Transcriptional expression ofcis-acting andtrans-acting splicing mutations cause autosomal dominant retinitis pigmentosa

Abstract

Two types of mutations may lead to deficient pre-mRNA splicing: cis-acting mutations that inactivate a constitutive or alternative splice site within the pre-mRNA, and trans-acting mutations that affect the function of a basal factor of the splicing machinery. Autosomal dominant retinitis pigmentosa (adRP) is caused by mutations in at least 12 genes, with mutations in rhodopsin being the most prevalent. Two cis-acting mutations, g.3811A>G and g.5167G>T at the splice site in the rhodopsin gene (RHO; GenBank U49742.1) are linked to adRP in a Spanish population; while a cis-acting mutation, g.4335G>T, has been linked to recessive RP (arRP). Transcriptional expression analysis showed that the cis-acting splicing mutations linked to adRP promoted alternative splice sites, while the arRP linked mutation results in exclusion of exon 4. Trans-acting splicing mutations associated with adRP have also been found, and mutations in the pre-mRNA splicing factors PRPF3, PRPF8, PRPF31, and RP9 are associated with adRP in several populations. This report describes a new mutation in PRPF3 in a Spanish adRP family. We also investigated the transcriptional patterns in Epstein-Barr virus (EBV)-transformed lymphoblastoid cells from patients carrying a mutation in PRPF8. Despite the role of PRPF8 in the minor U12 splicing processes, microarray analysis revealed that mutations in PRPF8 not only did not result in significant differences in splicing efficiency of rhodopsin, but no apparent changes in expression of U12-type intron genes and splicing processes was observed. Microarray analysis revealed a panel of differentially expressed genes mapped to the RP loci, and future work will determine their role in RP.

Prognosis for splicing factor PRPF8 retinitis pigmentosa, novel mutations and correlation between human and yeast phenotypes

“…Nevertheless, these mutations are highly clustered at the C-terminus and are either missense changes or are nonsense mutations that change or delete at most only the last 37 amino-acids of this remarkably conserved 2335 amino acid protein. This pattern of mutations makes it likely that mutated PRPF8 protein will be present in patient cells, a theory supported by the observation that a PRPF8 mRNA containing one of the C-terminal frameshift mutations was not subject to nonsense mediated decay (Gamundi, et al, 2008). Furthermore, a mouse knock-in model with the H2309P PRPF8 mutation exhibits sub-retinal pigment epithelium (RPE) deposits at two years of age (Graziotto et al 2010) but a PRPF8 knockout has no observable abnormalities (J.…”

“…However, splicing deficiency studies in human cell lines have been equivocal. Several laboratories reported that splicing factor RPcausing mutations caused measurable abnormalities in splicing in vitro (Ivings, et al, 2008;Mordes, et al, 2007;Wilkie, et al, 2008), but microarray analysis in vivo revealed no consistent splicing defects (Gamundi, et al, 2008;Rivolta, et al, 2006). Furthermore, the question as to why only the retina is affected in human patients remains unanswered.…”

“…These mutations account for approximately 3% of dominant RP or around 1% of all RP cases (Sullivan, et al, 2006). Real-time PCR analysis has shown similar expression of both wild type and mutant alleles in cell lines from RP patients carrying a nonsense mutation (Gamundi, et al, 2008). This suggests that this transcript (and by inference other mutant transcripts) is not removed by nonsense mediated decay.…”

“…Mutations in PRPF8 have been described as causing a severe form of dominant RP (Gamundi, et al, 2008;Walia, et al, 2008). However, in contrast to the phenotype for the commoner PRPF31 form of splicing-factor RP, which has been well documented in patients with a variety of mutations (Al-Maghtheh, et al, 1996;Evans, et al, 1995) there has been no published overview of PRPF8-RP phenotype across a range of mutations.…”

Progressive Sensorineural Hearing Loss and Normal Vestibular Function in a Dutch DFNB7/11 Family with a Novel Mutation in <i>TMC1</i>

“…A plasmid containing the genomic region encompassing exons 3-5 of RHO inserted at the EcoRI/SalI sites in the pCI-NEO vector was used for in vivo splicing assays [Gamundi et al, 2008]. The plasmid was digested with EcoNI and PflMI (New England Biolabs, Beverly, Mass., USA) according to the manufacturer's protocol, resulting in the removal of exon 4 and part of the flanking intronic sequences.…”