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1From the Division of Molecular Genetics, Institute of Ophthalmology, University College London, London, United Kingdom; the 3Moorfields Eye Hospital, London, United Kingdom; and the 4Academic Department of Surgery, St. Bartholomews and the Queen Mary’s School of Medicine and Dentistry, University of London, London, United Kingdom.
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Abstract |
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METHODS. Real-time quantitative RT-PCR was performed on RNA from lymphoblastoid cell lines derived from a large adRP family (RP856/AD5) that segregates an 11bp deletion in exon 11 of PRPF31. The mRNA levels from only the wild-type allele of PRPF31 were assayed using a probe designed across the deletion. The Mann-Whitney U test was used to compare the median mRNA copy numbers of the symptomatic with the asymptomatic carriers of the mutant PRPF31 allele. The PRPF31 protein levels from symptomatic and asymptomatic individuals were also assayed by Western blot analysis using an antibody specific to the wild-type PRPF31 protein.
RESULTS. The use of cell lines was validated by the observation that cell transformation did not alter PRPF31 expression in the cell lines compared with nucleated blood cells and donor retinas. A significant difference in wild-type PRPF31 mRNA levels was observed between symptomatic and asymptomatic individuals (P < 0.001) and was supported by Western blot analysis of the PRPF31 protein.
CONCLUSIONS. Partial penetrance in RP11 could be due to the coinheritance of a PRPF31 gene defect and a low-expressed wild-type allele. This study revealed a potential avenue for future therapy in that it appears the moderate overexpression of wild-type PRPF31 may prevent clinical manifestation of the disease.
The recently identified gene for autosomal dominant RP on chromosome 19q13.4 is a pre-mRNA splicing factor known as PRPF31.2 The gene encodes a 61 kDa protein (PRPF31, also referred to as splicing factor 61K), which is integral to the U4/U6+U5 trimer.3 4 The mutations identified to date include missense substitutions, splice-site mutations, deletions, and insertions.
Interestingly PRPF31 is one of three pre-mRNA splicing factors identified as causing adRP. Two other pre-mRNA splicing factors have also been implicated in adRP: PRPF3 on chromosome 1p13-q21 (RP18)5 and PRPF8 on chromosome 17p13.3 (RP13).6 Proteins encoded by these genes are essential for splicing in all cell types, yet the pathologic effects of mutations in all three genes is seen only in rod photoreceptors. The possible explanations for this specificity include the sensitivity of the photoreceptors to splicing stress during its disc shedding and outer segment renewal, an event linked to a surge in transcription for all genes involved in phototransduction,7 8 and the functional consequence of the mutations themselves. Functional studies on missense mutations of PRPF319 and the presence of large deletions resulting in hemizygosity for PRPF31 indicate that the overall effect of mutations is the reduction in the level of functional protein in the nucleus. This may cause an insufficiency in splicing function, which is revealed only under conditions of splicing stress as encountered in rod photoreceptors due to outer segment renewal. The apparent lack of any effect in all other cell types suggests that the cellular level of protein from one wild-type allele is sufficient to meet the basal splicing demand within these cells thus resulting in normal cell function.
The unique feature associated with mutations in PRPF31 is the nonpenetrance for symptoms and retinal changes in some obligate carriers of the disease allele. Therefore, in the case of PRPF31 haplo-insufficiency within photoreceptors does not adequately explain the clinical manifestation of disease. However, the lack of symptoms in some disease gene carriers can be explained if there is a rod photoreceptor-specific threshold for PRPF31 and the level of wild-type PRPF31 protein is modulated. In fact, an allelic effect has been suggested as the possible mechanism for nonpenetrance of PRPF31 mutations.10 Sib-pair analysis has shown a statistically significant correlation between the inheritance of the wild-type allele from the noncarrier/normal parent and the presence of disease in carrier offspring. Analysis of single nucleotide polymorphisms (SNPs) within the PRPF31 gene in sib-ships from two RP11-linked families, one of which is the family (RP856/AD5) investigated in this study, also demonstrated that asymptomatic individuals consistently inherited a different wild-type allele to the one inherited by their symptomatic siblings.2 This suggests the existence of differentially expressed wild-type alleles that can potentially determine the penetrance of the disease symptoms depending on whether or not a photoreceptor-specific PRPF31 activity threshold is surmounted. However, this still does not preclude the existence of a closely linked modifier gene that could influence the penetrance of the disease phenotype.
