Leber congenital amaurosis - a model for efficient genetic testing of heterogeneous disorders: LXIV Edward Jackson Memorial Lecture.
ABSTRACT 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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ABSTRACT: Spontaneous canine models exist for several inherited retinal dystrophies. This review will summarize the models and indicate where they have been used in translational gene therapy trials. The RPE65 gene therapy trials to treat childhood blindness are a good example of how studies in dogs have contributed to therapy development. Outcomes in human clinical trials are compared and contrasted with the result of the preclinical dog trials.02/2015; 26(1). DOI:10.1089/humc.2014.155
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ABSTRACT: Inherited retinal diseases are uncommon pathologies and one of the most harmful causes of childhood and adult blindness. Leber congenital amaurosis (LCA) is the most severe kind of these diseases accounting for approximately 5% of the whole retinal dystrophies and 20% of the children that study on blind schools. Clinical ophthalmologic findings including severe vision loss, nystagmus and ERG abnormalities should be suspected through the first year of life in this group of patients. Phenotypic variability is found when LCA patients have a full ophthalmologic examination. However, a correct diagnosis may be carried out; the determination of ophthalmologic clues as light sensibility, night blindness, fundus pigmentation, among other, join with electroretinographics findings, optical coherence tomography, and new technologies as molecular gene testing may help to reach to a precise diagnosis. Several retinal clinical features in LCA may suggest a genetic or gene particular defect; thus genetic-molecular tools could directly corroborate the clinical diagnosis. Currently, approximately 20 genes have been associated to LCA. In this review, historical perspective, clinical ophthalmological findings, new molecular-genetics technologies, possible phenotype-genotypes correlations, and gene therapy for some LCA genes are described.02/2015; 3(2):112-24. DOI:10.12998/wjcc.v3.i2.112
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ABSTRACT: Retinal membrane guanylyl cyclase 1 (RetGC1) regulated by guanylyl cyclase activating proteins (GCAPs) controls photoreceptor recovery and when mutated causes blinding disorders. We evaluated the principal models of how GCAP1 and GCAP2 bind RetGC1 - through a shared docking interface versus independent binding sites formed by distant portions of the cyclase intracellular domain. At near-saturating concentrations, GCAP1 and GCAP2 activated RetGC1 from HEK293 cells and RetGC2(-/-)GCAPs1,2(-/-) mouse retinas in a non-additive fashion. The Met26Arg GCAP1, which binds but does not activate RetGC1, suppressed activation of recombinant and native RetGC1 by competing with both GCAP1 and GCAP2. Untagged GCAP1 displaced both GCAP1-GFP and GCAP2-GFP from the complex with RetGC1 in HEK293 cells. The intracellular segment of a natriuretic peptide receptor A (NPRA) guanylyl cyclase failed to bind GCAPs, but replacing its kinase-homology (KHD) and dimerization (DD) domains with those from RetGC1 restored GCAP1 and GCAP2 binding by the hybrid cyclase and its GCAP-dependent regulation. Deletion of the Tyr1016-Ser1103 fragment in RetGC1 did not block GCAP2 binding to the cyclase. In contrast, substitutions in KHD, Trp708Arg and Ile734Thr, linked to Leber congenital amaurosis (LCA) prevented binding of both GCAP1GFP and GCAP2GFP. Our results demonstrate that GCAPs cannot regulate RetGC1 using independent primary binding sites. Instead, GCAP1 and GCAP2 bind with the cyclase molecule in a mutually exclusive manner using a common or overlapping binding site(s) in the Arg488-Arg851 portion of RetGC1, and mutations in that region causing congenital LCA blindness disrupt activation of the cyclase by both GCAP1 and GCAP2. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 01/2015; 290(11). DOI:10.1074/jbc.M114.629642 · 4.60 Impact Factor