October 2003
Volume 44, Issue 10
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Biochemistry and Molecular Biology  |   October 2003
Expression of PRPF31 mRNA in Patients with Autosomal Dominant Retinitis Pigmentosa: A Molecular Clue for Incomplete Penetrance?
Author Affiliations
  • Eranga N. Vithana
    From the Division of Molecular Genetics, Institute of Ophthalmology, University College London, London, United Kingdom; the
  • Leen Abu-Safieh
    From the Division of Molecular Genetics, Institute of Ophthalmology, University College London, London, United Kingdom; the
  • Lucia Pelosini
    Moorfields Eye Hospital, London, United Kingdom; and the
  • Elizabeth Winchester
    Moorfields Eye Hospital, London, United Kingdom; and the
  • Dan Hornan
    From the Division of Molecular Genetics, Institute of Ophthalmology, University College London, London, United Kingdom; the
  • Alan C. Bird
    Moorfields Eye Hospital, London, United Kingdom; and the
  • David M. Hunt
    From the Division of Molecular Genetics, Institute of Ophthalmology, University College London, London, United Kingdom; the
  • Stephen A. Bustin
    Academic Department of Surgery, St. Bartholomews and the Queen Mary’s School of Medicine and Dentistry, University of London, London, United Kingdom.
  • Shomi S. Bhattacharya
    From the Division of Molecular Genetics, Institute of Ophthalmology, University College London, London, United Kingdom; the
Investigative Ophthalmology & Visual Science October 2003, Vol.44, 4204-4209. doi:10.1167/iovs.03-0253
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      Eranga N. Vithana, Leen Abu-Safieh, Lucia Pelosini, Elizabeth Winchester, Dan Hornan, Alan C. Bird, David M. Hunt, Stephen A. Bustin, Shomi S. Bhattacharya; Expression of PRPF31 mRNA in Patients with Autosomal Dominant Retinitis Pigmentosa: A Molecular Clue for Incomplete Penetrance?. Invest. Ophthalmol. Vis. Sci. 2003;44(10):4204-4209. doi: 10.1167/iovs.03-0253.

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      © 2015 Association for Research in Vision and Ophthalmology.

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purpose. To investigate whether the incomplete penetrance phenotype characteristic of adRP families linked to chromosome 19q13.4 (RP11) with mutations in the PRPF31 gene is due to differentially expressed wild-type alleles in symptomatic and asymptomatic individuals.

 

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.