To compare mRNA copy numbers of wild-type alleles of PRPF31 from symptomatic and asymptomatic individuals we used a real-time quantitative reverse transcriptase PCR (RT-qPCR) assay.11 This Taqman assay utilizes the 5' nuclease activity of the DNA polymerase to hydrolyze a specific hybridization probe bound to the target amplicon, which causes an increase in fluorescence of the probe and allows progress of the PCR reaction to be quantified. RT-qRT-PCR has been used extensively for comparison of gene expression in tumor cells of many different cancers.12 RT-qRT-PCR has also been used to determine the levels of mRNA in a given tissue, for example the levels of phosphodiesterase 
- and ß-subunit messenger RNAs in neonatal retinal degeneration mouse retinas.13
Our study was carried out on immortalized lymphoblastoid cell lines from an adRP family, RP856/AD5 in which an 11bp deletion in exon 11 of PRPF31 segregates with the disease. Logistics precluded rebleeding of the entire family to obtain RNA from peripheral lymphocytes. To test the validity of using RNA from lymphoblastoid cell lines, PRPF31 expression in nucleated blood cells from control individuals and donor retinas was compared with expression in lymphoblastoid cells showing no significant difference. The deletion mutation in AD5 allowed the selective quantification of only the mRNA from the wild-type allele of PRPF31, because the specific probe spanned the deletion. Also being the largest RP11-linked family to date, AD5 has the most number of sib-ships with individuals of the two contrasting phenotypes enabling a comparison of a larger number of individuals than in any other RP11-linked family.
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Materials and Methods |
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RNA Extraction from Cell Lines, Postmortem Tissue, and Blood
RNA was isolated from liquid cultures of 20 cell lines (5 noncarrier, 7 asymptomatic, and 8 symptomatic individuals). All cells were harvested at the exponential phase of growth (5 x 105-2 x 106 cells/mL), and 
30 µg of total RNA was isolated with an RNA extraction kit (QIAGEN RNeasy Midi kit; Qiagen Ltd, Crawley, UK) from 106–107 cells. RNA quantity was measured photometrically (BioPhotometer; Eppendorf AG, Hamburg, Germany) and aliquots were stored at -80°C at 100 ng/µL. Two RNA extractions were carried out from two independent cell passages for each cell line.
Retinal RNA was extracted by use of the following protocol. The donor eyes were obtained from the Eye Bank of British Columbia. Whole globes were placed in an RNA protection reagent (RNAlater; Ambion, Austin, TX) after enucleation and corneal excision and stored at 4°C. The average time from death to dissection was 8 hours. Globes soaked in RNAlater were cut into segments and the retinas detached from the RPE. Approximately 10-mm diameter sections of retina were removed with Rnase-free instruments and frozen in fresh RNAlater, or processed immediately. Total RNA was isolated with an RNA extraction kit (Rnaqueous-4PCR; Ambion) according to the manufacturer’s protocol, including DNase treatment. RNA was analyzed for quantity and quality by gel electrophoresis. Nine RNA samples of equal quantity, from four donors (aged 52 to 64 years) were pooled to minimize intersample variation in gene expression.
Total RNA was extracted (Qiagen RNeasy Midi kit; Qiagen Ltd.) from 5 to 10 mL of fresh blood from four control individuals of white English origin.
