Genome-wide Association Study and Meta-Analysis Identify ISL1 as Genome-wide Significant Susceptibility Gene for Bladder Exstrophy

The bladder exstrophy-epispadias complex (BEEC) represents the severe end of the uro-rectal malformation spectrum, and is thought to result from aberrant embryonic morphogenesis of the cloacal membrane and the urorectal septum. The most common form of BEEC is isolated classic bladder exstrophy (CBE). To identify susceptibility loci for CBE, we performed a genome-wide association study (GWAS) of 110 CBE patients and 1,177 controls of European origin. Here, an association was found with a region of approximately 220kb on chromosome 5q11.1. This region harbors the ISL1 (ISL LIM homeobox 1) gene. Multiple markers in this region showed evidence for association with CBE, including 84 markers with genome-wide significance. We then performed a meta-analysis using data from a previous GWAS by our group of 98 CBE patients and 526 controls of European origin. This meta-analysis also implicated the 5q11.1 locus in CBE risk. A total of 138 markers at this locus reached genome-wide significance in the meta-analysis, and the most significant marker (rs9291768) achieved a P value of 2.13 × 10⁻¹². No other locus in the meta-analysis achieved genome-wide significance. We then performed murine expression analyses to follow up this finding. Here, Isl1 expression was detected in the genital region within the critical time frame for human CBE development. Genital regions with Isl1 expression included the peri-cloacal mesenchyme and the urorectal septum. The present study identified the first genome-wide significant locus for CBE at chromosomal region 5q11.1, and provides strong evidence for the hypothesis that ISL1 is the responsible candidate gene in this region.     

Systematic Cell-Based Phenotyping of Missense Alleles Empowers Rare Variant Association Studies: A Case for LDLR and Myocardial Infarction

A fundamental challenge to contemporary genetics is to distinguish rare missense alleles that disrupt protein functions from the majority of alleles neutral on protein activities. High-throughput experimental tools to securely discriminate between disruptive and non-disruptive missense alleles are currently missing. Here we establish a scalable cell-based strategy to profile the biological effects and likely disease relevance of rare missense variants in vitro. We apply this strategy to systematically characterize missense alleles in the low-density lipoprotein receptor (LDLR) gene identified through exome sequencing of 3,235 individuals and exome-chip profiling of 39,186 individuals. Our strategy reliably identifies disruptive missense alleles, and disruptive-allele carriers have higher plasma LDL-cholesterol (LDL-C). Importantly, considering experimental data refined the risk of rare LDLR allele carriers from 4.5- to 25.3-fold for high LDL-C, and from 2.1- to 20-fold for early-onset myocardial infarction. Our study generates proof-of-concept that systematic functional variant profiling may empower rare variant-association studies by orders of magnitude.     

The CD4+AT2R+ T cell subpopulation improves post‐infarction remodelling and restores cardiac function

Myocardial infarction (MI) is a major condition causing heart failure (HF). After MI, the renin angiotensin system (RAS) and its signalling octapeptide angiotensin II (Ang II) interferes with cardiac injury/repair via the AT1 and AT2 receptors (AT1R, AT2R). Our study aimed at deciphering the mechanisms underlying the link between RAS and cellular components of the immune response relying on a rodent model of HF as well as HF patients. Flow cytometric analyses showed an increase in the expression of CD4⁺ AT2R⁺ cells in the rat heart and spleen post‐infarction, but a reduction in the peripheral blood. The latter was also observed in HF patients. The frequency of rat CD4⁺ AT2R⁺ T cells in circulating blood, post‐infarcted heart and spleen represented 3.8 ± 0.4%, 23.2 ± 2.7% and 22.6 ± 2.6% of the CD4⁺ cells. CD4⁺ AT2R⁺ T cells within blood CD4⁺ T cells were reduced from 2.6 ± 0.2% in healthy controls to 1.7 ± 0.4% in patients. Moreover, we characterized CD4⁺ AT2R⁺ T cells which expressed regulatory FoxP3, secreted interleukin‐10 and other inflammatory‐related cytokines. Furthermore, intramyocardial injection of MI‐induced splenic CD4⁺ AT2R⁺ T cells into recipient rats with MI led to reduced infarct size and improved cardiac performance. We defined CD4⁺ AT2R⁺ cells as a T cell subset improving heart function post‐MI corresponding with reduced infarction size in a rat MI‐model. Our results indicate CD4⁺ AT2R⁺ cells as a promising population for regenerative therapy, via myocardial transplantation, pharmacological AT2R activation or a combination thereof.     

