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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Hum Mutat. Author manuscript; available in PMC 2010 September 1.
Published in final edited form as:
PMCID: PMC2753193

A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance


We report the study of a large American family displaying autosomal dominant retinitis pigmentosa with reduced penetrance, a form of progressive and hereditary retinal degeneration. Although the particular inheritance pattern and previous linkage mapping incriminated the PRPF31 gene, extensive screening of all its exons and their boundaries failed in the past to reveal any mutation. In this work, we sequenced the entire PRPF31 genomic region by both the classical Sanger method and ultra-high throughput (UHT) sequencing. Among the many variants identified, a single-base substitution (c.1374+654C>G) located deep within intron 13 and inside a repetitive DNA element was common to all patients and obligate asymptomatic carriers. This change created a new splice donor site leading to the synthesis of two mutant PRPF31 isoforms, degraded by nonsense-mediated mRNA decay. As a consequence, we observed an overall reduction in full-length PRPF31 mRNA and protein levels, with no evidence of mutant proteins being synthesized. Our results indicate that c.1374+654C>G causes retinitis pigmentosa via haploinsufficiency, similar to the vast majority of PRPF31 mutations described so far. We discuss the potential of UHT sequencing technologies in mutation screening and the continued identification of pathogenic splicing mutations buried deep within intronic regions.

Keywords: PRPF31, RP11, splicing, UHT sequencing


Retinitis pigmentosa (RP) is the name given to a group of inherited diseases that causes progressive degeneration of the retina. It has a cumulative prevalence of 1/4000, affecting over 1 million individuals worldwide [Hartong et al., 2006; Berson, 2007]. In early stages, it is characterized by night blindness, followed by a decrease in mid-peripheral vision and ultimately loss of central vision as well, due to the death of both rod and cone photoreceptor cells [Berson, 1993].

RP is genetically highly heterogeneous; it can be transmitted as a Mendelian autosomal dominant (adRP), autosomal recessive, or X-linked trait and also in small proportions as a digenic and non-Mendelian trait [Rivolta et al., 2002]. It can occur alone or as part of more complex syndromes. So far, 50 genes or loci have been shown to be involved in non-syndromic RP (, but together they account for only about half of all reported cases and thus many genes associated with RP remain unknown [Hartong et al., 2006]. The RP11 locus, located on 19q13.4 (MIM# 600138), was first linked to adRP from the analysis of a large British family [Al Maghtheh et al., 1994] and later confirmed to be responsible for the disease in several other pedigrees [Xu et al., 1995; Al Maghtheh et al., 1996; McGee et al., 1997; Vithana et al., 1998]. Mutations in the pre-mRNA splicing factor gene PRPF31 (MIM# 606419) were subsequently identified as the cause of adRP in RP11 families [Vithana et al., 2001]. So far, more than 40 PRPF31 mutations have been detected, some of which are characterized by large deletions [Abu-Safieh et al., 2006; Sullivan et al., 2006; Kohn et al., 2008]. However, the vast majority of PRPF31 mutations create premature termination codon (PTC) in the mRNA transcribed from the mutant allele, which in turn is degraded by nonsense-mediated mRNA decay (NMD). In such cases, the absence of PRPF31 mRNA and protein derived from the mutant allele has been shown to be the likely cause of the disease [Rio Frio et al., 2008b].

Analysis of several families with RP indicates that PRPF31 mutations may display incomplete penetrance, i.e. there are obligate carriers of mutations who do not manifest the disease (e.g. [Berson et al., 1969; Al Maghtheh et al., 1996]). Penetrance of the disorder in heterozygous carriers of PRPF31 mutations has been previously associated with the level of expression of the wild-type PRPF31 allele [Vithana et al., 2003; Rivolta et al., 2006; Liu et al., 2008]. Specifically, high expression levels of wild-type PRPF31 mRNA appear to offer protection from RP, possibly because they compensate for haploinsufficiency resulting from the degradation of mutant transcripts. By linkage studies, PRPF31 mRNA expression and therefore the penetrance of PRPF31 mutations has been linked to modifiers present within the PRPF31 locus itself and on chromosome 14q21-23 [McGee et al., 1997; Vithana et al., 2001; Rio Frio et al., 2008a].