Retinitis pigmentosa is a clinically and genetically heterogeneous disorder characterized by progressive degeneration of the peripheral retina leading to night blindness and visual field loss. Clinical manifestations include pigment deposition in the retina and attenuation of retinal blood vessels, with later depigmentation or atrophy of the RPE. ERG abnormalities are recordable in the early stages with attenuation of rod and cone responses. In advanced RP both rod and cone ERG responses are extinguished. 1 With an incidence of approximately 1 in 3500, RP can be inherited as an X-linked, autosomal dominant or autosomal recessive condition. 1  
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 PRPF31 9 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 α- and β-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. 
Materials and Methods
Patients
Affected and noncarrier members from family RP856/AD5 used in this study gave fully informed consent and the hospital ethics committee approved the study protocol. Furthermore, this research adheres to the tenets of the Declaration of Helsinki. Blood samples from family AD5 were submitted to the European Collection of Cell Cultures for the creation of immortalized lymphoblastoid cell lines using the Epstein-Barr virus. The cell lines were obtained as growing cultures and were maintained at 3 × 100,000 to 2 × 1,000,000 cells/mL: 5% CO2 37°C in RPMI 1640 media with 2 mM glutamine, 10% FCS and 1% penicillin/streptomycin. The cohort size was finally limited to 20 cell lines; originally a larger cohort was intended but sample numbers were restricted by the failure of cell transformation. 
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 × 105-2 × 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, C T ) 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 × 109 molecules to 10 molecules and used in RT-PCR reactions. One microgram of a 110-bp ssDNA contains 1.7 × 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 = (C T b)/m, where C T 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 α-rabbit secondary antibody diluted 1:3000 (BioRad Laboratories). Immunoreactive protein was detected using enhanced chemiluminescence (National Diagnostics). 
Results
Comparison of PRPF31 Copy Numbers from Tissue Culture Cells and Nucleated Blood Cells
PRPF31 mRNA levels determined from nucleated blood cells of four British Caucasians showed that PRPF31 copy numbers from noncarrier lymphoblastoid cell lines and nucleated blood cells were similar and therefore comparable, validating the use of RNA from lymphoblastoid cell lines (Table 1) . In this study PRPF31 copy numbers were related to total RNA concentration rather than to a single housekeeping gene. The use of internal standards comprising single housekeeping genes has been found to be inappropriate for studies involving tissue biopsies. 14 The PRPF31 copy numbers/μg of total RNA obtained from nucleated blood cells was comparable to copy numbers obtained for GAPDH in other studies. 12 This may reflect the relative abundance of PRPF31
Quantitative RT-PCR on Postmortem Retinal Tissue and Nucleated Blood Cells
PRPF31 mRNA levels in retina and nucleated blood cells were measured to determine whether the two tissues are comparable (Table 1) . PRPF31 copy numbers in retina compared with blood cells from four control individuals were similar, with little variability between the two tissues. Some variability was apparent in the PRPF31 copy numbers for blood lymphocytes from different control individuals; a large number of individuals would have to be assayed to investigate the amount of variation found in PRPF31 mRNA levels in nucleated blood cells in a given population. 
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. 
PRPF31 mRNA was detected in all 20 lymphoblastoid cell lines (Table 2) . The Mann-Whitney U test was used to compare the median copy number in pairs of the three groups, namely the symptomatic and asymptomatic carriers of the mutant PRPF31 allele and the noncarrier/normal individuals. We found the median PRPF31 mRNA copy number to be significantly higher in the noncarrier group compared with both the symptomatic and asymptomatic group (P < 0.01). This was expected, because the noncarrier individuals have two wild-type alleles of PRPF31 gene whereas both symptomatic and asymptomatic individuals have a single PRPF31 wild-type allele and the Taqman probe was designed to detect only wild-type alleles of PRPF31
Interestingly, the median mRNA copy number of the wild-ype PRPF31 allele was also higher in asymptomatic compared with symptomatic individuals (P < 0.001; Fig. 2 ). Indeed, a marked difference in mRNA copy number was observed between asymptomatic individual 298 and symptomatic individuals 319, 320, 323, and 325, who were part of the same sib-ship (Fig. 1) . Unfortunately, cell transformation failed for asymptomatic individual 316 whose mRNA copy number would have enabled the comparison of all symptomatic and asymptomatic individuals in the entire sib-ship. Symptomatic and asymptomatic individuals in this sib-ship inherit two different wild-type alleles from their noncarrier parent (Fig. 1) . The mean mRNA copy numbers of the four siblings (319, 320, 323, and 325) with identical wild-type allele show some variability (mean 2.47 × 107 ± 3.93 × 106) with individual 323 showing the lowest copy number. PRPF31 mRNA levels do not appear to be related to the age of the individuals although the mean age of the symptomatic group was higher than that of the asymptomatic and the noncarrier individuals. 
Western Blot Analysis of PRPF31 Protein Extracted from Cell Lines
To investigate PRPF31 protein levels between symptomatic and asymptomatic individuals, the soluble fraction of whole cell lysates was examined by SDS-polyacrylamide gel electrophoresis with Coomassie blue staining and Western blot analysis. The PRPF31 antibody (Anti-61K) used as a probe had been raised in a rabbit against a C-terminal peptide (amino acid residues 484–497) 4 and therefore only recognized the wild-type protein (499 a. a) seen as a band of 61 kDa on the immunoblot. Lack of additional bands suggests the specificity of the probe for the wild-type protein, because the smaller (469 a. a) putative mutant protein is likely to run at a different molecular weight. The PRPF31 protein band (61 kDa) was less intense in soluble cell extracts from symptomatic individuals compared with asymptomatic and noncarrier individuals. This demonstrates lower expression of the PRPF31 protein in symptomatic individuals (Figs. 3B1 3B2 ). 
The band intensities were analyzed using the Kodak digital science electrophoresis documentation analysis system 120 and the sum intensities of bands from the symptomatic group was ∼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
We have shown a statistically significant difference in PRPF31 mRNA copy numbers between symptomatic and asymptomatic individuals carrying a PRPF31 gene mutation in our largest adRP pedigree linked to chromosome 19q13.4. The mRNA copy number data were based on RNA from lymphoblastoid cell lines. 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. It is important to note that differences in PRPF31 copy numbers between the two phenotypes were considered and not absolute PRPF31 copy numbers. The difference in mRNA levels can be directly attributed to an underlying genetic variation that exists in the two phenotypic groups under investigation. 
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. 
 
