Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

Genet Med. 2012 Jun;14(6):576-85. doi: 10.1038/gim.2011.51. Epub 2012 Jan 26.


Purpose: 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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Antigens, Neoplasm / genetics
  • Biomarkers / analysis
  • Blindness / congenital
  • Blindness / genetics
  • Carrier Proteins / genetics
  • Case-Control Studies
  • Cell Cycle Proteins
  • Child
  • Child, Preschool
  • Consanguinity
  • Cytoskeletal Proteins
  • Exons / genetics
  • Eye Proteins / genetics
  • Genetic Heterogeneity
  • Guanylate Cyclase / genetics
  • Heterozygote
  • High-Throughput Nucleotide Sequencing / methods*
  • Homeodomain Proteins / genetics
  • Humans
  • Leber Congenital Amaurosis / diagnosis*
  • Leber Congenital Amaurosis / genetics
  • Membrane Proteins / genetics
  • Molecular Diagnostic Techniques / methods*
  • Mutation
  • Neoplasm Proteins / genetics
  • Nerve Tissue Proteins / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Receptors, Cell Surface / genetics
  • Trans-Activators / genetics
  • Validation Studies as Topic
  • cis-trans-Isomerases / genetics


  • AIPL1 protein, human
  • Adaptor Proteins, Signal Transducing
  • Antigens, Neoplasm
  • Biomarkers
  • CRB1 protein, human
  • Carrier Proteins
  • Cell Cycle Proteins
  • Cep290 protein, human
  • Cytoskeletal Proteins
  • Eye Proteins
  • Homeodomain Proteins
  • Membrane Proteins
  • Neoplasm Proteins
  • Nerve Tissue Proteins
  • Receptors, Cell Surface
  • Trans-Activators
  • cone rod homeobox protein
  • guanylate cyclase 1
  • retinoid isomerohydrolase
  • Guanylate Cyclase
  • cis-trans-Isomerases