During liver organogenesis, cellular transcriptional profiles are constantly reshaped by the action of hepatic transcriptional regulators, including FoxA1-3, GATA4/6, HNF1α/β, HNF4α, HNF6, OC-2, C/EBPα/β, Hex, and Prox1. These factors are crucial for the activation of hepatic genes that, in the context of compact chromatin, cannot access their targets. The initial opening of highly condensed chromatin is executed by a special class of transcription factors known as pioneer factors. They bind and destabilize highly condensed chromatin and facilitate access to other “non-pioneer” factors. The association of target genes with pioneer and non-pioneer transcription factors takes place long before gene activation. In this way, the underlying gene regulatory regions are marked for future activation. The process is called “bookmarking”, which confers transcriptional competence on target genes. Developmental bookmarking is accompanied by a dynamic maturation process, which prepares the genomic loci for stable and efficient transcription. Stable hepatic expression profiles are maintained during development and adulthood by the constant availability of the main regulators. This is achieved by a self-sustaining regulatory network that is established by complex cross-regulatory interactions between the major regulators. This network gradually grows during liver development and provides an epigenetic memory mechanism for safeguarding the optimal expression of the regulators. The liver participates in a variety of crucial biological processes such as hemopoiesis during embryonic life and metabolism, glycogen storage, detoxification, plasma protein secretion, acute phase reaction, and hormonal homeostasis in adulthood. The major cell type of the liver is the hepatocyte, which arises from endodermal precursors through a complex multistep differentiation process. During hepatocyte differentiation, the gene expression pattern of each intermediate cell type is generated by the action of transcription factors, which bind to the regulatory regions of their target genes and activate transcription at specific times during development. Developmental cell fate decisions are determined by cell-to-cell communication and the action of complex signaling pathways. Signaling molecules exert their function through the modulation of transcription factor activity, either directly or indirectly.In this review, we summarize current knowledge about the function of major transcription factors involved in different stages of liver development. Furthermore, we present our current understanding of the regulatory mechanisms of developmental gene activation.Albumin, Transthyretin, HNF4α, and α-fetoprotein are first activated at this stage [In early embryos, liver organogenesis is initiated from the definitive endoderm. In mice, during gastrulation at embryonic day 6.5 (E6.5), the primitive node is formed on the posterior side of the epiblast. Later on, this knot of cells forms a structure called a primitive streak. Cells migrating from the primitive streak give rise to the mesendoderm, which is the precursor of mesoderm and endoderm. Signaling factors such as Nodal lead these bipotential mesendoderm cells to segregate and generate the definitive endoderm. Through morphogenetic movements, the endoderm forms the primitive gut, which is surrounded by mesoderm. Subsequently, the gut tube is divided along the anterior–posterior axis into the foregut, midgut, and hindgut. At E8.5, cells in the ventral foregut endoderm receive BMP (bone morphogenic protein) signals from the septum transversum mesenchyme, parallel to FGF (fibroblast growth factor) signals from the adjacent developing heart, which facilitates their differentiation into hepatoblasts . Hepatocis stage ,2.After hepatoblast specification, the hepatic epithelium thickens and transforms into a pseudostratified epithelium, resulting in the formation of the liver diverticulum. By E9.5, the layer that surrounds the hepatoblasts is destroyed, enabling them to migrate into the septum transversum mesenchyme and produce the nascent liver bud. The septum transversum mesenchyme also contributes to the formation of hepatic stellate cells. From E9.5 until E15, hepatoblasts proliferate and the liver bud grows. Around E13, hepatoblasts begin to differentiate into hepatocytes or cholangiocytes. Hepatoblasts that are in contact with the portal vein will differentiate into cholangiocytes, whereas the rest of the hepatoblasts gradually differentiate into mature hepatocytes .Single-cell RNA-seq analyses revealed that cellular transcriptomes are very dynamic at the specification stage (E9.5–E15.5). On the other hand, gene expression profiles remain similar between E11.5 and E15.5, suggesting that liver specification mainly occurs prior to this time period . SpecifiThe functional importance of this set of transcription factors in hepatocyte specification and differentiation is best exemplified by the fact that the forced expression of certain combinations of these factors can convert other somatic cells such as fibroblasts into functional hepatocytes. Successful reprogramming of embryonic or adult fibroblasts has been reported by the co-overexpression of FoxA3, GATA4, and HNF1α  or HNF1βFoxA1 gene are characterized by low glucagon mRNA levels, hypoglycemia, weakness, and dehydration, leading to lethality between postnatal days 2 to 12 (P2 to P12). This study showed that FoxA1 has a significant role in the transcriptional control of genes regulating glucose homeostasis [FoxA proteins belong to the subfamily of the Forkhead box (FOX) transcription factors and are thought to be essential for the transcriptional regulation of virtually all genes expressed in the liver, lung, and pancreas ,10,11,12eostasis . Mouse meostasis ,24. FoxAeostasis . Initialeostasis ,28,29,30(A/T)GATA(A/G) in the cis-regulatory elements of target genes [GATA transcription factors participate in the regulation of embryonic morphogenesis and cellular differentiation . The GATet genes ,34. Initet genes ,35.Data from knockout and rescue experiments suggested that the function of GATA4 is important for ventral morphogenesis, especially for the expansion of the liver bud and the formation of the ventral pancreatic bud ,38,39,40N-terminus, and a highly acidic region in the C-terminus. The last two motifs are considered to be essential for the transcriptional activation [The haematopoietically expressed homeobox (Hex) transcription factor is a homeobox-containing protein essential for the development of the liver, and more specifically, for the expansion of the liver bud. It contains a DNA-binding domain, a proline-rich region in the tivation . Hex firtivation . On E10,tivation . In micetivation ,45. The tivation . Liver btivation ,48. Finativation . All theProspero-related homeobox 1 (Prox1) transcription factor is essential for the formation of several organs and tissues such as liver, pancreas, eye, lymphatic vessel, nerve, and cardiac muscle ,50,51. IHnf4α gene in embryonic hepatocytes showed that this transcription factor affects gluconeogenesis, glycogen synthesis, the architecture, and the functionality of hepatocytes [Hnf4α gene in the liver results in an aberrant accumulation of lipid, a reduction of serum cholesterol and triglyceride levels, and an increase in the serum bile acid concentration [HNF4α belongs to the orphan nuclear receptor family and constitutes the main transcriptional activator for many genes expressed in the liver . It contatocytes . HNF4α iatocytes . Finallyntration . All theHNF6 transcription factors belong to the ONECUT class, which contains two significant domains in the C-terminus that constitute the DNA-binding domain: a single-cut domain and a divergent homeodomain ,67,68,69N-terminal part of the proteins contains a transactivation and attenuation domain [N-terminus is relatively low, resulting in differences in their transactivation and attenuation properties [C/EBPα gene results in an abnormal liver phenotype due to the increased capacity of hepatocytes to proliferate [Two members of the CCAAT/enhancer-binding protein (C/EBP) family, C/EBPα and C/EBPβ, are important regulators of liver development . C/EBP pn domain ,90,91,92operties . During operties . In aduloperties . Mice laoperties ,96. The liferate ,96. The liferate ,98, and liferate .C-terminus [Hepatocyte nuclear factor 1 (HNF1) is a member of the POU homeobox gene family that mediates the transcriptional activation of cell type-specific genes in various organs, including the liver ,105,106.terminus ,104,109.terminus ,111,112.terminus . Apart fterminus .As mentioned above, the transcriptional activation of genes during development is mediated by several key hepatic regulators, which act in concert with specific signaling pathways to establish expression profiles that define differentiation-specific cellular states. Accumulating evidence suggests that regulatory regions (enhancers and promoters) of tissue-specific genes often reside in compacted genomic regions that cannot be accessed by transcription factors, thus acting as a barrier to transcription. Initial gene activation requires a defined sequence of transcription factor–DNA interactions and chromatin transitions, which can cope with the structural obstacle of chromatin condensation. This has become the prevailing view, following the discovery of a special class of transcription factors, now known as pioneer factors. These pioneer factors possess the ability to bind their recognition sequence when embedded into a highly condensed chromatin state.Alb gene was silent, and in the nascent liver bud, where the Alb gene was expressed [Pioneer factors were discovered in an attempt to uncover the first transcription factor that binds to the enhancer of the liver-specific albumin gene during embryogenesis. In vivo footprinting studies in an enhancer of the serum albumin gene showed that FoxA and GATA factors occupied their target sites both in pluripotent endoderm, where the xpressed ,116. Whexpressed . In thisxpressed ,119.C-terminal α-helical region of FoxA1, which is able to bind to core histones, is necessary for the opening of the chromatin [FoxA proteins bind to nucleosomal target sites via their H1-like DNA-binding domain ,120,121.hromatin ,123. Thehromatin ,125.Gene activation during development includes several steps. Initially, pioneer factors scan the genome and bind to particular regions as they encounter their binding sites . There aAs explained above, pioneer factors act as priming factors to establish the transcriptional competence of their target genes during development, but their binding is not accompanied by immediate transcription activation  ,140,141.A recent study has shown that the recruitment of two prominent hepatic regulators, HNF4α and C/EBPα, similarly to FoxA1, is not linked to concomitant gene activation during development . The timInsights into the abovementioned questions were provided by studying the dynamics of transcription factor recruitment and chromatin structure changes during developmental gene activation. It was observed that dynamic binding events, i.e., the transient binding of transcription factors, without gene activation is the most common phenomenon during development. The stable and transient association of transcription factors with different cis-regulatory elements in promoter and enhancer regions facilitates the recruitment of chromatin remodelers and the generation of active chromatin configurations. The length of time during which such dynamic interactions take place in a continuous fashion allows for the cumulative increase in histone modifications characteristic of active enhancers and the progressive expansion of stably open chromatin domains. In this way, bookmarking is part of a highly dynamic developmental maturation process during which regulatory regions are prepared for the acquisition of an optimal configuration that supports an efficient and stable transcription .The model above was supported by the analyses of mice that were deficient in the bookmarking factors HNF4α or C/EBPα. In both cases, a significant deregulation of transcription of most early-bound hepatic genes was observed in parallel to the blocking of acquisition in active chromatin states and the reciprocal accumulation of repressive histone modification marks .The link between the gene expression signature of embryonic and cancerous cells and tissues has been demonstrated by many previous studies ,145,146.Other studies have demonstrated the role of various pioneer factors  in establishing a gene expression profile that is permissive to cancer initiation and progression ,153,154.Liver regeneration relies on the intrinsic ability of the differentiated quiescent hepatocytes to enter the cell cycle, or on the cellular plasticity of other liver cells to transdifferentiate into hepatocytes in order to repopulate the damaged liver . It is kDuring mitosis, considerable alterations occur in the nuclear and chromosomal architecture. These include increased chromosome condensation, nuclear envelope breakdown, loss of long-range interactions between promoters and enhancers, and the displacement of many transcription regulators ,159. ThiThe first mechanistic insights into the process of transcription memory across many cell generations have been provided by the observations that histone modifications, characteristic of active chromatin states, are retained in parental nucleosomes following DNA replication . The perThe abovementioned features illustrated the model that the propagation of the proper reconstitution of transcription patterns during mitotic exit is assisted by epigenetic memory marks partially retained from the parental cells, which would guide transcription factors in their re-association with the previously established locations. These assisted global binding events may result in simultaneous transcriptional induction ,164. A rAs an analogy, one may assume that the mechanistic basis for the observed stable association of bookmarking factors with their targets during developmental gene activation involves cell cycle-mediated association/dissociation events in the same locations. This scenario has been challenged by several studies, demonstrating that a number of cell type-specific transcription factors remains bound to mitotic chromosomes ,168,169,The ever-growing list of transcription factors (TFs) that were found to be associated with mitotic chromatin includes C/EBPα, GBP, HSF1, HSF2, and HNF4α ,158,175,Taken together, the earliest pioneer factor in liver development, FoxA1, binds potently to mitotic chromatin; the subordinate pioneer factor GATA4 and non-pioneer factors are bound moderately; whereas other factors that act later in development bind very loosely or are excluded altogether from the chromosomes in mitosis. Thus, the analogy between the developmental and mitotic bookmarking mechanisms points to the common principles employed by living organisms for the execution of different processes, which utilize distinct functional properties of transcription factors.A common feature of all developmentally regulated hepatic gene regulatory regions is the combinatorial binding of many transcription factors. The stable association of multiple factors with different cis-regulatory elements is a prerequisite for high-level transcription. This depends on the excess availability of transcription factors. How is the required hepatocyte-specific and high concentration of the main regulators achieved?During liver development, the expression of the main hepatic regulators, described in a previous section, follows a sequential pattern. For instance, FoxA factors are highly expressed in all stages and their function is crucial, not only for the initial activation of developmental genes but also for the maintenance of hepatic gene expression . GATA faAt subsequent stages of hepatocyte maturation, a promoter occupancy analysis of the main hepatic regulators demonstrated multiple cross-regulatory interactions between a core set of six hepatic transcription factors, including HNF1α, HNF1β, HNF4α, HNF6, FoxA2, and LRH-1 . This reThe significance of the transcription factor network in setting up liver-specific transcriptional profiles during development and in preserving the hepatic gene expression program throughout the embryonic and adult life is demonstrated by the complexity that offers sustainability. The transient or permanent loss of one component of the circuit may have small effects on hepatic gene expression patterns once the complexity of the cross-regulatory network reaches a critical level. In this way, the hepatic network functions as a fundamental epigenetic memory mechanism, which secures the maintenance of the expression pattern in differentiated hepatocytes . In thisThe gene expression pattern of fully differentiated hepatocytes is generated by multiple regulatory signals involving the sequential action of hepatic transcription factors during embryonic and postnatal development. The process is initiated by pioneer factors that bind to and destabilize the chromatin at gene regulatory loci, which allows for the recruitment of additional transcription factors necessary for the activation of the target genes. The recruitment of transcription factors is not accompanied by immediate gene activation, but it initiates a lengthy maturation process involving the progressive expansion of active chromatin marks and the generation of a configuration that is competent for transcription initiation. A group of genes that are highly active in embryonic hepatocytes are fully shut down after birth, and many of them are reactivated in hepatocellular carcinoma. These genes are also marked by hepatic transcription factors throughout adult life. The association of transcription factors with their targets, without triggering immediate transcription activation either in embryonic or postnatal life, is called “bookmarking”. The bookmarking function of hepatic transcription factors is important for the developmental activation of the genes and the precise re-establishment of hepatic gene expression patterns following the mitotic phase of each cell duplication event. While we now have a good understanding of the chromatin maturation process, which accompanies bookmarking factor association, the critical step that determines the actual timing of the activation of hepatic genes is less understood.During the past years, it has been increasingly recognized that nuclear topology may be critical in determining the active and inactive states of genes. Given the high level of plasticity of the nuclear architecture in different cellular conditions, it is intriguing to assume that developmentally regulated loci may partition in different nuclear compartments at the priming, maturation, and activation stages. Such compartmentalization is likely to be virtual, generated by distinct long-range interactions with other genomic loci. We envisage that the contribution of gene topology and that of the different neighboring genomic regions may provide a novel regulatory layer that could influence the transcription factor binding and chromatin remodeling processes. The multiplicity of regulatory processes is expected to provide an additional level of plasticity to developmental decisions and orchestrate developmental gene expression patterns.


Colchicine has been proposed as a treatment for COVID-19 based on its anti-inflammatory actions. We aimed to evaluate the efficacy and safety of colchicine in patients admitted to hospital with COVID-19.2, and those with an estimated bodyweight of less than 70 kg. The primary outcome was 28-day mortality, secondary endpoints included time to discharge, the proportion of patients discharged from hospital within 28 days, and, in patients not on invasive mechanical ventilation at randomisation, a composite endpoint of invasive mechanical ventilation or death. All analyses were by intention-to-treat. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.In this streamlined, randomised, controlled, open-label trial, underway at 177 hospitals in the UK, two hospitals in Indonesia, and two hospitals in Nepal, several possible treatments were compared with usual care in patients hospitalised with COVID-19. Patients were eligible for inclusion in the study if they were admitted to hospital with clinically suspected or laboratory confirmed SARS-CoV-2 infection and had no medical history that might, in the opinion of the attending clinician, put the patient at significant risk if they were to participate in the trial. Eligible and consenting adults were randomly assigned (1:1) to receive either usual standard of care alone  or usual standard of care plus colchicine (colchicine group) using web-based simple (unstratified) randomisation with allocation concealment. Participants received colchicine 1 mg after randomisation followed by 500 μg 12 h later and then 500 μg twice a day by mouth or nasogastric tube for 10 days in total or until discharge. Dose frequency was halved for patients receiving a moderate CYP3A4 inhibitor , patients with an estimated glomerular filtration rate of less than 30 mL/min per 1·73mvs 1343 [25%] patients in the usual care group; risk ratio 1·02 [95% CI 0·96 to 1·09]; p=0·47).Between Nov 27, 2020, and March 4, 2021, 11 340 (58%) of 19 423 patients enrolled into the RECOVERY trial were eligible to receive colchicine; 5610 (49%) patients were randomly assigned to the colchicine group and 5730 (51%) to the usual care group. Overall, 1173 (21%) patients in the colchicine group and 1190 (21%) patients in the usual care group died within 28 days . Consistent results were seen in all prespecified subgroups of patients. Median time to discharge alive (10 days [IQR 5 to >28]) was