Protease‐activated receptor (PAR)‐2 is required for PAR‐1 signalling in pulmonary fibrosis

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease‐activated receptor (PAR)‐1 and PAR‐2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin‐induced lung fibrosis is diminished in both PAR‐1 and PAR‐2 deficient mice. We thus have been suggested that combined inactivation of PAR‐1 and PAR‐2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR‐1 and PAR‐2 agonists in the absence or presence of specific PAR‐1 or PAR‐2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild‐type and PAR‐2 deficient mice with or without a specific PAR‐1 antagonist (P1pal‐12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR‐1 and/or PAR‐2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR‐1 and PAR‐2 did not show any additive effects on these pro‐fibrotic responses. Strikingly, PAR‐2 deficiency as well as pharmacological PAR‐1 inhibition reduced bleomycin‐induced pulmonary fibrosis to a similar extent. PAR‐1 inhibition in PAR‐2 deficient mice did not further diminish bleomycin‐induced pulmonary fibrosis. Finally, we show that the PAR‐1‐dependent pro‐fibrotic responses are inhibited by the PAR‐2 specific antagonist. Targeting PAR‐1 and PAR‐2 simultaneously is not superior to targeting either receptor alone in bleomycin‐induced pulmonary fibrosis. We postulate that the pro‐fibrotic effects of PAR‐1 require the presence of PAR‐2.     

Reduction in Subventricular Zone-Derived Olfactory Bulb Neurogenesis in a Rat Model of Huntington’s Disease Is Accompanied by Striatal Invasion of Neuroblasts

Huntington’s disease (HD) is an inherited progressive neurodegenerative disorder caused by an expanded CAG repeat in exon 1 of the huntingtin gene (HTT). The primary neuropathology of HD has been attributed to the preferential degeneration of medium spiny neurons (MSN) in the striatum. Reports on striatal neurogenesis have been a subject of debate; nevertheless, it should be considered as an endogenous attempt to repair the brain. The subventricular zone (SVZ) might offer a close-by region to supply the degenerated striatum with new cells. Previously, we have demonstrated that R6/2 mice, a widely used preclinical model representing an early onset HD, showed reduced olfactory bulb (OB) neurogenesis but induced striatal migration of neuroblasts without affecting the proliferation of neural progenitor cell (NPCs) in the SVZ. The present study revisits these findings, using a clinically more relevant transgenic rat model of late onset HD (tgHD rats) carrying the human HTT gene with 51 CAG repeats and mimicking many of the neuropathological features of HD seen in patients. We demonstrate that cell proliferation is reduced in the SVZ and OB of tgHD rats compared to WT rats. In the OB of tgHD rats, although cell survival was reduced, the frequency of neuronal differentiation was not altered in the granule cell layer (GCL) compared to the WT rats. However, an increased frequency of dopamenergic neuronal differentiation was noticed in the glomerular layer (GLOM) of tgHD rats. Besides this, we observed a selective proliferation of neuroblasts in the adjacent striatum of tgHD rats. There was no evidence for neuronal maturation and survival of these striatal neuroblasts. Therefore, the functional role of these invading neuroblasts still needs to be determined, but they might offer an endogenous alternative for stem or neuronal cell transplantation strategies.     

Regression to the Mean and Predictors of MRI Disease Activity in RRMS Placebo Cohorts - Is There a Place for Baseline-to-Treatment Studies in MS?

BackgroundGadolinium-enhancing (GD+) lesions and T2 lesions are MRI outcomes for phase-2 treatment trials in relapsing-remitting Multiple Sclerosis (RRMS). Little is known about predictors of lesion development and regression-to-the-mean, which is an important aspect in early baseline-to-treatment trials.ObjectivesTo quantify regression-to-the-mean and identify predictors of MRI lesion development in placebo cohorts.Methods21 Phase-2 and Phase-3 trials were identified by a systematic literature research. Random-effects meta-analyses were performed to estimate development of T2 and GD+ after 6 months (phase-2) or 2 years (phase-3). Predictors of lesion development were evaluated with mixed-effect meta-regression.ResultsThe mean number of GD+-lesions per scan was similar after 6 months (1.19, 95%CI: 0.87-1.51) and 2 years (1.19, 95%CI: 1.00-1.39). 39% of the patients were without new T2-lesion after 6 month and 19% after 2 years (95%CI: 12-25%). Mean number of baseline GD+-lesions was the best predictor for new lesions after 6 months.ConclusionBaseline GD-enhancing lesions predict evolution of Gd- and T2 lesions after 6 months and might be used to control for regression to the mean effects. Overall, proof-of-concept studies with a baseline to treatment design have to face a regression to 1.2 GD+lesions per scan within 6 months.     

Development of a Novel Strategy to Isolate Lipophilic Allergens (Oleosins) from Peanuts

Background

Peanut allergy is one of the most severe class I food allergies with increasing prevalence. Especially lipophilic allergens, such as oleosins, were found to be associated with severe symptoms, but are usually underrepresented in diagnostic extracts. Therefore, this study focused on isolation, molecular characterization and assessment of the allergenicity of peanut oleosins.

