Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Hypoxia/reoxygenation impairs glucose absorption and cAMP-mediated secretion more profoundly than glutamine absorption and Ca2+/PKC-mediated secretion in rat ileum in vitro

BACKGROUND: Intestinal ischemia and reperfusion may lead to profuse secretion of water and electrolytes. The underlying mechanisms have been related to increased hydrostatic pressure, to denudation of intestinal villi, and, recently, to adenosine-mediated enhancement of chloride secretion. METHODS: We studied the effects of hypoxia and reoxygenation on baseline electrophysiologic parameters; on glucose- and glutamine-induced absorption; on secretion induced by carbachol, histamine, and forskolin; and on epithelial barrier function to disodium-fluorescein and horseradish peroxidase in rat ileum mounted in Ussing chambers. RESULTS: We observed that 30 minutes of hypoxia followed by 60 minutes of reoxygenation differentially affected glucose and glutamine absorption to 11% and 42%, respectively, of control values. Cyclic adenosine monophosphate-mediated secretion induced by forskolin was reduced to 9% of controls. In contrast, Ca(2+)/protein kinase C-mediated secretion induced by carbachol or histamine was reduced only to 35% to 48% of controls. Furthermore, the epithelium fully was capable of maintaining its barrier function to small and large permeability probes, even after 90 minutes of hypoxia. CONCLUSIONS: We conclude that hypoxia and reoxygenation differentially impair nutrient absorption, corroborating recent absorption data in in vivo models of ischemia, and that it differentially affects secretory capacity in crypts, dependent on the intracellular messenger pathway. The relative persistence of Ca(2+)/protein kinase C-mediated secretion to hypoxia and reoxygenation indicates that secretagogues that activate this pathway play a significant role in the intraluminal fluid sequestration and diarrhea observed after intestinal ischemia and reperfusio

Local intravascular coagulation and fibrin deposition on intestinal ischemia-reperfusion in rats

Background. This study investigates intravascular coagulation and thrombotic obstruction in the splanchnic vasculature after intestinal ischemia in relation to epithelial integrity and function. Methods. Intestinal ischemia was induced in rats by superior mesenteric artery occlusion for 20 or 40 minutes. Intestinal injury was assessed by histologic analysis, biochemical markers, and functional studies. During reperfusion, portal and systemic blood samples were collected to analyze activation of coagulation and fibrinolysis. Results. Superior mesenteric artery occlusion resulted in mild to moderate intestinal injury. Twenty and 40 minutes of ischemia and 3 hours of reperfusion resulted in local intestinal thrombin generation and conversion of fibrinogen to fibrin, reflected by 3- and 4-fold increases in thrombin-antithrombin complex levels and a 3-fold elevation of fibrin degradation products (D-dimer), respectively. During reperfusion, after a short-lasting initial, activation of local fibrinolysis, plasminogen activator activity was suppressed, as indicated by an approximately 4-fold increase in portal plasma levels of the plasminogen activator inhibitor D-dimer levels showed that activation of coagulation and depression of fibrinolysis resulted in fibrin formation, which was confirmed to be intravascular fibrin deposition by histologic examination. Conclusions. Intestinal ischemia-reperfusion results in local intravascular coagulation and fibrin depositio

Current treatment practice of functional abdominal pain disorders in children: A multicenter survey

Background: Approximately 90% of the children with chronic abdominal pain are diagnosed as having functional abdominal pain disorder (FAPD). The Dutch guideline “functional abdominal pain” provides a stepwise approach to treat FAPD. The aim of this survey was twofold first, to determine adherence to the Dutch guideline, and second to determine current management of FAPDs in clinical practice. Methods: A multicenter survey was designed. The survey was sent to pediatricians and pediatric residents in December 2020. The study ran from October 2020 until March 2021. Participants in ten hospitals in the western region of The Netherlands were invited to complete this survey. Respondents who indicated not to treat children with FAPDs or respondents who completed less than 3 steps of the survey were excluded. Results: In total, 85/174 (48.9%) respondents completed the survey. We included 80 respondents, 68 pediatricians and 12 pediatric residents, for analysis. Overall, self-reported guideline adherence was 85%. Self-reported adherence was higher than actual adherence. Only 50% of all respondents followed the first three steps of the guideline. The reported non-pharmacological and pharmacological treatments were diverse and varied between different age groups. The average follow-up duration was between 2 and 6 months, and the most regularly used outcome measures were attendance at school, quality of life, and adequate pain relief/reassurance. Conclusion: We reportedly observed a large variation in the management of children with FAPDs, due to low guideline adherence among clinicians. Improved guideline adherence may be accomplished by updating the guideline with specific recommendations per subtype, follow-up and outcome measures as well measures to improve guideline implementation

Pancreatic cyst surveillance imposes low psychological burden

Background/Objectives: For the currently recommended pancreatic cyst surveillance to be feasible, participant adherence is a prerequisite. Our objective was to evaluate the psychological burden of pancreatic cyst surveillance from a participant's perspective. Methods: The present participant survey is part of an international cohort study (PACYFIC study, www.pacyfic.net), which prospectively records the outcome of surveillance of asymptomatic pancreatic cysts. Participants are invited to complete questionnaires before and during cyst surveillance. Results: 109 participants, 31 enrolled before and 78 during surveillance (median time since cyst diagnosis 16.5 (IQR 36) months), returned a total of 179 questionnaires. The majority indicated that surveillance reduces concerns of developing pancreatic cancer (82%), gives a sense of certainty (81%) and is a good method to detect cancer (91%). Participants already undergoing surveillance reported more negative aspects than those still to commence, like sleeping worse (30% vs 13%, P = 0.035), postponing plans (32% vs 13%, P = 0.031), and finding the follow-up burdensome (33% vs 13%, P = 0.044). Overall, the vast majority (94%) deemed advantages to outweigh disadvantages. Anxiety and depression scores were low (median Hospital Anxiety and Depression Scale 4 for anxiety (IQR 6), 2 for depression (IQR 5)). Conclusion: The psychological burden of pancreatic cyst surveillance is low. Therefore, participant adherence is expected to be high and annual surveillance seems feasible

Molecular characterization of colorectal cancer related peritoneal metastatic disease

A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity