A splice-site mutation in a retina-specific exon of BBS8 causes nonsyndromic retinitis pigmentosa

Abstract

Tissue-specific alternative splicing is an important mechanism for providing spatiotemporal protein diversity. Here we show that an in-frame splice mutation in BBS8, one of the genes involved in pleiotropic Bardet-Biedl syndrome (BBS), is sufficient to cause nonsyndromic retinitis pigmentosa (RP). A genome-wide scan of a consanguineous RP pedigree mapped the trait to a 5.6 Mb region; subsequent systematic sequencing of candidate transcripts identified a homozygous splice-site mutation in a previously unknown BBS8 exon. The allele segregated with the disorder, was absent from controls, was completely invariant across evolution, and was predicted to lead to the elimination of a 10 amino acid sequence from the protein. Subsequent studies showed the exon to be expressed exclusively in the retina and enriched significantly in the photoreceptor layer. Importantly, we found this exon to represent the major BBS8 mRNA species in the mammalian photoreceptor, suggesting that the encoded 10 amino acids play a pivotal role in the function of BBS8 in this organ. Understanding the role of this additional sequence might therefore inform the mechanism of retinal degeneration in patients with syndromic BBS or other related ciliopathies.

Figure 1

Pedigree Drawing and Two LOD Scores Attained for Family PKRP179 during the Genome-wide Linkage Scan (A) Haplotypes for seven consecutive chromosome 14q microsatellite markers and the BBS8 splice-site mutation. The risk haplotype is shaded black, whereas alleles not cosegregating with risk haplotype are white. (B) A graphical illustration of the two-point LOD scores obtained during the genome-wide scan (note: a two-point LOD score of −4.34 corresponds to −∞). We identified significant linkage with a single marker, D14S256, which yielded a two-point LOD score of 3.12 at θ = 0.

Figure 2

Fundus Photographs and Electroretinography Responses Illustrating the Retinal Phenotype of Family PKRP179 (A–D) Fundus photographs of individual V:5 Oculus Dexter (OD) (A) and Oculus Sinister (OS) (B) and individual V:6 OD (C) and OS (D). Individual V:5 shows several features associated with RP, including a waxy pallor of the optic disc, attenuated arterioles, atrophy of RPE, and peripheral bone spicules, which are absent in unaffected individual V:6. (E–L) Electroretinography responses of individual V:5 are OD combined rod and cone response (E), OD cone response (F), OS combined rod and cone response (G), and OS cone response (H); and of individual V:6 are OD combined rod and cone response (I), OD cone response (J), OS combined rod and cone response (K) and OS cone response (L). Individual V:5 has typical RP changes, including loss of both rod and cone responses (E–H), whereas ERG readings of unaffected individual V:6 show no changes in the rod and cone response (I–L).

Figure 3

Sequence Chromatogram of the Splice-Site Mutation Identified in Family PKRP179, and the Predicted Effect of this Conserved Splice-Acceptor Site (A) Sequence chromatograms of a wild-type (top) and homozygote (bottom) for the IVS1-2A>G splice-site mutation. (B) The predicted effect of the mutation on the splicing of alternative exon 2a. The arrow points to the adenine residue mutated in family PKRP179. (C) Sequence conservation of the acceptor splice site and of exon 2a in BBS8 orthologs (primates are colored green; placental mammals are blue; vertebrates are purple). The acceptor site is conserved in 33 of 33 species with available genomic sequence for this locus, whereas the donor site at the 3′ end of the exon is conserved in 32 of 33 species. Arrow points to the splice acceptor site mutated in family PKRP179.

Figure 4

Expression Analysis of the Short and Long BBS8 mRNA Isoforms Expression levels of the short and long BBS8 mRNA isoforms (with or without exon 2a) in mouse tissue and retinal layers. (A) The BBS8 mRNA without exon 2 was detected in all tissues tested, whereas exon 2a was only found in the retinas. (B) Subsequent to laser capture microdissection and qRT-PCR, the BBS8 short message was detectable in all layers tested (GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; and RPE, retinal pigment epithelium), whereas the long isoform was found almost exclusively in the ONL. The y axis shows ratios of each transcript against GAPDH.