Here we report the study of a large family (ID #1562, Fig. 1) which was historically described as the first example of retinitis pigmentosa segregating as an autosomal dominant form with reduced penetrance [Berson et al., 1969; Berson and Simonoff, 1979]. Subsequent genotyping of selected members, using markers spanning the RP11 locus, strongly suggested that the PRPF31 gene was responsible for the disease [McGee et al., 1997]. Furthermore, PRPF31 expression analyses in lymphoblastoid cell lines (LCLs) from relevant members of this pedigree showed amounts of transcripts that were reduced with respect to controls and comparable to those present in individuals with known PRPF31 mutations [Rivolta et al., 2006]. Although all these data indicated that a dominant mutation in PRPF31 leading to haploinsufficiency was the likely cause of RP in family #1562, prior screens revealed no pathogenic changes [McGee et al., 2002] and no deletions [Rivolta et al., 2006] in any of the 14 PRPF31 exons.

Figure 1
Schematic pedigree of family #1562


Ascertainment of patients

Clinical evaluation of members of this family was originally described in 1969 and again in 1979 [Berson et al., 1969; Berson and Simonoff, 1979]. Following informed consent, DNA was extracted from lymphocytes collected from blood samples from family members as described previously [McGee et al., 1997].

Cell culture and drug treatments

Lymphoblastoid cell lines (LCLs) from individuals belonging to family #1562, from 10 members of the CEPH collection (normal controls), from 6 patients with known mutations in PRPF31 and one unaffected relative (cell line #14523, 14686, 14266, 12688, 14284, AG0293, and 14251) were obtained and cultured as previously described [Rivolta et al., 2006; Rio Frio et al., 2008b]. Inhibition of NMD was achieved by treating 5 million cells with 100 μM wortmannin or 300 μg/ml emetine (Sigma-Aldrich, St Louis, MO), dissolved in water, for a 6-hour period. Negative controls were performed by supplementing the cells with equivalent amounts of water and by incubating them in the same conditions.

Long-range PCR and DNA sequencing

Using previously-defined methods for long-range PCR of large genomic regions [Hinds et al., 2005], we selected 4 fragments containing the PRPF31 gene to amplify the genomic DNA from selected individuals (III-9, III-11, and IV-16, Fig.1). These fragments were 6893 bp, 10542 bp, 8124 bp, and 5690 bp in length, respectively, and encompassed a ~31 kb genomic region, from position 59′297′569 to 59′328′826 on chromosome 19 (NC_000019.8). The primers used in these 4 PCRs were in part previously published [Hinds et al., 2005] and in part newly-designed oligos (Supp. Table S1). PCR reactions were each performed in a final reaction volume of 50 μl, containing 1x GC Buffer I (TaKaRa, Otsu, Shiga, Japan), 0.4 mM of each dNTP, 0.2 μM of each primer, 0.5 U of TaKaRa LA Taq (TaKaRa) and 500 ng of DNA. Reactions were incubated at 94°C for 1 minute, followed by 30 cycles of 98°C for 5 seconds and 68°C for 15 minutes and a final elongation step of 70°C for 10 minutes. PCR products derived from each patient's DNA were pooled in an equimolar ratio.

Ultra-high throughput (UHT) sequencing reactions were performed on these long-range PCRs using an Illumina Genome Analyzer (Illumina, San Diego, CA). Small gaps between the first and second, and between the second and third regions (100 bp and 72 bp, respectively) were amplified and sequenced using the Sanger (dideoxy) method. The reads intensity files produced by the Illumina GA were processed through the Rolexa software package [Rougemont et al., 2008], which performs base calling to produce a maximal number of sequence reads with well defined quality information for each nucleotide. A total of 2′089′433 reads with sufficient length and quality were obtained. These reads were aligned to the human genome reference assembly 36 to find exact matches using the fetchGWI software [Iseli et al., 2007]: 718′883 reads found a single exact match (group U of unique reads), 235′273 reads found multiple exact matches (group R of repeated reads), and 1′135′277 reads remained with no exact match (group M of missed reads). A total of 689′957 among all U reads (96%) were mapped to the region of interest on chromosome 19. In order to discover polymorphisms in the sequenced region, the reads from group M were aligned using the global alignment software align0 [Myers and Miller, 1988] to the reference genomic sequence using the following method. First, we selected from all groups all the reads that shared a common 12-mer with any other 12-mer from the chromosomal region of interest, generating subsets of sequences termed Usel, Msel, and Rsel. Then, we performed a global alignment of all Msel sequences against the reference genome sequence, and discarded all alignments with more than 3 mismatches. Subsequently, we identified from Usel the reads mapping outside of the region of interest (Uout), performed a global alignment of Msel reads against the reads from Rsel and Uout, and discarded the reads from Msel which received a better alignment score in this latter global alignment. We predicted a SNP (or a variant) when an observed difference between the reads and the reference sequence occurred at a sufficient rate among all the reads that cover the position. In this case, we required at least 4 reads exhibiting the change and 20% of the total coverage.

