pathogenic event in vivo. In contrast, the truncation mutant retains its N-terminal half and is thus able to localize in the connecting cilium. We therefore favor the hypothesis that a truncated RPGR may interfere with a function at the connecting cilium. This is a minute subcellular compartment with essential roles in protein transport and disc morphogenesis, and it may be especially sensitive to interference by an abnormal protein.

This study raises the possibility that a subset of human RPGR mutations may similarly act as dominant gain-of-function alleles. If dominant human RPGR mutations indeed exist and confer a more severe phenotype than a loss-of-function allele, it may explain an apparent discrepancy in disease severity between human and murine RPGR mutations. Humans with RPGR mutations on average have a severe form of RP, whereas RPGR knockout mice have mild disease. Clearly, truncated RPGRs are not necessarily dominant mutants, as illustrated by the XLPRA1 mutant in dogs.⁷ A greater number of aberrant amino acid residues with positive charges at the C terminus could be one determinant for dominance. More data are needed before rules can be developed that enable reliable predictions.

It is noted that the term "dominant" in association with RPGR mutations has also been used in a different context. Families with dominant RP with RPGR mutations have been described in which overt disease develops in females.²⁵ Female carriers are mosaics for photoreceptors expressing either the mutant or the WT allele. In these families the mutant alleles are perhaps more severe, leading to early demise of photoreceptors expressing the mutant alleles. Cellular interactions within the retina²⁶ may in turn cause degeneration of photoreceptors expressing the WT allele. In this study, our use of the term refers to true dominance as classically defined, confirmed by directly testing allelic interactions. Further studies into the mechanism underlying dominance of RPGR mutants may provide insights into RPGR function and the molecular interactions at the connecting cilium. They are also important for the design of gene-replacement and mechanism-based therapeutic approaches.

	Acknowledgements

 
The authors thank Eliot L. Berson for commenting on the manuscript.

	Footnotes

 
Supported by grants from National Eye Institute Grant EY10309 and the Foundation Fighting Blindness.

Submitted for publication July 24, 2003; revised August 28, 2003; accepted September 17, 2003.

Disclosure: D.-H. Hong, None; B.S. Pawlyk, None; M. Adamian, None; T. Li, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.

Corresponding author: Tiansen Li, Berman-Gund Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114; tli{at}meei.harvard.edu.

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