Primers and Probes
PRPF31 sequence specific primers (forward 5'-AAGATGAAGGAGCGGCTGG-3' and reverse 5'-CCTCCTGGTAGGCGTCCTC-3') and the hydrolysis probe (5'- CCGGAAGCAGGCCAACCGTATG-3') were designed with the use of a commercial software (Primer Express, ver. 1.5; Applied Biosysytems, Warrington, UK) and synthesized by MWG Biotech (Ebersberg, Germany). The Taqman probe was labeled with a reporter dye (6-carboxy-fluorescein, FAM) at the 5'end and a quencher dye (6-carboxy-tetramethylrhodamine, TAMRA) at the 3' end. The primers binding to exon 11 and 12 of PRPF31 generated an amplicon of 91 bp with the probe hybridizing to sequence in exon 11 encompassing the deletion in the mutant allele. Therefore, the probe only hybridized to target molecules amplified from the wild-type PRPF31 mRNA.
RT-PCR Reactions
The one-tube/one enzyme RT-PCR protocol was used for the 5' nuclease assay. The volume for each reaction was 25 µL with 500 ng of RNA. Each sample was analyzed in duplicate; because RNA was extracted from each cell line twice, this resulted in four analyses per cell line. To prevent carryover of contaminating amplified DNA, the reaction was carried out in the presence of dUTP. Before RT the RNA template was heated for 2 minutes at 50°C in the presence of the enzyme uracil N-glycosylase at 0.01 U/µL (AmpErase UNG; Applied Biosystems). After 30 minutes of RT at 60°C and 5 minutes of denaturation at 92°C, PCR was carried out for 40 cycles of 20 seconds at 92°C and 1 minute at 62°C in the presence of the labeled probe. After the target amplification, the probe annealed to the amplicon and was displaced and cleaved between the reporter and quencher dyes by the nucleolytic activity of the polymerase. The amount of product resulting in detectable fluorescence at any given cycle within the exponential phase of PCR is proportional to the initial number of template copies. The number of PCR cycles (the threshold cycle, CT) needed to detect the amplicon is therefore a direct measure of template concentration. The RT-PCR reactions were performed, recorded, and analyzed using a real-time thermocycler (ABI7700Prism Sequence Detection systems; Applied Biosystems).
Generation of Standard Curves
Quantitation of PRPF31 gene expression in lymphoblastoid cell lines was carried out by relating the PCR threshold cycle obtained from samples to a PRPF31 standard curve. A 110-bp single-stranded sense oligonucleotide specifying the PRPF31 amplicon was synthesized (MWG Biotech) and serially diluted from 1 x 109 molecules to 10 molecules and used in RT-PCR reactions. One microgram of a 110-bp ssDNA contains 1.7 x 1013 molecules. RT for each dilution was carried out three times in duplicate. The standard curve was obtained by plotting the log (calculated copy number) against the threshold cycle. The copy numbers (N) of unknown samples were calculated from the regression line according to the formula: log N = (CT –b)/m, where CT is the threshold cycle, b is the y intercept, and m is the slope of the standard line. PRPF31 expression level is presented as the mRNA copy number per microgram of total RNA.
Preparation of Soluble Whole-Cell Extracts from Cell Lines
Total cellular protein was isolated from liquid cultures of 20 cell lines. All cell cultures were harvested at the exponential phase of cell growth. For each cell line a volume of culture (to obtain 108 cells) was gently centrifuged to obtain a pellet, which was resuspended in 1 mL of PBS and centrifuged again at 13,000 rpm for 3 minutes to repellet the cells. The cell pellet was resuspended in 100 µL of cell lysis buffer (20 mM HEPES, pH 7.8; 0.4 mM EDTA; 450 mM NaCl; 0.5 mM DTT; 0.5 mM PMSF) and incubated on ice for 10 minutes to lyse all membranes and release the proteins. The viscosity of the suspension was reduced by passing it 10 times through a 25-gauge needle. The suspension was then spun at 13,000 rpm for 40 minutes at 4°C and the supernatant containing soluble proteins was preserved. The protein concentration of all extracts was determined using the Bradford protein assay with BSA as the standard.