Sequencing of the PRPF31 gene was repeated on the same samples as well as long-range PCRs from 2 normal CEPH controls [Dausset et al., 1990] using the Sanger method, by amplifying its full genomic region (exon 1 to exon 14, corresponding to bases 5001 to 21361 from PRPF31 GenBank reference genomic sequence NG_009759.1) with specific primers (Supp. Table S1) and TaKaRa LA Taq (Takara), as described above. PCR products were purified using the Qiaquick PCR Purification kit (Qiagen, Venlo, The Netherlands) and sequenced using internal primers (Supp. Table S2). Sequencing reactions were performed by mixing 4.5 μl of a 1/10 dilution of purified PCR product, 0.75 μM of internal primer, 0.75 μl of BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and run on an ABI-3130XL (Applied Biosystems). Variants detected were named using cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the PRPF31 GenBank reference sequence NM_015629.3, according to the Human Genome Variation Society guidelines (

RNA extraction and cDNA synthesis

Approximately 5 million cells from each cell line were homogenized using QIAshredder columns and total RNA was isolated using the RNeasy kit (Qiagen) according to manufacturer's recommendations, with an on-column DNase treatment. To completely remove DNA, isolated RNA was incubated again with RNase-free DNase I (Roche, Indianapolis, IN) at 37°C for 30 minutes and 75°C for 10 minutes. RNA was retro-transcribed using anchored poly-dT oligos (dT20VN) and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). Negative control samples for PCR reactions were prepared by this same procedure, but reverse transcriptase was not added.

Mutation analysis by PCR and semi-quantitative PCR

Specific primers (Supp. Table S1) were designed to amplify the exon 13-exon 14 region of the cDNA derived from PRPF31 mRNA (NM_015629.3), except for LCLs from the patient with the PRPF31 c.323-2A>G change, for which previously developed mutation-specific primers were used [Rio Frio et al., 2008b]. PCR reactions were performed in a final volume of 10 μl, containing 1x GC Buffer I, 0.4 mM of each dNTP, 0.2 μM of each primer, 0.5 U of TaKaRa LA Taq (Takara) and 2 μl of a 1/10 dilution of retrotranscribed mRNA. Reactions were incubated at 94°C for 1 minute followed by 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 68°C for 2 minutes, and a final elongation step of 70°C for 10 minutes. A final stage of 95°C for 5 minutes followed by a slow cooling down to 4°C (0.01°C per second) was added to prevent the formation of heteroduplexes between PCR products containing similar sequences. PCR products were resolved on 1.5% agarose gels and, for semi-quantitative PCR, by capillary electrophoresis with the eGene HDA-GT12 Multi-Channel Genetic Analyzer (eGene Inc., Irvine, CA). Quantification was performed by 2 independent methods: the ImageJ software [Abramoff et al., 2004] to analyze the agarose gel image, and the Biocalculator software (eGene) for runs performed with the HDA-GT12 instrument. Data obtained from these 2 methods were combined to calculate the mean values of the ratio between mutant and wild-type band intensities, and the error propagation relative to this operation calculated accordingly.

To identify cDNA isoforms, PCR products were sub-cloned into TOPO TA vectors (Invitrogen) and propagated in the E. coli cells. PCR reactions to identify positive clones were performed directly on colonies and products were sequenced by the Sanger method.