the same in both groups, and there was no significant difference in the proportion of patients discharged from hospital alive within 28 days . In those not on invasive mechanical ventilation at baseline, there was no significant difference in the proportion meeting the composite endpoint of invasive mechanical ventilation or death , National Institute of Health Research, and Wellcome Trust. Inflammation is a key feature of severe COVID-19. Markedly raised concentrations of inflammatory markers, such as C-reactive protein (CRP), ferritin, interleukin-6 (IL-6), and other cytokines, are observed in severe cases and are associated with poor outcomes.Inflammasomes are a key part of the innate immune response to SARS-CoV-2 infection. These cytosolic pattern recognition receptor systems are activated in response to detection of pathogens in the cytosol and stimulate the release of proinflammatory cytokines.Evidence before this studyWe searched medRxiv, bioRxiv, Medline, Embase, and the WHO International Clinical Trials Registry Platform from Sept 1, 2019, to April 1, 2021, for clinical trials evaluating the effect of colchicine treatment in patients hospitalised with COVID-19, using the search terms (“SARS-CoV-2.mp” OR “COVID.mp” OR “COVID-19.mp” OR “2019-nCoV.mp” OR “Coronavirus.mp” OR “Coronavirinae/”) AND (“colchicine.mp” OR “colchicine/”), using validated filters to select for randomised controlled trials. No language restrictions were applied.We identified three relevant randomised trials that compared colchicine with usual care or placebo in patients hospitalised with COVID-19 (two at low risk of bias and one with some concerns because of limited information on randomisation process and poor clarity regarding the blinding in the study). Each trial suggested a potential favourable effect of colchicine on outcome measures of clinical improvement or duration of hospitalisation. The three trials combined included a total of 285 patients and seven deaths; even combined, these trials were not adequately powered to detect an effect on mortality.Added value of this studyThe RECOVERY trial is the first large, randomised trial to report results of the effect of colchicine in patients hospitalised with COVID-19. We found no significant effect of colchicine compared with usual care alone on 28-day mortality, the probability of discharge alive within 28 days, or the probability of progressing to the composite outcome of invasive mechanical ventilation or death in patients who were not receiving invasive mechanical ventilation at randomisation. We saw no evidence of benefit of colchicine in any patient subgroup.Implications of all the available evidenceThere is no good evidence that colchicine treatment is of clinical benefit for adults hospitalised with COVID-19 compared with current usual care.online.The RECOVERY trial is an investigator-initiated, streamlined, individually randomised, controlled, open-label, platform trial to evaluate the effects of potential treatments in patients hospitalised with COVID-19. Details of the trial design and results for other possible treatments  or P-glycoprotein inhibitors , or hypersensitivity to lactose were excluded from the colchicine comparison . Written2), and patients with an estimated bodyweight of less than 70 kg  convalescent plasma versus casirivimab and imdevimab versus usual care, 2) aspirin versus usual care, and 3) baricitinib versus usual care . Until JA single online follow-up form was completed when participants were discharged, had died, or 28 days after randomisation, whichever occurred first . InformaOutcomes were assessed 28 days after randomisation, with additional analyses specified at 6 months. The primary outcome was all-cause mortality. Secondary outcomes were time to discharge from hospital alive within 28 days and, in patients not on invasive mechanical ventilation at randomisation, receipt of invasive mechanical ventilation  or death. Prespecified subsidiary clinical outcomes were use of non-invasive respiratory support, time to successful cessation of invasive mechanical ventilation , use of haemodialysis or haemofiltration, cause-specific mortality, bleeding events, thrombotic events, and major cardiac arrhythmias. Information on suspected serious adverse reactions was collected in an expedited fashion to comply with regulatory requirements.The primary analysis for all outcomes was assessed according to the intention-to-treat principle by comparing patients randomly assigned to the colchicine group with those who were randomly assigned to the usual care group, but for whom colchicine was both available and a suitable treatment. For the primary outcome, all-cause 28-day mortality, the log-rank observed minus expected statistic and its variance were used to test both the null hypothesis of equal survival curves  and to calculate the one-step estimate of the average mortality rate ratio. We used Kaplan-Meier survival curves to display cumulative mortality over the 28-day period. We used the same method to analyse time to hospital discharge and successful cessation of invasive mechanical ventilation, with patients who died in hospital right-censored on day 29. Median time to discharge was derived from Kaplan-Meier estimates. For the prespecified composite secondary outcome of progression to invasive mechanical ventilation or death within 28 days in those not receiving invasive mechanical ventilation at randomisation, and the subsidiary clinical outcomes of receipt of ventilation and use of haemodialysis or haemofiltration, the precise dates were not available and so the risk ratio was estimated.Prespecified analyses were done for the primary outcome using the statistical test of interaction (test for heterogeneity or trend), in accordance with the prespecified analysis plan, defined by characteristics at randomisation: age, sex, ethnicity, level of respiratory support, days since symptom onset, and use of corticosteroids . An explEstimates of rate ratios and risk ratios are shown with 95% CIs. All p values are two-sided and are shown without adjustment for multiple testing. The full database is held by the study team, which collected the data from study sites and did the analyses at the Nuffield Department of Population Health, University of Oxford .As stated in the protocol, appropriate sample sizes could not be estimated when the trial was being planned at the start of the COVID-19 pandemic . As the On March 4, 2021, the independent data monitoring committee did a routine review of the available safety and efficacy data. The independent data monitoring committee notified the chief investigators that there was no convincing evidence that continued recruitment to the colchicine comparison would provide conclusive proof of worthwhile mortality benefit either overall or in any prespecified subgroup. Consequently, recruitment to the colchicine comparison was closed on March 5, 2021, and preliminary results were made available to the public.ClinicalTrials.gov, NCT04381936.Analyses were done using SAS (version 9.4) and (R version 3.4). The trial is registered with ISRCTN, 50189673, and The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.Between Nov 27, 2020, and March 4, 2021, 11 340 (58%) of 19 423 patients enrolled into the RECOVERY trial were eligible to receive colchicine  patients in the colchicine group and 5605 (98%) patients in the usual care group. Of the patients with a completed follow-up form, 5122 (93%) in the colchicine group received at least one dose of colchicine. Of those assigned to the colchicine group who received at least one dose, 3823 (75%) received all  of their scheduled doses during their hospital stay; 4576 (90%) received at least half of their scheduled doses . The medvs 1190 [21%] patients in the usual care group; rate ratio 1·01 [95% CI 0·93–1·10]; p=0·77; Primary outcome data are known for 11282 (99%) of the randomly assigned patients. There was no significant difference in the proportion of patients who died within 28-days between the two groups . Similar results were seen in all prespecified subgroups of patients  in both groups, and there was no significant difference in the probability of being discharged alive within 28 days between the two groups (3901 [70%] patients in the colchicine group and 4032 (70%) usual care group; rate ratio 0·98 [95% CI 0·94 to 1·03]; p=0·44; patients .Table 2EWe found no significant differences in the prespecified subsidiary clinical outcomes of cause-specific mortality , use of In this large, randomised trial involving more than 11 000 patients from three countries and more than 2000 deaths, use of colchicine was not associated with a reduction in mortality, duration of hospitalisation, or the risk of being ventilated or dying for those not on ventilation at baseline. These results were consistent across the prespecified subgroups of age, sex, ethnicity, duration of symptoms before randomisation, level of respiratory support at randomisation, and use of corticosteroids.The benefit of dexamethasone in patients with COVID-19 requiring respiratory support shows the importance of inflammation in this patient group and colchicine was proposed as a treatment for COVID-19 based on its anti-inflammatory activity.Strengths of this trial included that it was randomised, had a large sample size, broad eligibility criteria, was international, and more than 99% of patients were followed up for the primary outcome of all-cause mortality. However, there are some limitations. Detailed information on laboratory markers of inflammation and immune response and information on radiological features was not collected; therefore, it is not possible to assess if the effect of treatment varied between such subgroups of patients. Although this randomised trial is open-label, the outcomes are unambiguous and were ascertained without bias through linkage to routine health records.Three other randomised controlled trials have assessed the efficacy of colchicine for the treatment of patients hospitalised with COVID-19.The RECOVERY trial only studied patients who had been hospitalised with COVID-19; therefore, we are not able to provide any evidence on the safety and efficacy of colchicine used in other patient groups. In the COLCORONA trialIn summary, the results of this large, randomised trial do not support the use of colchicine in adults hospitalised with COVID-19.recoverytrial@ndph.ox.ac.ukCorrespondence to: Prof Peter W Horby and Prof Martin J Landray, RECOVERY Central Coordinating Office, Oxford OX3 7LF, United Kingdom. online form and sent to data.access@ndph.ox.ac.ukThe protocol, consent form, statistical analysis plan, definition and derivation of clinical characteristics and outcomes, training materials, regulatory documents, and other relevant study materials are available online. As described in the protocol , the triWe declare no competing interests.


