title: Advantage of Whole Exome Sequencing over Allele-Specific and Targeted Segment Sequencing in Detection of Novel TULP1 Mutation in Leber Congenital Amaurosis.
abstract: BACKGROUND
  Leber congenital amaurosis (LCA) is a severe form of retinal dystrophy with marked underlying genetic heterogeneity. Until recently, allele-specific assays and Sanger sequencing of targeted segments were the only available approaches for attempted genetic diagnosis in this condition. A broader next-generation sequencing (NGS) strategy, such as whole exome sequencing, provides an improved molecular genetic diagnostic capacity for patients with these conditions.
  
  
  METHODS
  In a child with LCA, an allele-specific assay analyzing 135 known LCA-causing variations, followed by targeted segment sequencing of 61 regions in 14 causative genes was performed. Subsequently, exome sequencing was undertaken in the proband, unaffected consanguineous parents and two unaffected siblings. Bioinformatic analysis used two independent pipelines, BWA-GATK and SOAP, followed by Annovar and SnpEff to annotate the variants.
  
  
  RESULTS
  No disease-causing variants were found using the allele-specific or targeted segment Sanger sequencing assays. Analysis of variants in the exome sequence data revealed a novel homozygous nonsense mutation (c.1081C > T, p.Arg361*) in TULP1, a gene with roles in photoreceptor function where mutations were previously shown to cause LCA and retinitis pigmentosa. The identified homozygous variant was the top candidate using both bioinformatic pipelines.
  
  
  CONCLUSIONS
  This study highlights the value of the broad sequencing strategy of exome sequencing for disease gene identification in LCA, over other existing methods. NGS is particularly beneficial in LCA where there are a large number of causative disease genes, few distinguishing clinical features for precise candidate disease gene selection, and few mutation hotspots in any of the known disease genes.
authors.affiliation: a Center for Applied Genomics, The Children's Hospital of Philadelphia , Philadelphia , PA , USA .
authors.forename: Yiran
authors.initials: Y
authors.lastname: Guo
authors.affiliation: b Eye and Developmental Genetics Research Group, Western Sydney Genetics Program, The Children's Hospital at Westmead , Sydney , NSW , Australia .|c Children's Medical Research Institute , Westmead , Sydney , NSW , Australia .
authors.forename: Ivan
authors.initials: I
authors.lastname: Prokudin
authors.affiliation: d College of Life Sciences, Sichuan University, Key Laboratory for Bio-resources and Eco-environment of Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology , Chengdu , PR China .|e BGI-Shenzhen , Shenzhen , China .
authors.forename: Cong
authors.initials: C
authors.lastname: Yu
authors.affiliation: e BGI-Shenzhen , Shenzhen , China .
authors.forename: Jinlong
authors.initials: J
authors.lastname: Liang
authors.affiliation: e BGI-Shenzhen , Shenzhen , China .
authors.forename: Yi
authors.initials: Y
authors.lastname: Xie
authors.affiliation: f Department of Ophthalmology , The Children's Hospital at Westmead , Sydney , NSW , Australia .
authors.forename: Maree
authors.initials: M
authors.lastname: Flaherty
authors.affiliation: a Center for Applied Genomics, The Children's Hospital of Philadelphia , Philadelphia , PA , USA .
authors.forename: Lifeng
authors.initials: L
authors.lastname: Tian
authors.affiliation: f Department of Ophthalmology , The Children's Hospital at Westmead , Sydney , NSW , Australia .
authors.forename: Stephanie
authors.initials: S
authors.lastname: Crofts
authors.affiliation: a Center for Applied Genomics, The Children's Hospital of Philadelphia , Philadelphia , PA , USA .
authors.forename: Fengxiang
authors.initials: F
authors.lastname: Wang
authors.affiliation: a Center for Applied Genomics, The Children's Hospital of Philadelphia , Philadelphia , PA , USA .
authors.forename: James
authors.initials: J
authors.lastname: Snyder
authors.affiliation: f Department of Ophthalmology , The Children's Hospital at Westmead , Sydney , NSW , Australia .
authors.forename: Craig
authors.initials: C
authors.lastname: Donaldson
authors.affiliation: a Center for Applied Genomics, The Children's Hospital of Philadelphia , Philadelphia , PA , USA .
authors.forename: Nada
authors.initials: N
authors.lastname: Abdel-Magid
authors.affiliation: a Center for Applied Genomics, The Children's Hospital of Philadelphia , Philadelphia , PA , USA .
authors.forename: Lyam
authors.initials: L
authors.lastname: Vazquez
authors.affiliation: a Center for Applied Genomics, The Children's Hospital of Philadelphia , Philadelphia , PA , USA .|g Division of Human Genetics , The Children's Hospital of Philadelphia , Philadelphia , PA , USA .|h Department of Pediatrics , The Perelman School of Medicine, University of Pennsylvania , Philadelphia , PA , USA .
authors.forename: Brendan
authors.initials: B
authors.lastname: Keating
authors.affiliation: a Center for Applied Genomics, The Children's Hospital of Philadelphia , Philadelphia , PA , USA .|g Division of Human Genetics , The Children's Hospital of Philadelphia , Philadelphia , PA , USA .|h Department of Pediatrics , The Perelman School of Medicine, University of Pennsylvania , Philadelphia , PA , USA .
authors.forename: Hakon
authors.initials: H
authors.lastname: Hakonarson
authors.affiliation: e BGI-Shenzhen , Shenzhen , China .|i Department of Biology , University of Copenhagen , Copenhagen , Denmark .|j King Abdulaziz University , Jeddah , Saudi Arabia .
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: b Eye and Developmental Genetics Research Group, Western Sydney Genetics Program, The Children's Hospital at Westmead , Sydney , NSW , Australia .|c Children's Medical Research Institute , Westmead , Sydney , NSW , Australia .|k Discipline of Ophthalmology & Save Sight Institute, University of Sydney , Sydney , Australia , and.|l Disciplines of Paediatrics and Child Health & Genetic Medicine, University of Sydney , Sydney , NSW , Australia.
authors.forename: Robyn V
authors.initials: RV
authors.lastname: Jamieson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002648:Child; D018389:Codon, Nonsense; D003241:Consanguinity; D004252:DNA Mutational Analysis; D004596:Electroretinography; D059472:Exome / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057130:Leber Congenital Amaurosis / Q000175:diagnosis / Q000235:genetics* / Q000503:physiopathology; D008969:Molecular Sequence Data; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide; D014792:Visual Acuity / Q000502:physiology
chemical_list: D018389:Codon, Nonsense; D005136:Eye Proteins; C105443:TULP1 protein, human
keywords: Leber congenital amaurosis; TULP1; retinitis pigmentosa; whole exome sequencing