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Mutations in AIPL1 are associated with Leber Congenital Amaurosis (LCA), a major cause of childhood blindness, yet the cellular function of the encoded protein has yet to be fully elucidated. In order to investigate the biochemistry of AIPL1, we have developed a system for the expression of the recombinant protein in bacteria and its subsequent purification. The secondary structure and thermostability of wild-type and mutant proteins have been examined by circular dichroism (CD) spectroscopy. Some of the variants, notably W278X and P376S, had markedly different secondary structure compositions, indicating that the proteins had not folded properly, whilst W278X and T114I were particularly thermolabile. When eukaryotic cells were transfected with the AIPL1 expression constructs, we show by immunofluorescence microscopy that wild-type protein is distributed throughout the nucleus and cytoplasm. Several of the mutants give similar results. With two of the disease-associated variants (W278X and A336Δ2), however, the protein remains in the cytoplasm in aggresome-like particles. These particles were shown to be ubiquitinated, indicating that the mutant protein had been tagged for proteosomal degradation. On this basis, we can conclude that wild-type protein is expressed in a soluble and folded manner, and that some of the disease-associated mutant proteins are nonfunctional because they are insoluble and are degraded by the cell. Other mutations appear to have a more localised effect on secondary structure, which does not result in insolubility or affect protein targeting, but reduces the stability of the protein at human body temperature.

Background: Leber's congenital amaurosis (LCA) represents the earliest and severest form of retinal dystrophy leading to congenital blindness. A total of 20% of children attending blind schools have this disease. LCA has a multigenic basis and is proving central to our understanding of the development of the retina. We describe the clinical and molecular genetic features of four inbred pedigrees from neighbouring remote villages in northern Pakistan, in which some of the affected members have concurrent keratoconus. Methods: History-taking and physical and eye examinations were performed in the field. Venipuncture, DNA extraction, studies of linkage to known LCA genes, automated sequencing and polymorphism analyses for haplotype assessments were done. Results: We examined 12 affected and 15 unaffected family members. By history, there were an additional nine blind people in the four pedigrees. In each pedigree a consanguineous marriage was evident. We found a homozygous nonsense mutation in the AIPLI gene, which replaces a tryptophan with a stop codon (Trp278X). The phenotype is severe and variable, despite the common molecular genetic etiology in each family. Affected patients had hand motion to no light perception vision and fundus findings ranging from maculopathy to diffuse pigmentary retinopathy. Three affected members had definite keratoconus, and two were suspects based on mild cone formation in the cornea of at least one eye. Interpretation: We have identified four Pakistani families with a severe form of LCA that is associated with severe keratoconus in some affected members. The molecular etiology in all four families is a homozygous nonsense mutation, Trp278X, in the photoreceptor—pineal gene AIPLI . To our knowledge, this is one of the first phenotype—genotype correlations of AIPLI -associated LCA.

Inherited retinopathies are a genetically and phenotypically heterogeneous group of diseases affecting approximately one in 2000 individuals worldwide. For the past 10 years, the Laboratory for Molecular Diagnosis of Inherited Eye Diseases (LMDIED) at the University of Texas-Houston Health Science Center has screened subjects ascertained in the United States and Canada for mutations in genes causing dominant and recessive autosomal retinopathies. A combination of single strand conformational analysis (SSCA) and direct sequencing of five genes (rhodopsin, peripherin/RDS, RP1, CRX, and AIPL1) identified the disease-causing mutation in approximately one-third of subjects with autosomal dominant retinitis pigmentosa (adRP) or with autosomal dominant cone-rod dystrophy (adCORD). In addition, the causative mutation was identified in 15% of subjects with Leber congenital amaurosis (LCA). Overall, we report identification of the causative mutation in 105 of 506 (21%) of unrelated subjects (probands) tested; we report five previously unreported mutations in rhodopsin, two in peripherin/RDS, and one previously unreported mutation in the cone-rod homeobox gene, CRX. Based on this large survey, the prevalence of disease-causing mutations in each of these genes within specific disease categories is estimated. These data are useful in estimating the frequency of specific mutations and in selecting individuals and families for mutation-specific studies.

