Molecular characterisation of sporadic endolymphatic sac tumours and comparison to von Hippel–Lindau disease‐related tumours

Aims: Although inactivation of the von Hippel-Lindau gene (VHL) on chromosome 3p25 is considered to be the major cause of hereditary endolymphatic sac tumours (ELSTs), the genetic background of sporadic ELST is largely unknown. The aim of this study was to determine the prevalence of VHL mutations in sporadic ELSTs and compare their characteristics to VHL-disease-related tumours. Methods: Genetic and epigenetic alterations were compared between 11 sporadic and 11 VHL-disease-related ELSTs by targeted sequencing and DNA methylation analysis. Results: VHL mutations and small deletions detected by targeted deep sequencing were identified in 9/11 sporadic ELSTs (82%). No other cancer-related genetic pathway was altered except for TERT promoter mutations in two sporadic ELST and one VHL-disease-related ELST (15%). Loss of heterozygosity of chromosome 3 was found in 6/10 (60%) VHL-disease-related and 10/11 (91%) sporadic ELSTs resulting in biallelic VHL inactivation in 8/10 (73%) sporadic ELSTs. DNA methylation profiling did not reveal differences between sporadic and VHL-disease-related ELSTs but reliably distinguished ELST from morphological mimics of the cerebellopontine angle. VHL patients were significantly younger at disease onset compared to sporadic ELSTs (29 vs. 52 years, p < 0.0001, Fisher's exact test). VHL-disease status was not associated with an increased risk of recurrence, but the presence of clear cells was found to be associated with shorter progression-free survival (p = 0.0002, log-rank test). Conclusion: Biallelic inactivation of VHL is the main mechanism underlying ELSTs, but unknown mechanisms beyond VHL may rarely be involved in the pathogenesis of sporadic ELSTs

Genetic and epigenetic characterization of posterior pituitary tumors

Pituicytoma (PITUI), granular cell tumor (GCT), and spindle cell oncocytoma (SCO) are rare tumors of the posterior pituitary. Histologically, they may be challenging to distinguish and have been proposed to represent a histological spectrum of a single entity. We performed targeted next-generation sequencing, DNA methylation profiling, and copy number analysis on 47 tumors (14 PITUI; 12 GCT; 21 SCO) to investigate molecular features and explore possibilities of clinically meaningful tumor subclassification. We detected two main epigenomic subgroups by unsupervised clustering of DNA methylation data, though the overall methylation differences were subtle. The largest group (n = 23) contained most PITUIs and a subset of SCOs and was enriched for pathogenic mutations within genes in the MAPK/PI3K pathways (12/17 [71%] of sequenced tumors: FGFR1 (3), HRAS (3), BRAF (2), NF1 (2), CBL (1), MAP2K2 (1), PTEN (1)) and two with accompanying TERT promoter mutation. The second group (n = 16) contained most GCTs and a subset of SCOs, all of which mostly lacked identifiable genetic drivers. Outcome analysis demonstrated that the presence of chromosomal imbalances was significantly associated with reduced progression-free survival especially within the combined PITUI and SCO group (p = 0.031). In summary, we observed only subtle DNA methylation differences between posterior pituitary tumors, indicating that these tumors may be best classified as subtypes of a single entity. Nevertheless, our data indicate differences in mutation patterns and clinical outcome. For a clinically meaningful subclassification, we propose a combined histo-molecular approach into three subtypes: one subtype is defined by granular cell histology, scarcity of identifiable oncogenic mutations, and favorable outcome. The other two subtypes have either SCO or PITUI histology but are segregated by chromosomal copy number profile into a favorable group (no copy number changes) and a less favorable group (copy number imbalances present). Both of the latter groups have recurrent MAPK/PI3K genetic alterations that represent potential therapeutic targets

In vitro hepatotoxicity of Petasites hybridus extract (Ze 339) depends on the concentration, the cytochrome activity of the cell system, and the species used

