Downes SM, Packham E, Cranston T, Clouston P, Seller A, Németh AH. Detection Rate of Pathogenic Mutations in ABCA4 Using Direct Sequencing: Clinical and Research Implications. Arch Ophthalmol. 2012;130(11):1486-1490. doi:10.1001/archophthalmol.2012.1697
Author Affiliations: Oxford Eye Hospital (Dr Downes), Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital (Drs Downes and Németh), Oxford Regional Molecular Genetics Service (Mss Packham and Cranston and Drs Clouston and Seller), and Department of Clinical Genetics, Churchill Hospital (Dr Németh), Oxford, England.
We set out to determine the mutation detection rate in 50 subjects referred with possible Stargardt disease (STGD) using direct sequencing of the ABCA4 gene (GenBank NM_000350). Pathogenic mutations in ABCA4 have been found to cause Stargardt disease (STGD)/fundus flavimaculatus as well as some cases of cone-rod dystrophy, autosomal recessive retinitis pigmentosa, and bull’s-eye maculopathy.1,2
Mutation detection rates reported in patients are highly variable, depending on multiple factors including the phenotype and mutation detection method used.3- 5 Because next-generation sequencing is likely to be introduced into clinical diagnostics, we revisited direct (Sanger) sequencing, currently the gold standard for mutation detection, in a large group of patients with STGD to determine the number of patients receiving a confident molecular diagnosis using a sequencing approach.
Fifty patients with STGD or other possible ABCA4 retinopathies were recruited. All patients (and involved relatives) were counseled and consented according to the Declaration of Helsinki, and the study was approved by the local ethics committee. Full ophthalmic history, examination, and electrophysiology were performed. The patients' DNA was extracted, and direct (dideoxy/Sanger) sequencing of all ABCA4 exons and a 25–base pair flanking sequence was performed using standard protocols. The pathogenicity of all sequence variants was determined using standard software (Alamut version 1.5; Interactive Biosoftware). Multiplex ligation-dependent probe amplification analysis (P151 and P152 kits; MRC-Holland) was used to identify copy number variants. We determined phase and segregation when possible. A confident molecular diagnosis was defined as the presence of 2 or more pathogenic mutations.
Of 50 patients, 34 had a phenotype compatible with STGD. Of these 34 patients, 27 (79%) had at least 2 pathogenic mutations; 7 of the 34 patients had a single mutation identified, 2 of the 34 had premature termination codons (stop codon and frameshift), and 5 of the 34 had known missense mutations. The identification of a single mutation supports the clinical diagnosis but is not conclusive; however, the carrier rate is estimated to be 1 in 35 to 1 in 50 patients, so we would not expect as many as one-fifth of our patients to be carriers by chance alone. Among the 50 patients, 11 were diagnosed as having STGD by other centers in the past but on current clinical review were reclassified as shown in the Table, reflecting both progression of the disorder and the presence of phenocopies or misdiagnoses. In 5 of the 11 patients, the identification of 2 pathogenic mutations confirmed the historical diagnosis and all had chorioretinal atrophy on current clinical examination, consistent with progression of the disorder.5 One of the 11 patients with chorioretinal atrophy (subject 40) had a single stop codon, again strongly supporting the original clinical diagnosis. Six of the 11 patients did not have pathogenic mutations in ABCA4. Two of the 6 have since been found to have mutations in other genes: CDH3 in subject 416 and CRX in subject 47.7 Subject 49 (with late-onset pattern dystrophy) has a disease-associated allele of unknown mechanism and a missense variant of unknown significance, making the molecular diagnosis in this patient uncertain. In 3 of the 6 patients with a historical diagnosis of STGD and atypical phenotypes (subjects 42, 48, and 50), the molecular diagnosis remains unknown and further investigations are ongoing. Two of 5 patients with bull’s-eye maculopathy had 2 pathogenic mutations, 1 of the 5 had a single known missense mutation, and 2 of the 5 had no mutations, confirming that bull’s-eye maculopathy is genetically heterogeneous. A total of 10 novel mutations were identified (Table).
A range of phenotypes can be associated with mutations in ABCA4 ; therefore, genetic testing is important in establishing a firm diagnosis. Direct sequencing in our cohort resulted in a confident molecular diagnosis in 79% of patients with an STGD phenotype, supporting its use as a diagnostic tool. By comparison, the highest reported detection rate in a smaller group of patients was 68% using direct sequencing4 and 63.5% using arrayed primer extension.5 In patients with a historical diagnosis of STGD, more than 50% had mutations in ABCA4 testing but the remainder did not and will require further investigation. Among our patients with STGD, 21% had only a single mutation; further research is required to determine the explanation for this. In our cohort, no cases could be explained by copy number variants. Sequencing identified several novel mutations and has the highest detection rate of available technologies. Despite this, further research on the underlying genetic mechanisms in ABCA4 retinopathies is required.
Correspondence: Dr Németh, Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Level 6 West Wing, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, England (firstname.lastname@example.org).
Author Contributions: Dr Downes and Ms Packham contributed equally to the work.
Conflict of Interest Disclosures: None reported.
Funding/Support: This work was supported by the Oxford Partnership Comprehensive Biomedical Research Centre funded by the Department of Health NIHR Biomedical Research Centre Programme and by Oxford Radcliffe Hospital Flexibility and Sustainability Funding.
Disclaimer: The views expressed in this article are those of the authors and not necessarily those of the Department of Health.
Additional Contributions: We thank the subjects and their families for participating in this study and the staff of the Oxford Eye Hospital for assistance with data collection, in particular Alexina Fantato, RGN, OND, MBACP, and Clare Arnisson-Newgass, RGN.