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration that affects approximately 1 in 3500 people worldwide. Recently we identified the gene responsible for the RP1 form of autosomal dominant retinitis pigmentosa (adRP) at 8q11-12 and found two different nonsense mutations in three families previously mapped to 8q. The RP1 gene is an unusually large protein, 2156 amino acids in length, but is comprised of four exons only. To determine the frequency and range of mutations in RP1 we screened probands from 56 large adRP families for mutations in the entire gene. After preliminary results indicated that mutations seem to cluster in a 442 nucleotide segment of exon 4, an additional 194 probands with adRP and 409 probands with other degenerative retinal diseases were tested for mutations in this region alone. We identified eight different disease-causing mutations in 17 of the 250 adRP probands tested. All of these mutations are either nonsense or frameshift mutations and lead to a severely truncated protein. Two of the eight different mutations, Arg677X and a 5 bp deletion of nucleotides 2280-2284, were reported previously, while the remaining six mutations are novel. We also identified two rare missense changes in two other families, one new polymorphic amino acid substitution, one silent substitution and a rare variant in the 5'-untranslated region that is not associated with disease. Based on this study, mutations in RP1 appear to cause at least 7% (17/250) of adRP. The 5 bp deletion of nucleotides 2280-2284 and the Arg677X nonsense mutation account for 59% (10/17) of these mutations. Further studies will determine whether missense changes in the RP1 gene are associated with disease, whether mutations in other regions of RP1 can cause forms of retinal disease other than adRP and whether the background variation in either the mutated or wild-type RP1 allele plays a role in the disease phenotype.

Two frequent protein variants of glutamate pyruvate transaminase (GPT) (E.C.2.6.1.2) have been used as genetic markers in humans for more than two decades, although chromosomal mapping of the GPT locus in the 1980s produced conflicting results. To resolve this conflict and develop useful DNA markers for this gene, we isolated and characterized cDNA and genomic clones of GPT. We have definitively mapped human GPT to the terminus of 8q using several methods. First, two cosmids shown to contain the GPT sequence were derived from a chromosome 8-specific library. Second, by fluorescencein situhybridization, we mapped the cosmid containing the human GPT gene to chromosome band 8q24.3. Third, we mapped the ratgptcDNA to the syntenic region of rat chromosome 7. Finally, PCR primers specific to human GPT amplify sequences contained within a “half-YAC” from the long arm of chromosome 8, that is, a YAC containing the 8q telomere. The human GPT genomic sequence spans 2.7 kb and consists of 11 exons, ranging in size from 79 to 243 bp. The exonic sequence encodes a protein of 495 amino acids that is nearly identical to the previously reported protein sequence of human GPT-1. The two polymorphic GPT isozymes are the result of a nucleotide substitution in codon 14, coding for a histidine in GPT-1 and an asparagine in GPT-2, which causes a gain or loss of anNlaIII restriction site. In addition, a cosmid containing the GPT sequence also contains a previously unmapped, polymorphic microsatellite sequence, D8S421. The cloned GPT gene and associated polymorphisms will be useful for linkage and physical mapping of disease loci that map to the terminus of 8q, including atypical vitelliform macular dystrophy (VMD1) and epidermolysis bullosa simplex, type Ogna (EBS1). In addition, this will be a useful system for characterizing the telomeric region of 8q. Finally, determination of the molecular basis of the GPT isozyme variants will permit PCR-based detection of this worldwide polymorphism.

Mutations in AIPL1 cause Leber congenital amaurosis (LCA), the most severe form of inherited blindness in children; however, the function of this protein in normal vision remains unknown. To determine amino acid subsequences likely to be important for function, we have compared the protein sequence of several species. Sequence conservation is highest across the three Aipl1 tetratricopeptide (TPR) motifs and extends across the protein, except for a proline-rich amino acid sequence present only at the C-terminus of primate Aipl1. The length of the proline-rich region varies within primates; however, the length differences between human and primate Aipl1 do not correlate with evolutionary distance. These observations reinforce the importance of the TPR domains for function, the similarity of Aipl1 to a family of proteins that act as molecular chaperones, and the importance of comparative sequencing data for determination of whether AIPL1 sequence variants in patients are likely to cause retinopathy.