Table 1.
 
PRPF31 Expression in Retina, Nucleated Blood Cells and Lymphoblastoid Cells from Normal/Noncarrier Individuals
Table 1.
 
PRPF31 Expression in Retina, Nucleated Blood Cells and Lymphoblastoid Cells from Normal/Noncarrier Individuals
Tissue PRPF31 Copy Number/μg of Total RNA (Mean/SD)
Peripheral retina (pool of 4 retinas) 1.32 × 108
Blood lymphocyte
 BL 1 1.35 × 108
 BL2 1.27 × 108
 BL3 1.43 × 108
 BL4 1.05 × 108
Lymphoblastoid cell line
 NCL1 1.09 × 108
 NCL2 1.06 × 108
 NCL3 1.12 × 108
 NCL4 1.18 × 108
Mean 1.21 × 108 ± 1.39 × 107
Figure 1.
 
The schematic diagram of the AD5/RP856 pedigree depicting the individuals enrolled for the PRPF31 mRNA quantitation study with their mean mRNA copy number/μg of total RNA (×107) shown within parentheses. Symptomatic and asymptomatic disease gene carriers are drawn in solid black and check, respectively; all bear the deletion mutation (1115 to 1125 del) in PRPF31. Noncarrier individuals are drawn in white. In all disease gene carriers the disease allele is depicted as a solid black bar; for all individuals only the wild-type allele marker data is shown (an arrow indicates the position of PRPF31 gene within the RP11 markers). The different wild-type haplotype alleles inherited by asymptomatic individuals 316, 298 and symptomatic individuals 319, 320, 325, and 323 belonging to one sib-ship is distinguished from each other by the shading of the asymptomatic haplotype.
Figure 1.
 
The schematic diagram of the AD5/RP856 pedigree depicting the individuals enrolled for the PRPF31 mRNA quantitation study with their mean mRNA copy number/μg of total RNA (×107) shown within parentheses. Symptomatic and asymptomatic disease gene carriers are drawn in solid black and check, respectively; all bear the deletion mutation (1115 to 1125 del) in PRPF31. Noncarrier individuals are drawn in white. In all disease gene carriers the disease allele is depicted as a solid black bar; for all individuals only the wild-type allele marker data is shown (an arrow indicates the position of PRPF31 gene within the RP11 markers). The different wild-type haplotype alleles inherited by asymptomatic individuals 316, 298 and symptomatic individuals 319, 320, 325, and 323 belonging to one sib-ship is distinguished from each other by the shading of the asymptomatic haplotype.
Table 2.
 
PRPF31 mRNA Expression in Symptomatic (S), Asymptomatic (AS) Individuals Carrying the (1115–1125 del) PRPF31 Mutation, and in Normal/Noncarrier (N) Individuals of the ADS Family
Table 2.
 
PRPF31 mRNA Expression in Symptomatic (S), Asymptomatic (AS) Individuals Carrying the (1115–1125 del) PRPF31 Mutation, and in Normal/Noncarrier (N) Individuals of the ADS Family
Cell Line Clinical Status/ Age (y) mRNA Copy Number/ μg RNA
307 S 58 2.56 × 107
306 S 47 2.97 × 107
305 S 23 2.63 × 107
319 S 64 2.66 × 107
320 S 56 2.75 × 107
323 S 49 1.89 × 107
325 S 55 2.58 × 107
271 S 22 2.62 × 107
Mean 47ys Mean 2.58 × 107 ± 3.09 × 106
297 AS 76 5.25 × 107
298 AS 61 5.06 × 107
255 AS 56 3.47 × 107
311 AS 23 3.26 × 107
353 AS 23 3.48 × 107
299 AS 26 4.04 × 107
340 AS 21 5.25 × 107
Mean 41 * Mean 4.26 × 107 ± 9.02 × 106
300 N 32 1.09 × 108
343 N 24 1.06 × 108
295 N 50 1.12 × 108
296 N 50 1.18 × 108
341 N 50 1.0 × 108
Mean 41 , † Mean 1.09 × 108 ± 6.71 × 106
Figure 2.
 
Scatter plot showing PRPF31 mRNA copy numbers in symptomatic (S), asymptomatic (AS), and noncarrier (N) individuals of the AD5/RP856 pedigree. The horizontal bar indicates the mean copy number.
Figure 2.
 