Methods and Results

A comprehensive method adapted for the isolation of peanut oil bodies of high purity was developed comprising a stepwise removal of seed storage proteins from oil bodies. Further separation of the oil body constituents, including the allergens Ara h 10, Ara h 11, the presumed allergen oleosin 3 and additional oleosin variants was achieved by a single run on a preparative electrophoresis cell. Protein identification realized by N-terminal sequencing, peptide mass fingerprinting and homology search revealed the presence of oleosins, steroleosins and a caleosin. Immunoblot analysis with sera of peanut-allergic individuals illustrated the IgE-binding capacity of peanut-derived oleosins.

Conclusion

Our method is a novel way to isolate all known immunologically distinct peanut oleosins simultaneously. Moreover, we were able to provide evidence for the allergenicity of oleosins and thus identified peanut oleosins as probable candidates for component-resolved allergy diagnosis.     

Long-Term Sequelae of Severe Acute Kidney Injury in the Critically Ill Patient without Comorbidity: A Retrospective Cohort Study

Background and Objectives

Acute kidney injury (AKI) necessitating renal replacement therapy (RRT) is associated with high mortality and increased risk for end stage renal disease. However, it is unknown if this applies to patients with a preliminary unremarkable medical history. The purpose of this study was to describe overall and renal survival in critically ill patients with AKI necessitating RRT stratified by the presence of comorbidity.

Design, Setting, Participants, and Measurements

A retrospective cohort study was performed, between 1994 and 2010, including all adult critically ill patients with AKI necessitating RRT, stratified by the presence of comorbidity. Logistic regression, survival curve and cox proportional hazards analyses were used to evaluate overall and renal survival. Standardized mortality rate (SMR) analysis was performed to compare long-term survival to the predicted survival in the Dutch population.

Results

Of the 1067 patients included only 96(9.0%) had no comorbidity. Hospital mortality was 56.6% versus 43.8% in patients with and without comorbidity, respectively. In those who survived hospitalization 10-year survival was 45.0% and 86.0%, respectively. Adjusted for age, sex and year of treatment, absence of comorbidity was not associated with hospital mortality (OR=0.74, 95%-CI=0.47-1.15), while absence of comorbidity was associated with better long-term survival (adjusted HR=0.28, 95%-CI = 0.14-0.58). Compared to the Dutch population, patients without comorbidity had a similar mortality risk (SMR=1.6, 95%-CI=0.7-3.2), while this was increased in patients with comorbidity (SMR=4.8, 95%-CI=4.1-5.5). Regarding chronic dialysis dependency, 10-year renal survival rates were 76.0% and 92.9% in patients with and without comorbidity, respectively. Absence of comorbidity was associated with better renal survival (adjusted HR=0.24, 95%-CI=0.07-0.76).

Conclusions

While hospital mortality remains excessively high, the absence of comorbidity in critically ill patients with RRT-requiring AKI is associated with a relative good long-term prognosis in those who survive hospitalization.     

Paravertebral Catheter for Three-Level Injection in Radical Mastectomy: A Randomised Controlled Study

Introduction

Paravertebral block (PVB) is an alternative to general anaesthesia (GA) for breast surgery. However, for extensive surgery multiple punctures are needed increasing the immanent risk of the method. The purpose of this study was to evaluate PVB via catheter and injections at three different levels. Primary outcome was the quality of postoperative analgesia, in particular, the number of patients requiring additional morphine.

Methods

In a randomised single blinded clinical study patients scheduled for breast surgery including axillary approach, were randomly allocated to different anaesthetic techniques, n = 35 each. Patients received either GA with sevoflurane or PVB with catheter at level Th 4. In PVB-patients a 1:2 mixture of bupivacaine 0.5% and lidocaine 2% with adrenaline was injected sequentially 10 ml each at three different levels.

Results

Complication-free catheter insertion was possible in all 35 scheduled patients. The need for postoperative analgesics was higher after GA compared to PVB (22 vs.14 patients); p = 0.056. Postoperative morphine consumption was 1.55 (GA) and 0.26 mg (PVB) respectively (p < 0.001). Visual rating score (VRS) for pain at rest and at movement was higher in GA patients on post anaesthesia care unit (PACU) as well as on the ward at 1 - 6h and 6 - 12h. Readiness for discharge was earlier after PVB (4.96 and 6.52 hours respectively). After GA the incidence and severity of postoperative nausea and vomiting (PONV) was higher, though not significantly. Patients’ satisfaction was comparable in both groups.

Conclusions

Three-level injection PVB via catheter for extensive mastectomy was efficient and well accepted. Using a catheter may enhance safety by avoiding multiple paravertebral punctures when extended spread of analgesia is required.

Trial Registration

www.ClinicalTrial.gov NCT02065947