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
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
[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.