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

Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy

Background In previous studies, Propionibacterium acnes was cultured from intervertebral disc tissue of similar to 25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of P. acnes as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate P. acnes prevalence in resected disc cultures, while providing microscopic evidence of P. acnes biofilm in the intervertebral discs. Methods Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and P. acnes phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH. Results Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with P. acnes. In 89 cases, P. acnes was cultured exclusively;in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative Staphylococcus spp.) Among positive specimens, the median P. acnes bacterial burden was 350 CFU/g (12 - similar to 20,000 CFU/g). Thirtyeight P. acnes isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA1, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed P. acnes in situ. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. P. acnes bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013). Conclusions This study confirms that P. acnes is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of P. acnes biofilms within such specimens, consistent with infection rather than microbiologic contamination

ATAD3A-related pontocerebellar hypoplasia: new patients and insights into phenotypic variability

Abstract Background Pathogenic variants in the ATAD3A gene lead to a heterogenous clinical picture and severity ranging from recessive neonatal-lethal pontocerebellar hypoplasia through milder dominant Harel-Yoon syndrome up to, again, neonatal-lethal but dominant cardiomyopathy. The genetic diagnostics of ATAD3A-related disorders is also challenging due to three paralogous genes in the ATAD3 locus, making it a difficult target for both sequencing and CNV analyses. Results Here we report four individuals from two families with compound heterozygous p.Leu77Val and exon 3–4 deletion in the ATAD3A gene. One of these patients was characterized as having combined OXPHOS deficiency based on decreased complex IV activities, decreased complex IV, I, and V holoenzyme content, as well as decreased levels of COX2 and ATP5A subunits and decreased rate of mitochondrial proteosynthesis. All four reported patients shared a strikingly similar clinical picture to a previously reported patient with the p.Leu77Val variant in combination with a null allele. They presented with a less severe course of the disease and a longer lifespan than in the case of biallelic loss-of-function variants. This consistency of the phenotype in otherwise clinically heterogenous disorder led us to the hypothesis that the severity of the phenotype could depend on the severity of variant impact. To follow this rationale, we reviewed the published cases and sorted the recessive variants according to their impact predicted by their type and the severity of the disease in the patients. Conclusion The clinical picture and severity of ATAD3A-related disorders are homogenous in patients sharing the same combinations of variants. This knowledge enables deduction of variant impact severity based on known cases and allows more accurate prognosis estimation, as well as a better understanding of the ATAD3A function

Effects of Sunitinib and Other Kinase Inhibitors on Cells Harboring a PDGFRB Mutation Associated with Infantile Myofibromatosis

Infantile myofibromatosis represents one of the most common proliferative fibrous tumors of infancy and childhood. More effective treatment is needed for drug-resistant patients, and targeted therapy using specific protein kinase inhibitors could be a promising strategy. To date, several studies have confirmed a connection between the p.R561C mutation in gene encoding platelet-derived growth factor receptor beta (PDGFR-beta) and the development of infantile myofibromatosis. This study aimed to analyze the phosphorylation of important kinases in the NSTS-47 cell line derived from a tumor of a boy with infantile myofibromatosis who harbored the p.R561C mutation in PDGFR-beta. The second aim of this study was to investigate the effects of selected protein kinase inhibitors on cell signaling and the proliferative activity of NSTS-47 cells. We confirmed that this tumor cell line showed very high phosphorylation levels of PDGFR-beta, extracellular signal-regulated kinases (ERK) 1/2 and several other protein kinases. We also observed that PDGFR-beta phosphorylation in tumor cells is reduced by the receptor tyrosine kinase inhibitor sunitinib. In contrast, MAPK/ERK kinases (MEK) 1/2 and ERK1/2 kinases remained constitutively phosphorylated after treatment with sunitinib and other relevant protein kinase inhibitors. Our study showed that sunitinib is a very promising agent that affects the proliferation of tumor cells with a p.R561C mutation in PDGFR-beta

Distribution of <i>P</i>. <i>acnes</i> colony counts in culture-positive disc tissue specimens.

<p>Distribution of <i>P</i>. <i>acnes</i> colony counts in culture-positive disc tissue specimens.</p

<i>Propionibacterium acnes</i> biofilm is present in intervertebral discs of patients undergoing microdiscectomy

<div><p>Background</p><p>In previous studies, <i>Propionibacterium acnes</i> was cultured from intervertebral disc tissue of ~25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of <i>P</i>. <i>acnes</i> as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate <i>P</i>. <i>acnes</i> prevalence in resected disc cultures, while providing microscopic evidence of <i>P</i>. <i>acnes</i> biofilm in the intervertebral discs.</p><p>Methods</p><p>Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and <i>P</i>. <i>acnes</i> phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH.</p><p>Results</p><p>Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with <i>P</i>. <i>acnes</i>. In 89 cases, <i>P</i>. <i>acnes</i> was cultured exclusively; in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative <i>Staphylococcus spp</i>.) Among positive specimens, the median <i>P</i>. <i>acnes</i> bacterial burden was 350 CFU/g (12 - ~20,000 CFU/g). Thirty-eight <i>P</i>. <i>acnes</i> isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA₁, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed <i>P</i>. <i>acnes in situ</i>. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. <i>P</i>. <i>acnes</i> bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013).</p><p>Conclusions</p><p>This study confirms that <i>P</i>. <i>acnes</i> is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of <i>P</i>. <i>acnes</i> biofilms within such specimens, consistent with infection rather than microbiologic contamination.</p></div

Summary of the microorganisms isolated in 162 cases from 368 patient intervertebral disc specimens by anaerobic culture.

<p>Summary of the microorganisms isolated in 162 cases from 368 patient intervertebral disc specimens by anaerobic culture.</p

Visualization of bacterial biofilm in the disc tissue by CSLM and confirmation of <i>P</i>. <i>acnes</i> by FISH.

<p>A. Three dimensional reconstructed CSLM image of biofilm bacteria stained with a DNA stain (SYTO9, green) in a disc tissue sample (#4, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174518#pone.0174518.t002" target="_blank">Table 2</a>). B-C. The presence of <i>P. acnes</i> biofilms in this sample verified using FISH. Epifluorescence micrographs of a biofilm cluster showing red fluorescence from the CY5-labeled EUB338 general eubacterial probe (B) and green fluorescence from the CY3-labled <i>P. acnes</i>-specific probe (C). Co-localization of the red and green fluorescence indicates that all of the bacteria in this biofilm were <i>P. acnes</i>.</p