April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Targeted Sequence Capture and Next Generation Sequencing of a 5Mb Region on Chromosome 15q Previously Linked to Keratoconus
Author Affiliations & Notes
  • C. E. Willoughby
    Centre for Vision and Vascular Science,
    Queen's University Belfast, Belfast, United Kingdom
    Dept of Ophthalmology, Royal Victoria Hospital, Belfast, United Kingdom
  • D. P. Dash
    Centre for Vision and Vascular Science,
    Queen's University Belfast, Belfast, United Kingdom
  • G. Silvestri
    Centre for Vision and Vascular Science,
    Queen's University Belfast, Belfast, United Kingdom
    Dept of Ophthalmology, Royal Victoria Hospital, Belfast, United Kingdom
  • J. Jackson
    Centre for Vision and Vascular Science,
    Queen's University Belfast, Belfast, United Kingdom
    Dept of Ophthalmology, Royal Victoria Hospital, Belfast, United Kingdom
  • D. G. Frazer
    Dept of Ophthalmology, Royal Victoria Hospital, Belfast, United Kingdom
  • A. E. Hughes
    Centre for Public Health,
    Queen's University Belfast, Belfast, United Kingdom
  • D. A. C. Simpson
    Centre for Vision and Vascular Science,
    Queen's University Belfast, Belfast, United Kingdom
  • Footnotes
    Commercial Relationships  C.E. Willoughby, None; D.P. Dash, None; G. Silvestri, None; J. Jackson, None; D.G. Frazer, None; A.E. Hughes, None; D.A.C. Simpson, None.
  • Footnotes
    Support  RDO (NI) RRG4.46
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 4644. doi:
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      C. E. Willoughby, D. P. Dash, G. Silvestri, J. Jackson, D. G. Frazer, A. E. Hughes, D. A. C. Simpson; Targeted Sequence Capture and Next Generation Sequencing of a 5Mb Region on Chromosome 15q Previously Linked to Keratoconus. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4644.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Our group previously mapped a large Northern Irish family affected by autosomal dominant, clinically severe keratoconus and anterior polar cataract to a 5.5Mb region on chromosome 15q22. The linkage region contains 85 positional candidate genes, 28 of which have been Sanger sequenced with no pathogenic mutation identified. To identify the molecular genetic defect in this family targeted sequence capture and next generation sequencing was performed.

Methods: : A custom Nimblegen sequence capture array was designed to capture 5Mb of the 5.5Mb region (a repetitive gene devoid 0.5Mb region was excluded). Next generation sequencing (NGS) of the captured and enriched 5Mb region was performed on the Illumina Genome Analyzer platform. NGS was performed in one affected family member, pooled DNA from affected (‘super-affected’) and unaffected (‘super-unaffected’) family members. Reference sequence assembly was completed using the ‘Genomics Workbench’ software (CLC bio). Coding sequence variants were prioritised for Sanger sequencing in family members if unique to the single and pooled affected samples and absent from pooled unaffected samples.

Results: : Sequencing coverage ranged from 8-63 fold and the heterozygote call parameters were manually adjusted using the ‘Genomics Workbench’ software. Twenty-five potentially-pathogenic coding sequence variants were identified in the single and pooled affected family members’ captured and enriched DNA from the 5Mb region on chr15q, and absent from the pooled unaffected family members’ sample and dbSNP (NCBI). These variants were present in eleven known and predicted genes within the linkage region. Variants in seven genes were excluded following Sanger sequencing on the basis of non-segregation, presence in population controls and failure to replicate NGS data. Analysis of the remaining four genes is ongoing, in addition to intronic variants surrounding splice sites.

Conclusions: : Custom targeted sequence capture followed by next generation sequencing of a linkage region in single and pooled DNA is an effective strategy to facilitate molecular characterisation and disease gene discovery.

Keywords: keratoconus • genetics • mutations 
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