Leber congenital amaurosis (LCA) is the most severe inherited retinopathy, with the earliest age of onset. Because this currently incurable disease is present from birth and is a relatively rare disorder, the development of animal models that closely resemble the phenotype in patients is especially important. Our previous genetic analyses of LCA patients identified mutations in the aryl-hydrocarbon interacting protein-like 1 (AIPL1) gene. Here we present development of an animal model of AIPL1-associated LCA, the Aipl1-deficient mouse. Aipl1 is expressed at low levels throughout human and mouse retinal development and is rapidly upregulated as photoreceptors differentiate. The mouse displays rapid retinal degeneration and massive Muller cell gliosis, resembling the phenotype of the rd mouse, which is caused by a mutation in the gene for the β-subunit of the rod-specific phosphodiesterase. We confirm that this phenotype is consistent with the human disease using electroretinograms, and document the disease pathogenesis by analyzing the development of all retinal cell types and synaptogenesis during retinal histogenesis. Ectopic expression of AIPL1 led to deregulated retinal progenitor cell proliferation and alterations in cell fate specification; however, no gross abnormalities of proliferation during retinal development were detected. Data from analysis of proliferation and cell fate specification during retinal development of Aipl1-deficient mice suggests that there may be redundancy or compensation for Aipl1 loss by other related proteins. Because this mouse model closely mimics the human retinopathy caused by homozygous mutations in this gene, it provides a preclinical model for testing therapies to rescue the vision of children whose blindness is caused by AIPL1 mutations.

Objectives: To describe the phenotype of Leber congenital amaurosis (LCA) in 26 probands with mutations in aryl hydrocarbon receptor interacting protein-like 1 protein (AIPL1) and compare it with phenotypes of other LCA-related genes. To describe the electroretinogram (ERG) in heterozygote carriers. Methods: Patients with AIPL1-related LCA were identified in a cohort of 303 patients with LCA by polymerase chain reaction single-strand confirmational polymorphism mutation screening and/or direct sequencing. Phenotypic characterization included clinical and ERG evaluation. Seven heterozygous carrier parents also underwent ERG testing. Results: Seventeen homozygotes and 9 compound heterozygotes were identified. The W278X mutation was most frequent (48% of alleles). Visual acuities ranged from light perception to 20/400. Variable retinal appearances, ranging from near normal to varying degrees of chorioretinal atrophy and intraretinal pigment migration, were noted. Atrophic and/or pigmentary macular changes were present in 16 (80%) of 20 probands. Keratoconus and cataracts were identified in 5 (26%) of 19 patients, all of whom were homozygotes. The ERG of a parent heterozygote carrier revealed significantly reduced rod function, while ERGs for 6 other carrier parents were normal. Conclusions: The phenotype of LCA in patients with AIPL1 mutations is relatively severe, with a maculopathy in most patients and keratoconus and cataract in a large subset. Rod ERG abnormalities may be present in heterozygous carriers of AIPL1 mutations. Clinical Relevance: Understanding and recognizing the phenotype of LCA may help in defining the course and severity of the disease. Identifying the gene defect is the first step in preparation for therapy since molecular diagnosis in LCA will mandate the choice of treatment.

Leber congenital amaurosis (LCA) is often considered the most severe inherited retinopathy, and AIPL1 was the fourth gene identified as associated with LCA. Although the function of AIPL1 is unknown, it has been reported to interact with NUB1. Here, we searched for a NUB1-binding site on AIPL1 and located it between amino acid residues 181 and 330 in AIPL1. Importantly, many LCA-associated mutations of AIPL1 have been found at this site. Hence, we hypothesized that the interaction between NUB1 and AIPL1 is affected in patients with LCA. To test this possibility, we used three different assays to investigate the interaction between NUB1 and the AIPL1 mutants associated with LCA. Some of the AIPL1 mutants did not interact with NUB1, suggesting that abolishment of this interaction is involved in the pathogenesis of LCA. Other AIPL1 mutants, however, did interact with NUB1, suggesting that other molecules are also involved in the pathogenesis.

Leber congenital amaurosis (LCA) is a genetically heterogeneous, autosomal recessive retinal degenerative disease responsible for approximately 5% of all inherited retinopathies (Kaplan et al., 1990). LCA is often considered the most severe form of childhood retinopathy, and infants with this disease are usually blind at birth. To date, mutations in six genes have been found to cause LCA and two additional loci have been mapped (www.sph.uth.tmc.edu/RetNet/). Recently, we identified the fourth LCA associated gene, the aryl hydrocarbon receptor interacting protein-like 1(AIPL1)(Sohocki et al., 2000a). Mutations in AIPL1 are estimated to account for approximately 7-9% of LCA cases worldwide and have been found to cause autosomal dominant cone-rod dystrophy (Sohocki et al., 2000b). Mutations in the aryl hydrocarbon receptor-interacting protein-like 1(AIPL1)gene have been found to cause autosomal recessive Leber congenital amaurosis (LCA), an early onset form of retinal degeneration. AsAIPL1is a novel gene, it is necessary to determine its normal function to better understand how mutations in this gene lead to disease.

