Dataset Information

Identification of Potentially Pathogenic Variants in the Posterior Polymorphous Corneal Dystrophy 1 Locus.

ABSTRACT: Posterior polymorphous corneal dystrophy 1 (PPCD1) is a genetic disorder that affects corneal endothelial cell function and leads to loss of visual acuity. PPCD1 has been linked to a locus on chromosome 20 in multiple families; however, Sanger sequencing of protein-coding genes in the consensus region failed to identify any causative missense mutations. In this study, custom capture probes were utilized for targeted next-generation sequencing of the linked region in a previously reported family with PPCD1. Variants were detected through two bioinformatics pipelines and filtered according to multiple criteria. Additionally, a high-resolution microarray was used to detect copy number variations. No non-synonymous variants in the protein-coding region of annotated genes were identified. However, 12 single nucleotide variants in 10 genes, and 9 indels in 7 genes met the filtering criteria and were considered candidate variants for PPCD1. Eleven single nucleotide variants were confirmed by Sanger sequencing, including 2 synonymous variants and 9 non-coding variants, in 9 genes. One microdeletion was detected in an intron of OVOL2 by microarray but was subsequently not identified by PCR. Using a comprehensive next-generation sequencing approach, a total of 16 genes containing single nucleotide variants or indels that segregated with the affected phenotype in an affected family previously mapped to the PPCD1 locus were identified. Screening of these candidate genes in other families previously mapped to the PPCD1 locus will likely result in the identification of the genetic basis of PPCD1.

SUBMITTER: Le DJ

PROVIDER: S-EPMC4927100 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Identification of Potentially Pathogenic Variants in the Posterior Polymorphous Corneal Dystrophy 1 Locus.

Le Derek J DJ Chung Duk-Won D DW Frausto Ricardo F RF Kim Michelle J MJ Aldave Anthony J AJ

PloS one 20160629 6

Posterior polymorphous corneal dystrophy 1 (PPCD1) is a genetic disorder that affects corneal endothelial cell function and leads to loss of visual acuity. PPCD1 has been linked to a locus on chromosome 20 in multiple families; however, Sanger sequencing of protein-coding genes in the consensus region failed to identify any causative missense mutations. In this study, custom capture probes were utilized for targeted next-generation sequencing of the linked region in a previously reported family ...[more]

PMID: 27355326

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Project description:To identify the genetic basis of posterior polymorphous corneal dystrophy 1 (PPCD1) using next-generation sequencing (NGS) of the common PPCD1 support interval, in which Sanger sequencing failed to identify a pathogenic mutation.Enrichment of the portion of chromosome 20 containing the common PPCD1 interval was performed on DNA extracted from an affected and an unaffected member of a family previously linked to the PPCD1 locus. NGS using the Roche 454 Titanium platform was performed, followed by computational analysis using NextGENe Software.NGS of the selectively enriched chromosomal 20 region between markers D20S48 and D20S190 produced over 400,000 DNA sequence reads with an average of 350 bases for each of the two DNA samples. Alignment of the DNA sequence reads with the reference sequence from the National Center of Biotechnology Information (NCBI) resulted in over 119 million matched bases per sample. Approximately 68,000 DNA sequence variants were identified in the common PPCD1 support interval in the affected individual, which was approximately twice the number of sequence variants identified in the unaffected individual. In both individuals, approximately 0.5% of the identified variants mapped to the 13 known and 16 predicted genes in the PPCD1 support interval, including 16 of the 17 (94%) variants previously identified by Sanger sequencing in the 13 known genes. In both individuals, the variant not identified by NGS was located in a region of inadequate coverage.NGS identified all of the exonic sequence variants that were previously identified by Sanger sequencing in known genes in adequately covered regions of the common PPCD1 interval, although the pathogenic variant is yet to be discovered. Given adequate coverage of a selectively enriched chromosomal region of interest, NGS represents a useful technique to screen for sequence variants in candidate gene loci that has multiple advantages over previously employed techniques for mutation discovery.

Project description:ImportanceThe identification of steep corneal curvatures in a significant percentage of patients with posterior polymorphous corneal dystrophy (PPCD) confirms this previously reported association and suggests a role for the ZEB1 protein in keratocyte function.ObjectiveTo determine whether PPCD is characterized by significant corneal steepening.Design, setting, and participantsCross-sectional study at university-based and private ophthalmology practices of 38 individuals (27 affected and 11 unaffected) from 23 families with PPCD.ExposureSlitlamp examination and corneal topographic imaging were performed for individuals with PPCD and unaffected family members. Saliva or blood samples were obtained from each individual for DNA isolation and ZEB1 sequencing. Corneal ZEB1 expression was measured using immunohistochemistry.Main outcomes and measuresPercentage of individuals affected with PPCD and controls with an average keratometric value greater than 48.0 diopters (D) in each eye; the mean keratometric value averaged for both eyes of individuals with PPCD and controls; and the correlation of ZEB1 mutation with keratometric value.ResultsZEB1 coding region mutations were identified in 7 of the 27 affected individuals. Ten of the 38 individuals (26.3%) had average keratometric values greater than 48.0 D OU: 10 of 27 individuals with PPCD (37.0%; 6 of 7 individuals with ZEB1 mutations [85.7%] and 4 of 20 individuals without ZEB1 mutations [20.0%]) and 0 of 11 unaffected individuals (P = .04 for unaffected vs affected individuals; P = .004 for individuals with PPCD with vs without ZEB1 mutation). The mean keratometric value of each eye of affected individuals (48.2 D) was significantly greater than that of each eye of unaffected family members (44.1 D) (P = .03). Affected individuals with ZEB1 mutations demonstrated a mean keratometric value of 53.3 D, which was significantly greater than that of affected individuals without ZEB1 mutations (46.5 D; P = .004). Fluorescence immunohistochemistry demonstrated ZEB1 expression in keratocyte nuclei.Conclusions and relevanceAbnormally steep corneal curvatures are identified in 37% of all individuals with PPCD and 86% of affected individuals with PPCD secondary to ZEB1 mutations. ZEB1 is present in keratocyte nuclei, suggesting a role for ZEB1 in keratocyte function. Therefore, ZEB1 may play a role in both corneal stromal and endothelial development and function, and PPCD should be considered both an endothelial dystrophy and an ectatic disorder.

Dataset Information

Identification of Potentially Pathogenic Variants in the Posterior Polymorphous Corneal Dystrophy 1 Locus.

Publications

Identification of Potentially Pathogenic Variants in the Posterior Polymorphous Corneal Dystrophy 1 Locus.

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