Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis

Abstract

Leber congenital amaurosis (LCA, MIM 204000) accounts for at least 5% of all inherited retinal disease and is the most severe inherited retinopathy with the earliest age of onset. Individuals affected with LCA are diagnosed at birth or in the first few months of life with severely impaired vision or blindness, nystagmus and an abnormal or flat electroretinogram (ERG). Mutations in GUCY2D (ref. 3), RPE65 (ref. 4) and CRX (ref. 5) are known to cause LCA, but one study identified disease-causing GUCY2D mutations in only 8 of 15 families whose LCA locus maps to 17p13.1 (ref. 3), suggesting another LCA locus might be located on 17p13.1. Confirming this prediction, the LCA in one Pakistani family mapped to 17p13.1, between D17S849 and D17S960-a region that excludes GUCY2D. The LCA in this family has been designated LCA4 (ref. 6). We describe here a new photoreceptor/pineal-expressed gene, AIPL1 (encoding aryl-hydrocarbon interacting protein-like 1), that maps within the LCA4 candidate region and whose protein contains three tetratricopeptide (TPR) motifs, consistent with nuclear transport or chaperone activity. A homozygous nonsense mutation at codon 278 is present in all affected members of the original LCA4 family. AIPL1 mutations may cause approximately 20% of recessive LCA, as disease-causing mutations were identified in 3 of 14 LCA families not tested previously for linkage.

Fig. 1

Fluorescence in situ hybridization (FISH). AIPL1-containing bacterial artificial chromosome (BAC), shown in red, hybridizes to 17p13.1, consistent with placement of AIPL1 in the Stanford G3 radiation hybrid panel. These data refute the original placement of AIPL1 to 17p13.3 by placement in the GeneBridge 4.0 radiation hybrid panel. Chromosome 17 α-satellite DNA is indicated (green).

Fig. 2

Gene and protein structure of AIPL1. a, AIPL1 consists of six exons, with alternate polyadenylation sites in the 3′ UTR shown by arrows. Cys239Arg denotes the location of the TGC→CGC missense mutation in exon 5 of the RFS128 family. Trp278X denotes the location of the TGG→TGA nonsense mutation in exon 6 of the KC, MD, RFS127 and RFS121 families. Ala336Δ2 denotes the location of the 2-bp deletion in exon 6 of RFS121. Benign coding sequence substitutions identified were Phe37Phe (TTT/TTC; 0.98/0.02 frequency), Cys89Cys (TGC/TGT; 0.99/0.01), Asp90His (GAC/CAC; 0.84/0.16), Leu100Leu (CTG/CTA; 0.57/0.43) and Pro217Pro (CCG/CCA; 0.61/0.39) b, Protein sequence of AIPL1. The alignment demonstrates the high level of sequence conservation between rat and human AIPL1, and mouse and human AIP. Identical residues in the four sequences are noted with an asterisk; identical residues in three of the sequences are indicated with a period.

Fig. 3

Expression of AIPL1 in human tissues. Northern blots from adult tissues were incubated with an AIPL1 probe. Total retinal RNA blot (top left) and poly(A)⁺ RNA multi-tissue northern (MTN, top right) are shown. No signal was observed in MTN at 4-, 24- or 48-h exposure. 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. Both blots were incubated with a β-actin probe as a control (bottom). Bold arrows indicate mRNA molecules of the predicted sizes, 1,538 and 2,247 bp, in retina.

Fig. 4

Retina and pineal expression of Aipl1. a, Digoxygenin in situ hybridization of Aipl1 in adult mouse retina, with expression throughout the outer nuclear layer and photoreceptor inner segments. b, Sense control of (a) with same reaction time. A slight background signal is observed across photoreceptor outer segments. c, Short (16 h) colour reaction of Aipl1 in adult mouse retina, showing a high level of mRNA in photoreceptor inner segments. d, Expression of Aipl1 in adult mouse pineal gland.e, Sense control of (d), with same reaction time. f, Expression of Aipl1 mRNA in P14 rat pineal. g, Sense control of (f), with same reaction time. Scale bar for a–c, 30 μm; d,e, 50 μm; f,g, 70 μm. RPE, retinal pigment epithelium; OS, outer photoreceptor segment; IS, inner photoreceptor segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Immunolocalization of the AIPL1 protein has not been performed; therefore, site of AIPL1 protein localization is currently unknown.

Fig. 5

Pedigrees and mutation screen of AIPL1 in families. a, The Trp278X mutation is homozygous in three families: KC, MD and RFS127. SSCA of all living individuals of the KC pedigree demonstrate segregation of the mutant allele. Top electropherogram, an unaffected control (TGG/TGG); middle, heterozygous G/A mutation at codon 278; bottom, DNA sequence of a homozygous, affected member of MD (TGA/TGA). b, The RFS121 affected individuals are compound heterozygotes for the Trp278X and Ala336Δ2 bp mutations. Top electropherogram, unaffected control; bottom, heterozygous G/A mutation at codon 278 (left) and heterozygous 2-bp deletion beginning in codon 336 (right) in an affected individual of RFS121. c, The Cys239Arg mutation found in family RFS128. Top electopherogram, unaffected control (TGC/TGC); bottom, DNA sequence of a homozygous affected individual (CGC/CGC).

Fig. 6

Fundus photograph of affected LCA patient (11 y). Typical symptoms of LCA are present: widespread retinal pigment epithelium changes with pigment clumping, attenuated retinal vessels, pale optic disk and macular atrophy. Members of the KC family also display keratoconus. Because AIPL1 is not expressed in the cornea, it is possible that this symptom is secondary to LCA in this family, due to eye rubbing and so on.