Mutations in a novel retina-specific gene cause autosomal dominant retinitis pigmentosa

Abstract

Inherited retinal diseases are a common cause of visual impairment in children and young adults, often resulting in severe loss of vision in later life. The most frequent form of inherited retinopathy is retinitis pigmentosa (RP), with an approximate incidence of 1 in 3,500 individuals worldwide. RP is characterized by night blindness and progressive degeneration of the midperipheral retina, accompanied by bone spicule-like pigmentary deposits and a reduced or absent electroretinogram (ERG). The disease process culminates in severe reduction of visual fields or blindness. RP is genetically heterogeneous, with autosomal dominant, autosomal recessive and X-linked forms. Here we have identified two mutations in a novel retina-specific gene from chromosome 8q that cause the RP1 form of autosomal dominant RP in three unrelated families. The protein encoded by this gene is 2,156 amino acids and its function is currently unknown, although the amino terminus has similarity to that of the doublecortin protein, whose gene (DCX) has been implicated in lissencephaly in humans. Two families have a nonsense mutation in codon 677 of this gene (Arg677stop), whereas the third family has a nonsense mutation in codon 679 (Gln679stop). In one family, two individuals homozygous for the mutant gene have more severe retinal disease compared with heterozygotes.

Fig. 1

Gene and protein structure of RP1. a, RP1 consists of four exons, three of which (exons 2–4) contain coding sequence. All disease-causing mutations and polymorphic sites are in exon 4. b, Protein sequence of RP1 (2,156 aa). c, Protein alignment of RP1. The partial alignment demonstrates a conserved region in the first 370 residues of RP1 that shares similarity with several eukaryotic proteins, including: a unique protein fragment deduced from a retina-specific EST cluster consensus (STACK accession number eye2931), with 50% identity over 86 residues; human DCX, with 25% identity over 328 residues; a human protein of unknown function (KIAA0369), with 20% identity over 707 residues; mouse Dcx, with 25% identity over 328 residues; a C. elegans predicted gene product (W07G1.5), with 25% identity over 332 residues; and Schizosaccharomyces pombe predicted protein product CAB9768.1, with 17% identity over 195 residues. Colouring represents degree of physiochemical conservation of residues as follows: purple, proline/glycine; yellow, cysteine; blue, hydrophilic; light blue, aromatic; green, negative; red, positive; pink, aliphatic/hydrophobic. A continuous line over the RP1 sequence represents matches to Prosite nuclear localization signal PS50079.

Fig. 2

Sequence electropherograms of mutations found in three adRP families. a, The Arg677stop mutation found in American family UCLA-RP01 and Australian family D. Top, an unaffected control (CGA/CGA). Middle, heterozygous C/T mutation at codon 677 (CGA/TGA). Bottom, DNA sequence of a homozygous, affected member of UCLA-RP01 (TGA/TGA). This mutation destroys a TaqI site. b, The Gln679stop mutation in English family UK-RP1. Normal sequence (top; CAA/CAA) and the heterozygous C/T mutation (bottom; CAA/TAA) are shown. This mutation destroys a Cac8I site.

Fig. 3

Pedigree and mutation screen of a consanguineous nuclear family in UCLA-RP01. a, Pedigree. Disease phenotypes in this portion of the family suggest that two individuals are homozygous for the disease mutation. Open symbols are unaffected individuals (dd), hatched symbols are affected heterozygotes (Dd) and filled symbols are severely affected homozygotes (DD). The parents of the two severely affected individuals are third cousins once removed. b, Segregation of the Arg677 stop mutation. Digestion of amplimer F with TaqI in an unaffected individual produces a 248-bp fragment and an 81-bp fragment. The CGA→TGA change at codon 677 eliminates the TaqI site, producing an undigested band of 329 bp. Unaffected individuals have the two lower bands, heterozygous affected individuals have three bands and homozygous affected individuals have one large, undigested fragment. M, molecular weight marker.

Fig. 4

Expression of RP1 transcript in human tissues. We incubated northern blots containing poly(A)⁺ RNA from human adult tissues with an RP1 probe. Lane 1, adult retina; lane 2, heart; lane 3, whole brain; lane 4, placenta; lane 5, lung; lane 6, liver; lane 7, skeletal muscle; lane 8, kidney; lane 9, pancreas. A 2-h exposure is shown. A 42-h exposure reveals nothing over background in lanes 2–9. As control, the same blots were incubated with a human β-actin probe.