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Sushil Dubey, Perumalsamy Vijayalakshmi, Sharwan Kedia, Periasamy Sundaresan; Identification of Mutations in Candidate Genes in Patients with Globe Anomalies: A Targeted Next-Generation Sequencing Approach. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3366.
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© ARVO (1962-2015); The Authors (2016-present)
To find out the spectrum of genetic variations in candidate genes in patients with microphthalmia, anophthalmia and coloboma (MAC) in South Indian cohort.
We evaluated 85 MAC patients’ DNA samples for mutations and sequence variants in the candidate genes by two different approaches. We performed targeted re-sequencing to determine the efficacy of next-generation sequencing for the rapid screening of multiple genes in MAC patients. Targeted re-sequencing of 67 genes was done in 20 pooled samples without bar-coding. Targeted regions were captured on a 244K microarray platform and sequencing was performed on Illumina/ Solexa platform. Data analysis was done using SeqQC tool and reads were aligned against Homo sapiens genome using BWA tool. Variation detection was done using Sam tools. In addition, exons along with flanking exon- introns boundaries of RAX, OTX2, and SOX2 genes were screened in 65 MAC samples by direct sequencing. Hundred ethnically-matched control samples were sequenced to confirm the identified variants as patient specific.
The re-sequencing of 67 genes detected 80 insertions, 108 deletions and 988 SNPs/ substitution mutations and nine of these variants were confirmed by direct sequencing as population specific non-pathogenic changes. Screening by direct sequencing identified, five novel mutations in RAX, OTX2, and SOX2 genes in this cohort. A homozygous substitution mutation p. Arg179Trp, found in one bilateral anophthalmia patient, was identified in RAX gene. Three heterozygous mutations; p. Pro142Fs [c. 426insC (in a proband with bilateral anophthalmia)], p. Thr186Fs [c. 558delC (in one proband with microphthalmia in one eye and anophthalmia in other)] and a compound heterozygote p. Gln104X, p. Gln106 His (in one patient with microphthalmia) were identified in OTX2 gene. These three mutations in OTX2 gene cause premature termination of the coding sequence. Mutation p. Val303Val was identified in SOX2 gene in only one patient with microphthalmia and iris coloboma.
>96% of the targeted regions were captured and sequenced by NGS technology. Targeted re-sequencing identifies 1176 variants. Most of the variations would be Indian population specific changes and may not be playing a role in causing the disease. Five novel mutations identified in RAX, OTX2, and SOX2 suggest the role of these genes in ocular development.
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