Western Blot Analyses
Aliquots containing equal amounts of total proteins were electrophoresed in 10% SDS-polyacrylamide gels in duplicate. One gel was stained with Coomassie blue stain to confirm equal loading. In addition, 5 µL (1.5 µg) of a total snRNP preparation containing PRPF31 was loaded as a positive control. The proteins on the other gel were transferred by electroblotting to a nirocellulose membrane (BioRad) using transfer buffer (50 mM Tris, pH 9.1; 390 mM glycine; 0.04% SDS; 20% methanol). Blots were blocked with 5% (w/v) milk powder proteins in PBS and then probed with PRPF31 antibody (Anti-61K). Anti-61K raised in a rabbit against a C-terminal peptide (amino acid residues 484–497)4 was diluted 1:500 for Western blot analysis. Anti-61K antibody should only recognize the wild-type protein (499 a. a) because the smaller (469 a. a) mutant protein produced from the deleted allele has 98 novel amino acids after codon 371. After washing, the blots were probed with a horseradish peroxidase (HRP)-conjugated -rabbit secondary antibody diluted 1:3000 (BioRad Laboratories). Immunoreactive protein was detected using enhanced chemiluminescence (National Diagnostics).
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Results |
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Quantitative RT-PCR on Cell Line RNA
Figure 1 shows the abridged pedigree AD5 depicting all the individuals whose cell lines were included in this study. The disease status of the individuals enrolled in the study had been established in clinical examinations and has been reported previously.15 16 PRPF31 mRNA levels of duplicate samples from each lymphoblastoid cell line was measured twice, thus providing a mean estimate of four analyses. Dilutions of the oligonucleotide standard were also included in each RT-qPCR assay to test for reproducibility and sensitivity.
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50% to 60% that of the asymptomatic group (where sum intensities are defined as the sum of all pixel intensities in the band rectangle). There were also differences in the sum band intensities between the noncarrier group and the asymptomatic group with the asymptomatic band strength being 80% that of the noncarrier group, although by eye the band intensities appeared similar. The corresponding Coomassie blue-stained gel showed banding patterns of approximately equal intensity for all samples, confirming uniform loading of samples and thus suggesting that differing PRPF31 band intensities were due to differences in the level of protein expression (Figs. 3A1 and 3A2 ). Similar data were reproduced in another independent experiment (data not shown). |
Discussion |
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The mRNA data from cell lines indicated that symptomatic patients inherit a relatively poorly expressed PRPF31 wild-type allele from their noncarrier parent compared with asymptomatic patients. The mRNA data were supported by the Western blot analysis, which showed corresponding lower levels of PRPF31 protein in symptomatic cell lines compared to asymptomatic cell lines. Therefore it appears that the clinical manifestation of RP in AD5 is modulated by the low expression of wild-type PRPF31 allele in trans with the mutant allele.
On Western blot analysis noncarrier and asymptomatic cell line PRPF31 protein levels appeared similar although the copy numbers of PRPF31 in noncarrier individuals were approximately two times that of asymptomatic individuals. In fact based on the sum intensity values of the bands it appeared that asymptomatic bands were 80% as intense as the normal/non carrier PRPF31 protein bands. The correlation between the number of mRNA and protein molecules is generally not strong enough to predict one value from the measurement of the other.17 18 Therefore a high mRNA copy number as seen in the noncarrier individuals may not necessarily translate to a correspondingly high level of protein. It is possible that there are mechanisms in place to maintain a certain steady state or basal level of PRPF31 protein within cells. Therefore the asymptomatic PRPF31 protein levels may be close to the noncarrier PRPF31 protein levels, as was observed by Western blot analysis.