Quantification of wild-type mRNA derived from the mutant allele

cDNA from individuals carrying the c.1374+654C>G intronic mutation, who were also heterozygotes for SNP rs1058572:G>A alleles in exon 7, was amplified from exon 7 to exon 14 using specific oligos (Supp. Table S1). The A allele of this SNP was demonstrated to be in linkage disequilibrium with the mutation and was thus used as a marker to quantify allele-specific expression. The oligo annealing to exon 7 was designed with one mismatch (base 14, T instead of G) to prevent digestion of cDNA at a site located 18 bp upstream of the site of interest by BseRI, used for quantification of the wild-type mRNA derived from the mutant allele. PCR reactions were performed in a total volume of 25 μl, containing 1x Expand High Fidelity PCR System (Roche) buffer with MgCl2, 100 μM dNTP mix, 200 nM of each primer, 1 U of Expand High Fidelity PCR System (Roche) and 500 ng of retrotranscribed mRNA. The thermal profile used was optimized to prevent the amplification of other cDNAs than those derived from wild-type mRNA. Specifically, PCR reactions were incubated at 94°C for 15 minutes, followed by 40 cycles of 94°C for 15 seconds, 63°C for 30 seconds and 68°C for 1 minute.

Quantification of wild-type mRNA allelic expression using the SNP rs1058572:G>A was performed by interpolation, via the use of calibration standards. Two different cDNA templates from CEPH LCLs that were homozygous for either the major or minor SNP alleles were mixed to obtain quantification standards in the following ratios (G/A): 90/10, 80/20, 70/30, 60/40 and 50/50 at concentrations comparable with cDNA templates (to avoid differences in signal strength). cDNA from samples, quantification standards and controls (cDNA derived from homozygotes for either SNP alleles) were amplified on the same plate.

PCR products were first assessed to be unique products on a 2% agarose gel, and then digested using two restriction enzymes. BssSI (New England Biolabs, Ipswich, MA), which cuts all PCR products regardless of their allelic origin, was used to reduce the size in order to increase resolution for quantification; BseRI (New England Biolabs), which specifically recognizes the G allele of SNP rs1058572:G>A and cuts 14 bp away from it (towards the internal part of the PCR product), was used to distinguish PCR products derived from each allele. Digested products were run again on a 2% agarose gel and quantified by the ImageJ software. PCR products from transcripts derived from homozygous genotypes were used as digestion controls.

Antibodies and western blotting

Total protein was extracted from LCLs in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1% Tx-100, 0.1% SDS, 0.5% sodium deoxycholate) with Complete, Mini EDTA-free Protease Inhibitor Tablets (Roche), and protein content was estimated using the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL). Detection of PRPF31 protein was performed via western blotting using a specific N-terminal PRPF31 antibody and analyzed with the Odyssey Infra-Red Detector (LI-COR Biosciences, Lincoln, NE) as previously described [Rio Frio et al., 2008b]. Individual extractions were performed on 3 independent cell cultures of LCLs derived from 1 affected (IV-16) and 2 asymptomatic (III-9, III-11) carriers of the mutation from family #1562 and from the normal control cell line #14251. Equal amounts were run on denaturing SDS-PAGE gels with the Precision Prestained Protein Standards (Bio-Rad, Hercules, CA). For each extraction, 4 gels were run independently.


Identification of a novel intronic mutation in the PRPF31 gene

We amplified by long-range PCR a region of approximately 31 kb that encompasses the PRPF31 gene (including flanking regions 13 kb upstream and 2 kb downstream) by using as template material the genomic DNA of 1 affected patient (IV-16) and 2 asymptomatic carriers of the disease (III-9, III-11) from family #1562 (Fig. 1). Ultra-high throughput (UHT) sequencing of these amplicons, performed independently for each individual, revealed the presence of 102 SNPs, 56 of which already recorded in dbSNP. The median coverage of the chromosomal region of interest was 620 X, with an interquartile range of 324 to 1185 X. None of the variants specifically present within the 16 kb genomic region spanning PRPF31 exons 1 to 14 were deemed pathogenic as they were referenced in SNP databases, were found in DNA from control CEPH LCLs, or did not cosegregate with the disease haplotype.