A Dutch family with autosomal dominant retinitis pigmentosa (adRP) displayed a phenotype characterized by an early age of onset, a diffuse loss of rod and cone sensitivity, and constricted visual fields (type I). One male showed a mild progression of the disease. Linkage analysis showed cosegregation of the genetic defect with markers from chromosome 17p13.1-p13.3, a region overlapping the RP13 locus. The critical interval of the RP locus as defined in this family was flanked by D17S926 and D17S786, with a maximal lod score of 4.2 (theta = 0.00) for marker D17S1529. Soon after the mapping of the underlying defect to the 17p13 region, a missense mutation (6970G>A; R2310K) was identified in exon 42 of the splicing factor gene PRPC8 in one patient of this family. Diagnostic restriction enzyme digestion of exon 42 amplified from genomic DNA of all family members revealed that the R2310K mutation segregated fully with the disease. The type I phenotype observed in this family is similar to that described for three other RP13 families with mutations in PRPC8.

Leber congenital amaurosis (LCA, OMIM No. 204000) is a severe, early-onset inherited retinopathy that accounts for approximately 5% of all inherited retinal disease. To date, four genes associated with Leber congenital amaurosis have been identified, the most recent of which is the aryl-hydrocarbon interacting-like 1 gene, AIPL1 (OMIM No. 604392,604393) (Sohocki et al., 2000). Mutations in AIPL1 are the cause of 7 to 9% of cases of autosomal recessive LCA and may cause dominant cone-rod dystrophy (Sohocki et al., 2000a) (Figures 1 and 2).

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy and the most frequent cause of inherited blindness in children. LCA is usually inherited in an autosomal recessive fashion, although rare dominant cases have been reported. One form of LCA, LCA4, maps to chromosome 17p13 and is genetically distinct from other forms of LCA, We recently identified the gene associated with LCA4, AIPL1 (aryl-hydrocarbon interacting protein-like 1) and identified three mutations that were the cause of blindness in five families with LCA, In this study, AIPL1 was screened for mutations in 512 unrelated probands with a range of retinal degenerative diseases to determine if AIPL1 mutations cause other forms of inherited retinal degeneration and to determine the relative contribution of AIPL1 mutations to inherited retinal disorders in populations worldwide. We identified 11 LCA families whose retinal disorder is caused by homozygous or compound heterozygous AIPL1 mutations. We also identified affected individuals in two apparently dominant families, diagnosed with juvenile retinitis pigmentosa or dominant cone-rod dystrophy, respectively, who are heterozygous for a 12-bp AIPL1 deletion. Our results suggest that AIPL1 mutations cause approximately 7% of LCA worldwide and may cause dominant retinopathy, (C) 2000 Academic Press.

