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

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

Pedigrees of 3 families with PACD.

<p><b>A</b>. Family 1 (abbreviated). <b>B</b>. Family 2. <b>C</b>. Family 3. Families 1<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095037#pone.0095037-Aldave1" target="_blank">[1]</a> and 3<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095037#pone.0095037-Johnson1" target="_blank">[7]</a> have been reported previously. Asterisks indicate individuals in whom genetic testing was performed. Filled symbols represent affected individuals; open symbols represent unaffected individuals; ? represents individuals of undetermined affected status; arrowhead indicates proband.</p

Quantitative PCR oligonucleotides.

<p>Quantitative PCR oligonucleotides.</p

Confirmation of the <i>OVOL2</i> Promoter Mutation c.-307T>C in Posterior Polymorphous Corneal Dystrophy 1

<div><p>Purpose</p><p>To identify the genetic basis of posterior polymorphous corneal dystrophy (PPCD) in families mapped to the PPCD1 locus and in affected individuals without <i>ZEB1</i> coding region mutations.</p><p>Methods</p><p>The promoter, 5’ UTR, and coding regions of <i>OVOL2</i> was screened in the PPCD family in which linkage analysis established the PPCD1 locus and in 26 PPCD probands who did not harbor a <i>ZEB1</i> mutation. Copy number variation (CNV) analysis in the PPCD1 and PPCD3 intervals was performed on DNA samples from eight probands using aCGH. Luciferase reporter assays were performed in human corneal endothelial cells to determine the impact of the identified potentially pathogenic variants on <i>OVOL2</i> promoter activity.</p><p>Results</p><p><i>OVOL2</i> mutation analysis in the first PPCD1-linked family demonstrated segregation of the c.-307T>C variant with the affected phenotype. In the other 26 probands screened, one heterozygous coding region variant and five promoter region heterozygous variants were identified, though none are likely pathogenic based on allele frequency. Array CGH in the PPCD1 and PPCD3 loci excluded the presence of CNV involving either <i>OVOL2</i> or <i>ZEB1</i>, respectively. The c.-307T>C variant demonstrated increased promoter activity in corneal endothelial cells when compared to the wild-type sequence as has been demonstrated previously in another cell type.</p><p>Conclusions</p><p>Previously identified as the cause of PPCD1, the <i>OVOL2</i> promoter variant c.-307T>C was herein identified in the original family that established the PPCD1 locus. However, the failure to identify presumed pathogenic coding or non-coding <i>OVOL2</i> or <i>ZEB1</i> variants, or CNV involving the PPCD1 and PPCD3 loci in 26 other PPCD probands suggests that other genetic loci may be involved in the pathogenesis of PPCD.</p></div

Results of genomic CNV analysis using the CytoScan® HD Array in 3 families with PACD.

<p>A schematic of the genes in the region of chromosome 12q21.33 is shown at the top. In Family 1, an unaffected individual (IV-17) without a deletion in the region of chromosome 12q21.33 is shown above an affected individual (III-4) who demonstrates a 701 Kb heterozygous deletion involving <i>CCER1, KERA</i>, <i>LUM</i>, <i>DCN</i>, and <i>EPYC</i>. In Family 2, a 1.318 Mb heterozygous deletion involving <i>CCER1, KERA</i>, <i>LUM</i>, <i>DCN</i>, and <i>EPYC</i> was identified in an affected individual (IV-2). In Family 3, a 701 Kb heterozygous deletion in chromosome 12q21.33 with identical boundaries to that identified in Family 1 was identified in an affected individual (III-4).</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

Pedigree of the original family mapped to the PPCD1 locus.

<p>Females are represented by circles, males by squares. Affected individuals are shown with filled symbols and unaffected individuals are shown with open symbols. Diagonal lines across symbols indicate individuals who are deceased. An asterisk (*) indicates individuals who underwent <i>OVOL2</i> promoter screening by Sanger sequencing.</p

The c.-307T>C variant increases <i>OVOL2</i> promoter activity.

<p><i>OVOL2</i> promoter constructs containing either wild type (<i>OVOL2 P</i> ^<i>WT</i>) or mutant (<i>OVOL2 P</i> ^{<i>c</i>.<i>-307T</i>>C}) promoter sequences were transfected into HCEnC-21T cells and the relative luciferase activities of each construct was measured. The <i>OVOL2 P</i> ^{<i>c</i>.<i>-307T</i>>C} promoter construct produced significantly higher levels of luminescence compared to the <i>OVOL2 P</i> ^<i>WT</i> construct. (* p value < 0.05, n = 3, error bars = SEM).</p

<i>OVOL2</i> promoter and 5’ UTR variants identified in 27 probands with PPCD.

<p><i>OVOL2</i> promoter and 5’ UTR variants identified in 27 probands with PPCD.</p