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John Kuchtey, Rachel W. Kuchtey; Exome Sequencing A Family With Primary Congenital Glaucoma. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4511.
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The purpose of this study is to identify the causative genetic variant in a family with recessive inheritance of primary congenital glaucoma (PCG).
DNA samples were obtained from a non-consanguineous family of European ancestry comprised of the unaffected parents, the proband and his 3 siblings, one of which is also affected by PCG. The known PCG-causing genes, CYP1B1 and LTBP2, were sequenced in the affected siblings by conventional Sanger DNA sequencing. The exons and flanking intronic sequence of all known genes were sequenced for all 6 family members by targeted capture of exons and next generation sequencing. Exome capture and library preparation was carried with the Agilent 50 Mb exome capture kit. The exome libraries were sequenced using the Illumina HiSeq instrument with 75 base pair reads and paired ends. Each patient’s library was sequenced in a single flow cell lane, with all samples sequenced in a single flow cell run. The program BWA was used to align paired-end sequence reads to the reference human genome. The GATK analysis package was used for SNP and indel calling.
Initial screening of CYP1B1 and LTBP2 by Sanger DNA sequencing revealed no potentially causative variants. Exome sequencing resulted in an average across samples of 220 million 75-bp reads aligning to the genome, 84% of which aligned to the capture target, resulting in an average read depth of 227-fold. Using a simple autosomal recessive model of inheritance and filtering based on genotype quality score >30, initial analysis revealed 80 SNPs that segregate with disease and are not present in dbSNP build 132. Of these 80 SNPs, only 2 resulted in amino acid changes.
The absence of causative variants in the known PCG-associated genes suggests that the disease allele in this family will identify a novel glaucoma gene. Filtering exome sequence data based on novelty and inheritance within this 6-member pedigree results in a manageable number of variants for analysis and may identify a novel gene involved in PCG. Exome sequencing to identify a PCG-causing gene has not been previously reported.
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