The aim of this retrospective chart review study was to examine the cost effectiveness of angiotensin-converting enzyme inhibitors (ACEIs); angiotensin receptor blockers (ARBs); and dihydropyridine calcium channel blockers (CCBs) such as amlodipine, monotherapies in the management of essential hypertension among adult patients (≥18 years) without cancer, cardiovascular disease, and chronic kidney disease in the primary care clinics of a university-affiliated tertiary care hospital. Patients were followed up for at least 12 months from the initiation of therapy. Propensity score bin bootstrapping with 10,000 replications was conducted to generate the 95% confidence intervals (CI) for both treatment outcome  and diastolic blood pressures (DBP) in mmHG) and the cost ). Among the 153 included patients who met the inclusion criteria, 111 patients were on ACEIs/ARBs, while 44 patients were on amlodipine. On the basis of the bootstrap distribution, we found that the use of ACEIs/ARBs was associated with an incremental reduction of SBP of up to 4.46 mmHg but with an incremental cost of up to SAR 116.39 (USD 31.04), which results in an incremental cost effectiveness ratio (ICER) of SAR 26.09 (USD 6.95) per 1 mmHg reduction with 55.26% level of confidence. With regard to DBP, ACEIs/ARBs were associated with an incremental reduction of DBP of up to 5.35 mmHg and an incremental cost of up to SAR 144.96 (USD 38.66), which results in an ICER of SAR 27.09 (USD 7.23) per 1 mmHg reduction with 68.10% level of confidence. However, ACEIs/ARBs were less effective and costlier than amlodipine in reducing SBP and DBP with 44.74% and 31.89% levels of confidence, respectively. The findings of this study indicate that the use of ACEI or ARB as a monotherapy seems to be more effective than amlodipine monotherapy in the management of essential hypertension in primary care settings with minimal incremental cost. Hypertension (HTN) is one of the most prevalent chronic diseases worldwide. It results in serious consequences, such as retinopathy, neuropathy, nephropathy, stroke, and myocardial infarction if left untreated ,2. This The use of antihypertensive drugs as monotherapy in the management of essential HTN is uncommon since different antihypertensive medications are mostly used in combination with other antihypertensive medications belonging to different classes or with diuretics . HoweverIn Saudi Arabia, the utilization rates of different antihypertensive medications are high due to the high prevalence of essential HTN among the Saudi population, which is believed to be over 50% among those aged 55 years and above . ACEIs/AThis was a single-center retrospective chart review study that was conducted at King Saud University Medical City in Riyadh, Saudi Arabia. Electronic health records of adult patients (≥18 years) visiting family practice clinics were reviewed to identify patients with essential HTN treated with a single antihypertensive medication for at least 12 months. Patients with cancer, cardiovascular disease, chronic kidney disease, and/or stroke, as well as those with secondary HTN  were excluded. Age, gender, comorbidities , number of prescription medications, baseline SBP and DBP, duration of follow-up in months from the time where patients were diagnosed with HTN to the next annual clinic visit, follow-up SBP and DBP, and antihypertensive medications  were reviewed and collected. Moreover, the utilization of different health services, such as labs and imaging studies, were collected. The electronic health record system also known as E-Sihi was implemented at King Saud University Medical City in mid-2015. Therefore, the investigators decided to recruit patients who were first diagnosed with essential HTN and started on amlodipine or ACEIs/ARBs  anytime between July 2015 and June 2018 to ensure complete data collection with no missing observations since it is highly unlikely to find patients with complete data prior to the implementation of E-Sihi. The retrospective data collection started on 21 January 2018 and ended on 27 June 2019.t-test, and one-way ANOVA were conducted as appropriate to compare the characteristics of patients on amlodipine and ACEIs/ARBs . Propensity score matching was conducted on the basis of baseline SBP and DBP, age, gender, follow-up period, body mass index (BMI), and Charlson’s comorbidity index (CCI) score [Using Wilcoxon–Mann–Whitney test, allocation ratio of 1 (amlodipine) to 3 (ACEIs/ARBs), α = 0.05, β = 0.2, power of 0.8, effect size of d = 0.52, we estimated the minimum sample size to be 144 patients (36 patients on amlodipine and 108 patients on ACEIs/ARBs). The outcome was defined as the difference between the baseline and follow-up SBP and DBP in mmHG. On the other hand, the costs of prescription medications and healthcare services  were retrieved from the Saudi Ministry of Health cost center. Chi-squared test, Fisher’s exact test, Student’s I) score . Bias-co® version 9.4 .All statistical analyses were conducted using SASn = 42), and 111 patients (72.55%) were taking ACEIs or ARBs , as shown in p < 0.05), as shown in Out of the 2000 medical records that were reviewed for patients who have been diagnosed with HTN, 153 patients met the inclusion criteria and were included in the analysis. A total of 42 patients were taking amlodipine (27.45%)  and SAR 144.96 (USD 38.66) per 1 mmHg reduction in SBP or DBP. These findings are consistent with the previously published studies which found that ACEIs/ARBs, such as lisinopril and irbesartan, were more cost-effective than amlodipine in the management of essential HTN .16 and S. These iAlthough this is the first study, to our knowledge, to compare the cost effectiveness of ACEI/ARB to CCB monotherapy using real-world data in Saudi Arabia, several limitations must be acknowledged. First, this is a single-centered study with a relatively small sample size, which limits the generalizability of its findings. However, the study was conducted in a well-known public tertiary care hospital in the Saudi capital where patients from all segments of the Saudi population are cared for. Secondly, information bias cannot be ruled out as the data were retrieved from the electronic healthcare records during a specified timeframe , which also increases the risk of selection bias. Nevertheless, obtaining real-world data from electronic healthcare records in Saudi Arabia where multiple barriers to access to healthcare data suitable for conducting economic evaluation exist is a daunting task . MoreoveEffective management of HTN is essential in preventing major complications, such as stroke and myocardial infarction. Therefore, early detection and treatment of HTN using the most effective and affordable antihypertensive medication is important. In this study, ACEIs/ARBs were shown to be more effective with reasonable incremental cost in managing essential HTN in comparison to amlodipine. Future studies should examine the cost effectiveness of different monotherapies as well as combination therapies in the management of HTN using larger sample sizes and more robust study designs.


This study aimed to evaluate the effect of vitamin D deficiency on sleep quality, depression, anxiety, and physiological stress in patients with chronic obstructive pulmonary disease (COPD). We screened for COPD patients with normal (NorVD) (n=24) and insufficient (InsVD) (n=7) vitamin D levels. The Pittsburgh sleep quality index (PSQI), the Beck anxiety inventory (BAI), the Beck depression inventory (BDI) and the Baevsky’s stress index were used for the sleep and psychometric evaluation. The evaluation of sleep quality by PSQI showed that NorVD individuals had higher duration and quality of sleep when compared with the InsVD group. Additionally, the group InsVD presented higher risk of developing sleep quality . BDI was higher in the InsVD, and this group had a higher risk of developing moderate and severe depression . The stress index indicated higher values in the InsVD in relation to the NorVD group (InsVD=24±0.8 vs. NorVD=16±0.9), and the group InsVD showed higher risk of developing high and very high physiological stress . The stress and sleep quality effects were negatively correlated with vitamin D levels. Insufficient levels of vitamin D negatively affect sleep quality and psychometric variables. Furthermore, poor indexes of quality of life are complications commonly observed in these patients due to conditions as depression, anxiety, stress, and worse sleep quality2.Chronic obstructive pulmonary disease (COPD) is a chronic disease strongly associated with severe morbidity and is currently the third leading cause of death globally3. In COPD, vitamin D deficiency often occurs due to smoking-induced skin aging, reduced outdoor activity, and poor-quality food intake4.Another relevant factor in COPD patients is vitamin D deficiency. Vitamin D deficiency is highly prevalent in a general population and has been associated with aggravating autoimmune diseases, allergies, endocrine and metabolic disorders, cancer, infections, cardiovascular diseases, low sleep quality and psychological disorders5. Accordingly, vitamin D receptors were identified in brain regions related to emotional-affective disorders such as the prefrontal cortex, hypothalamus and substantia nigra6.The increased susceptibility to psychological stress and poor sleep quality related to vitamin D deficiency might play a vital role in the pathophysiology of depression and anxiety7. In this sense, vitamin D was demonstrated to increase the gene expression of factors regulating the production of serotonin, dopamine, and a nerve growth factor8. Therefore, it is suggested that the maintenance of normal vitamin D levels plays a fundamental role in mental health8.The role of vitamin D in neuropsychiatric disorders seem to be mediated by its action as a neuroactive hormone involved in crucial functions, such as neuroprotection, neuroimmunomodulation, brain development, and regular brain function1. Mental disorders compromise not only the quality of life of COPD patients but impose a critical economic burden, as they are important risk factors for several other diseases9.The maintenance of mental health has received more and more attention and has been an important focus of study by health professionals as it affects the general quality of life and chronic distressAlthough patients with COPD are more likely to develop depression, anxiety, stress, and poor sleep quality, it is not completely clear whether vitamin D deficiency in these patients may interfere with these variables. Therefore, since vitamin D deficiency appears to be common in COPD patients and its levels seems to be related to mental health and quality of life in the general population, this study investigated the effect of serum vitamin D levels on sleep quality, stress, depression, and anxiety in COPD patients.This was a cross-sectional study of COPD patients from the Outpatient Clinic of the Asthmatic Patient Assistance Program of the Maranhão Federal University Hospital. The sample consisted of 39 COPD patients. A sample error of 5% and a confidence interval of 95% were adopted. The COPD patients were divided into two groups: insufficient vitamin D (InsVD) and normal vitamin D (NorVD).Patients were excluded if they had any of the following conditions: having other nutritional problems besides vitamin D deficit, a history of hypercalcemia, cancer, presence of pulmonary infection, tuberculosis, pleural effusion, congestive heart failure, primary pulmonary hypertension, pulmonary embolism, restrictive airway disease, vitamin D supplementation, orthopedic disorders affecting physical performance, and inability to walk.2).Weight measurement was performed using a digital scale on the kilogram scale . For stature, the EST 23 Trena Compact Stadiometer was used in the millimeter scale. Also, the body mass index (BMI) was