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Brett Deml, Linda Reis, Elena Semina; Whole exome sequencing identifies novel mutations in OTX2 , CRYBA4, and PAX6 in microphthalmia patients. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3364.
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© ARVO (1962-2015); The Authors (2016-present)
Anophthalmia and microphthalmia (A/M) are defined as the complete absence or reduction in size of the eye and has a combined incidence between 1-3.2 cases per 10,000 live births. A/M is a genetically heterogeneous disorder that has been associated with mutations in at least 35 genes with SOX2 representing the major causative factor. Approximately 60% of A/M patients lack a molecular diagnosis. We submitted nine SOX2-negative A/M patients for whole exome sequencing to screen for mutations in known genes as well as identify novel genes involved in A/M.
DNA samples from nine A/M patients were analyzed by whole exome sequencing using Agilent Sure Select v4+UTR platform for exome capture and PerkinElmer sequencing service. Base/variant calls were made using the Geospiza software package and patients were examined for mutations in 100 known ocular genes.
Heterozygous mutations, confirmed with Sanger sequencing, were identified in three of the nine patients. A novel deletion, c.651delC, predicted to lead to a frameshift, p.(Thr218Hisfs*76), was identified in OTX2 in a patient with bilateral anophthlamia. A novel substitution, c.158+1 G>A, predicted to lead to aberrant splicing was identified in intron 3 of CRYBA4 in a patient with microphthalmia, congenital cataracts, and coloboma of the iris, cornea, and retina. Another novel mutation was identified in the homeodomain of PAX6, c.767 T>C (p.Val256Ala), in a patient, and his similarly affected brother, with microphthalmia, iris hypoplasia, sclerocornea, coloboma, congenital glaucoma, and aphakia.
The deletion in OTX2 is the most C-terminal deletion reported to date. The c.158+1 G>A mutation is the first splice site mutation identified in CRYBA4. To the best of our knowledge, this is the first report of coloboma of the iris, cornea and retina in association with CRYBA4 mutation. The p.Val256Ala PAX6 allele is associated with a more severe phenotype than typically reported for missense mutations and represents only the sixth missense variant to be reported in the PAX6 homeodomain. Due to the severity of the phenotype the absence of a second mutation was confirmed by sequencing the complete PAX6 gene. Analysis of the remaining six unexplained patients is ongoing. These results demonstrate the efficiency of whole exome sequencing to identify causative mutations for a genetically heterogeneous disorder, such as A/M.
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