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Open Access Research article

Targeted 'Next-Generation' sequencing in anophthalmia and microphthalmia patients confirms SOX2, OTX2 and FOXE3 mutations

Nelson Lopez Jimenez1, Jason Flannick2, Mani Yahyavi1, Jiang Li3, Tanya Bardakjian4, Leath Tonkin5, Adele Schneider4, Elliott H Sherr3 and Anne M Slavotinek1*

Author Affiliations

1 Department of Pediatrics, Division of Genetics, University of California, San Francisco, 533 Parnassus St, Room U585P, San Francisco CA 94143-0748 USA

2 Broad Institute of Harvard and MIT, Cambridge MA Massachusetts General Hospital, Boston, Massachusetts USA

3 Department of Neurology, University of California, San Francisco, San Francisco, California 94143-0114 USA

4 Division of Genetics, Department of Pediatrics, Albert Einstein Medical Center, Philadelphia, Pennsylvania 19141 USA

5 Vincent J. Coates Genomics Sequencing Laboratory (GSL) QB3/University of California, Berkeley USA B206 Stanley Hall MC 3220 Berkeley, CA 94720-3220

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BMC Medical Genetics 2011, 12:172  doi:10.1186/1471-2350-12-172

Published: 28 December 2011



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.


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


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.


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.

anophthalmia; microphthalmia; next-generation sequencing; SOX2; OTX2; FOXE3