calculated using the formula weight (kg)/height , three measurements of BP were performed on the right arm, with at least a 2-min interval between each one.The procedures for the measurement of blood pressure (BP) were made according to the guidelines of the seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC7)7.One morning was reserved for data collection of spirometry tests  by a qualified technician. Subjects underwent the spirometry test in the sitting position, wearing a nose clip to obtain the following parameters: forced vital capacity (FVC); forced expiratory volume in 1sec (FEV1); as well as the ratio of FEV1 to FVC , and all his predicted values according to the American Thoracic Society (ATS)In addition to the automatic evaluation, the quality of spirometric tests was assessed according to some criteria, including the number of acceptable maneuvers according to American Thoracic Society, ranging from 0 to 3, the highest kept by the spirometry software; the reproducibility .Physiological stress was evaluated by heart rate variability , using a 12-lead electrocardiogram, with a sampling frequency of the electrocardiogram signal of 600Hz , and obtaining moment by moment R-R intervals. The subjects remained lied down for at least 20 minutes before the recording onset, and the electrocardiogram was monitored for 10 minutes..txt through the same analysis software  for analysis of the heart rate (HR) variability of the R-R interval in the time domain. The index used was mean R-R, mean HR, RMSSD, SD1 (%), and SD2 (%). The stress index was the square root  of the Baevsky’s stress index17. The Baevsky’s stress index values between 50-150 are considered normal, 150-500 elevated, 500-900 high, and >900 very high10.Then, the series of R-R intervals were extracted in Serum vitamin D level was extracted from a small blood sample and evaluated by measuring 25-hydroxyvitamin D (25-OH-D) using the chemiluminescence method according to the manufacturer’s instructions. Serum 25-OH-D levels <20ng/mL was considered deficient, from 20 to 30ng/mL was considered insufficient levels (InsVD) and ≥30ng/mL was appropriate values (NorVD). The analysis was performed in the laboratory of the Maranhão Federal University Hospital.14. The PSQI used seven components: i) subjective sleep quality, ii) sleep latency, iii) duration of sleep, iv) habitual sleep efficiency; v) sleep disorders; vi) use of medication for sleep; and vii) daytime sleepiness and disorders during the day. Each element was individually determined and the score of each component was added to give an overall score ranging from 0 to 21 points. The higher the value obtained, the worse the quality of sleep . For good sleep quality, the sum of the scores is only 5.The quality of sleep and the presence of sleep disorders were evaluated using the Pittsburgh sleep quality index (PSQI), as initially described by Buysse11. BAI is a 21-item checklist developed with large clinical samples to measure anxiety symptoms according to the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders of the American Psychiatric Association (APA)12. Application and scoring are rapid and the patient rates symptom intensity during the last seven days on a scale from 0  to 3 (severely). The BAI adequately covers the significant cognitive, affective, and physiological symptoms of anxiety13.The Beck anxiety inventory (BAI) and the Beck depression inventory (BDI) were used to evaluate anxiety and depression levels, respectively, as initially described by Beck12.To verify the presence of self-reported depression, patients answered the BDI and minimal, mild, moderate, and severe scores for major depressive episodes were obtained, according to the APA. The BDI is scored by summing the highest ratings for each of the 21 items. Each item is rated on a 4-point scale ranging from 0 to 3, and the total scores can range from 0 to 63. BDI total scores ranging from 0 to 13 represent “minimal” depression; total scores from 14 to 19 are “mild;” total scores from 20 to 28 are “moderate;” and total scores from 29 to 63 are “severe”p<0.05. We used GraphPad Prism 5 for statistical analysis. Effect size higher than 0.8 were considered very strong; values ranging from 0.6-0.8 were considered strong; values ranging from 0.4-0.6 were considered moderate; and lower than 0.4 were considered negligible11.Data were reported as mean±standard deviation. The normality of data was tested using the Kolmogorov-Smirnov test. An unpaired t-test was used to compare parametric data. The chi-square was used to compare the groups when data were distributed in frequency. Pearson correlation was used in parametric data. Differences were considered significant when Blood pressure, spirometry test and body composition variables in COPD individuals with normal and insufficient vitamin D.As presented in the The assessment of pulmonary function indicated that the NorVD group showed similar values of FVC  and FEV  when compared to the InsVD group. Additionally, there was no significant difference in FEV1/FVC and FEF 25-75 % .p=0.009). Subjective sleep quality, sleep latency, sleep efficiency, sleep disorders, use of sleeping medication, and daytime sleepiness were similar .We present in the p=0.0007) (p=0.01).The stress index was higher in the InsVD group in relation to the NorVD group . The dat=0.0007) . AdditioCorrelation analysis indicated the stress and sleep quality indexes negatively corrected with vitamin D levels, suggesting that individuals with lower levels of vitamin D present higher levels of stress and impaired sleep quality .This study aimed to investigate the effect of serum vitamin D levels on sleep quality, depression, anxiety, and physiological stress in COPD patients. The main findings of the present study are that insufficient vitamin D levels in patients with COPF evokes impaired sleep quality, depression, and higher levels of physiological stress. Additionally, stress index and sleep quality negatively correlated with vitamin D levels.14. Besides, vitamin D supplementation improved sleep latency and sleep duration in subjects with vitamin D deficiency15. However, these studies did not investigate patients with COPD, so that present study provide the first evidence that this relationship is also observed in patients susceptible to sleep disorders.These data corroborate with other studies that have shown a relationship between sleep quality and vitamin D levels. For instance, a meta-analysis discussed the relationship between vitamin D deficiency and sleep disorders, including poor sleep quality, short sleep duration, and drowsiness. Accordingly, low serum 25-(OH)-D levels has been reported to be associated with an increased risk of sleep disorders16. Besides, adequate levels of vitamin D can act as a modulator of inflammatory response, thus presenting neuroprotective actions18. Additionally, vitamin D regulates the production of serotonin, dopamine, and a nerve growth factor in the brain. These substances have been prominent in the prophylaxis and therapy of psychiatric disorders19. Supporting these findings, several studies have identified vitamin D deficiency as an essential factor for depression22. However, these previous studies have not explored the interactions between stress, depression, anxiety, and sleep quality in patients with COPD.In addition to changes in sleep quality, we observed a higher score in depression questionaries’ (BDI) in COPD patients with insufficient vitamin D levels. Interest in role of vitamin D in depression has increased. This is because of evidence of increased expression of vitamin D receptors (VDRs) in brain areas that play a crucial role in regulating mood24. Furthermore, although we did not find changes in blood pressure, elevated psychosocial stress can contribute to cardiovascular disease as endothelial dysfunction, atherosclerosis, and impaired autonomic control26.Another variable evaluated in this study was physiological measures of stress by the Baevsky stress index. The InsVD group showed higher stress levels than NorVD groups. This finding is possibly related to the impaired sleep quality and increased levels of depression. In fact, the stress has been documented as a prominent environmental factor involved in etiology and development of mood disorders27 and autonomic dysfunction has been recognized as a significant predictor of mortality. Additionally, vitamin D supplementation is associated with improved autonomic modulation in healthy humans28. Therefore, adequate levels of vitamin D can be crucial to cardiovascular health, preventing endothelial cell death in human cells, inhibiting the generation of superoxide anion, and maintaining mitochondrial function21.This idea is supported by the fact that the Baevsky stress index is strongly linked to increased cardiac sympathetic response during stressful situations. Also, some authors used this method to assess the level of stress along with psychometric variablesAlthough studies have suggested that psychological variables are associated with sleep disorders and low levels of vitamin D can lead to chronic physiological stress, few reports investigated this relationship in patients with COPD. In this sense, the identification of population subgroups that have increased level of depression and, perhaps, have a more significant benefit in vitamin D supplementation is a relevant aspect.We believe that this work will encourage professionals to consider the relationship between low levels of vitamin D and depression, sleep quality, and stress when treating patients with COPD. However, further studies are necessary to standardize dose, frequency, and duration of supplementation or administration of vitamin D in these patients. Besides, the association of changes in vitamin D levels with inflammatory markers over time as well as adjust confounding factors like lifestyle and demographic factors are issues to be addressed in further studies.We conclude that levels of vitamin D are negatively correlated with sleep quality, depression, and stress in patients with COPD.


SNRPN) gene locus encompassing the PWS IC and was identified by using a variety of techniques. Haplotype studies suggest that the IC microdeletion was vertically transmitted from an unaffected paternal grandmother to an unaffected father and then caused PWS in two sibling grandchildren when the IC microdeletion was inherited paternally. Based on the results of our study, preimplantation genetic diagnosis (PGD) was applied successfully to exclude imprinting deficiency in preimplantation embryos before transfer into the mother’s uterus. Our study may be especially instructive regarding accurate diagnosis, differential diagnosis, genetic counseling, and PGD for familial PWS patients.Prader–Willi syndrome (PWS) is a complex genetic syndrome caused by the loss of function of genes in 15q11-q13 that are subject to regulation by genomic imprinting and expressed from the paternal allele only. The main clinical features of PWS patients are hypotonia during the neonatal and infantile stages, accompanied by delayed neuropsychomotor development, hyperphagia, obesity, hypogonadism, short stature, small hands and feet, mental disabilities, and behavioral problems. However, PWS has a clinical overlap with other disorders, especially those with other gene variations or chromosomal imbalances but sharing part of the similar clinical manifestations with PWS, which are sometimes referred to as Prader–Willi syndrome-like (PWS-like) disorders. Furthermore, it is worth mentioning that significant obesity as a consequence of hyperphagia in PWS usually develops between the ages of 1 and 6 years, which makes early diagnosis difficult. Thus, PWS is often not clinically recognized in infants and, on the other hand, may be wrongly suspected in obese and intellectually disabled patients. Therefore, an accurate investigation is necessary