Autosomal Recessive Rod-Cone Dystrophy with Mild Extra-Ocular Manifestations Due to a Splice-Affecting Variant in BBS9

Abstract

Bardet-Biedl syndrome (BBS), one of the most common forms of syndromic inherited retinal diseases (IRDs), is characterized by the combination of retinal degeneration with additional extra-ocular manifestations, including obesity, intellectual disability, kidney disease, polydactyly and other skeletal abnormalities. We observed an Israeli patient with autosomal recessive apparently non-syndromic rod-cone dystrophy (RCD). Extra-ocular findings were limited to epilepsy and dental problems. Genetic analysis with a single molecule molecular inversion probes-based panel that targets the exons and splice sites of 113 genes associated with retinitis pigmentosa and Leber congenital amaurosis revealed a homozygous rare missense variant in the BBS9 gene (c.263C>T;p.(Ser88Leu)). This variant, which affects a highly conserved amino acid, is also located in the last base of Exon 3, and predicted to be splice-altering. An in vitro minigene splice assay demonstrated that this variant leads to the partial aberrant splicing of Exon 3. Therefore, we suggest that this variant is likely hypomorphic. This is in agreement with the relatively mild phenotype observed in the patient. Hence, the findings in our study expand the phenotypic spectrum associated with BBS9 variants and indicate that variants in this gene should be considered not only in BBS patients but also in individuals with non-syndromic IRD or IRD with very mild extra-ocular manifestations.

Keywords: BBS9; Bardet–Biedl syndrome; retina; retinitis pigmentosa; rod–cone dystrophy.

Figure 1

Identification of the BBS9 c.263C>T;p.(Ser88Leu) variant. (A) Pedigree of Patient R2011. The black-filled symbol represents an affected individual, whereas clear symbols represent unaffected individuals. A double line represents a consanguineous marriage. (B) Mutant and wild-type (wt) nucleotide sequence traces of the boundary between BBS9 Exon and Intron 3. The c.263C>T variant (marked by an arrow) affects the last base of Exon 3, which is part of the conserved donor splice-site. (C) Evolutionary conservation of BBS9 serine 88. The analysis was performed with The ConSurf Server [29] based on 272 BBS9 orthologues. The background color of each amino acid indicates its conservation score (see scale). Serine at position 88 has the highest conservation score, e, exposed amino acid; b, buried amino acid; f, functionally important amino acid (conserved and exposed); s, structurally important amino acid (conserved and buried). (D) Multiple sequence alignment of the region spanning the Ser88 amino acid of the BBS9 protein (highlighted in red and marked by an arrow) in various organisms. Conserved amino acids are indicated by a black background. Similar amino acids are indicated by a gray background. Also shown is the predicted sequence of the mutant protein harboring the p.Ser88Leu variant (first line).

Figure 2

Clinical findings in Patient R2011. (A,B) Color fundus photographs (OD/OS), showing arterial attenuation, diffuse peripheral nasal depigmentation and inferior macular atrophy. (C,D) Fundus autofluorescence (OD/OS), revealing a bilateral symmetric pattern of the perifoveal hyperautofluorescent ring (red arrows) and midperipheral nasal hypoautofluorescent specks (yellow arrows). (E,F) Optical coherence tomography (OCT) of the macula (OD/OS), demonstrating preserved fovea (yellow brackets) with perifoveal ellipsoid zone loss (blue arrows). The green arrows on the left images indicate the location of the cross-sections shown on the right.

Figure 3

Humphrey visual field 24-2 of left eye (A) and right eye (B) eye, revealing mild symmetric constriction, characterized by bilateral upper arcuate scotoma, corresponding to the anatomic disruption in the macula.

Figure 4

Minigene constructs and products obtained in the in vitro splicing assay. (A) An agarose gel image is shown, demonstrating the RT-PCR products derived from WT and mutant (Mut) constructs. M, size marker. (B) Shown is a schematic representation of the constructs, which include BBS9 Exons 2, 3, and 4 (represented by blue boxes) and the introns between them (represented by straight lines). Either a C or a T is present at the last base of Exon 3. The locations of primers used for RT-PCR analysis are indicated by arrows. Vector-derived sequences are shown in orange. Also shown are the obtained products, and the relative abundance of each product in each sample. Product 1 is correctly spliced; Product 2 is an aberrantly spliced product, in which a cryptic donor site located within Exon 3 was used; Product 3 is an aberrantly spliced product in which Exon 3 was completely skipped.