13 research outputs found
Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2
Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.−339_361dup for CHED1 and c.−370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.−274T>G and c.−307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies
Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2
Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes - Fig 7
<p><b>Comparison of variant detection (shown as values of variant allelic fraction; AF) in DNA reference standards</b> (NA12878, NA24149, NA24385, NA24631 and NA24143) obtained from CZECANCA analysis (x-axis) and AF from VCF files for these standards downloaded from <a href="http://jimb.stanford.edu/giab/" target="_blank">http://jimb.stanford.edu/giab/</a> (y-axis). The graph shows all variants with GATK quality >100 reached in CZECANCA analysis (including FP variants) and undetected (FN) variants. Heterozygote variants clustered in the center, while homozygote variants in right upper corner. Variant distribution was partially influenced by the differences in mean sequencing coverage targeting 100X and 300X in CZECANCA and DNA reference standards VCFs, respectively. The number of TP, TN, FP, FN, and total number of variant (= CZECANCA target) was used to calculate of sensitivity, specificity, and accuracy of CZECANCA analysis.</p
Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes
<div><p>Background</p><p>Carriers of mutations in hereditary cancer predisposition genes represent a small but clinically important subgroup of oncology patients. The identification of causal germline mutations determines follow-up management, treatment options and genetic counselling in patients’ families. Targeted next-generation sequencing-based analyses using cancer-specific panels in high-risk individuals have been rapidly adopted by diagnostic laboratories. While the use of diagnosis-specific panels is straightforward in typical cases, individuals with unusual phenotypes from families with overlapping criteria require multiple panel testing. Moreover, narrow gene panels are limited by our currently incomplete knowledge about possible genetic dispositions.</p><p>Methods</p><p>We have designed a multi-gene panel called CZECANCA (CZEch CAncer paNel for Clinical Application) for a sequencing analysis of 219 cancer-susceptibility and candidate predisposition genes associated with frequent hereditary cancers.</p><p>Results</p><p>The bioanalytical and bioinformatics pipeline was validated on a set of internal and commercially available DNA controls showing high coverage uniformity, sensitivity, specificity and accuracy. The panel demonstrates a reliable detection of both single nucleotide and copy number variants. Inter-laboratory, intra- and inter-run replicates confirmed the robustness of our approach.</p><p>Conclusion</p><p>The objective of CZECANCA is a nationwide consolidation of cancer-predisposition genetic testing across various clinical indications with savings in costs, human labor and turnaround time. Moreover, the unified diagnostics will enable the integration and analysis of genotypes with associated phenotypes in a national database improving the clinical interpretation of variants.</p></div
Identification of c.661_662delinsTA double substitution (p.Val221*) in <i>PALB2</i> (NM_024675).
<p>The BAM file displayed in IGV shows the <i>cis</i>-position of both substitutions in approximately 50% of forward (pink bars) and reverse (blue bars) reads, respectively.</p
Coverage (y-axis) of coding sequences (x-axis) of 219 CZECANCA target genes from a routine, randomly selected run targeting 100X coverage.
<p>Note: Fully covered genes are depicted in green letters, genes with coverage <20X in a single exon are in orange letters, and genes with uncovered regions exceeding single exon or >10% of coding sequence are in red letters. Green horizontal bars (below individual graphs constructed using “Boudalyzer” script) indicate coverage ≥ 20X; red horizontal bars indicate regions covered <20X and uncovered regions.</p
CNV detection is influenced by a DNA preparation method.
<p>Panels show analyses of remaining ACMG genes (not shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195761#pone.0195761.g005" target="_blank">Fig 5B and 5C</a>) from four runs performed in laboratory 1 (116 DNA samples fragmented by ultrasound) and laboratory 3 (125 DNA samples fragmented enzymatically). The numbers in parentheses express number of samples with possible CNVs from all analyzed samples in contributing laboratories. *indicate samples analyzed by MLPA negatively (FP–black) or positively (TP–red). Bin set covering exon 1 in <i>RET</i> was excluded from the analysis due to the large coverage variability.</p
Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes - Fig 4
<p><b>Analysis of intra-run (A), inter-run (B), and inter-laboratory (C) replicates.</b> The panels show sequencing coverages (y-axis) of the identified variants arranged according to chromosomal localizations (x-axis). We used moving average curves (average of 3 values) to compare trends in coverages. Panel (A) describes the results of an analysis of three independently processed intra-run replicates from an identical DNA sample pooled in 33 ng (considered as 100%), 24.75 ng (75%), and 16.5 ng (50%), respectively. Panel (B) demonstrates variant coverages identified in two independent inter-run (run 8 and 14) replicates. All coverage values of sample #3647 in run 14 were corrected by a factor of 1.3880 to normalize coverages between samples (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195761#pone.0195761.s004" target="_blank">S4 Table</a>). Panel (C) shows coverages of variants identified in an inter-laboratory control sequenced in four laboratories (Lab) participating in panel validation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195761#pone.0195761.s005" target="_blank">S5 Table</a>). The coverages of variants identified in Lab 2, 3, and 4 were normalized to the average coverage of Lab 1 for better comparisons of coverages.</p
Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes - Fig 3
<p><b>Coverage of selected genes from the CZECANCA (A-E) and TruSight Cancer sequencing (F) panels.</b> The pictures show coverage (at y-axis) alongside the coding sequences of <i>BRCA1</i> (NM_007294), <i>BRCA2</i> (NM_000059), <i>PALB2</i> (NM_024675), and <i>TP53</i> (NM_000546), the vertical lines represent exon boundaries. Panels A–D show results obtained from a CZECANCA NGS analysis of various samples performed in four participating laboratories using the ultrasound (A, B) or enzymatic (C, D) DNA fragmentation protocol. Examples of the identified CNV aberrations in the depicted genes (deletions in <i>BRCA1</i>, <i>BRCA2</i> and <i>TP53</i> and duplication in <i>PALB2</i>) are shown in panel E. For comparison, panel F demonstrates the uneven coverage of the depicted genes by sequencing using the TruSight Cancer panel (Illumina).</p