STSs designed to the retina/pineal-expressed EST clusters THC220430 and THC90422 were originally mapped to 17p13.3 (ref. 7) near a retinitis pigmentosa (RP13) candidate region8. Further testing refined the localization to 17p13.1, between SHGC-2251 and SHGC-6095, within the LCA4 candidate region and approximately 2.5 Mb distal to GUCY2D. Fluorescence in situ hybridization (Fig. 1) confirmed the localization. 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 ... cDNA sequencing of the two clusters indicated that the ESTs represent transcripts of one gene. THC90422 transcripts bypass the THC220430 polyadenylation signal, resulting in a 3′ UTR longer by 709 bp. The 180-bp 5′ UTR and coding sequence encoded by the six-exon gene are identical in the 1,538-bp and 2,247-bp transcripts (Fig. 2a). 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 ... The protein encoded by AIPL1 was named human aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) due to its similarity (49% identity, 69% positive) to human aryl hydrocarbon receptor-interacting protein (AIP), a member of the FK506-binding protein (FKBP) family9 (Fig. 2b). The predicted protein consists of 384 amino acids, with a 43,865-dalton molecular mass, and a 5.57 pI. The protein sequence includes three tetratricopeptide repeats (TPR), a 34–amino-acid motif found in proteins with nuclear transport or protein chaperone activity9. Northern-blot hybridization identified mRNA molecules of the predicted sizes in total retinal RNA. The probe also cross-hybridized to 18s rRNA (Fig. 3) in the retina. We detected a weaker signal in skeletal muscle and heart on a poly(A)+ RNA multi-tissue northern blot after very long exposure. It is likely that this signal represents cross-hybridization, as the transcripts differ in size from the retinal mRNAs and are faint. The northern-blot did not indicate AIPL1 expression in brain, but only cerebral tissue was included in the blot. In situ hybridization indicates expression in rat and mouse pineal gland, a high level of expression in adult mouse photoreceptors (Fig. 4) and no expression in cornea (data not shown). 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 ... 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 ... Sequencing of the rat Aipl1 cDNA revealed amino acid sequence conservation (87% identity and 96% similarity) between rat and human AIPL1. Rat Aipl1, mouse Aip and human AIP lack a 56–amino-acid carboxy-terminal extension present in AIPL1 (Fig. 2b). This extension includes a ‘hinge’ motif of high flexibility, with multiple O-glycosylation sites, and a casein kinase II (CK2) phosphorylation site, which may be involved in protein complex regulation (as is the CK2 site within the hinge of another FKBP family member, FKBP52; ref. 10). The hinge appears to be conserved in primates, as it is also present in the squirrel monkey (Saimiri sciureus; data not shown). Single-stranded conformational analysis (SSCA) identified three benign nucleotide substitutions within the AIPL1 exon 3 amplimer: G/A at −14, G/A at −10 bp and G/A at codon 100 (Leu100Leu, CTG/CTA). We identified four haplotypes for the combined polymorphisms; the most common, GCG and GAA, have frequencies of 55% and 41%, respectively. Sequencing of AIPL1 from the DNA of one affected individual of the original LCA4 family (Fig. 5a) revealed a homozygous nonsense mutation (Trp278X, TGG→TGA). This allele, if expressed, encodes a protein shorter by 107 amino acids than wild-type AIPL1. The truncated protein includes only 20 of the 34 amino acids of the third TPR motif, a region conserved between human, rat and mouse AIPL1, and AIP. SSCA in other family members confirmed that all affected family members are homozygous for this mutation (Fig. 5a) and that 100 ethnically matched controls did not carry this mutation. 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 ... AIPL1 was next analysed in another Pakistani family, MD (Fig. 5a), whose LCA had been mapped to 17p13.1, with GUCY2D excluded by mutational analysis. Sequencing of AIPL1 indicated that affected individuals of this family are homozygous for the Trp278X mutation (Fig. 5a). The MD and KC families differ in haplotype (GCG and GAA, respectively) of the AIPL1 exon 3 polymorphisms, as well as for microsatellite markers tightly linked to AIPL1. These results suggest that the Trp278X mutations causing the LCA in these two families are not derived from a recent, common ancestor. Assay of AIPL1 in 14 families of European descent with LCA that had not been tested previously for linkage to 17p identified apparent disease-causing mutations in three additional families, as follows. Direct sequencing of AIPL1 in the two affected individuals of family RFS121 indicated two mutations, a 2-bp deletion in codon 336 (Ala336Δ2 bp; Fig. 5b) and Trp278X. The deletion results in a frameshift and a termination delayed by 47 codons. The termination signal used in the deletion transcript is upstream of the first AIPL1 polyadenylation signal; therefore, the alternate transcripts from this allele are not predicted to encode alternate proteins. Allele-specific PCR in one affected individual confirmed that the 2-bp deletion and Trp278X are on opposite chromosomes. Therefore, the affected individuals in RFS121 are compound heterozygotes, having received the Trp278X mutation from one parent and the Ala336Δ2 mutation from the other. No unaffected RFS121 family members inherited both mutations. The Ala336Δ2 bp mutation was not observed in 55 unrelated control individuals of European descent. AIPL1 sequencing in two affected individuals from family RFS127 (Fig. 5a) indicated homozygous Trp278X mutations—the same mutation identified in the KC and MD families. Haplo-type analysis of tightly linked microsatellite markers and of the AIPL1 exon 3 polymorphisms suggest that the mutations in the RFS127 and MD families are likely to have descended from a common ancestor; however, there is no indication of Pakistani origin for members of this family. The three affected individuals of family RFS128 (Fig. 5c) are homozygous for a T→C nucleotide substitution predicted to encode a Cys239Arg substitution. This cysteine is conserved in human and rat AIPL1, and in AIP (Fig. 2). This mutation was not identified in over 55 ethnically matched control individuals. Affected members of this family are homozygous for microsatellite markers D17S796 and D17S1881, which are tightly linked, flanking markers of AIPL1. In contrast, affected family members are heterozygous for microsatellite markers D17S960 and D17S1353, which flank GUCY2D. We have identified a new gene that causes LCA4. We detected homozygous AIPL1 mutations in three families in which GUCY2D was excluded as the cause of the disease by linkage or mutation screening: KC, MD and RFS128. AIPL1 is the fourth gene to be associated with LCA. Mutations in AIPL1 may be a common cause of LCA, as an AIPL1 mutation was identified as the apparent cause of the retinal disease in 3 of 14 (21±8%, 90% C.I.) unmapped LCA families. AIPL1 should be assayed in LCA families whose disease locus maps to 17p13 but do not carry disease-causing mutations in GUCY2D, as in 7 of 15 original LCA1 families4. Due to the proximity of AIPL1 and GUCY2D on 17p13, linkage mapping may not distinguish between the genes. Further, is possible that LCA patients who are identical by descent (IBD) at one locus are also IBD at the other. Therefore, both AIPL1 and GUCY2D should be screened for mutations in families whose LCA locus maps to 17p13 or in families with affected individuals who are homozygous for mutations in either gene, unless linkage excludes one of the genes. Of the five families reported here, GUCY2D was excluded by linkage testing or mutation screening in three, the fourth is a compound heterozygote and the fifth is homozygous for a disease-causing mutation confirmed in other families. The similarity of AIPL1 to AIP and the presence of three TPR motifs suggest that it may be involved in retinal protein folding or trafficking. Its role in the pineal gland is also uncertain. The pineal gland contributes to resetting circadian rhythm by diurnal release of melatonin. Additionally, children with destructive pinealomas often display precocious puberty, suggesting a role in long-term periodicity11. Because LCA patients with AIPL1 mutations have grossly abnormal photoreceptors at an early age, the pineal gland also may be affected. Careful clinical characterization of LCA4 patients may reveal pineal-associated abnormalities. Therefore, identifying the exact role of AIPL1 in photoreceptors and the pineal gland will improve our understanding of disease pathology in these patients, and contribute to our understanding of the biology of normal vision and pineal activity.

