SNV annotation differs between annotation databases.

<p>Comparison of functional annotations between the RefSeq (RS-Feb15) and the Ensembl (Enbl75) databases for SNVs detected by both bioinformatics pipelines. (A) Number of annotated SNVs detected by the BowTie2-SAMtools pipeline. (B) Number of annotated SNVs detected by the BWA-GATK HaplotypeCaller pipeline.</p

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

<div><p>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 <i>OVOL2</i> 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.</p></div

Annotated candidate indels in the PPCD1 interval.

<p>Annotated candidate indels in the PPCD1 interval.</p

Coverage and read-depth of next-generation sequencing reads of the PPCD1 locus.

<p>Histogram depicts the number of reads aligning to the PPCD1 candidate region on chromosome 20 for a representative individual (hg19 reference sequence; histogram produced using Partek® Genomics Suite®).</p

Detection of OVOL2, CCM2L and THBD in normal donor and PPCD corneal endothelium by F-IHC.

<p>H&E: Hematoxylin and eosin stain (row 1). Primary antibodies directed against the proteins encoded by <i>OVOL2</i> (row 2), <i>CCM2L</i> (row 3) and <i>THBD</i> (row 4) were used to detect protein expression in the corneal endothelium of a normal donor (column 1) and two PPCD corneas (columns 2 and 3). A secondary antibody conjugated to a fluorescent moiety (Alexa Fluor 594, red) was used to visualize the localization of the primary antibodies. The sections were counterstained with DAPI, which stained the nuclei blue. Numbers located at lower right corner of each panel represent the quantification of the fluorescent signal in fluorescence units per pixel (FU/px), corrected for autofluorescence.</p

Abbreviated ideogram of chromosome 20 with PPCD1-associated intervals.

<p>Relative position of previously reported intervals associated with PPCD1 are on the left of the ideogram. Relative position of the interval enriched for NGS in this study is on the right of the ideogram. Ideogram and genomic coordinates are based on the hg19 reference build. *The interval reported by Hosseini et al. refines the original interval reported by Heon et al.</p

Annotated candidate SNVs in the PPCD1 interval.

<p>Annotated candidate SNVs in the PPCD1 interval.</p

Quantitative PCR results for <i>EPYC</i> and <i>DCN</i> for 3 families with PACD.

<p>The cutoff between 1 and 2 copies is represented by a horizontal red line at 1.414, the geometric mean between 1 and 2. Results are grouped by affected status and arranged in order of location on pedigree. Data are represented as the mean± SEM. A =  affected; U =  unaffected; *Genomic CNV analysis also performed.</p

Clinical findings from Family 2.

<p><b>A</b>. Slit lamp photomicrograph demonstrating peripheral corneal opacification in individual IV-5. <b>B</b>–<b>C</b>. Slit lamp photomicrograph of central and peripheral corneal opacification in individuals III-6 (B) and III-2 (C). <b>D</b>. Corneal topographic imaging demonstrates significant flattening of the corneal curvature, with a steep K value of 38.79 D, in individual III-2.</p

Expression of the SLRP gene cluster and <i>CCER1</i> in the corneal stroma by qPCR.

<p><b>A</b>. The transcript levels for the keratocyte markers <i>CD34</i> and <i>VIM</i> were significantly higher in the corneal stroma than in the corneal endothelium. Statistical analysis was performed using a two-tailed unpaired t-test. <b>B</b>. Expression of <i>LUM</i> and <i>KERA</i> was significantly higher than <i>DCN</i>, which in turn was higher than the undetectable expression levels of <i>EPYC</i> and <i>CCER1</i>. Statistical analyses were performed using one-way ANOVA and Bonferroni's Multiple Comparison Test (*p≤0.05; **p≤0.01; ***p≤0.001 (error bars  =  SEM)). Non-significant results are not represented.</p