Targeted 'next-generation' sequencing in anophthalmia and microphthalmia patients confirms SOX2, OTX2 and FOXE3 mutations.
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.
Department of Pediatrics, Division of Genetics, University of California, San Francisco, 533 Parnassus St, Room U585P, San Francisco, CA 94143-0748 USA.
Nelson Lopez
NL
Jimenez
Jason
J
Flannick
Mani
M
Yahyavi
Jiang
J
Li
Tanya
T
Bardakjian
Leath
L
Tonkin
Adele
A
Schneider
Elliott H
EH
Sherr
Anne M
AM
Slavotinek
D000595:Amino Acid Sequence; D000853:Anophthalmos / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D051858:Forkhead Transcription Factors / Q000235:genetics*; D017353:Gene Deletion; D020440:Gene Duplication; D006801:Humans; D008850:Microphthalmos / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D051857:Otx Transcription Factors / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D055748:SOXB1 Transcription Factors / Q000235:genetics*; D017422:Sequence Analysis, DNA
C404285:FOXE3 protein, human; D051858:Forkhead Transcription Factors; C497659:OTX2 protein, human; D051857:Otx Transcription Factors; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors