Deep intronic mutation in OFD1, identified by targeted genomic next-generation sequencing, causes a severe form of X-linked retinitis pigmentosa (RP23)

Abstract

X-linked retinitis pigmentosa (XLRP) is genetically heterogeneous with two causative genes identified, RPGR and RP2. We previously mapped a locus for a severe form of XLRP, RP23, to a 10.71 Mb interval on Xp22.31-22.13 containing 62 genes. Candidate gene screening failed to identify a causative mutation, so we adopted targeted genomic next-generation sequencing of the disease interval to determine the molecular cause of RP23. No coding variants or variants within or near splice sites were identified. In contrast, a variant deep within intron 9 of OFD1 increased the splice site prediction score 4 bp upstream of the variant. Mutations in OFD1 cause the syndromic ciliopathies orofaciodigital syndrome-1, which is male lethal, Simpson-Golabi-Behmel syndrome type 2 and Joubert syndrome. We tested the effect of the IVS9+706A>G variant on OFD1 splicing in vivo. In RP23 patient-derived RNA, we detected an OFD1 transcript with the insertion of a cryptic exon spliced between exons 9 and 10 causing a frameshift, p.N313fs.X330. Correctly spliced OFD1 was also detected in patient-derived RNA, although at reduced levels (39%), hence the mutation is not male lethal. Our data suggest that photoreceptors are uniquely susceptible to reduced expression of OFD1 and that an alternative disease mechanism can cause XLRP. This disease mechanism of reduced expression for a syndromic ciliopathy gene causing isolated retinal degeneration is reminiscent of CEP290 intronic mutations that cause Leber congenital amaurosis, and we speculate that reduced dosage of correctly spliced ciliopathy genes may be a common disease mechanism in retinal degenerations.

Figure 1.

RP23 pedigree showing segregation of the OFD1 mutation IVS9+706A>G. Electropherograms of intron 9 OFD1 sequence are shown below individuals in the pedigree. Position IVS9+706 is highlighted with an asterisk. Affected males (II:1, IV:4 and V:2) carry the mutation A>G. Obligate carriers (III:2 and IV:2) are heterozygous. Individual IV:9 is also a carrier.

Figure 2.

RP23 is caused by a deep intronic OFD1 mutation resulting in the insertion of a cryptic exon. (A) RT–PCR of OFD1 exons 8–10 reveals an additional aberrant, larger transcript in RP23 patient-derived RNA. (B) Sequence analysis of control and patient transcripts showing that the aberrant larger transcript in RP23 patient RNA is due to the insertion of a cryptic exon (exon X) between exons 9 and 10. Schematic of normal and aberrant transcripts is shown below the electropherograms. (C) RT–PCR using specific primers to exon X produces a product in RP23 patient-derived RNA only. (D) Quantification of normal OFD1 transcript and transcript containing cryptic exon X in RP23-derived RNA (n = 5); values are mean ± 1 standard error, ***P< 0.001. (E) Quantification of the normal OFD1 8-9-10 transcript in control- and patient-derived RNA by real-time PCR. RNA levels were normalized using GAPDH (n = 6); values are mean ± 1 standard deviation, *P< 0.05.

Figure 3.

Consequence of RP23 mutation on the OFD1 gene and protein structure. (A) Intron 9-derived cryptic exon X sequence (in capitals) showing new splice acceptor (chr X: 13 768 290–13 768 291) and donor (chr X: 13 768 354–13 768 355) sites used (lowercase bold) and the IVS9+705A>G RP23 mutation (bold and underlined). Exon X causes a frameshift and premature stop codon (protein translation shown below DNA sequence). (B) The RP23 cryptic exon X transcript results in a severely truncated protein product lacking important coiled coil domains. (C) Point mutations identified in the OFD1 gene as a cause of male lethal OFD1 (arrows and arrowheads), Simpson–Golabi–Behmel syndrome type 2 (SGBS2) and Joubert syndrome (JBTS10). The position of the RP23 mutation in intron 9 is indicated.