Scatter plot showing PRPF31 mRNA copy numbers in symptomatic (S), asymptomatic (AS), and noncarrier (N) individuals of the AD5/RP856 pedigree. The horizontal bar indicates the mean copy number.
Figure 3.
 
Western blot analysis of PRPF31 protein. Soluble cell lysate (∼5 μg/lane) was separated by 10% SDS/PAGE and transferred to nitro cellulose and was analyzed with Anti 61K antibody. (A1) and (A2) Coomassie Blue staining of the gel separated by SDS-PAGE showing equal loading. (B1) and (B2) Immunoblotting of PRPF31 protein. Lane 1, recombinant protein molecular weight marker (Amersham); lane 2, total snRNP preparation containing PRPF31 loaded as a positive control; lanes 3 to 5 and 12 to 15 symptomatics (S) 271, 306, 305, 307, 323, 320, and 319, respectively; lanes 6 to 8 and 16 to 18 asymptomatics (AS) 298, 311, 340, 299, 353, and 297, respectively; lanes 9 to 11 and 19 to 20 noncarriers (N) 296, 295, 343, 341 and 300. A single band of ∼61 kDa was detected in all lanes.
Figure 3.
 
Western blot analysis of PRPF31 protein. Soluble cell lysate (∼5 μg/lane) was separated by 10% SDS/PAGE and transferred to nitro cellulose and was analyzed with Anti 61K antibody. (A1) and (A2) Coomassie Blue staining of the gel separated by SDS-PAGE showing equal loading. (B1) and (B2) Immunoblotting of PRPF31 protein. Lane 1, recombinant protein molecular weight marker (Amersham); lane 2, total snRNP preparation containing PRPF31 loaded as a positive control; lanes 3 to 5 and 12 to 15 symptomatics (S) 271, 306, 305, 307, 323, 320, and 319, respectively; lanes 6 to 8 and 16 to 18 asymptomatics (AS) 298, 311, 340, 299, 353, and 297, respectively; lanes 9 to 11 and 19 to 20 noncarriers (N) 296, 295, 343, 341 and 300. A single band of ∼61 kDa was detected in all lanes.
The authors thank Reinhard Lurmann for supplying the PRPF31 antibody and Rebecca Hands for helping with instrumentation. 
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Figure 1.
 
The schematic diagram of the AD5/RP856 pedigree depicting the individuals enrolled for the PRPF31 mRNA quantitation study with their mean mRNA copy number/μg of total RNA (×107) shown within parentheses. Symptomatic and asymptomatic disease gene carriers are drawn in solid black and check, respectively; all bear the deletion mutation (1115 to 1125 del) in PRPF31. Noncarrier individuals are drawn in white. In all disease gene carriers the disease allele is depicted as a solid black bar; for all individuals only the wild-type allele marker data is shown (an arrow indicates the position of PRPF31 gene within the RP11 markers). The different wild-type haplotype alleles inherited by asymptomatic individuals 316, 298 and symptomatic individuals 319, 320, 325, and 323 belonging to one sib-ship is distinguished from each other by the shading of the asymptomatic haplotype.
Figure 1.
 
The schematic diagram of the AD5/RP856 pedigree depicting the individuals enrolled for the PRPF31 mRNA quantitation study with their mean mRNA copy number/μg of total RNA (×107) shown within parentheses. Symptomatic and asymptomatic disease gene carriers are drawn in solid black and check, respectively; all bear the deletion mutation (1115 to 1125 del) in PRPF31. Noncarrier individuals are drawn in white. In all disease gene carriers the disease allele is depicted as a solid black bar; for all individuals only the wild-type allele marker data is shown (an arrow indicates the position of PRPF31 gene within the RP11 markers). The different wild-type haplotype alleles inherited by asymptomatic individuals 316, 298 and symptomatic individuals 319, 320, 325, and 323 belonging to one sib-ship is distinguished from each other by the shading of the asymptomatic haplotype.
Figure 2.
 
Scatter plot showing PRPF31 mRNA copy numbers in symptomatic (S), asymptomatic (AS), and noncarrier (N) individuals of the AD5/RP856 pedigree. The horizontal bar indicates the mean copy number.
Figure 2.
 
Scatter plot showing PRPF31 mRNA copy numbers in symptomatic (S), asymptomatic (AS), and noncarrier (N) individuals of the AD5/RP856 pedigree. The horizontal bar indicates the mean copy number.
Figure 3.
 
