Leber Congenital Amaurosis–A Model for Efficient Genetic Testing of Heterogeneous Disorders: LXIV Edward Jackson Memorial Lecture

Department of Ophthalmology, The University of Iowa College of Medicine, Carver Family Center for Macular Degeneration, Iowa City, IA 52242, USA.
American Journal of Ophthalmology (Impact Factor: 3.87). 01/2008; 144(6):791-811. DOI: 10.1016/j.ajo.2007.08.022
Source: PubMed


To critically evaluate our experience in molecular testing of Leber congenital amaurosis (LCA) and to use this information to devise a general approach to heterogeneous recessive disorders. Careful clinical and molecular characterization of large cohorts of patients affected with inherited eye diseases will be an essential step in the development of effective therapy for these diseases, especially when the therapy involves gene replacement.
A molecular genetic case-control study.
Six hundred forty-two unrelated individuals with the clinical diagnosis of LCA and 200 unrelated control individuals were screened for disease-causing sequence variations in eight genes using various combinations of single-strand conformational polymorphism analysis (SSCP), automated DNA sequencing, multiplex allele-specific ligation analysis (SNPlex), and high-density solid-phase single nucleotide polymorphism genotyping.
Four hundred forty instances of 189 different disease-causing sequence variations were observed in this study, 98 of which have not been previously reported. One hundred forty-six of the 189 variations (77%) were observed in only a single individual. The observed variations were not evenly distributed among the LCA patients or among the eight genes. Empirical analysis of this uneven distribution was used to devise a multi-platform mutation detection strategy that is four times more efficient than a more conventional strategy of completely sequencing all of the coding regions of all LCA genes in all subjects. Hardy-Weinberg analysis of the observed mutations suggests that these eight genes are collectively responsible for about 70% of the cases of LCA in North America. The carrier frequency of the most common LCA allele (an intron 26 variation in CEP290) was found to be 2/3,248, which suggests that the overall prevalence of LCA in this population is about 1/81,000. An allele-specific ligation assay (SNPlex) was designed to detect 68 of the most common LCA-causing alleles, and semi-quantitative analysis of the data from this assay also revealed examples of gene deletion and isodisomy in the cohort.
The data demonstrates that a tiered screening strategy combining allele-specific detection with automated DNA sequencing can increase the efficiency of autosomal recessive mutation detection four-fold when compared with DNA sequencing alone. However, the very high rate of unique mutations observed in this study (77%) suggests that DNA sequencing will remain an important part of the overall strategy if high sensitivity is to be achieved.

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    • "Uniquely, AIPL1 proteins in primates contain a third proline-rich region (PRD) located C-terminally to the TPR domain (Fig. 1a). Mutations associated with LCA are found in all three domains of AIPL1 (Sohocki et al. 2000b; Dharmaraj et al. 2004; Stone 2007; Tan et al. 2012). The key role of AIPL1 in rods and cones is its function as a specialized chaperone for cGMP-specific phosphodiesterase- 6 (PDE6), the effector enzyme in the phototransduction cascade (Liu et al. 2004; Ramamurthy et al. 2004). "
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    ABSTRACT: Mutations in the primate-specific proline-rich domain (PRD) of aryl hydrocarbon receptor-interacting protein-like 1(AIPL1) are thought to cause Leber congenital amaurosis or dominant cone-rod dystrophy. The role of PRD and the mechanisms of PRD mutations are poorly understood. Here, we have examined properties of hAIPL1 and effects of the PRD mutations on protein structure and function. Solution structures of hAIPL1, hAIPL11-316 with PRD truncation, and the P351Δ12 and P376S mutants were examined by Small Angle X-ray Scattering. Our analysis suggests that PRD assumes an extended conformation and does not interact with the FK506-binding and tetratricopeptide domains. The PRD truncation, but not PRD mutations, reduced the molecule's radius of gyration and maximum dimension. We demonstrate that hAIPL1 is a monomeric protein, and its secondary structure and stability are not affected by the PRD mutations. PRD itself is an extended monomeric random coil. The PRD mutations caused little or no changes in hAIPL1 binding to known partners, phosphodiesterase-6A and HSP90. We also identified the γ-subunit of phosphodiesterase-6 as a novel partner of hAIPL1 and hypothesize that this interaction is altered by P351Δ12. Our results highlight the complexity of mechanisms of PRD mutations in disease and the possibility that certain mutations are benign variants. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Jul 2015 · Journal of Neurochemistry
    • "LCA is a clinically and genetically heterogeneous disease predominantly inherited in an autosomal recessive manner. The population frequency is estimated between 1/30,000 and 1/81,000 (Koenekoop, 2004; Stone, 2007) and it represents 5% of all retinal dystrophies. The clinical features of LCA include nystagmus, photophobia, amaurotic pupils, and the absence of, or severely reduced, rod and cone responses on the electroretinogram (ERG). "
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    ABSTRACT: Leber congenital amaurosis (LCA) causes severe visual impairment and blindness very early in life. Mutant alleles of several genes acting in different pathways, of which all have critical roles for normal retinal function, were involved in LCA development. The purpose of this study was to use genome-wide genotyping to identify LCA-causing loci in two Turkish families. Genome-wide genotyping and haplotype analysis were performed for prioritization of candidate genes for mutation screening in families with LCA. Identified informative critical choromosomal regions obtained by homozygosity mapping from the families were searched for overlapping of any LCA causative genes. Corresponding clinical phenotypes of the patients with identified mutations were evaluated. In this study, two families were shown to be linked to two different LCA loci covering retinol dehydrogenase 12 (RDH12) and aryl-hydrocarbon-interacting protein-like1 (AIPL1) genes. Mutation screening revealed a novel p.Gln141* mutation in the AIPL1 gene and a previously described p.Thr49Met mutation in the RDH12 gene in a homozygous state. Our patients with the RDH12 mutation had the distinct macular coloboma sign, and the patient with the AIPL1 mutation developed microphthalmia and severe widespread retinal pigment epithelial atrophy, in contrast to previously reported cases. It is currently evident that mutation screening needs to be done in at least 18 genes known to be associated with LCA. Thus, homozygosity mapping is an alternative technique to improve the molecular diagnosis in LCA, which is a group of genetically and clinically heterogeneous diseases causing retinal degeneration. The patients without mutation in known genes may further be analyzed by using next-generation sequencing.
    No preview · Article · Aug 2014 · DNA and Cell Biology
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    • "Leber congenital amaurosis (LCA; OMIM 204000) is a group of rare and severe inherited retinal dystrophies with a prevalence of ~1:50,000 individuals worldwide [1,2]. The clinical characteristics of LCA include severe and early vision loss that appears in the first year of life, amaurotic pupils, sensory nystagmus and the absence of electrical signals on electroretinogram (ERG) [3]. "
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    ABSTRACT: Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations.
    Full-text · Article · Nov 2013 · PLoS ONE
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