GWAS meta-analysis of 16 790 patients with Barrett's oesophagus and oesophageal adenocarcinoma identifies 16 novel genetic risk loci and provides insights into disease aetiology beyond the single marker level.

OBJECTIVE: Oesophageal cancer (EC) is the sixth leading cause of cancer-related deaths. Oesophageal adenocarcinoma (EA), with Barrett's oesophagus (BE) as a precursor lesion, is the most prevalent EC subtype in the Western world. This study aims to contribute to better understand the genetic causes of BE/EA by leveraging genome wide association studies (GWAS), genetic correlation analyses and polygenic risk modelling. DESIGN: We combined data from previous GWAS with new cohorts, increasing the sample size to 16 790 BE/EA cases and 32 476 controls. We also carried out a transcriptome wide association study (TWAS) using expression data from disease-relevant tissues to identify BE/EA candidate genes. To investigate the relationship with reported BE/EA risk factors, a linkage disequilibrium score regression (LDSR) analysis was performed. BE/EA risk models were developed combining clinical/lifestyle risk factors with polygenic risk scores (PRS) derived from the GWAS meta-analysis. RESULTS: The GWAS meta-analysis identified 27 BE and/or EA risk loci, 11 of which were novel. The TWAS identified promising BE/EA candidate genes at seven GWAS loci and at five additional risk loci. The LDSR analysis led to the identification of novel genetic correlations and pointed to differences in BE and EA aetiology. Gastro-oesophageal reflux disease appeared to contribute stronger to the metaplastic BE transformation than to EA development. Finally, combining PRS with BE/EA risk factors improved the performance of the risk models. CONCLUSION: Our findings provide further insights into BE/EA aetiology and its relationship to risk factors. The results lay the foundation for future follow-up studies to identify underlying disease mechanisms and improving risk prediction.

[Therapy of esophageal motility disorders]. / Therapieoptionen von primären Motilitätsstörungen des Ösophagus.

Esophageal motility disorders are diseases in which there are malfunctions of the act of swallowing due to a change in neuromuscular structures. The main symptom is therefore dysphagia for solid and/or liquid foods, often accompanied by symptoms such as chest pain, regurgitation, heartburn, and weight loss. Esophageal manometry is the gold standard in diagnostics. Endoscopy and radiology serve to exclude inflammatory or malignant changes. With the introduction of high-resolution esophageal manometry (HRM), the diagnosis of esophageal motility disorders has improved and led to a new classification with the Chicago Classification, which has been modified several times in the last decade, most recently in 2020 with the Chicago Classification v4.0. Compared to the previous version 3.0, there are some important changes that are presented based on the most important esophageal motility disorders in everyday clinical practice.

[Diagnosis of esophageal motility disorders - Significance of the new Chicago classification v4.0]. / Diagnostik von Motilitätsstörungen des Ösophagus ­ Bedeutung der neuen Chicago-Klassifikation v4.0.

Esophageal motility disorders are diseases in which there are malfunctions of the act of swallowing due to a change in neuromuscular structures. The main symptom is therefore dysphagia for solid and/or liquid foods, often accompanied by symptoms such as chest pain, regurgitation, heartburn, and weight loss. Esophageal manometry is the gold standard in diagnostics. Endoscopy and radiology serve to exclude inflammatory or malignant changes. With the introduction of high-resolution esophageal manometry (HRM), the diagnosis of esophageal motility disorders has improved and led to a new classification with the Chicago Classification, which has been modified several times in the last decade, most recently in 2021 with the Chicago Classification v4.0. Compared to the previous version 3.0, there are some important changes that are presented based on the most important esophageal motility disorders in everyday clinical practice.

[Diagnosis of esophageal motility disorders - Significance of the new Chicago classification v4.0]. / Diagnostik von Motilitätsstörungen des Ösophagus ­ Bedeutung der neuen Chicago-Klassifikation v4.0.

Esophageal motility disorders are diseases in which there are malfunctions of the act of swallowing due to a change in neuromuscular structures. The main symptom is therefore dysphagia for solid and/or liquid foods, often accompanied by symptoms such as chest pain, regurgitation, heartburn, and weight loss. Esophageal manometry is the gold standard in diagnostics. Endoscopy and radiology serve to exclude inflammatory or malignant changes. With the introduction of high-resolution esophageal manometry (HRM), the diagnosis of esophageal motility disorders has improved and led to a new classification with the Chicago Classification, which has been modified several times in the last decade, most recently in 2021 with the Chicago Classification v4.0. Compared to the previous version 3.0, there are some important changes that are presented based on the most important esophageal motility disorders in everyday clinical practice.

[Therapy of esophageal motility disorders]. / Therapieoptionen von primären Motilitätsstörungen des Ösophagus.

Esophageal motility disorders are diseases in which there are malfunctions of the act of swallowing due to a change in neuromuscular structures. The main symptom is therefore dysphagia for solid and/or liquid foods, often accompanied by symptoms such as chest pain, regurgitation, heartburn, and weight loss. Esophageal manometry is the gold standard in diagnostics. Endoscopy and radiology serve to exclude inflammatory or malignant changes. With the introduction of high-resolution esophageal manometry (HRM), the diagnosis of esophageal motility disorders has improved and led to a new classification with the Chicago Classification, which has been modified several times in the last decade, most recently in 2020 with the Chicago Classification v4.0. Compared to the previous version 3.0, there are some important changes that are presented based on the most important esophageal motility disorders in everyday clinical practice.

Maternal inflammation has a profound effect on cortical interneuron development in a stage and subtype-specific manner.

Severe infections during pregnancy are one of the major risk factors for cognitive impairment in the offspring. It has been suggested that maternal inflammation leads to dysfunction of cortical GABAergic interneurons that in turn underlies cognitive impairment of the affected offspring. However, the evidence comes largely from studies of adult or mature brains and how the impairment of inhibitory circuits arises upon maternal inflammation is unknown. Here we show that maternal inflammation affects multiple steps of cortical GABAergic interneuron development, i.e., proliferation of precursor cells, migration and positioning of neuroblasts, as well as neuronal maturation. Importantly, the development of distinct subtypes of cortical GABAergic interneurons was discretely impaired as a result of maternal inflammation. This translated into a reduction in cell numbers, redistribution across cortical regions and layers, and changes in morphology and cellular properties. Furthermore, selective vulnerability of GABAergic interneuron subtypes was associated with the stage of brain development. Thus, we propose that maternally derived insults have developmental stage-dependent effects, which contribute to the complex etiology of cognitive impairment in the affected offspring.

Lack of APP and APLP2 in GABAergic Forebrain Neurons Impairs Synaptic Plasticity and Cognition.

Amyloid-ß precursor protein (APP) is central to the pathogenesis of Alzheimer's disease, yet its physiological functions remain incompletely understood. Previous studies had indicated important synaptic functions of APP and the closely related homologue APLP2 in excitatory forebrain neurons for spine density, synaptic plasticity, and behavior. Here, we show that APP is also widely expressed in several interneuron subtypes, both in hippocampus and cortex. To address the functional role of APP in inhibitory neurons, we generated mice with a conditional APP/APLP2 double knockout (cDKO) in GABAergic forebrain neurons using DlxCre mice. These DlxCre cDKO mice exhibit cognitive deficits in hippocampus-dependent spatial learning and memory tasks, as well as impairments in species-typic nesting and burrowing behaviors. Deficits at the behavioral level were associated with altered neuronal morphology and synaptic plasticity Long-Term Potentiation (LTP). Impaired basal synaptic transmission at the Schafer collateral/CA1 pathway, which was associated with altered compound excitatory/inhibitory synaptic currents and reduced action potential firing of CA1 pyramidal cells, points to a disrupted excitation/inhibition balance in DlxCre cDKOs. Together, these impairments may lead to hippocampal dysfunction. Collectively, our data reveal a crucial role of APP family proteins in inhibitory interneurons to maintain functional network activity.

Influence of oral premedication and prewarming on core temperature of cardiac surgical patients: a prospective, randomized, controlled trial.

BACKGROUND: Perioperative hypothermia is still very common and associated with numerous adverse effects. The effects of benzodiazepines, administered as premedication, on thermoregulation have been studied with conflicting results. We investigated the hypotheses that premedication with flunitrazepam would lower the preoperative core temperature and that prewarming could attenuate this effect. METHODS: After approval by the local research ethics committee 50 adult cardiac surgical patients were included in this prospective, randomized, controlled, single-centre study with two parallel groups in a university hospital setting. Core temperature was measured using a continuous, non-invasive zero-heat flux thermometer from 30 min before administration of the oral premedication until beginning of surgery. An equal number of patients was randomly allocated via a computer-generated list assigning them to either prewarming or control group using the sealed envelope method for blinding. The intervention itself could not be blinded. In the prewarming group patients received active prewarming using an underbody forced-air warming blanket. The data were analysed using Student's t-test, Mann-Whitney U-test and Fisher's exact test. RESULTS: Of the randomized 25 patients per group 24 patients per group could be analysed. Initial core temperature was 36.7 ± 0.2 °C and dropped significantly after oral premedication to 36.5 ± 0.3 °C when the patients were leaving the ward and to 36.4 ± 0.3 °C before induction of anaesthesia. The patients of the prewarming group had a significantly higher core temperature at the beginning of surgery (35.8 ± 0.4 °C vs. 35.5 ± 0.5 °C, p = 0.027), although core temperature at induction of anaesthesia was comparable. Despite prewarming, core temperature did not reach baseline level prior to premedication (36.7 ± 0.2 °C). CONCLUSIONS: Oral premedication with benzodiazepines on the ward lowered core temperature significantly at arrival in the operating room. This drop in core temperature cannot be offset by a short period of active prewarming. TRIAL REGISTRATION: This trial was prospectively registered with the German registry of clinical trials under the trial number DRKS00005790 on 20th February 2014.

Outcomes of pneumatic dilation in achalasia: Extended follow-up of more than 25 years with a focus on manometric subtypes.

BACKGROUND AND AIM: Pneumatic dilation (PD) is the most popular nonsurgical treatment for achalasia. This study investigated predicting factors, including manometric subtypes for symptom recurrence in the long term, in patients with achalasia treated with a single PD. METHODS: Between 1983 and 2013, a total of 107 patients were treated initially with a single PD and included in this longitudinal cohort study. Outcomes were correlated with demographics, symptoms (Eckardt score), and esophagographic and manometric features. Manometric tracings were retrospectively classified according to the three subtypes of the Chicago classification. RESULTS: Ninety-one (85%) patients were successfully treated after the first PD. The median follow-up was 13.8 years (interquartile range 7-20). During follow-up, 54% of the patients experienced a clinical relapse. The overall cumulative success rates at 2, 5, 10, 15, 20, and 25 years were 64%, 53%, 49%, 42%, 36%, and 36%, respectively. Age < 40 years, lower esophageal sphincter pressure > 15 mmHg, a cardia width < 5 mm, and an esophageal barium column height > 1 cm 4 to 12 weeks post-dilation significantly correlated with symptom recurrence, whereas achalasia subtypes did not significantly correlate with the treatment results. CONCLUSION: Pneumatic dilation in achalasia is an effective therapy in the short term, but its effect wanes in the very long term. Young age at presentation, a high lower esophageal sphincter pressure, a narrow cardia, and an esophageal barium column of > 1 cm after PD are predictive factors for the need of repeated treatment.

A randomised, double-blind trial comparing budesonide formulations and dosages for short-term treatment of eosinophilic oesophagitis.

OBJECTIVE: To investigate the efficacy and safety of two different budesonide formulations (effervescent tablet for orodispersible use (BET) and viscous suspension (BVS)) with different daily dosages for short-term treatment of eosinophilic oesophagitis (EoE). DESIGN: Adults with active EoE (n=76) randomly received 14 days' treatment with either BET 2×1 mg/day (BET1, n=19) or BET 2×2 mg/day (BET2, n=19), or BVS 2×5 mL (0.4 mg/mL)/day (BVS, n=19) or placebo (n=19) in a double-blind, double-dummy fashion, with a 2-week follow-up. Primary end point was histological remission (mean of <16 eosinophils/mm(2 )hpf). Secondary end points included endoscopy score, dysphagia score, drug safety and patient's preference for drug formulation. RESULTS: Histological remission occurred in 100%, 94.7% and 94.7% of budesonide (BET1, BET2, BVS, respectively) and in 0% of placebo recipients (p<0.0001). The improvement in total endoscopic intensity score was significantly higher in the three budesonide groups compared with placebo. Dysphagia improved in all groups at the end of treatment; however, improvement of dysphagia persisted only in those treated with BET1 (p=0.0196 vs placebo). There were no serious adverse events. Local fungal infection (stained fungi) occurred in two patients of each budesonide group (10.5%). The effervescent tablet was preferred by 80% of patients. CONCLUSIONS: BET or BVS was highly effective and safe for short-term treatment of EoE. The 1 mg (twice daily) dosage was equally effective as the 2 mg twice daily dosage. The majority of patients preferred the effervescent tablet formulation. CLINICALTRIALSGOV NUMBER: NCT02280616; EudraCT number, 2009-016692-29.

Classification of ADAMTS binding sites: The first step toward selective ADAMTS7 inhibitors.

Genome-wide association studies identified ADAMTS7 as a risk locus for coronary artery disease. In carotid arteries of rats, neointima formation after balloon-mediated injury goes along with enhanced Adamts7 expression. Vice versa, Adamts7-deficient mice display reduced neointima formation following vascular injury. Although a causal link between ADAMTS7 and coronary artery disease remains to be proven, inhibition of ADAMTS7 represents a potential new target for intervention in this disease. ADAMTS7, a member of the 'a disintegrin and metalloproteinase with thrombospondin motifs' (ADAMTS) family of proteins, contains a catalytic zinc ion in the binding site of its metalloproteinase domain. The structure of ADAMTS7 and its inhibitors are unknown. In this study, we used in silico methods, including homology modeling and pharmacophore modeling, to analyze the ADAMTS7 metalloproteinase domain, particularly its binding site. The results revealed structural and sequence differences relative to the binding sites of the other ADAMTS proteins; these non-conserved regions represent potential binding regions for selective ADAMTS7 inhibitors. The main contribution of this study is the proposal of a pharmacophore for ADAMTS7. The characterization of the ADAMTS7 binding site and definition of a pharmacophore are the first step toward developing a new therapeutic target for coronary artery disease.

Murine genetic deficiency of neuronal nitric oxide synthase (nNOS(-/-) ) and interstitial cells of Cajal (W/W(v) ): Implications for achalasia?

BACKGROUND AND AIM: Nitric oxide (NO) is an important inhibitory mediator of esophageal function, and its lack leads to typical features of achalasia. In contrast, the role of intramuscular interstitial cells of Cajal (ICC-IM) and vasoactive intestinal peptide (VIP) in lower esophageal sphincter (LES) function is still controversial. Therefore, we examined the function and morphology of the LES in vivo in NO-deficient (nNOS(-/-) ), ICC-IM-deficient (W/W(v) )-, and wild-type (WT) mice. METHODS: Esophageal manometry was performed with a micro-sized transducer catheter to quantify LES pressure, swallow evoked LES relaxation, and esophageal body motility. The LES morphology was examined by semiquantitative analysis of the immunoreactivity (reduction grade I-IV) of neuronal NOS (nNOS), ICC-IM, and VIP and their correlation with esophageal function. RESULTS: nNOS(-/-) in comparison to WT mice showed a significantly higher LES mean resting pressure with an impaired swallow induced relaxation, whereas W/W(v) mice had a hypotensive LES with decreased relaxation. W/W(v) and nNOS(-/-) mice demonstrated differing degrees of tubular esophageal dysfunction. The reduced immunoreactivity of nNOS correlated with an increased LES pressure and decreased LES relaxation, respectively. Cajal-cell reduction correlated with impaired LES relaxation, whereas VIP reduction revealed no correlation with esophageal function. CONCLUSIONS: The reduction of ICC-IM and nNOS can cause dysfunction of the LES and esophageal peristalsis, whereas VIP reduction seems to have no effect. ICC-IM and nNOS deficiency might be independent relevant causes of esophageal dysfunction similar to that seen in human achalasia.

Prospective evaluation of the routine use of a nasopharyngeal airway (Wendl Tube) during endoscopic propofol-based sedation.

BACKGROUND/AIMS: Insertion of a nasopharyngeal airway (NPA) during endoscopic sedation is only recommended in the event of respiratory problems. We evaluated the safety and efficacy of routine insertion of an NPA during sedation in gastrointestinal (GI) endoscopy. METHODS: Between July 2009 and April 2012, patients with colonoscopy or expected longer-lasting or therapeutic upper GI endoscopy were pseudo-randomized in a weekly alternating fashion to perform sedation (midazolam in combination with propofol) with or without NPA insertion. The primary outcome measure was respiratory depression (oxygen saturation <90%). Secondary measures included hypotension (systolic blood pressure <90 mm Hg), bradycardia (heart rate <40 beats/min) or nasopharyngeal damage after NPA insertion. RESULTS: 216 (106 females, mean age 60.7 ± 9.65 years) were enrolled. Colonoscopy was performed in 131 patients and upper endoscopy in 85 patients. In 105 patients an NPA was used (intervention group). Five (4.7%) of those patients showed minor nasopharyngeal injury. Respiratory depression (13.5 vs. 1.9%, p = 0.002) and hypotension (11 vs. 5%, p = 0.09) occurred more frequently in the control than in the intervention group. CONCLUSION: The routine placement of an NPA can reduce the frequency of hypoxemic events during endoscopic sedation with minor risks for nasopharyngeal injury.

Effectiveness of Biologics, Patient-Reported Outcomes, and Clinical Photography in a Subset of Patients with Moderate-to-Severe Psoriasis: Week 12 Results from the Psoriasis Study of Health Outcomes (PSoHO).

Purpose: Since skin is highly accessible, clinical photography is a useful tool to visually substantiate the real-world effectiveness outcomes of biologic-treated adults with moderate-to-severe psoriasis (PsO). We report the effectiveness and patient-reported outcomes at Week 12 between anti-interleukin (IL)-17A biologics and other biologics as well as ixekizumab and guselkumab in patients with available clinical photography at baseline and Week 12. Patients and Methods: The Psoriasis Study of Health Outcomes (PSoHO) is an international, non-interventional, cohort study investigating the effectiveness of biologics in adults with moderate-to-severe psoriasis at Week 12. Outcomes included the proportion of patients who achieved 90% improvement in Psoriasis Area and Severity Index (PASI90) and/or static Physician Global Assessment (sPGA) 0/1 (primary endpoint), PASI100, PASI90, Dermatology Life Quality Index (DLQI), and Itch Numeric Rating Scale (NRS) (secondary endpoints) at Week 12. Data are reported descriptively. Results: This analysis included 59 biologic-treated (23 anti-IL-17A; 36 other biologics) patients with available clinical photographs from the overall PSoHO study (n=1981). At baseline, the mean (standard deviation [SD]) age was 45.7 (11.1) years, 71.2% were male, 52.5% were bio-experienced and the median (interquartile range) duration of disease was 10.5 (12.4) years. Mean (SD) PASI was 16.9 (9.3) and sPGA was 3.5 (0.8). At Week 12, 65.2%/47.2% of the anti-IL-17A/other biologics cohort achieved the primary outcome. Response rates for PASI90/100 were numerically higher with anti-IL-17A than with other biologics. Patients receiving anti-IL-17A had numerically better outcomes for DLQI 0/1 and Itch NRS than those receiving other biologics at Week 12. Clinical photographs confirmed skin improvements in ixekizumab- and guselkumab-treated patients. Conclusion: This subgroup analysis showed that anti-IL-17A biologics are effective at rapidly improving signs and symptoms of PsO and improving quality of life. Additionally, serial photography provided visual evidence of biologic treatment response over time.

Clinical and manometric course of nonspecific esophageal motility disorders.

