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

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 is found in 1 in 5,000 to 10,000 individuals and is a devastating birth defect because of severe visual impairment [1]. Genetic testing to identify the cause of anophthalmia and/or microphthalmia (A/M) is frequently requested. The transcription factor SOX2 is mutated in 10-20% of patients with bilateral A/M and genomic sequencing and deletion analysis of this gene is the first test to determine the cause of severe bilateral A/M [2,3]. However, the remaining pathogenic genes implicated in A/M, such as OTX2 or GDF6, are each mutated in a small percentage of patients and more than 60% of patients with A/M do not receive a molecular diagnosis after currently available clinical genetic testing (Table 1) [4-16]. In addition, screening of all of the known A/M genes is rarely completed on a clinical basis because there is no currently ava