to differentiate classical PWS from PWS-like phenotypes, which is imperative for further treatment. For PWS, it is usually sporadic, and very rare family history and affected siblings have been described. Here, we report the clinical and molecular findings in a three-generation family with a novel 550-kb microdeletion affecting the chromosome 15 imprinting center (IC). Overall, the present study finds that the symptoms of our patient are somewhat different from those of typical PWS cases diagnosed and given treatment in our hospital. The familial occurrence and clinical features were challenging to our diagnostic strategy. The microdeletion included a region within the complex small nuclear ribonucleoprotein polypeptide protein N ( The Prader–Willi syndrome (PWS) is characterized by hypotonia and feeding problems in early infancy, as well as hypogonadism, small hands and feet, craniofacial signs, and hyperphagia leading to profound obesity. It is triggered by the loss of function of genes in 15q11-q13 that are regulated by genomic imprinting and expressed from the paternal allele only. In approximately 70% of the cases, PWS is the result of deletion of 5–7 Mb in the paternal 15q11-13 region; nearly 28% attributes to maternal uniparental disomy (mUPD); and in < 2%, it is developed as the consequence of mutation, microdeletion, or translocation disrupting the imprinting center (IC) . In PWS The 1-day-old male proposita was the second child of healthy non-consanguineous Chinese parents. Parental ages at birth of the child were 31 years (father) and 30 years (mother). During the pregnancy, ultrasound screening did not reveal any structural abnormalities except for decreased fetal activity. The first child of the couple was a baby girl who died 4 days after birth for unknown causes. Considering the family history of unexplained neonatal death, interventional prenatal diagnosis was recommended by the genetic counselor but refused by the couple. At 40 weeks + 5 days’ gestation, the patient was hospitalized with fetal distress. An elective cesarean section was performed on the day of admission due to breech presentation. The neonatal evaluation recorded 2,950 g in birth weight (17.8th centile), 50 cm in length (41.7th centile), and 35 cm in head circumference (65.6th centile), with an Apgar score of 10, 7, and 5 at 1, 5, and 10 min, respectively. The boy was transferred to the neonatal intensive care unit directly after birth because of severe respiratory distress, poor suck, and hypotonia. A thin male baby with poor response to external stimuli was noticed from a first glimpse at the patient. Physical examination revealed weak cry, flat nose, big nostrils, bilateral epicanthus, wide-spaced nipples, long slender fingers, and slight maldescent of the left testis . NeuroloPeripheral blood samples from the patient and his family members were obtained for karyotyping, with their information anonymized prior to submission. Standard G-banded chromosome analysis at a 550-band resolution was performed using phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes prepared from the samples according to standard procedures. Chromosomal abnormalities were designated based on the International System for Human Cytogenetic Nomenclature guidelines (ISCN 2016).To isolate genomic DNA, the QIAamp DNA blood mini kit  was used according to the manufacturer’s protocol.Proband DNA was sequenced to identify the causal gene. The DNA was isolated from peripheral blood with CWE9600 Automated Nucleic Acid Extraction System using CWE2100 Blood DNA Kit V2 . Here, 750 ng of genomic DNA was fragmented into 200–300 bp by Scientz08-III Ultrasonic Homogenizer . The DNA fragments were then processed by end-repairing, A-tailing, and adaptor ligation using KAPA Library Preparation Kit , followed by an eight-cycle pre-capture PCR amplification. Then, the amplified DNA sample was captured in the Agilent SureSelect XT2 Target Enrichment System , with the captured DNA fragments purified by Dynabeads MyOne Streptavidin T1  and amplified by 13 cycles of post-capture PCR. The final products were further purified by Agencourt AMPure XP  and quantitated with Life Invitrogen Qubit 3.0 using Qubit dsDNA HS Assay Kit . Eventually, quantified DNA was sequenced with 150-bp paired-end reads on Illumina Novaseq 6000 platform  according to the standard manual. The coverage contained the exon regions and adjacent intron regions (50 bp) of all human genes by SeqCap EZ Choice XL Library (Roche NimbleGen). The average sequencing depth of the target region for the proband was 123.02×, among which 96.49% of the target sequence had a sequencing depth of more than 20×. The father was 140.85×, of which 97.90% of the target sequence had a sequencing depth of more than 20×. The mother was 160.89×, of which 97.83% of the target sequence had a sequencing depth of more than 20×.1 after being evaluated with Illumina Sequence Control Software (SCS). The single-nucleotide polymorphisms (SNPs) were called by using GATK software 2. Variants were annotated using ANNOVAR3, and the effects of single-nucleotide variants (SNVs) were predicted by SIFT, Polyphen-2, and Mutation_Taster programs  using Burrows–Wheeler Aligner (BWA)programs . All varHere, 750 ng of genomic DNA was fragmented to an average size of 200–300 bp, and DNA libraries were constructed using KAPA Library Preparation Kit, with reagent employed for one of the libraries. The constructed DNA library samples were then taken for high-throughput sequencing with Illumina Nova Seq 6000. The sequencing depth is 0.6×, whole-genome low-depth sequencing. High-quality double-ended sequencing reads were aligned to the human reference genome sequence from the UCSC database using the BWA tool. The window width was preset at 50 Kb, with an adjustment amount of 5 Kb. A two-step calibration of guanine-cytosine (GC) and population model was performed across each of the windows. After removing the abnormal windows, the standard deviation between the copy ratio and the reference set of each window was calculated. A standard deviation of less than 0.15 determined by the software was considered to be in accordance with the quality control. The size and copy ratio of the final copy number variation (CNV) segments were calculated by identifying the break point. Afterward, identified and mapped CNVs were interrogated against publicly available databases, including Decipher, Database of Genomic Variants (DGV), 1,000 Genomes, and Online Mendelian Inheritance in Man (OMIM).4, DECIPHER5, DGV6, and ISCA7.DNA from the patient, his father, and paternal grandmother was genotyped using InfiniumOmniZhongHua-8 array . In addition to a genome-wide functional resolution of approximately 20 kb for deletions and 50 kb for duplications, the array also had a higher density coverage of the 15q11-q13 region. The experiments were carried out under the manufacturer’s instructions. Genotype calling, quality control, and identification of CNV were performed using Illumina KaryoStudio software and cnvPartition algorithm, with various databases employed for array data evaluation and genotype–phenotype correlation analysis, including OMIM®) reagents and kits obtained from MRC-Holland  were used to verify the methylation status of chromosomes 15, including ME028-B2 kit containing sequence-specific probes that was applied for testing along the length of the 15q11.2-q13 region for the patient, his father, and paternal grandmother. In the presence of methylation-sensitive restriction enzymes, the B2 kit equipped with 48 MLPA probes was employed for copy number detection and methylation status verification. Approximately 50 ng of genomic DNA was introduced for each of the MS-MLPA reactions according to manufacturer’s instructions. The PCR products were analyzed by capillary electrophoresis on an ABI 3100 sequencer  using GeneScan 500 LIZ dye Size Standard and formamide , and GeneMarker version 2.64  was used to determine the copy number and methylation status associated with the critical region of Prader–Willi syndrome/Angelman syndrome (PWS/AS) patients. The fluorescent signals from the copy number probes, by comparing with the normal controls, showed the ratios of 0.5 for deletions and 1.5 for duplications. Since the methylation probes were maternally imprinted , the ratio of methylated probes to normal controls would increase accordingly in the presence of additional copies from maternal alleles but not paternal alleles.Methylation-Specific Multiplex Ligation-dependent Probe Amplification .SNRPN, SNHG14, PWAR6, SNORD115, and SNORD116) according to UCSC Genome Browser on Human February 2009 Assembly. We extended the genetic analysis to the family members of the patient . The same deletion was detected in the paternal grandmother and the father of the patient but not in the mother, supporting the paternal origin of the deletion  did not show findings of variants with pathogenic significance in clinically relevant candidate genes causing PWS phenotype in this patient and other family members .Since all of the other family members are in good health, the results suggested vertical transmission of the IC microdeletion from the unaffected paternal grandmother to the unaffected father, which consequently resulted in PWS in these two sibling grandchildren when the IC microdeletion was inherited paternally.SNRPN gene. The same deletion was also verified in the father and the paternal grandmother, but negative in the mother, which was corresponding to the paternal origin of the deletion as well. Methylation patterns within this region of this family revealed that the paternal grandmother and the father displayed an abnormal hypomethylation pattern in the SNRPN region due to complete loss on the maternal chromosome 15, whereas the patient presented with the typical PWS hypermethylation pattern in four SNRPN probes as a result of complete loss on his paternal chromosome 15  mapping revealed a 398-kb region of the chromosome 15 microdeletion, with the proximal breakpoint at 24,966,348 bp and the distal one at 25,364,551 bp . No addiOEP001280 and OEP001281)]. The microdeletion of PWS IC was transmitted silently through two generations prior to being expressed in the third generation via the female germline—the paternal grandmother, the father, and then the two affected sibling grandchildren. With the aid of these genetic tools, PGD was therefore applied successfully to exclude imprinting deficiency in preimplantation embryos before transfer into the mother’s uterus . As expected, the mother has given birth to a healthy boy.In view of the clinical manifestation, karyotype, WES, CNV, CMA, and MS-MLPA, a diagnosis of PWS resulting from ∼550-kb loss on his paternal chromosome 15, which the breakpoint refers to the CNV calling for WGS, can eventually be confirmed for this patient [the sequencing reads for variant calling and the data for CMA had been deposited with NODE Bioproject (MAGEL2 gene (Schaaf-Yang syndrome) (RAI1 gene (Smith–Magenis syndrome)   and RAI1yndrome) , 1p36 moyndrome) , 2pter dyndrome) , deletioyndrome) , deletioyndrome) , 10q26 dyndrome) , 12q subyndrome) , chromosyndrome) , paracenyndrome) , and dupyndrome) . TherefoAs a sporadic genetic disorder with remarkable developmental consequences, PWS is usually triggered by a paternal deletion, maternal uniparental disomy, or imprinting defect of the chromosome region 15q11-q13, which could be diagnosed using the standard methylation tests (MS-MLPA) with higher accuracy. The current genetic epidemiology indicates that approximately 65–75% PWS cases have a detectable deletion in this region, 20–30% cases are caused by mUPD, and imprinting errors have been observed in 1%  . Since tSNRPN, SNHG14, PWAR6, SNORD115, and SNORD116 on the maternal chromosome 15 of the paternal grandmother and the father. The IC deletion caused atypical PWS-like phenotype in the proposita and his elder