Another important question to consider is whether the mRNA and protein expression patterns seen in the lymphocytes of symptomatic and asymptomatic individuals can be extended to the rod photoreceptors of these individuals. Comparison of PRPF31 expression in the peripheral retina and blood lymphocytes showed similar results for these very different tissues. It is reasonable to infer that relatively lower PRPF31 protein levels are likely to be present within the rod photoreceptors of symptomatic individuals. The clinical manifestation of RP may subsequently arise because of the sensitivity of rod photoreceptors to the level of the PRPF31 protein at times of increased mRNA synthesis, for example in the event of disc shedding and turnover.2
There are several possible mechanisms for the difference in mRNA and protein levels between symptomatic and asymptomatic individuals. Firstly, different transcriptional activity of the promoter may be a factor. Also, posttranscriptional regulatory events such as mRNA translation and decay may also vary for different wild-type alleles of PRPF31. Other factors include protein isoforms (resulting from different wild-type alleles) with different biological half-lives.
Scanning of the PRPF31 genomic sequence of symptomatic and asymptomatic individuals from AD5 identified several polymorphisms, both in coding and noncoding sequences. Analysis of these polymorphisms clearly showed that in a given sib-ship the two contrasting phenotypes inherit a different haplotype from their noncarrier parent. However, to date only one change in PRPF31, located in intron 1 (IVS1+14A>G), has been shown to segregate concordantly among all the symptomatic and asymptomatic individuals within the AD5 pedigree. It remains to be proven that this sequence variation is directly involved in the low expression of PRPF31 or exists in linkage disequilibrium with an as yet unidentified sequence variation(s).
A phenomenon similar to that described in this study is encountered in Erythropoietic protoporphyria (EPP). EPP is a rare autosomal dominant disorder of heme biosynthesis, characterized by partial decrease in ferrochelatase (FECH) activity. FECH is the terminal enzyme of the heme biosynthetic pathway and catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. EPP, like RP caused by mutations in PRPF31, exhibits incomplete penetrance. It has been demonstrated that clinical expression of EPP requires the coinheritance of a wild-type FECH allele with low expression and a mutant FECH allele.19 Furthermore, the underlying cause for low expression has been identified as an intronic single nucleotide polymorphism, IVS3-48T/C, which modulates the use of a constitutive aberrant acceptor splice site. The aberrantly spliced mRNA is degraded by a nonsense-mediated decay mechanism, producing a decreased steady-state level of mRNA resulting in decreased FECH enzyme activity necessary for EPP phenotypic expression.20
In conclusion, we have shown that in our largest adRP pedigree with a deletion in PRPF31, the clinical manifestation of RP could be due to coinheritance of a PRPF31 mutation and a wild-type low-expressed allele. To show that this phenomenon is generally involved in adRP caused by mutations in PRPF31 and not restricted to a single family (AD5), more adRP families need to be studied. This study also revealed a potential avenue for future therapy for this adRP locus, as it appears that increased expression of wild-type PRPF31 may prevent clinical manifestation of the disease. The identification of the genetic basis for differential expression of wild-type alleles would be helpful in determining the prognosis of the children from carrier parents.
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Acknowledgements |
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Footnotes |
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Supported by Medical Research Council Grants G0000011 and G9301094, The Foundation for Fighting Blindness, USA, The British Retinitis Pigmentosa Society and Fight For Sight. DH is funded by Research into Ageing Fellowship (Ref 217).
Submitted for publication March 12, 2003; revised April 22, 2003; accepted April 24, 2003.
Disclosure: E.N. Vithana, None; L. Abu-Safieh, None; L. Pelosini, None; E. Winchester, None; D. Hornan, None; A.C. Bird, None; D.M. Hunt, None; S.A. Bustin, None; S.S. Bhattacharya, 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: Eranga N. Vithana, Division of Molecular Genetics, Institute of Ophthalmology, University College London, 11–43 Bath Street, London EC1V 9EL, United Kingdom; evithana{at}hgmp.mrc.ac.uk.
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