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Subhabrata Chakrabarti, Meha Kabra, Anil K Mandal, Sirisha Senthil, Inderjeet Kaur; Whole Exome Sequencing in Trios Indicate Novel Candidate Genes in Primary Congenital Glaucoma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2542.
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© ARVO (1962-2015); The Authors (2016-present)
Primary congenital glaucoma (PCG) is an autosomal recessive disorder of the eye and has been largely attributed to mutations in the CYP1B1 gene ranging from 20-100% worldwide. The present study aimed identify novel genes in PCG cases that are devoid of CYP1B1 mutations.
From a large number of clinically well characterized PCG cases who were devoid of CYP1B1 mutations (n=321), five trios consisting of an affected child along with parents born out of consanguineous marriages (first cousins) were selected for whole exome sequencing (WES). The WES was performed on an Ion Proton platform (Life Tech) using the whole exome AmpliSeq chemistry following the manufacturer's guidelines for library preparation and enrichment and sequencing. We sequenced at an average depth of 100X and the raw data comprising the BAM files were uploaded in the Ion reporter software for initial analysis. Further, we devised an algorithm to determine the potential homozygous candidate gene variant(s) segregating in the affected children across these 5 trios wherein, their parents were heterozygous for the variant. The variants belonging to the novel candidate genes are being replicated in the additional PCG cases (n=316) devoid of CYP1B1 mutations.
The overall sequence data revealed >50,000 variants for each trio. This was cleaned up following standard quality control methods, wherein, polymorphic variants reported in the dbSNP and 1000 genomes were excluded. Further filtering based on ClinVar database and variant effects significantly reduced the number of potential variants (n~150) Following this, only the common homozygous loci (n=12) in all the PCG affected children across these trios that were heterozygous in their unaffected parents were retained. Further functional assessment based on bioinformatic analysis of these missense variants (SIFT, polyphen and Grantham) led to the identification of two novel genes that may be involved in PCG. These variants were hihly conserved across species and absent in 300 unrelated normal controls.
These novel genes belong to the family of transcription factors (Heat shock proteins), one of which have earlier been implicated in anterior segment anomalies in an animal model. Further functional validation of these genes would help in understanding their precise role in PCG.
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