title: Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis.
abstract: OBJECTIVE
  Leber congenital amaurosis (LCA) is a rare congenital retinal dystrophy associated with 16 genes. Recent breakthroughs in LCA gene therapy offer the first prospect of treating inherited blindness, which requires an unequivocal and early molecular diagnosis. While present genetic tests do not address this due to a tremendous genetic heterogeneity, massively parallel sequencing (MPS) strategies might bring a solution. Here, we developed a comprehensive molecular test for LCA based on targeted MPS of all exons of 16 known LCA genes.
  
  
  METHODS
  We designed a unique and flexible workflow for targeted resequencing of all 236 exons from 16 LCA genes based on quantitative PCR (qPCR) amplicon ligation, shearing, and parallel sequencing of multiple patients on a single lane of a short-read sequencer. Twenty-two prescreened LCA patients were included, five of whom had a known molecular cause.
  
  
  RESULTS
  Validation of 107 variations was performed as proof of concept. In addition, the causal genetic defect and a single heterozygous mutation were identified in 3 and 5, respectively, of 17 patients without previously identified mutations.
  
  
  CONCLUSIONS
  We propose a novel targeted MPS-based approach that is suitable for accurate, fast, and cost-effective early molecular testing in LCA, and easily applicable in other genetic disorders.
authors.affiliation: Center for Medical Genetics Ghent, Ghent University, Ghent, Belgium.
authors.forename: Frauke
authors.initials: F
authors.lastname: Coppieters
authors.forename: Bram
authors.initials: B
authors.lastname: De Wilde
authors.forename: Steve
authors.initials: S
authors.lastname: Lefever
authors.forename: Ellen
authors.initials: E
authors.lastname: De Meester
authors.forename: Nina
authors.initials: N
authors.lastname: De Rocker
authors.forename: Caroline
authors.initials: C
authors.lastname: Van Cauwenbergh
authors.forename: Filip
authors.initials: F
authors.lastname: Pattyn
authors.forename: Françoise
authors.initials: F
authors.lastname: Meire
authors.forename: Bart P
authors.initials: BP
authors.lastname: Leroy
authors.forename: Jan
authors.initials: J
authors.lastname: Hellemans
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
authors.forename: Elfride
authors.initials: E
authors.lastname: De Baere
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000951:Antigens, Neoplasm / Q000235:genetics; D015415:Biomarkers / Q000032:analysis; D001766:Blindness / Q000151:congenital / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D016022:Case-Control Studies; D018797:Cell Cycle Proteins; D002648:Child; D002675:Child, Preschool; D003241:Consanguinity; D003598:Cytoskeletal Proteins; D005091:Exons / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics; D018740:Genetic Heterogeneity; D006162:Guanylate Cyclase / Q000235:genetics; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D057130:Leber Congenital Amaurosis / Q000175:diagnosis* / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D011956:Receptors, Cell Surface / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D054928:Validation Studies as Topic; D019745:cis-trans-Isomerases / Q000235:genetics; D000099178:Retinoid Isomerohydrolase
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000951:Antigens, Neoplasm; D015415:Biomarkers; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006162:Guanylate Cyclase; D018398:Homeodomain Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D015534:Trans-Activators; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C403625:AIPL1 protein, human; C448939:CRB1 protein, human; C493903:Cep290 protein, human; C109288:cone rod homeobox protein; C110715:guanylate cyclase 1