sister. The microdeletion was transmitted silently through the female germline  but impaired the erasure of the maternal imprint and/or the establishment of a paternal imprint in the male germline (the proposita). So, the proposita inherited a paternal chromosome with a missing paternal imprint, leading to the development of PWS. The proposita and his elder sister exhibited part of the major clinical manifestations of PWS, including neonatal and infantile hypotonia, weak cry, feeding difficulties, and hypoplastic external genitalia, followed by recurrent respiratory infections. Interestingly, additional features such as low birth weight, characteristic facial features , and hypopigmentation (fair skin and hair) that are common clinical features of PWS were not seen in this patient. These findings, accompanied by early neonatal death, once misled us to the consideration of neonatal lethal monogenic diseases , mitochondrial diseases, or chromosomal diseases in the first place. Therefore, the familial history of neonatal death and atypical clinical features, though presented as rare events in PWS patients, could lead clinical judgment astray, thus should arouse vigilance among pediatricians.In our research, we described a rare familial occurrence and atypical clinical features of PWS with a 550-kb microdeletion at 15q11.2: a typical IC deletion spanning NPAP1, PWRN1, SNHG14, SNORD116, and SNRPN. Also, patient 288417 showed a core phenotype characterized by obesity, aggressive behavior, intellectual disability, and psychosis (information of infant period was not provided), pointing toward a causative role of the genes in the minimal critical region in the broader phenotype of typical PWS. Furthermore, in animal models of PWS, knockout Snord116 mice displayed cognitive deficits  and contains two C/D box snoRNAs—the SNORD116 cluster and SNORD109A—as the only putative functional genes . In revideficits , growth deficits , hyperphdeficits . So, allOverall, the present study finds that the symptoms of our patient are distinct from, and may be more severe than, those of typical PWS cases. The familial occurrence and atypical clinical features were challenging to our diagnostic strategy. Based on the results of our study, PGD was applied successfully to exclude imprinting deficiency in preimplantation embryos before transfer into the mother’s uterus. As expected, the mother gave birth to a healthy boy. Our study may be especially instructive regarding accurate diagnosis, differential diagnosis, genetic counseling, treatment, and PGD for familial PWS patients.OEP001280 and OEP001281.The data presented in the study are deposited in the NODE BioProject repository, accession numbers are The studies involving human participants were reviewed and approved by the Ethics Committee of Qilu Children’s Hospital of Shandong University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.YLiu, YY, and SZ supervised the project. KZ and SL wrote the manuscript. WG, KZ, YLiu, and YLv were involved in the clinical diagnosis and whole-exome sequencing and bioinformatics analysis. DW and MG performed the karyotype analysis and chromosome microarray analysis. XL and ZG participated in case follow-up. All authors were involved in the conception, experiment design, and data analysis and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Macrocystis pyrifera) dominate the nearshore ecosystem and provide habitat for myriad benthic organisms. In the fjords, salinity was low and both turbidity and nutrients from terrigenous sources were high, with benthic communities dominated by active suspension feeders . Areas closer to the Pacific Ocean showed more oceanic conditions with higher salinity and lower turbidity, with benthic communities experiencing more open benthic physical space in which predators  and herbivorous browsers  were more conspicuous components of the community compared to the inner fjords. Hagfish (Myxine sp.) was the most abundant and frequently occurring fish taxa observed on deep-sea cameras (80% of deployments), along with several taxa of sharks , which collectively were also observed on 80% of deep-sea camera deployments. The kelp forests, deep fjords, and other nearshore habitats of the KNR represent a unique ecosystem with minimal human impacts at present. The KNR is part of the ancestral territory of the indigenous Kawésqar people and their traditional knowledge, including the importance of the land-sea connection in structuring the marine communities of this region, is strongly supported by our scientific findings.The newly created Kawésqar National Park (KNP) and National Reserve (KNR) in southern Chile consists of diverse terrestrial and marine habitats, which includes the southern terminus of the Andes, the Southern Patagonia Ice Fields, sub-Antarctic rainforests, glaciers, fjords, lakes, wetlands, valleys, channels, and islands. The marine environment is influenced by wide ranging hydrological factors such as glacier melt, large terrigenous inputs, high precipitation, strong currents, and open ocean water masses. Owing to the remoteness, rugged terrain, and harsh environmental conditions, little is known about this vast region, particularly the marine realm. To this end, we conducted an integrated ecological assessment using SCUBA and remote cameras down to 600 m to examine this unique and largely unexplored ecosystem. Kelp forests (primarily  The newly created Kawésqar National Park (KNP) and the marine component—the Kawésqar National Reserve (KNR)—is one of the largest park systems in the world and the second largest in Chile after the adjacent Bernardo O’Higgins National Park. It consists of a diverse range of terrestrial and marine habitats that includes the southern terminus of the Andes, the Southern Patagonia Ice Fields, sub-Antarctic rainforests, glaciers, fjords, lakes, wetlands, valleys, channels, and islands. The region consists of largely unfragmented ecosystems, with relatively low anthropogenic impacts, and low human population density . The marMacrocystis pyrifera) is a ubiquitous component of the coastal seascape [Repeated abrasive expansions and retreats of glaciers, which occurred during the Quaternary Period, along with the formation of the Southern Patagonian Icefield (SPI), have given this region its dramatic geomorphology, which is characterized by deep valleys, rugged terrains, glaciers, streams, channels, rivers, and fjords –6. This seascape , and is seascape , 13. Theseascape . The steseascape , 16, butThe Strait of Magellan is a 565 km interoceanic route and a major hydrological feature that separates the South American continent from Tierra del Fuego. It is the confluence of water masses from the Pacific and Atlantic oceans, with influence from the Southern Ocean , 18. TheThe KNR is part of the ancestral territory of the Kawésqar or Kawésqar Wæs people, which extends from the Gulf of Penas to the Strait of Magellan . EvidencOwing to its remoteness, vast areas of this region have yet to be explored and remain understudied. Little is known about the marine communities of this vast remote area, particularly at deeper depths, and what role these diverse habitats and oceanographic conditions play in structuring this unique ecosystem. To this end, we conducted a comprehensive quantitative survey of the health of this largely unknown marine environment, highlighting the drivers that influence this unique ecosystem, and examining these in the context of the traditional ecological knowledge of the Kawésqar people.doi.org/10.5061/dryad.f7m0cfxvj.Data were collected by all authors in a collaborative effort. Non-invasive research was conducted, which included photographs, video, and visual estimates described in the methods below. The Republic of Chile granted all necessary permissions to conduct this research. No vertebrate sampling was conducted and therefore no approval was required by any Animal Care and Use Committee. Our data are publicly available at Data Dryad: 2 of protected terrestrial land, while the KNR protecting > 26,000 km2 of surrounding marine areas under the Chile National Reserve category. Collectively they incorporate the former Alacalufes Forest Reserve, new fiscal land, and land donation from Tompkins Conservation.The KNP and KNR include large swaths of the archipelagos in the provinces of Magallanes and Última Esperanza, as well as half of Isla Riesco. The KNP includes over 34,000 kmThe expedition originated in Punta Arenas on 21 February 2020 and covered a total of 2,352 km . We surv2). For colonial organisms  colonies, rather than individuals, were counted. Only non-cryptic invertebrates > 1 cm were enumerated. A second diver counted the number of kelp stipes (M. pyrifera and Lessonia spp.) within 1-m on either side of these transects. M. pyrifera holdfast diameters were measured by a third diver as an indication of plant size and age [Loxechinus albus in the general vicinity of the transect.Two transects of 25-m length, carried out on SCUBA, were conducted parallel to shore, towards the lower edge of the kelp zone. For sessile and mobile invertebrates, the number of individuals was estimated within 1-m of either side of the transect line (50 mH’ = − ∑(pi ln pi), where pi is the proportion of all individuals counted that were of species i. Pielou’s evenness was calculated as: J = H´/ln(S), where S is the total number of species present.Species diversity was calculated from the Shannon-Weaver Diversity Index : H’ = − 2) at each survey site (N = 2 transects). The transect column extended from the benthos to the surface, or as far as visibility allowed, to include species associated with the kelp canopy and water column. Total fish lengths were estimated to the nearest cm. In addition, photographs were taken in situ to assist with species identification, and document underwater coloration and associated habitat. Fish species biogeographic affinities and trophic group designations were obtained from published literature [For fish surveys, a scuba diver counted and sized all fishes within 1-m of either side of the 25-m transect lines  in a 33-cm diameter borosilicate glass sphere that is rated to ~7,000 m depth . ViewingVideo footage was annotated for taxa present (identified to the lowest possible taxonomic level), as well as for maximum number of individuals of a given taxon per video frame (MaxN), which provides a metric of relative abundance. Frequency of occurrence (Freq. occ. %) for each taxon observed was calculated as the percentage of incidence across all deployments. The substrata for each camera deployment were classified into standard geological categories consisting of mud, pebble, cobble, and boulder , 31. SeaM. pyrifera and Lessonia spp. at each station were compared using a one sample t-test. The correlation between the densities of M. pyrifera and Lessonia spp. was compared using Spearman’s rank-order correlation. The correlation of M. pyrifera stipe density with temperature and salinity was also tested using Spearman’s rank-order correlation. The densities of Lessonia spp. and temperature and salinity were compared in a similar manner. The correlation of M. pyrifera holdfast diameter with temperature and salinity was tested using Pearson’s product moment correlation. Benthic taxa richness, numerical abundance, Shannon-Weaver diversity, and Pielou’s evenness were all compared with temperature and salinity using Spearman’s rank-order correlation. Fish species richness, numerical abundance, biomass, and Shannon-Weaver diversity were all compared with temperature and salinity using Spearman’s rank-order correlation.Densities of M. pyrifera and Lessonia spp. stipe densities (No. m-2), and M. pyrifera holdfast diameter (cm). To rank explanatory environmental variables in their importance for being associated with the structure of the benthic community, we used a forward selection where the statistical significance