The goal of our research is to determine the genes and mutations causing autosomal dominant retinitis pigmentosa (adRP) and related diseases. As is now common knowledge, this deceptively-simple goal is confounded by the exceptional heterogeneity of retinitis pigmentosa and other forms of retinal degeneration. This heterogeneity includes allelic heterogeneity, i.e., different mutations in the same gene causing different clinical phenotypes or modes of inheritance; genetic heterogeneity, i.e., different genes causing similar diseases; and clinical heterogeneity, i.e., the same gene—even the same allele—causing disimilar diseases in different individuals, even within the same family.

Mutations in the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) gene have been found in patients with Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. To determine the normal function of AIPL1 and to better understand how mutations in this gene cause disease, we performed a yeast two-hybrid screen to identify AIPL1-interacting proteins in the retina. One of the identified interacting proteins corresponds to NUB1 (NEDD8 Ultimate Buster 1), which is thought to control many biological events, especially cell cycle progression, by downregulating NEDD8 expression. The AIPL1–NUB1 interaction was verified by co-immunoprecipitation studies in Y79 retinoblastoma cells, demonstrating that this interaction occurs within cells that share a number of features with retinal progenitor cells. Furthermore, we examined the localization of the AIPL1 protein within developing and adult retinas, and found that AIPL1 is present in the developing photoreceptor layer of the human retina and within the photoreceptors of the adult retina. Similar to AIPL1, NUB1 is also expressed in the developing and adult retina. Therefore, it is possible that the early-onset form of retinal degeneration seen in LCA patients with AIPL1 mutations may be due to a defect in the regulation of cell cycle progression during photoreceptor maturation. These data raise the possibility that AIPL1 is important for appropriate photoreceptor formation during development and/or survival following differentiation.

SummaryMutations in the retinal-expressed gene CRX (cone-rod homeobox gene) have been associated with dominant cone-rod dystrophy and with de novo Leber congenital amaurosis. However, CRX is a transcription factor for several retinal genes, including the opsins and the gene for interphotoreceptor retinoid binding protein. Because loss of CRX function could alter the expression of a number of other retinal proteins, we screened for mutations in the CRX gene in probands with a range of degenerative retinal diseases. Of the 294 unrelated individuals screened, we identified four CRX mutations in families with clinical diagnoses of autosomal dominant cone-rod dystrophy, late-onset dominant retinitis pigmentosa, or dominant congenital Leber amaurosis (early-onset retinitis pigmentosa), and we identified four additional benign sequence variants. These findings imply that CRX mutations may be associated with a wide range of clinical phenotypes, including congenital retinal dystrophy (Leber) and progressive diseases such as cone-rod dystrophy or retinitis pigmentosa, with a wide range of onset.