Article

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: 4.02). 01/2008; 144(6):791-811. DOI: 10.1016/j.ajo.2007.08.022
Source: PubMed

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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    • "A deficiency of functional RPE65 results in failure of the retinoid cycle and a lack of 11-cis-retinal supply from the retinal pigment epithelium to the photoreceptors. Normal visual pigment formation cannot occur; resulting in a phenotype of severely reduced rod and cone photoreceptor sensitivity and associated visual impairment (Stone, 2007). Briard dogs with a null mutation in Rpe65 develop a similar phenotype to that of RPE65-LCA patients (Narfströ m et al., 1989; Wrigstad et al., 1994; Aguirre et al., 1998; Veske et al., 1999). "
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    ABSTRACT: Aims: Young Rpe65-deficient dogs have been used as a model for human RPE65 Leber congenital amaurosis (RPE65-LCA) in proof-of-concept trials of recombinant adeno-associated virus (rAAV) gene therapy. However, there are relatively few reports of the outcome of rAAV gene therapy in Rpe65-deficient dogs older than two years of age. The purpose of this study was to investigate the success of this therapy in older Rpe65-deficient dogs. Results: Thirteen eyes were treated in dogs aged between 2 and 6 years. A rAAV2 vector expressing the human RPE65 cDNA driven by the human RPE65 promoter was delivered by subretinal injection. Twelve of the thirteen eyes had improved retinal function as assessed by electroretinography and all showed improvement in vision at low lighting intensities. Histologic examination of 5 of the eyes was performed but found no correlation between ERG rescue and numbers of remaining photoreceptors. Conclusions: We conclude that functional rescue is still possible in older dogs and that the use of older Rpe65-deficient dogs, rather than young Rpe65-deficient dogs that have very little loss of photoreceptors more accurately models the situation when treating human RPE65-LCA patients.
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    • "Leroy & Dharmaraj (2003) on the other hand reports that the vision loss in individual with LCA may vary greatly from relatively mild acuity problems (20 ⁄ 80) to no light perception , depending on the genes associated with LCA. Retinitis Pigmentosa is a term used to refer to another clinically and genetically heterogeneous group of retinal degenerations that are closely related to LCA (Stone 2007). Lotery et al. (2001) claimed that 'the distinction between LCA and RP is largely based on age of onset of visual dysfunction . "
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    • "The p.Tyr142Cys variant was previously described as a mutation (Vallespin et al., 2007a). Stone and colleagues, however, considered this variant as a polymorphism based on the estimate of pathogenic probability and the identification of this variant in a patient with two disease-causing alleles in another LCA gene (Stone, 2007) (LCA-63). The pathogenicity of the second variant p.Val242Met also remains unclear (LCA-64) (Swain et al., 1997; Rivolta et al., 2001; Chen et al., 2002). "
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