Targeted 'next-generation' sequencing in anophthalmia and microphthalmia patients confirms SOX2, OTX2 and FOXE3 mutations

BMC Med Genet. 2011 Dec 28:12:172. doi: 10.1186/1471-2350-12-172.


Background: Anophthalmia/microphthalmia (A/M) is caused by mutations in several different transcription factors, but mutations in each causative gene are relatively rare, emphasizing the need for a testing approach that screens multiple genes simultaneously. We used next-generation sequencing to screen 15 A/M patients for mutations in 9 pathogenic genes to evaluate this technology for screening in A/M.

Methods: We used a pooled sequencing design, together with custom single nucleotide polymorphism (SNP) calling software. We verified predicted sequence alterations using Sanger sequencing.

Results: We verified three mutations - c.542delC in SOX2, resulting in p.Pro181Argfs*22, p.Glu105X in OTX2 and p.Cys240X in FOXE3. We found several novel sequence alterations and SNPs that were likely to be non-pathogenic - p.Glu42Lys in CRYBA4, p.Val201Met in FOXE3 and p.Asp291Asn in VSX2. Our analysis methodology gave one false positive result comprising a mutation in PAX6 (c.1268A > T, predicting p.X423LeuextX*15) that was not verified by Sanger sequencing. We also failed to detect one 20 base pair (bp) deletion and one 3 bp duplication in SOX2.

Conclusions: Our results demonstrated the power of next-generation sequencing with pooled sample groups for the rapid screening of candidate genes for A/M as we were correctly able to identify disease-causing mutations. However, next-generation sequencing was less useful for small, intragenic deletions and duplications. We did not find mutations in 10/15 patients and conclude that there is a need for further gene discovery in A/M.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Anophthalmos / genetics*
  • Base Sequence
  • DNA Mutational Analysis
  • Forkhead Transcription Factors / genetics*
  • Gene Deletion
  • Gene Duplication
  • Humans
  • Microphthalmos / genetics*
  • Molecular Sequence Data
  • Mutation*
  • Otx Transcription Factors / genetics*
  • Polymorphism, Single Nucleotide
  • SOXB1 Transcription Factors / genetics*
  • Sequence Analysis, DNA


  • FOXE3 protein, human
  • Forkhead Transcription Factors
  • OTX2 protein, human
  • Otx Transcription Factors
  • SOX2 protein, human
  • SOXB1 Transcription Factors