Expression of PROKR1 and PROKR2 in Human Enteric Neural Precursor Cells and Identification of Sequence Variants Suggest a Role in HSCR

Background The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung's disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS. Principal Findings In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function. Conclusions Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.This work was supported by Fondo de Investigación Sanitaria, Spain (PI070080, PI1001290 and PI071315 for the E-Rare project), Consejería de Innovación Ciencia y Empresa de la Junta de Andalucía (CTS 2590) and Consejería de Salud de la Junta de Andalucía (PI0249-2008). The CIBER de Enfermedades Raras is an initiative of the ISCIII. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Background: Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. Results: We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. Conclusions: Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases

Prospective individual patient data meta-analysis of two randomized trials on convalescent plasma for COVID-19 outpatients

Data on convalescent plasma (CP) treatment in COVID-19 outpatients are scarce. We aimed to assess whether CP administered during the first week of symptoms reduced the disease progression or risk of hospitalization of outpatients. Two multicenter, double-blind randomized trials (NCT04621123, NCT04589949) were merged with data pooling starting when = 50 years and symptomatic for <= 7days were included. The intervention consisted of 200-300mL of CP with a predefined minimum level of antibodies. Primary endpoints were a 5-point disease severity scale and a composite of hospitalization or death by 28 days. Amongst the 797 patients included, 390 received CP and 392 placebo; they had a median age of 58 years, 1 comorbidity, 5 days symptoms and 93% had negative IgG antibody-test. Seventy-four patients were hospitalized, 6 required mechanical ventilation and 3 died. The odds ratio (OR) of CP for improved disease severity scale was 0.936 (credible interval (CI) 0.667-1.311); OR for hospitalization or death was 0.919 (CI 0.592-1.416). CP effect on hospital admission or death was largest in patients with <= 5 days of symptoms (OR 0.658, 95%CI 0.394-1.085). CP did not decrease the time to full symptom resolution

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Genetic Basis of Hirschsprung¿s Disease

Editors: Ramón Núñez and Maria Angelica Fabbro (C/ Fco. Vaca Morales, Badajoz, Spain, and others).Hirschsprung disease (HSCR, OMIM: 142623), or aganglionic megacolon, is a developmental disorder characterised by the absence of intramural ganglion cells in the submucosal and myenteric plexuses (Meissner and Auerbach plexuses, respectively) along a variable length of the distal gastrointestinal tract, leading to the most common form of functional intestinal obstruction in neonates and children [1]. The disorder was first reported in 1888 by the Danish pediatrician Harald Hirschsprung in two unrelated boys treated until 7 and 11 month of age for severe constipation and abdominal distension resulting in congenital megacolon [2]. But was not till the 1940s, when histological studies revealed the absence of ganglion cells downstream of the dilated part of the colon [1]. HSCR is regarded as a failure of neural crest cells to migrate, proliferate, differentiate or survive in the wall of the gastrointestinal tract between the fifth and 12th week of gestation to form the enteric nervous system (ENS) [3]. The classification of HSCR is according to the length of the aganglionic segment [4]. While the internal anal sphincter is the constant inferior limit, patients could be classified as short-segment HSCR (S-HSCR: 80% of cases) when the aganglionic segment does not extend beyond the upper sigmoid, and long-segment HSCR (L-HSCR: 20% of cases) when aganglionosis extends proximal to the sigmoid. A less common HSCR variety is total colonic aganglionosis (TCA, 3–8% of cases) in which the entire colon and the terminal portion of the ileum is involved [5]. Three rare HSCR variants have also been reported: (1) total intestinal HSCR when the whole bowel is involved [5]; (2) ultra-short segment HSCR involving the distal rectum below the pelvic floor and the anus, proximal to the normal aganglionic zone [6]; (3) suspended HSCR, a controversial condition, where a portion of the colon is aganglionic above a normal distal segment. The incidence of HSCR is estimated at 1/5000 live births [7]. However, the incidence varies significantly among ethnic groups (1.0, 1.5, 2.1, and 2.8 per 10 000 live births in Hispanics, Caucasian-Americans, African-Americans, and Asians, respectively) [8]. S-HSCR is far more frequent than L-HSCR (80% and 20%, respectively) [9, 10]. There is a sex bias with a preponderance of affected males and a sex ratio of 4/1 [11]. Interestingly, the male: female ratio is significantly higher for S-HSCR (4.2–4.4) than for L-HSCR (1.2–1.9) [8, 11]. HSCR most commonly presents sporadically, although it can be familial and may be inherited as autosomal dominant or autosomal recessive, with reduced penetrance and male predominance [9, 11]. In 70% of cases, HSCR occurs as an isolated trait and in the other 30% HSCR is associated with other congenital malformation syndromes [12].Peer Reviewe

Hirschsprung’s disease as a model of complex genetic etiology

Hirschsprung disease (HSCR), or aganglionic megacolon, is a developmental disorder characterised by the absence of ganglion cells along variable length of the distal gastrointestinal tract, leading to the most common form of functional intestinal obstruction in neonates and children. Aganglionosis is attributed to a failure of neural crest cells to migrate, proliferate, differentiate or survive during enteric nervous system (ENS) development in the embryonic stage. The incidence of HSCR is estimated at 1/5000 live births and most commonly presents sporadically with reduced penetrance and male predominance, although it can be familial and may be inherited as autosomal dominant or autosomal recessive. In 70% of cases, HSCR occurs as an isolated trait and in the other 30% HSCR is associated with other congenital malformation syndromes. HSCR has a complex genetic etiology with several genes and loci being described as associated with either isolated or syndromic forms. These genes encode for receptors, ligands (especially those participating in the RET and EDNRB signaling transduction pathways), transcriptional factors or other cell elements that are usually involved in the neural crest cell development and migration that give rise to ENS. Nevertheless, the RET proto-oncogene is considered the major disease causing gene in HSCR. A common RET variant within the conserved transcriptional enhancer sequence in intron 1 has been shown to be associated with a great proportion of sporadic cases and could act as a modifier by modulating the penetrance of mutations in other genes and possibly of those mutations in the RET proto-oncogene itself