Ze 339, a CO2 extract prepared from the leaves of Petasites hybridus, possesses antispasmodic and anti‐inflammatory effects and is proven to be effective in the treatment of allergic rhinitis. To study possible hepatotoxic effects of Ze 339, its main constituents and metabolites, a series of in vitro investigations were performed. Furthermore, different reconstituted fractions of extract (petasins and fatty acid fraction) were examined in three in vitro test systems using hepatocytes: Two human cell lines, with lower and higher activity of cytochrome P450 enzymes (HepG2, HepaRG) as well as a rodent cell line with high cytochrome P450 activity (H‐4‐II‐E), were used. Metabolic activity, assessed by the WST‐1 assay, was chosen as indicator of cytotoxicity. To assess potential bioactivation of Ze 339 compounds, metabolic experiments using S9 fractions from rats, dogs, and humans and isolated cytochromes (human/rat) were performed, and the formation of reactive metabolites was assessed by measuring cellular concentrations of glutathione and glutathione disulphide.Our data revealed that the cytotoxicity of Ze 339, its single constituents, and main metabolites depends on the concentration, the cytochrome activity of the cell system, and the species used.publishe

Generation and Analysis of Draft Sequences of 'Stolbur' Phytoplasma from Multiple Displacement Amplification Templates

Phytoplasma-associated diseases are reported for more than 1,000 plant species worldwide. Only a few genome sequences are available in contrast to the economical importance of these bacterial pathogens. A new strategy was used to retrieve phytoplasma strain-specific genome data. Multiple displacement amplification was performed on DNA obtained from <3 g of plant tissue from tobacco and parsley samples infected with ‘stolbur' strains. Random hexamers and Phi29 polymerase were evaluated with and without supplementation by group-assigned oligonucleotides providing templates for Illumina's sequencing approach. Metagenomic drafts derived from individual and pooled strain-specific de novo assemblies were analyzed. Supplementation of the Phi29 reaction with the group-assigned oligonucleotides resulted in an about 2-fold enrichment of the percentage of phytoplasma-assigned reads and thereby improved assembly results. The obtained genomic drafts represent the largest datasets available from ‘stolbur' phytoplasmas. Sequences of the two strains (558 kb, 448 proteins and 516 kb, 346 proteins, respectively) were annotated allowing the identification of prominent membrane proteins and reconstruction of core pathways. Analysis of a putative truncated sucrose phosphorylase provides hints on sugar degradation. Furthermore, it is shown that drafts obtained from repetitive-rich genomes allow only limited analysis on multicopy regions and genome completeness

Phylogenetic tree constructed by parsimony analyses of deduced malate dehydrogenase peptide sequences of available phytoplasmas, <i>Clostridium botulinum</i> strains ATCC3502 and ATCC19397, <i>Bacillus subtilis</i> subsp. <i>subtilis</i>, and <i>Bacillus cereus</i> employing <i>Escherichia coli</i> strain K12 as outgroup.

<p>Accession numbers are given in parentheses. Numbers on the branches are bootstrap values obtained for 1,000 replicates (only values above 60% are shown). The tree is drawn to scale, with branch lengths calculated using the average pathway method, and are in the units of the number of changes over the whole sequence. The scale bar represents 20 amino acid substitutions.</p

Experimental hosts and genes used in qRT-PCR experiments with their correlating average (Ø) C_T and ΔC_T values after normalization (long amplicons).

<p>Experimental hosts and genes used in qRT-PCR experiments with their correlating average (Ø) C_T and ΔC_T values after normalization (long amplicons).</p

RT-PCR confirming the expression of <i>pgi</i>, <i>pfkA</i>, <i>fba</i>, <i>tpiA</i>, <i>pduL</i>, <i>ackA</i>, <i>degV</i> and SAP11-like gene.

<p>RNA was obtained from <i>Nicotiana occidentalis</i>, <i>Malus domestica</i>, <i>Catharanthus roseus</i> infected by ‘<i>Ca</i>. P. mali’ strain AT. The RT-PCR products were separated on a 1.4% TAE agarose gel. Lane number nine was used for negative control applying water as template (example SAP11-like gene). The product size of around 200 bp was estimated by the 50 bp DNA ladder (Lifetechnologies) loaded on first and last lane of each gel.</p

COG categories assigned to expressed gene products identified by RNA-Seq and/or proteome analysis (orange) versus the deduced protein content of '<i>Ca</i>. P. mali' strain AT (green).

<p>Values indicate total number reached in each category.</p

Experimental hosts and genes used in qRT-PCR experiments with their correlating average (Ø) C_T and ΔC_T values after normalization (short amplicons).

<p>Experimental hosts and genes used in qRT-PCR experiments with their correlating average (Ø) C_T and ΔC_T values after normalization (short amplicons).</p