Abstract
BackgroundAnophthalmia/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.MethodsWe used a pooled sequencing design, together with custom single nucleotide polymorphism (SNP) calling software. We verified predicted sequence alterations using Sanger sequencing.ResultsWe 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.ConclusionsOur 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.
Highlights
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
The remaining pathogenic genes implicated in A/M, such as OTX2 or GDF6, are each mutated in a small percentage of patients and more
For ANOP1, we sequenced 9 known A/M genes (FOXE3, SIX3, SOX2, PAX2, PAX6, BMP4, OTX2, VSX2, and CRYBA4; gene order dictated by chromosome location)
Summary
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. Anophthalmia is found in 1 in 5,000 to 10,000 individuals and is a devastating birth defect because of severe visual impairment [1]. 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]. Next-generation sequencing has primarily been applied to Mendelian disorders in order to simplify analysis and few birth defects or
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