RUNX3 transcript variants have distinct roles in ovarian carcinoma and differently influence platinum sensitivity and angiogenesis

The prognosis of late-stage epithelial ovarian cancer (EOC) patients is affected by chemotherapy response and the malignant potential of the tumor cells. In earlier work, we identified hypermethylation of the runt-related transcription factor 3 gene (RUNX3) as a prognostic biomarker and contrary functions of transcript variants (TV1 and TV2) in A2780 and SKOV3 cells. The aim of the study was to further validate these results and to increase the knowledge about RUNX3 function in EOC. New RUNX3 overexpression models of high-grade serous ovarian cancer (HGSOC) were established and analyzed for phenotypic (IC50 determination, migration, proliferation and angiogenesis assay, DNA damage analysis) and transcriptomic consequences (NGS) of RUNX3 TV1 and TV2 overexpression. Platinum sensitivity was affected by a specific transcript variant depending on BRCA background. RUNX3 TV2 induced an increased sensitivity in BRCA1wt cells (OVCAR3), whereas TV1 increased the sensitivity and induced a G2/M arrest under treatment in BRCA1mut cells (A13-2-12). These different phenotypes relate to differences in DNA repair: homologous recombination deficient A13-2-12 cells show less γH2AX foci despite higher levels of Pt-DNA adducts. RNA-Seq analyses prove transcript variant and cell-line-specific RUNX3 effects. Pathway analyses revealed another clinically important function of RUNX3—regulation of angiogenesis. This was confirmed by thrombospondin1 analyses, HUVEC spheroid sprouting assays and proteomic profiling. Importantly, conditioned media (CM) from RUNX3 TV1 overexpressing A13-2-12 cells induced an increased HUVEC sprouting. Altogether, the presented data support the hypothesis of different functions of RUNX3 transcript variants related to the clinically relevant processes—platinum resistance and angiogenesis

Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies

MiR-145 expression and rare NOTCH1 variants in bicuspid aortic valve-associated aortopathy

MicroRNAs (miRNAs) may serve as elegant tool to improve risk stratification in bicuspid aortic valve (BAV)-associated aortopathy. However, the exact pathogenetic pathway by which miRNAs impact aortopathy progression is unknown. Herewith, we aimed to analyze the association between circulating miRNAs and rare variants of aortopathy-related genes. 63 BAV patients (mean age 47.3±11.3 years, 92% male) with a root dilatation phenotype, who underwent aortic valve+/-proximal aortic surgery at a single institution (mean post-AVR follow-up 10.3±6.9 years) were analyzed. A custom-made HaloPlex HS panel including 20 aortopathy-related genes was used for the genetic testing. miRNAs were extracted from whole blood and miRNA analysis was performed using miRNA-specific assay. Study endpoint was the association between circulating miRNAs and rare genetic variants in the aortopathy gene panel. The study cohort was divided into a subgroup with rare variants vs. a subgroup without rare variants based on the presence of rare variants in the respective genes (i.e., at least one variant present). The genetic analysis yielded n = 6 potentially and likely pathogenic rare variants within the NOTCH1 gene as being the most common finding. Univariate analysis between blood miRNAs and NOTCH1 variants revealed a significantly lower expression of miR-145 in the subgroup of patients with NOTCH1 variants vs. those without NOTCH1 variants (i.e., delta Ct 4.95±0.74 vs. delta Ct 5.57±0.78, p = 0.04). Our preliminary data demonstrate a significant association between blood miR-145 expression and the presence of rare NOTCH1 variants. This association may be indicative of a specific pathogenetic pathway in the development of genetically-triggered bicuspid aortopathy

Sepsis induces long-lasting impairments in CD4+ T-cell responses despite rapid numerical recovery of T-lymphocyte populations

