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Molecular diagnosis for heterogeneous genetic diseases with targeted high-throughput DNA sequencing applied to retinitis pigmentosa
  1. David A Simpson,
  2. Graeme R Clark,
  3. Sharon Alexander,
  4. Giuliana Silvestri,
  5. Colin E Willoughby
  1. Centre for Vision and Vascular Science, Queen's University Belfast, Belfast, Northern Ireland, UK
  1. Correspondence to Dr David Simpson, Queen's University Belfast, Centre for Vision and Vascular Science, Ophthalmic Research Centre, Institute of Clinical Science A, Royal Victoria Hospital, Belfast BT12 6BA, N Ireland, UK; david.simpson{at}


Background The genetic heterogeneity of many Mendelian disorders, such as retinitis pigmentosa which results from mutations in over 40 genes, is a major obstacle to obtaining a molecular diagnosis in clinical practice. Targeted high-throughput DNA sequencing offers a potential solution and was used to develop a molecular diagnostic screen for patients with retinitis pigmentosa.

Methods A custom sequence capture array was designed to target the coding regions of all known retinitis pigmentosa genes and used to enrich these sequences from DNA samples of five patients. Enriched DNA was subjected to high-throughput sequencing singly or in pools, and sequence variants were identified by alignment of up to 10 million reads per sample to the normal reference sequence. Potential pathogenicity was assessed by functional predictions and frequency in controls.

Results and conclusions Known homozygous PDE6B and compound heterozygous CRB1 mutations were detected in two patients. A novel homozygous missense mutation (c.2957A→T; p.N986I) in the cyclic nucleotide gated channel β1 (CNGB1) gene predicted to have a deleterious effect and absent in 720 control chromosomes was detected in one case in which conventional genetic screening had failed to detect mutations. The detection of known and novel retinitis pigmentosa mutations in this study establishes high-throughput DNA sequencing with DNA pooling as an effective diagnostic tool for heterogeneous genetic diseases.

  • Diagnostics test
  • clinical genetics
  • genetic screening/counselling
  • molecular genetics
  • ophthalmology

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  • Funding This research was supported by the Health and Social Care Northern Ireland R&D of the Public Health Agency (project grant RRG 4·43).

  • Competing interests None.

  • Ethics approval This study was conducted with the approval of the Northern Ireland Research Ethics Committee.

  • Provenance and peer review Not commissioned; externally peer reviewed.