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Frauke Coppieters, Bram De Wilde, Ellen De Meester, Steve Lefever, Nina De Rocker, Bart P. Leroy, Jo Vandesompele, Elfride De Baere; Leber Congenital Amaurosis: Development Of A Comprehensive Molecular Genetic Test Panel Using Next-generation Sequencing. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3315.
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Leber Congenital Amaurosis (LCA) is caused by mutations in 16 known genes, which together account for approximately 70% of cases. Most often genetic testing for LCA is limited to screening of a selected number of mutations and/or Sanger sequencing of a subset of genes. The goal of this study was to design an accurate, fast and affordable molecular test for all known LCA genes using next-generation sequencing (NGS).
We developed a novel protocol consisting of quantitative PCR (qPCR) amplification followed by ligation and fragmentation, sequencing on a Genome Analyzer IIx run, data analysis by NextGENe software. Ten LCA patients were included, five of which had a known molecular defect through prior screening.
Our proof-of-concept study consisted of resequencing of all exons of 16 LCA genes (RD3, RPE65, CRB1, MERTK, IQCB1, LRAT, LCA5, TULP1, IMPDH1, CEP290, RPGRIP1, RDH12, SPATA7, AIPL1, GUCY2D and CRX) in 10 LCA patients. Using in-house developed primer design, 375 primer pairs were designed to cover 236 exons and their intron-exon boundaries. Following amplification, ligation and shearing, all amplicon pools were indexed and together sequenced in a single lane of a Genome Analyzer IIx run (1x100bp). This yielded sufficient coverage for 93-95%% of exons. In total, 104 out of 107 previously identified variants were detected using the NextGENe software, including the known mutations in the positive control group. In addition, mutations were found in two out of five mutation-negative patients.
We developed a novel, cost-efficient workflow for enrichment and parallel sequencing of all currently known LCA genes. The protocol combines reliable and efficient high-throughput qPCR amplification with reproducible ligation and shearing steps, thereby enabling sequencing of regions of interest with variable length, such as exons, on a short-read sequencer. Moreover, the flexibility of the workflow allows easy expansion of the panel with other genes, providing an excellent basis for molecular testing of other retinal dystrophies. Finally, our protocol will allow an early molecular diagnosis in LCA patients, which is essential with respect to reproductive issues, prognosis and eligibility to gene therapy, given recent breakthroughs in gene therapy for RPE65-related LCA.
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