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

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

Nelson Lopez Jimenez  1 , Jason Flannick, Mani Yahyavi, Jiang Li, Tanya Bardakjian, Leath Tonkin, Adele Schneider, Elliott H Sherr, Anne M Slavotinek
Affiliations

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

Nelson Lopez Jimenez et al. BMC Med Genet. .

Abstract

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.

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Figures

Figure 1
Figure 1
Mutations in the coding sequence of anophthalmia genes in ANOP1 and ANOP2 patients. Figure 1A. Chromatogram showing c.542delC in SOX2 in patient 09-122, predicted as c.539A > D. Figure 1B. Chromatogram showing c.313C > T in OTX2, predicting p.Gln105X in patient 792-508. Figure 1C. Chromatogram showing c. 720C > A in FOXE3, predicting p.Cys240X in patient 792-563.
See this image and copyright information in PMC

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