Western blot analysis of PRPF31 protein. Soluble cell lysate (∼5 μg/lane) was separated by 10% SDS/PAGE and transferred to nitro cellulose and was analyzed with Anti 61K antibody. (A1) and (A2) Coomassie Blue staining of the gel separated by SDS-PAGE showing equal loading. (B1) and (B2) Immunoblotting of PRPF31 protein. Lane 1, recombinant protein molecular weight marker (Amersham); lane 2, total snRNP preparation containing PRPF31 loaded as a positive control; lanes 3 to 5 and 12 to 15 symptomatics (S) 271, 306, 305, 307, 323, 320, and 319, respectively; lanes 6 to 8 and 16 to 18 asymptomatics (AS) 298, 311, 340, 299, 353, and 297, respectively; lanes 9 to 11 and 19 to 20 noncarriers (N) 296, 295, 343, 341 and 300. A single band of ∼61 kDa was detected in all lanes.
Figure 3.
 
Western blot analysis of PRPF31 protein. Soluble cell lysate (∼5 μg/lane) was separated by 10% SDS/PAGE and transferred to nitro cellulose and was analyzed with Anti 61K antibody. (A1) and (A2) Coomassie Blue staining of the gel separated by SDS-PAGE showing equal loading. (B1) and (B2) Immunoblotting of PRPF31 protein. Lane 1, recombinant protein molecular weight marker (Amersham); lane 2, total snRNP preparation containing PRPF31 loaded as a positive control; lanes 3 to 5 and 12 to 15 symptomatics (S) 271, 306, 305, 307, 323, 320, and 319, respectively; lanes 6 to 8 and 16 to 18 asymptomatics (AS) 298, 311, 340, 299, 353, and 297, respectively; lanes 9 to 11 and 19 to 20 noncarriers (N) 296, 295, 343, 341 and 300. A single band of ∼61 kDa was detected in all lanes.
Table 1.
 
PRPF31 Expression in Retina, Nucleated Blood Cells and Lymphoblastoid Cells from Normal/Noncarrier Individuals
Table 1.
 
PRPF31 Expression in Retina, Nucleated Blood Cells and Lymphoblastoid Cells from Normal/Noncarrier Individuals
Tissue PRPF31 Copy Number/μg of Total RNA (Mean/SD)
Peripheral retina (pool of 4 retinas) 1.32 × 108
Blood lymphocyte
 BL 1 1.35 × 108
 BL2 1.27 × 108
 BL3 1.43 × 108
 BL4 1.05 × 108
Lymphoblastoid cell line
 NCL1 1.09 × 108
 NCL2 1.06 × 108
 NCL3 1.12 × 108
 NCL4 1.18 × 108
Mean 1.21 × 108 ± 1.39 × 107
Table 2.
 
PRPF31 mRNA Expression in Symptomatic (S), Asymptomatic (AS) Individuals Carrying the (1115–1125 del) PRPF31 Mutation, and in Normal/Noncarrier (N) Individuals of the ADS Family
Table 2.
 
PRPF31 mRNA Expression in Symptomatic (S), Asymptomatic (AS) Individuals Carrying the (1115–1125 del) PRPF31 Mutation, and in Normal/Noncarrier (N) Individuals of the ADS Family
Cell Line Clinical Status/ Age (y) mRNA Copy Number/ μg RNA
307 S 58 2.56 × 107
306 S 47 2.97 × 107
305 S 23 2.63 × 107
319 S 64 2.66 × 107
320 S 56 2.75 × 107
323 S 49 1.89 × 107
325 S 55 2.58 × 107
271 S 22 2.62 × 107
Mean 47ys Mean 2.58 × 107 ± 3.09 × 106
297 AS 76 5.25 × 107
298 AS 61 5.06 × 107
255 AS 56 3.47 × 107
311 AS 23 3.26 × 107
353 AS 23 3.48 × 107
299 AS 26 4.04 × 107
340 AS 21 5.25 × 107
Mean 41 * Mean 4.26 × 107 ± 9.02 × 106
300 N 32 1.09 × 108
343 N 24 1.06 × 108
295 N 50 1.12 × 108
296 N 50 1.18 × 108
341 N 50 1.0 × 108
Mean 41 , † Mean 1.09 × 108 ± 6.71 × 106
Copyright 2003 The Association for Research in Vision and Ophthalmology, Inc.
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