BACKGROUND AND AIMS: The evolution of nonspecific esophageal motility disorders remains unclear. The aim of this study was to investigate whether nonspecific esophageal motility disorders progress into specific motility disorders and whether such progression is predictable. METHODS: Seventy-six symptomatic patients (49 males, 27 females, mean age 57 ± 16 years) with newly diagnosed nonspecific esophageal motility disorders were prospectively registered and followed-up. Follow-up visits, with structured interviews and manometric re-evaluation, were recommended biannually and whenever symptoms exacerbated. RESULTS: Forty-three patients were followed for up to 4 years, symptoms worsened in 30% of patients, resolved in 26%, improved in 14% and were unchanged in 30%. Twenty-eight patients agreed to undergo manometric re-evaluation. Fifteen (53.6%) of these patients showed a progression to achalasia. The remaining patients continued to display features of nonspecific esophageal motility disorders (32%) or had normal motility (11%). The only significant association could be determined between age and progression to achalasia reaching nearly 100% in patients' ≤46 years of age. In contrast, none of the patients' ≥68 years progressed. CONCLUSION: More than half of the patients in our cohort with nonspecific esophageal motility disorders showed a transition into achalasia. Neither manometric nor clinical findings predicted the progression of nonspecific esophageal motility disorders. However, young patients were more likely to progress to achalasia.

Rapid decay of unstable Leishmania mRNAs bearing a conserved retroposon signature 3'-UTR motif is initiated by a site-specific endonucleolytic cleavage without prior deadenylation.

We have previously shown that the Leishmania genome possess two widespread families of extinct retroposons termed Short Interspersed DEgenerated Retroposons (SIDER1/2) that play a role in post-transcriptional regulation. Moreover, we have demonstrated that SIDER2 retroposons promote mRNA degradation. Here we provide new insights into the mechanism by which unstable Leishmania mRNAs harboring a SIDER2 retroposon in their 3'-untranslated region are degraded. We show that, unlike most eukaryotic transcripts, SIDER2-bearing mRNAs do not undergo poly(A) tail shortening prior to rapid turnover, but instead, they are targeted for degradation by a site-specific endonucleolytic cleavage. The main cleavage site was mapped in two randomly selected SIDER2-containing mRNAs in vivo between an AU dinucleotide at the 5'-end of the second 79-nt signature (signature II), which represents the most conserved sequence amongst SIDER2 retroposons. Deletion of signature II abolished endonucleolytic cleavage and deadenylation-independent decay and increased mRNA stability. Interestingly, we show that overexpression of SIDER2 anti-sense RNA can increase sense transcript abundance and stability, and that complementarity to the cleavage region is required for protecting SIDER2-containing transcripts from degradation. These results establish a new paradigm for how unstable mRNAs are degraded in Leishmania and could serve as the basis for a better understanding of mRNA decay pathways in general.

Temporal hyper-precision of brainstem neurons alters spatial sensitivity of binaural auditory processing with cochlear implants.

The ability to localize a sound source in complex environments is essential for communication and navigation. Spatial hearing relies predominantly on the comparison of differences in the arrival time of sound between the two ears, the interaural time differences (ITDs). Hearing impairments are highly detrimental to sound localization. While cochlear implants (CIs) have been successful in restoring many crucial hearing capabilities, sound localization via ITD detection with bilateral CIs remains poor. The underlying reasons are not well understood. Neuronally, ITD sensitivity is generated by coincidence detection between excitatory and inhibitory inputs from the two ears performed by specialized brainstem neurons. Due to the lack of electrophysiological brainstem recordings during CI stimulation, it is unclear to what extent the apparent deficits are caused by the binaural comparator neurons or arise already on the input level. Here, we use a bottom-up approach to compare response features between electric and acoustic stimulation in an animal model of CI hearing. Conducting extracellular single neuron recordings in gerbils, we find severe hyper-precision and moderate hyper-entrainment of both the excitatory and inhibitory brainstem inputs to the binaural comparator neurons during electrical pulse-train stimulation. This finding establishes conclusively that the binaural processing stage must cope with highly altered input statistics during CI stimulation. To estimate the consequences of these effects on ITD sensitivity, we used a computational model of the auditory brainstem. After tuning the model parameters to match its response properties to our physiological data during either stimulation type, the model predicted that ITD sensitivity to electrical pulses is maintained even for the hyper-precise inputs. However, the model exhibits severely altered spatial sensitivity during electrical stimulation compared to acoustic: while resolution of ITDs near midline was increased, more lateralized adjacent source locations became inseparable. These results directly resemble recent findings in rodent and human CI listeners. Notably, decreasing the phase-locking precision of inputs during electrical stimulation recovered a wider range of separable ITDs. Together, our findings suggest that a central problem underlying the diminished ITD sensitivity in CI users might be the temporal hyper-precision of inputs to the binaural comparator stage induced by electrical stimulation.