We therefore resequenced this 16 kb DNA fragment in the same individuals by the Sanger method. In IV-16, chosen as reference, we identified 17 heterozygous variants that were also detected by UHT processing, and an additional change in intron 13 (Table 1). This novel change was a C>G transition at position 654 of the intron, c.1374+654C>G, located within the first of 7 consecutive and almost identical sequences of 56 bp composing a VNTR (variable number of tandem repeats) element (Fig. 2). The new variant was in fact present in the list of potential changes detected by UHT sequencing, but the coverage was much lower than the median in this region, and hence it was discarded as noise. Specifically, UHT reads could not be reliably aligned on this VNTR since they were in average 30-35 bp-long, i.e. shorter than the size of the repeated module itself.

Figure 2
Genomic localization and sequence of the VNTR containing the c.1374+654C>G variant
PRPF31 heterozygous variants detected in individual IV-16

The c.1374+654C>G change represented a good pathogenic candidate since it was the only one detected in all 3 individuals who had the disease haplotype, was not detected in DNA from CEPH cell lines, and was not referenced in dbSNP ( or the Ensembl SNP ( database. Sequencing of DNA extracted from blood samples from remaining members of family #1562 showed that c.1374+654C>G was present in all affected members of the family, as well as all obligate asymptomatic carriers from previous haplotype analyses ([McGee et al., 1997], Fig. 1). Furthermore, this variant was not detected in 300 control chromosomes, suggesting that it was likely to be the causative mutation in this family. Previously clinically uncharacterized family members (IV-2, 8, 10, 11, 23 and 33) were found to carry the mutation and likely represent asymptomatic individuals.

Effect of the c.1374+654C>G mutation on PRPF31 pre-mRNA splicing

In silico analyses of the PRPF31 allele carrying the c.1374+654C>G mutation, using the NNSPLICE software [Pedersen and Nielsen, 1997], predicted that this variant would very likely create a new splice site. Specifically, the likelihood score for splicing donor sites at this position increased from <0.1 in the wild-type sequence to 0.96 in the mutant sequence, out of a maximum of 1.00. PCR amplification of cDNA from LCLs from individuals carrying the c.1374+654C>G variant and a normal control using primers in exons 13 and 14 yielded a 158 bp product in all of the samples, as well as 2 supplementary PCR products of 333 bp (MUT1) and 811 bp (MUT2), present in lower amounts, only in LCLs from the 3 carriers of c.1374+654C>G (Fig. 3A). Cloning and sequencing of these PCR products revealed that the common 158 bp PCR product contained the wild-type exon 13-exon 14 junction. The MUT1 mRNA isoform (r.1770_1771ins1770+479_1770+653) was comprised of exon 13 spliced with bases 479 to 653 of intron 13, followed by exon 14, indicating that a cryptic acceptor site at position 478 was activated and an additional exon of 175 bp was inserted (Fig. 3B). The MUT2 mRNA (r.1770_1771ins1770+1_1770+653) did not use the native intron 13 splice donor site and instead retained bases 1 to 653 of intron 13 as part of exon 13, which was then joined with exon 14 (Fig. 3B). These aberrant isoforms were not observed in transcripts present in LCLs from 26 control individuals and 6 RP patients with different mutations in PRPF31 (data not shown). Altogether, these data indicate that splicing of pre-mRNAs derived from the PRPF31 allele containing the c.1374+654C>G mutation is specifically affected by the presence of a newly-created strong donor splice site and results in partial retention of intron 13.

Figure 3
PRPF31 mRNA isoforms in LCLs from members of family #1562

Analysis of mRNA derived from the mutant allele

By in silico analysis, MUT1 and MUT2 mRNAs were predicted to produce transcripts with 37 and 11 mutant codons after codon 458, respectively, followed by a premature termination codon (PTC) (Fig. 3B). Since these PTCs are located before the last exon, both mutant PRPF31 transcripts are potential target for NMD-driven degradation, a phenomenon that could explain their presence in reduced amounts with respect to the wild-type isoform (Fig. 3A).

