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Jonathan Han, Ricardo Frausto, Michelle Kim, Anthony Aldave; Identification by whole-exome next-generation sequencing of coding region mutations as candidates for posterior amorphous corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4733.
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To identify the causative coding region variant of posterior amorphous corneal dystrophy (PACD) by whole-exome sequencing of the 12q21.33-q23.1 PACD locus.
Slit-lamp examination was performed on 54 members of a multigenerational pedigree with PACD to determine their affected status. Peripheral blood samples were collected from each family member, and extracted genomic DNA from selected affected and unaffected individuals were used for whole-exome sequencing. Both the alignment of the paired-end reads and the variant discovery were conducted within the Partek Flow software. A list of candidate variants comprising those identified within the PACD locus (bounded by the D12S1812 and D12S1051 genetic markers) was generated using the Partek Genomic Suite software. Twenty-eight variants were then compared to previous Sanger sequencing results using a spreadsheet application to determine the number of false positives and false negatives at two different coverage thresholds.
Fifteen individuals were diagnosed as affected based on characteristic clinical features. Whole-exome sequencing was performed on genomic DNA from 5 affected and 1 unaffected individuals. Analysis of the sequencing data revealed 12 single nucleotide variants (SNV) within 9 genes that were present in each of the affected samples but not in the unaffected sample. Six of the 12 SNVs were coding region variants. In addition, two indels were identified that were present only in the affected individuals: a 3’UTR insertion and a coding region deletion. By comparing variants previously identified in the same 5 affected and 1 unaffected individuals using Sanger sequencing to those identified with whole-exome sequencing, 23/30 variants (23.3% false negative rate) were identified at a coverage threshold 10 and 27/30 variants (10.0% false negative rate) were identified at a coverage threshold of 5. No false positives occurred at either threshold.
We report one of the initial applications of next-generation resequencing technology to resequence a candidate region previously associated with a corneal dystrophy. The relatively low number of identified variants presents a reasonable cohort of candidate variants that will be confirmed by Sanger sequencing, followed by determination of segregation in the remaining 10 affected and 38 unaffected individuals.
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