title: Detection of novel mutations that cause autosomal dominant retinitis pigmentosa in candidate genes by long-range PCR amplification and next-generation sequencing.
abstract: OBJECTIVE
  To devise an effective method for detecting mutations in 12 genes (CA4, CRX, IMPDH1, NR2E3, RP9, PRPF3, PRPF8, PRPF31, PRPH2, RHO, RP1, and TOPORS) commonly associated with autosomal dominant retinitis pigmentosa (adRP) that account for more than 95% of known mutations.
  
  
  METHODS
  We used long-range PCR (LR-PCR) amplification and next-generation sequencing (NGS) performed in a GS Junior 454 benchtop sequencing platform. Twenty LR-PCR fragments, between 3,000 and 10,000 bp, containing all coding exons and flanking regions of the 12 genes, were obtained from DNA samples of patients with adRP. Sequencing libraries were prepared with an enzymatic (Fragmentase technology) method.
  
  
  RESULTS
  Complete coverage of the coding and flanking sequences of the 12 genes assayed was obtained with NGS, with an average sequence depth of 380× (ranging from 128× to 1,077×). Five previous known mutations in the adRP genes were detected with a sequence variation percentage between 35% and 65%. We also performed a parallel sequence analysis of four samples, three of them new patients with index adRP, in which two novel mutations were detected in RHO (p.Asn73del) and PRPF31 (p.Ile109del).
  
  
  CONCLUSIONS
  The results demonstrate that genomic LR-PCR amplification together with NGS is an effective method for analyzing individual patient samples for mutations in a monogenic heterogeneous disease such as adRP. This approach proved effective for the parallel analysis of adRP and has been introduced as routine. Additionally, this approach could be extended to other heterogeneous genetic diseases.
authors.affiliation: Molecular Genetics Unit, Hospital of Terrassa, Barcelona, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: de Sousa Dias
authors.forename: Imma
authors.initials: I
authors.lastname: Hernan
authors.forename: Beatriz
authors.initials: B
authors.lastname: Pascual
authors.forename: Emma
authors.initials: E
authors.lastname: Borràs
authors.forename: Begoña
authors.initials: B
authors.lastname: Mañé
authors.forename: Maria José
authors.initials: MJ
authors.lastname: Gamundi
authors.forename: Miguel
authors.initials: M
authors.lastname: Carballo
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D015723:Gene Library; D005799:Genes, Dominant / Q000235:genetics*; D056726:Genetic Association Studies; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods*; D012174:Retinitis Pigmentosa / Q000235:genetics*
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