A homozygous missense mutation in the IRBP gene (RBP3) associated with autosomal recessive retinitis pigmentosa

Abstract

Purpose: Interphotoreceptor retinoid-binding protein (IRBP) has been considered essential for normal rod and cone function, as it mediates the transport of retinoids between the photoreceptors and the retinal pigment epithelium. This study was performed to determine whether mutations in the IRBP gene (RBP3) are associated with photoreceptor degeneration.

Methods: A consanguineous family was ascertained in which four children had autosomal recessive retinitis pigmentosa (RP). Homozygosity mapping performed with SNP microarrays revealed only one homozygous region shared by all four affected siblings. Sequencing of RBP3, contained in this region, was performed in this family and others with recessive RP. Screening was also performed on patients with various other forms of retinal degeneration or malfunction.

Results: Sequence analysis of RBP3 revealed a homozygous missense mutation (p.Asp1080Asn) in the four affected siblings. The mutation affects a residue that is completely conserved in all four homologous modules of the IRBP protein of vertebrate species and in C-terminal-processing proteases, photosynthesis enzymes found in bacteria, algae, and plants. Based on the previously reported crystal structure of Xenopus IRBP, the authors predict that the Asp1080-mediated conserved salt bridge that appears to participate in scaffolding of the retinol-binding domain is abolished by the mutation. No RBP3 mutations were detected in 395 unrelated patients with recessive or isolate RP or in 680 patients with other forms of hereditary retinal degeneration.

Conclusions: Mutations in RBP3 are an infrequent cause of autosomal recessive RP. The mutation Asp1080Asn may alter the conformation of the IRBP protein by disrupting a conserved salt bridge.

Figure 1

Molecular genetic analysis in a consanguineous family with autosomal recessive RP. (A) Pedigree of the family; the parents were first cousins and had no history of visual loss. All four affected siblings shared a 9-Mb homozygous region on chromosome 10, whereas the unaffected sibling was not homozygous for alleles in this interval. (B) Sequence analysis of RBP3 identified a homozygous missense mutation (c.3238G>A, p.Asp1080Asn) in exon 2 in all affected individuals. Arrow: mutant base.

Figure 2

Alignment of the four IRBP repeat modules of vertebrate species, and C-terminal-processing proteases of bacteria, algae, and plants. The black highlighting indicates identical residues, the gray highlighting indicates conserved residues, and no highlighting indicates residues that are not conserved. Asp1080 is a completely identical residue among all these sequences. The multiple sequence alignment was made with ClustalW using the Blosum scoring matrix, and shaded with BoxShade 3.21 (all provided in the public domain by the EMBnet, Swiss Institute of Bioinformatics, Lausanne, Switzerland, http://www.ch.embnet.org/index.html).

Figure 3

Clinical characteristics of two affected siblings demonstrated variability in the severity of the disease. The older brother (II-1) was less severely affected than the younger brother was (II-3). (A) More clumped and bone spicule pigment was noted on ophthalmoscopy in the younger (right) versus the older (left) brother. (B) Visual fields of the right eyes of the patients are illustrated. Visual fields were more severely diminished in the younger brother (II-3; right) than in the older brother (II-1; left). (C) Full-field ERGs from both brothers (columns 2 and 3) reveal profound loss of rod function (0.5 Hz blue) and some remaining cone function (30-Hz flicker) compared with normal (column 1). The panels show the right eye of these patients at their most recent visits. Three consecutive sweeps are overlaid to illustrate ERG response reproducibility, three successive responses to a flash of light, or, in the case of the cone, the flicker.

Figure 4

Molecular modeling of the Asp1080Asn mutation. (A) Structural modeling suggests that the highly conserved aspartic acid (D) and arginine (R) residues form a salt bridge in the scaffold of the retinol-binding pocket. This ribbon structure represents the second module of Xenopus IRBP (X2IRBP) docked with all-trans retinol (carbons, magenta). Blue: the X2IRBP N-terminal region; red: C-terminal region. D462, the aspartic acid residue corresponding to D1080 in human IRBP, and R464 are shown in stick representation (oxygen, red; nitrogen, blue). A probable salt bridge forms between the carboxamide side group of D462 and guanidinium group of R464 (dotted line). (B) Stereo view showing the relationship between the retinol-binding pocket and the conserved D and R residues (The image may be viewed in 3-D without specialized stereo glasses. Suggestions for viewing molecular stereo images are found at http://spdbv.vital-it.ch/themolecularlevel/0help/stereoview.html).

Figure 5

Haplotype analysis in the family members of an isolate patient with sector RP. (A) A heterozygous nonsense mutation (c.1216G>T, p.Glu406X) was identified in the patient and in his unaffected brother, who carried the same haplotype on the other allele. (B) Sequence chromatogram showing the heterozygous nonsense mutation identified in this family.