Clinical implementation of RNA sequencing for Mendelian disease diagnostics.

BACKGROUND: Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics. METHODS: We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage. RESULTS: We detected on average 12,500 genes per sample including around 60% of all disease genes-a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions. CONCLUSION: Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

Selective inactivation of SIDER2 retroposon-mediated mRNA decay contributes to stage- and species-specific gene expression in Leishmania.

Despite their high genomic synteny, the Leishmania major and Leishmania infantum species exhibit extensive differences in mRNA expression patterns throughout the parasite's development. Yet, the underlying mechanisms for this species-specific differential gene expression are largely unknown. Here we report that Short Interspersed DEgenerated Retroposons of the SIDER2 subfamily, shown previously to promote rapid mRNA turnover, confer differential regulation of orthologous transcripts resulting in a stage- and species-specific gene expression. We demonstrate that SIDER2-mediated decay of two L. major transcripts encoding a hypothetical protein and an aminomethyltransferase to a similar extent in promastigote and amastigote developmental forms results in a constitutive low expression of the corresponding proteins. In contrast, their L. infantum orthologs are differentially expressed due to the selective inactivation of SIDER2 in intracellular amastigotes. Inactivation of the SIDER2 function blocks the SIDER2-mediated deadenylation-independent decay pathway, and stabilized transcripts are degraded by a slower, deadenylation-dependent mechanism. Sequence variations in SIDER2 retroposons between orthologous transcripts do not contribute to SIDER2 inactivation. Our data suggest that SIDER2 inactivation is 3'-untranslated region context-dependent and that involves possibly species- and stage-specific trans-acting factor(s). These findings further emphasize the important contribution of SIDER retroposons in the control of gene expression across the Leishmania genus.

Long-term recurrence rates following dilation of symptomatic Schatzki rings.

BACKGROUND AND AIMS: This study investigated the long-term clinical course of patients with Schatzki rings, who were treated by single bougie dilation. Furthermore, it analyzed possible predictors for the time of recurrence. PATIENTS AND METHODS: A total of 133 patients (100 males, 33 females) with a mean age of 57 ± 14.6 years who were treated by single dilation with the use of Maloney bougies without the aid of fluoroscopy were prospectively registered and followed-up for a mean duration of 58.3 months (range 12-240 months). Duration of remission was evaluated by Kaplan-Meier estimates with regard to recurrence. Log-rank test was performed to analyze possible predictors for the time to second dilation (recurrence). RESULTS: No complications occurred and all patients were symptom-free at the first follow-up examination 4 weeks after dilation. However, later on, 73 patients required a second dilation. The estimate remission rates were 63.8% (95% CI: 55.6-72.0%) after 2 years, 44.3% (95% CI: 35.4-53.4%) after 5 years, and 39.9% (95% CI: 30.5-49.3%) after 10 years. Neither the initial morphological findings, nor age or gender determined the need for repeated dilation. Only patients treated with a large bougie diameter (≥52 F) seemed to have a tendency for a longer time until symptomatic recurrence. CONCLUSIONS: Single dilation of symptomatic Schatzki rings is a safe and effective therapy. However, more than half of the patients will need a second treatment. Recurrences are unrelated to initial morphological findings, age, or gender. Only the treatment with a large bougie diameter (≥52 F) showed a tendency for a longer time of remission.