To test this hypothesis, we analyzed LCLs derived from the 3 carriers of the c.1374+654C>G mutation, one control CEPH individual, and one patient who is a carrier of a different PRPF31 mutation (c.323-2A>G) known to result in an NMD-sensitive transcript [Rio Frio et al., 2008b]. Treatment of these LCLs with 2 potent NMD inhibitors, emetine and wortmannin, rescued mutant mRNA from LCLs with the c.323-2A>G mutation, indicating that inhibition of NMD was effective (data not shown). Similarly, amounts of both mutant mRNA isoforms MUT1 and MUT2 significantly increased relative to wild-type transcripts in LCLs carrying the c.1374+654C>G mutation. Meanwhile, only wild-type mRNA was amplified from control LCLs (CEPH), even when NMD was blocked. This indicates that the MUT1 or MUT2 isoforms are not naturally present or, if present, would be produced in extremely low amounts in absence of the mutation (Fig. 4A). Although precise quantification was not possible, since PCR efficiencies were product-specific, MUT1 and MUT2 mRNA isoforms represented on average 5% and 10%, respectively, of the wild-type mRNA in untreated LCLs, which increased to 38% and 83%, respectively, in LCLs grown in the presence of wortmannin and to 15% and 51%, respectively, in emetine-treated LCLs (Fig. 4B).

Figure 4
Rescue of NMD-sensitive mRNA isoforms MUT1 and MUT2 by wortmannin and emetine

Unlike other PRPF31 mutations that affect canonical splicing sites, the c.1374+654C>G mutation creates a new donor splice site but does not abolish the natural donor site of exon 13, which in fact is used to create the MUT1 isoform (Fig. 3). Consequently, it is conceivable that some wild-type mRNA could be derived from mutant alleles. To test this hypothesis, we searched for a heterozygous SNP in the PRPF31 coding sequence that could be used to tag the mutant allele. Individuals III-9 and IV-16 were found to be heterozygotes (G/A) for rs1058572:G>A, located in exon 7, whereas III-11 was a homozygote for the A allele, indicating that the A allele was in linkage disequilibrium with the mutation. We amplified PRPF31 cDNA derived from the 2 heterozygotes from exon 7 to exon 14, using PCR conditions optimized to prevent the amplification of the MUT1 and MUT2 isoforms. Analysis of BseRI-digested PCR products, cleaved when the rs1058572:G>A G allele is present, revealed that approximately 10% of the wild-type mRNA was derived from the mutant PRPF31 allele (Supp. Figure S1). Overall, the c.1374+654C>G mutation leads then to the formation of 3 different mRNA forms r.[=, 1770_1771ins1770+479_1770+653, 1770_1771ins1770+1_1770+653].

Protein analysis in LCLs from carriers of the c.1374+654C>G mutation

Western blots in LCLs from individuals with the c.1374+654C>G mutation, by using an antibody recognizing the N-terminal PRPF31 moiety, revealed a single 61-kDa band corresponding to the expected size of wild-type PRPF31. Importantly, no truncated proteins resulting from the possible translation of the MUT1 or MUT2 isoforms were detected (Fig. 5).

Figure 5
Protein analysis in 3 members from family #1562 and one control


In this study we report the identification of a mutation in the PRPF31 gene, which has been elusive for almost 40 years, in a large family with autosomal dominant RP with reduced penetrance. The c.1374+654C>G change is the first reported PRPF31 mutation located deep within an intron, confirming the suspicion that the genetic defect in this family was nonconventional. This sequence variant creates a very strong new donor splice site at position 654 of intron 13, resulting in the production of 2 novel mRNA isoforms, MUT1 and MUT2, that retain different parts of intron 13 during mRNA splicing and maturation. This new donor splice site was never used in control cell lines and, consequently, we conclude that such isoforms are the result of aberrant splicing, rather than natural alternative splicing.