Massive apoptosis of lymphocytes is a hallmark of sepsis. The resulting immunosuppression is associated with secondary infections, which are often lethal. Moreover, sepsis-survivors are burdened with increased morbidity and mortality for several years after the sepsis episode. The duration and clinical consequences of sepsis induced-immunosuppression are currently unknown. We have used the mouse model of peritoneal contamination and infection (PCI) to investigate the quantitative and qualitative recovery of T lymphocytes for 3.5 months after sepsis with or without IL-7 treatment. Thymic output and the numbers of naive and effector/memory CD4+ and CD8+ lymphocytes quickly recovered after sepsis. IL-7 treatment resulted in an accelerated recovery of CD8+ lymphocytes. Next generation sequencing revealed no significant narrowing of the T cell receptor repertoire 3.5 months after sepsis. In contrast, detailed functional analyses of T helper (Th)-cell responses towards a fungal antigen revealed a significant loss of Th cells. Whereas cytokine production was not impaired at the single cell level, the absolute number of Th cells specific for the fungal antigen was reduced. Our data indicate a clinically relevant loss of pathogen-specific T cell clones after sepsis. Given the small number of naive T lymphocytes specific for a given antigen, this decrement of T cell clones remains undetected even by sensitive methods such as deep sequencing. Taken together, our data are compatible with long lasting impairments in CD4+ T-cell responses after sepsis despite rapid recovery of T lymphocyte populations

MiR-145 expression and rare NOTCH1 variants in bicuspid aortic valve-associated aortopathy

<div><p>MicroRNAs (miRNAs) may serve as elegant tool to improve risk stratification in bicuspid aortic valve (BAV)-associated aortopathy. However, the exact pathogenetic pathway by which miRNAs impact aortopathy progression is unknown. Herewith, we aimed to analyze the association between circulating miRNAs and rare variants of aortopathy-related genes. 63 BAV patients (mean age 47.3±11.3 years, 92% male) with a root dilatation phenotype, who underwent aortic valve+/-proximal aortic surgery at a single institution (mean post-AVR follow-up 10.3±6.9 years) were analyzed. A custom-made HaloPlex HS panel including 20 aortopathy-related genes was used for the genetic testing. miRNAs were extracted from whole blood and miRNA analysis was performed using miRNA-specific assay. Study endpoint was the association between circulating miRNAs and rare genetic variants in the aortopathy gene panel. The study cohort was divided into a subgroup with rare variants vs. a subgroup without rare variants based on the presence of rare variants in the respective genes (i.e., at least one variant present). The genetic analysis yielded n = 6 potentially and likely pathogenic rare variants within the <i>NOTCH1</i> gene as being the most common finding. Univariate analysis between blood miRNAs and <i>NOTCH1</i> variants revealed a significantly lower expression of miR-145 in the subgroup of patients with <i>NOTCH1</i> variants vs. those without <i>NOTCH1</i> variants (i.e., delta Ct 4.95±0.74 vs. delta Ct 5.57±0.78, p = 0.04). Our preliminary data demonstrate a significant association between blood miR-145 expression and the presence of rare <i>NOTCH1</i> variants. This association may be indicative of a specific pathogenetic pathway in the development of genetically-triggered bicuspid aortopathy.</p></div

Sepsis induces long-lasting impairments in CD4+ T-cell responses despite rapid numerical recovery of T-lymphocyte populations

Massive apoptosis of lymphocytes is a hallmark of sepsis. The resulting immunosuppression is associated with secondary infections, which are often lethal. Moreover, sepsis-survivors are burdened with increased morbidity and mortality for several years after the sepsis episode. The duration and clinical consequences of sepsis induced-immunosuppression are currently unknown. We have used the mouse model of peritoneal contamination and infection (PCI) to investigate the quantitative and qualitative recovery of T lymphocytes for 3.5 months after sepsis with or without IL-7 treatment. Thymic output and the numbers of naive and effector/memory CD4+ and CD8+ lymphocytes quickly recovered after sepsis. IL-7 treatment resulted in an accelerated recovery of CD8+ lymphocytes. Next generation sequencing revealed no significant narrowing of the T cell receptor repertoire 3.5 months after sepsis. In contrast, detailed functional analyses of T helper (Th)-cell responses towards a fungal antigen revealed a significant loss of Th cells. Whereas cytokine production was not impaired at the single cell level, the absolute number of Th cells specific for the fungal antigen was reduced. Our data indicate a clinically relevant loss of pathogen-specific T cell clones after sepsis. Given the small number of naive T lymphocytes specific for a given antigen, this decrement of T cell clones remains undetected even by sensitive methods such as deep sequencing. Taken together, our data are compatible with long lasting impairments in CD4+ T-cell responses after sepsis despite rapid recovery of T lymphocyte populations

Correlation of individual microRNAs.

<p>Correlation of individual microRNAs.</p