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

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

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

ZEB1 insufficiency causes corneal endothelial cell state transition and altered cellular processing.

The zinc finger e-box binding homeobox 1 (ZEB1) transcription factor is a master regulator of the epithelial to mesenchymal transition (EMT), and of the reverse mesenchymal to epithelial transition (MET) processes. ZEB1 plays an integral role in mediating cell state transitions during cell lineage specification, wound healing and disease. EMT/MET are characterized by distinct changes in molecular and cellular phenotype that are generally context-independent. Posterior polymorphous corneal dystrophy (PPCD), associated with ZEB1 insufficiency, provides a new biological context in which to understand and evaluate the classic EMT/MET paradigm. PPCD is characterized by a cadherin-switch and transition to an epithelial-like transcriptomic and cellular phenotype, which we study in a cell-based model of PPCD generated using CRISPR-Cas9-mediated ZEB1 knockout in corneal endothelial cells (CEnCs). Transcriptomic and functional studies support the hypothesis that CEnC undergo a MET-like transition in PPCD, termed endothelial to epithelial transition (EnET), and lead to the conclusion that EnET may be considered a corollary to the classic EMT/MET paradigm

Scheme of typing of <i>M</i>. <i>tuberculosis</i> strains using 13 genes of type II TA systems.

<p>The algorithm for determining the genotype is presented. The scheme shows that, after the first iteration to determine the genotype, the number of genes for the analysis is decreased twofold. Each gene in the brackets is given its position that is replaced, and the appropriate nucleotide is indicated. All replacements are calculated relative to the reference strain H37Rv.</p

<i>Mycobacterium tuberculosis</i> Type II Toxin-Antitoxin Systems: Genetic Polymorphisms and Functional Properties and the Possibility of Their Use for Genotyping

<div><p>Various genetic markers such as IS-elements, DR-elements, variable number tandem repeats (VNTR), single nucleotide polymorphisms (SNPs) in housekeeping genes and other groups of genes are being used for genotyping. We propose a different approach. We suggest the type II toxin-antitoxin (TA) systems, which play a significant role in the formation of pathogenicity, tolerance and persistence phenotypes, and thus in the survival of <i>Mycobacterium tuberculosis</i> in the host organism at various developmental stages (colonization, infection of macrophages, etc.), as the marker genes. Most genes of TA systems function together, forming a single network: an antitoxin from one pair may interact with toxins from other pairs and even from other families. In this work a bioinformatics analysis of genes of the type II TA systems from 173 sequenced genomes of <i>M</i>. <i>tuberculosis</i> was performed. A number of genes of type II TA systems were found to carry SNPs that correlate with specific genotypes. We propose a minimally sufficient set of genes of TA systems for separation of <i>M</i>. <i>tuberculosis</i> strains at nine basic genotype and for further division into subtypes. Using this set of genes, we genotyped a collection consisting of 62 clinical isolates of <i>M</i>. <i>tuberculosis</i>. The possibility of using our set of genes for genotyping using PCR is also demonstrated.</p></div

The type II TA systems of mycobacteria were investigated. Schematic diagram of the toxin-antitoxin system.

<p>(A) TA systems are annotated according to the GenBank database, excluding VapBC50 (rv3750c-rv3749c), VapBC49 (rv3180c-rv3181c), HigBA3 (rv3182-rv3183), HigBA2 (rv2022c-rv2021c), MazEF10 (rv0298-rv0299) and VapBC45 (rv2018-rv2019) systems; these systems are annotated according to Sala et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#pone.0143682.ref032" target="_blank">32</a>]. The system RelBE3 (rv3358-rv3357, GenBank database, NCBI) is called the YefM/YoeB system by Sala. All of the TA systems depicted here are type II (systems marked with an asterisk are novel TA systems that are not classified to any family, but for which functional activity has been shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#pone.0143682.ref032" target="_blank">32</a>]). The 13 genes, our proposed set for genotyping, are highlighted in bold. (B) Type II TA systems are encoded by two genes, a toxin and an antitoxin, that form one operon with a promoter located upstream of the first antitoxin gene. PIN domain is the functional part of the toxin gene, the four conserved acidic residues marked at the picture: the three well-conserved acidic residues, at positions 4[D], 40[E] and 93[D], and with fourth acidic residue is less well conserved at position 112[D].</p

Detection of the Ural genotype by qPCR.

<p>Fluorescence in the FAM channel (blue): (1)13_2978, (2) 13–3114, (3) 13–3086, (4) 13_3158, (5) 13_4178, (6) 13_3539, (7) 13_2566, (8) 13_3632, (9) 13_3599, (10) 13_3896, (11) 13_3582, (12) 13_4189, (13) 13_3535, (15) 13_3147; Fluorescence in the HEX channel (green): (14) 13_3147, (16) 13_2978. Fluorescence of the channel FAM (blue) indicates the accumulation of the PCR product containing cytosine (C); the fluorescence of the channel HEX (green) indicates the accumulation of the PCR product containing thymine (T, the variable nucleotide) and indicates the SNP in the <i>vapC10</i> gene (C394→T394) characteristic of the Ural genotype. Line 14 (13_3147) and 16 (13_2978) belong to the Ural genotype. For isolate 13_2978 fluorescence is detected on the two channels (FAM and HEX), this can indicate the presence of impurities (coinfection). qPCR fluorescence in RFU (relative fluorescence units) vs. PCR cycles. Intensity of fluorescence depending on the number of qPCR cycles for strains belonging to the Euro-American lineage.</p