Both the MUT1 and MUT2 mRNA isoforms harbour premature termination codons before the last exon and, similar to the majority of PRPF31 mutations described so far, have been shown to be present in reduced amounts compared to wild-type PRPF31 mRNA. This can be attributed to the action of NMD, since NMD inhibitors significantly rescued these transcripts. Analyses of PRPF31 proteins in carriers of the c.1374+654C>G mutation revealed no detectable amounts of mutant proteins derived from MUT1 and MUT2 mRNA. Considering the demonstrated high sensitivity of the methods used (3-4% of the full-length PRPF31 were detectable [Rio Frio et al., 2008b]), it is highly unlikely that significant quantities of mutant PRPF31 protein were indeed present in these patient cell lines. These results are consistent with previous findings on 6 other PRPF31 mutations that lead to PTC-containing and NMD-sensitive transcripts [Rio Frio et al., 2008b], reinforcing the notion that haploinsufficiency is likely the primary cause of PRPF31-linked RP.

The canonical donor splice site in exon 13 was not abolished by c.1374+654C>G and retained some functional activity, in conjunction with the natural acceptor site for exon 14 or with a cryptic site at position 478 in intron 13. Specifically, approximately 1/10 of the pre-mRNA derived from the mutant allele was correctly spliced, generating wild-type transcripts. This represents the first demonstrated example of some residual wild-type mRNA being derived from an allele carrying a PRPF31 mutation and may explain the slightly higher mRNA levels found in patients from family #1562, compared with all other affected and asymptomatic carriers from other families with different PRPF31 mutations [Rivolta et al., 2006]. Interestingly, many asymptomatic individuals in this family (IV-2, IV-8, IV-10, IV-11, IV-23 and IV-33) were only identified by molecular genetic evidence, reinforcing the notion that asymptomatic carriers of PRPF31 mutations are truly clinically unaffected. Furthermore, the large number of asymptomatic and affected individuals in this family would be a great asset for the future characterization of the penetrance factors influencing clinical manifestations of PRPF31-linked RP if additional lymphoblast cell lines were to be created and analyzed from these family members.

Splicing mutations, most of which lead to exon skipping and intron retention [Cooper et al., 2009], account for approximately 10% of all diseases caused by point mutations [Wang and Cooper, 2007] and the vast majority have been shown to affect natural splice sites and their surrounding canonical sequences [Teraoka et al., 1999]. The creation of additional splice sites in the middle of an intron which leads to the formation of pseudo exons is relatively rare [Kralovicova et al., 2005], with only a small number of genes having been reported so far (including CEP290, associated with Leber congenital amaurosis [den Hollander et al., 2006], β-globin [Treisman et al., 1983], factor VIII [Bagnall et al., 1999], CFTR [Highsmith et al., 1994; Chillon et al., 1995], and a few others). This is perhaps because screening for deep intronic mutations is not routinely conducted, despite, as demonstrated here, such mutations can have dramatic effects on splicing and consequently be harmful for the cell. Our data support therefore the relatively recent concept that mutational screens should not be limited to coding regions, but extended to intronic sequences as well.

Since intronic sequencing requires large processing capacity, we primarily utilized UHT sequencing and, as a complementary method, conventional Sanger sequencing. Although the genomic variations identified using these two methods largely overlapped, the pathogenic mutation was surprisingly not identified using the UHT method. Alignments of the ~30 bp sequences that were generated by UHT sequencing could not identify the c.1374+654C>G change, since it was present in the first of 7 nearly-identical repetitive sequences of 56 base pairs and therefore could not be correctly matched. Specifically, the mutation was only present in 1/14 of the sequences aligned with the repeat consensus, and this low signal was undistinguishable from noise. Therefore, despite the numerous advantages of the new UHT system, small read-length and complex repetitive genomic elements remain an issue for UHT sequencing methods [Hardiman, 2008; Pop and Salzberg, 2008]. However, projected improvements in sequence length and alignment assembly for UHT technologies are likely to overcome these problems in the near future, allowing the possibility to routinely screen entire candidate genes to identify disease-causing mutations.

Supplementary Material

Supp Data 01


We would like to acknowledge investigators at Fasteris SA, Plan-les-Ouates, Switzerland for help and fruitful discussion on UHT sequencing.

Our work was supported by the Swiss National Science Foundation (grants # 310000-109620 and 320000-121929), the National Institutes of Health (NIH-EY00169, P30-EY014104), Research to Prevent Blindness (Harvard Medical School, Dept. of Ophthalmology, Unrestricted Grant), and The Foundation Fighting Blindness.


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