of each variable was judged by a Monte-Carlo unrestricted permutation test with 499 permutations [To describe the pattern of benthic community structure among locations and their relationship to environmental variables, we performed direct gradient analysis  using the ordination program CANOCO version 5.0 . The RDAutations . Deep-seStations varied in oceanographic conditions based on their proximity to open ocean or inland locations, with locations closer to the Pacific Ocean having cooler temperatures and higher salinity compared to more inland locations, which were influenced by freshwater runoff resulting in lower salinity . Mean seGaimardia trapesina, Spirorbidae, erect and encrusting bryozoans, Sycozoa gaimardi), which caused the kelp fronds to be brittle and sunken from the weight. Lessonia spp. were almost totally absent in the fjords, only becoming abundant at the locations exposed to oceanic conditions . The density of M. pyrifera was significantly and negatively correlated with temperature  but not salinity . The density of Lessonia spp. was not significantly correlated with temperature  or salinity , although results are suggestive for salinity.Kelp beds in the sheltered fjords tended to be narrow and shallow (< 10 m) with numerous epiphytic taxa  holdfast diameter, which is a measure of plant size and age, averaged 24.2 cm (± 10.7) and was significantly and negatively correlated with temperate  and marginally positively correlated with salinity . The average test diameter of the sea urchin, Loxechinus albus, was 8.9 cm (± 1.5) and was not significantly correlated with temperature or salinity (p > 0.05 for both).Kelp  accounted for 74.2% of total abundance among all feeding groups, followed by carnivores , herbivorous browsers , passive suspension feeders , deposit feeders , and omnivores .-2 was 41.9 (± 67.8), with a minimum of 88 individuals and a maximum of 28,204. The number of individuals was positively correlated with temperature  and negatively correlated with salinity . Shannon-Weaver diversity averaged 1.9 (± 0.8) with a minimum of 0.4 and a maximum of 3.2. Diversity was negatively correlated with temperature  and positively correlated with salinity . Pielou’s evenness averaged 0.6 (± 0.2) with a minimum of 0.1 and a maximum of 0.9. Evenness was also negatively correlated with temperature  and positively correlated with salinity .There was an average of 30.2 (± 9.3) benthic taxa per transect, with a minimum of 11 and a maximum of 54 taxa . The numLessonia spp. contributed an additional 13% but was not significant in the model.Sampling stations were well separated in ordination space based on abundance of benthic taxa, with salinity and temperature accounting for much of the separation . ExplanaMytilus chilensis and the orange trumpet sponge Amphimedon maresi. Locations in the Strait of Magellan  clula Gaeta , 11 and la Gaeta  were at -2 was 0.23 (± 0.17), with a minimum of 0.04 individuals and a maximum of 0.80. The number of individuals was positively correlated with temperature  and negatively correlated with salinity . Most individuals were small, with a mean size of only 12.5 (± 5.5) cm, and therefore biomass only averaged 5.3 (± 4.0) g m-2. Biomass was positively correlated with temperature  and negatively correlated with salinity . Shannon-Weaver diversity averaged 0.81 (± 0.40) and was positively correlated with temperature  and negatively correlated with salinity .A total of 19 species of fishes from 9 families and 4 orders were observed on shallow water transects . OverallPatagonotothen cornucola was the most abundant and common species comprising 36% of both total abundance and total biomass and occurring in 76% of all transects. This was followed by Patagonotothen tessellata, which accounted for 27% of total abundance, 35% of total biomass, and occurred on 42% of all transects. Macrocystis fronds were an important habitat for many juvenile and cryptic fish species.The family Nototheniidae was the most specious with 8 species and accounted for 94% of total numerical abundance and 95% of total biomass . PatagonMacrocystis forests harbored unique communities that varied depending on oceanographic conditions. We observed a total of 69 invertebrate taxa from 19 classes and 9 phyla in these habitats. Of these taxa, 29 were not recorded on transects in the kelp forests , whip corals (Primnoella chilensis), and bryozoans (Carbasea ovoidea). In these protected fjords, fine sediments covered much of the substrate, and increased with depth. In the areas more exposed to oceanic conditions, the coral-water hydrocoral Errina antarctica, which is considered a vulnerable marine ecosystem (VME) species, was observed at several locations. The most extensive community was found off Isla de los Lobos at depths from 20 to 40 m. Within the hydrocoral beds we observed several species that were uncommon in the shallows such as Actinostola chilensis, Carbasea ovoidea, Adeonella sp. 2, Gorgonocephalus chilensis, Florometra magellanica, Grammaria abietina, Ophiuroglypha lymani, Microporella hyadesi, Labidiaster radiosus, a tubular sponge Haliclona and a large cervicorn black bryozoan. Below 30 m, the density of Errina antarctica decreased but soft corals such as Acanthogorgia sp., Thouarella koellikeri and Thouarella sp. 2 became more abundant.In the fjords, the steep slopes below the kelp beds were dominated by large sponges  fish taxa were observed on deep-sea camera deployments.Myxine sp.) was the most abundant and frequently occurring fish taxa observed, occurring in 80% of the deployments, with an average MaxN of 5.4 (± 7.2) and a maximum of 20 individuals per frame , Islas Caceres (600 m), and Islas Lobos (250 m)—had particularly high diversity of observed sharks, and these locations were the most exposed to open ocean influences. No other fish taxa occurred in > 30% of the deployments.Hagfish  are the primary drivers of the variability we observed in these marine communities. The high heterogeneity in the benthic communities that we observed is likely due to the extreme environmental conditions and the extraordinarily complex coastline of the region. The continental fjords and channels to the east are strongly influenced by freshwater inputs received from the Southern Patagonian Ice Fields. These are the largest ice fields on earth outside the polar regions , 35, andThe fjords and channels of the KNR are characterized by complex geomorphologies where water inputs from terrestrial and marine ecosystems overlap and mix. This interaction between oceanic waters and freshwater from multiple sources  produces strong vertical and horizontal gradients in temperature, salinity, nutrient ratios, and light availability , 38. TheSalinity explained much of the variation in benthic communities and ranged from 16.7 ppt in the inland fjords to 31.1 ppt at sites closer to the Pacific Ocean. While temperature only varied by 3.6°C (39% difference between high and low), the pattern was not as expected, with colder temperatures at sites more exposed to the open ocean and sites farther from the ocean, and closer inland having warmer temperatures.Kelp forests are the dominant nearshore ecosystem in the region. Deeper and wider kelp zones were associated with more oceanic conditions , while shallower and narrower kelp forests were found in the interior fjords, where salinity was lower. Benthic communities in the fjords were dominated by active suspension feeders such as mussels and sponges. High nutrient inputs from terrigenous sources and strong pelagic-benthic coupling create a favorable environment for this trophic guild to flourish , 41. BenLithodes santolla), which is one of the most important fisheries in the region. Juvenile L. santolla were observed on 42% of our transects, with an average density of 204.0 individuals (± 319.4) ha-1. According to the Chilean ministry of fisheries annual report on the status of fisheries, the king crab fishery is fully exploited and trending towards over-exploitation [Harpagifer bispinis and Patagonotothen spp. [Doryteuthis (Amerigo) gahi uses the kelp stipes to attach its egg masses in this region [The kelp forests of Southern Chile are some of the healthiest on earth , 3, 42 aoitation . These khen spp. , and thes region .Qualitative surveys of deep reefs (20–40 m) revealed a diverse assemblage of species. On the rocky slopes and boulder habitat, tunicates, echinoderms, mollusks, bryozoans, and sponges were abundant, some of which were not found in shallower water. The fjord region is a dynamic mixing zone, resulting from deep-water emergence, where typically deep-water organisms can be found in comparably shallow water , 47. ThiErrina antarctica found within the KNR provides habitat for numerous macroepibenthic species and plays an important role for the biodiversity of the Chilean fjord region, but colonies are slow-growing and easily damaged [The coral-water hydrocoral  damaged . It is w damaged , 49. Des damaged  and else damaged . RecoverMerluccius australis), Patagonian grenadier (Macruronus magellanicus), southern blue whiting , and Chilean deep-sea king crab (Lithodes turkayi) were observed on deep-sea camera footage, but their distributions were highly variable. All these species are targeted by industrial and artisanal fisheries, with the Austral hake stock having collapsed and the stocks of Patagonian grenadier and southern blue whiting over-exploited in the region [The deeper habitats of this region (200–600 m) were dominated by sandy and silty benthic substrate with low structural complexity of the benthos. As a result, species diversity was low, particularly for benthic invertebrates. Commercially important species such as Austral hake  became common [The Kawésqar people divided the territory into two parts, based on the geographical and biological characteristics of the territory . Jáutok  beaches , 52, 53.e common , 53, 54.Kelps dominate cold-water coastal zones and can become physiologically stressed at high sea temperatures, particularly when nutrient availability is low , 56. CliM. pyrifera to successfully colonize new habitats resulting from deglaciation [The unique climatic conditions in the fjords and channels of Chilean Patagonia’s fragmented coastline have made this part of the world one of the last refuges for giant kelp. Recent research has shown that kelp forests in the region can acclimate to new environmental conditions caused by increasing glacial retreat due to global warming by producing anti-stress compounds, which may make it possible to cope with increased ultraviolet radiation, temperature, and the presence of herbivorous invertebrates . Therefoaciation , which c2-enriched environment can lead to imbalances in kelp ecosystems by reversing the dominance of producers  to subordinates  and by suppressing the abundance and feeding rate of the primary grazers of turf algae (sea urchins), which can dramatically alter the competitive dominance within the ecosystem [While kelp forests in the fjord region may be able to cope with increasing water temperatures and nutrients, other future changes in ocean chemistry may be a cause for concern. It has been shown that ocean acidification and a COcosystem , 61. Addcosystem , and thicosystem –65 and tcosystem .Chile is the world’s second largest producer of farmed salmon and the industry is currently expanding into the Aysén and Magellan regions due in part to a large-scale outbreak of an infectious salmon anemia virus between 2008 and 2010 around Chiloé Island , 68. SalEscaped salmon, which are not native to Chile, have invaded nearly the entire Patagonia region , constitLoxechinus albus), red marine algae (Gigartina skottsbergii), and Chilean king crab (Lithodes santolla), with annual landings between 3,000 and 6,000 t. In addition, southern scallop (Chlamys vitrea), Magellan mussel (Aulacomya atra), Chilean mussel (Mytilus chilensis), razor clam (Ensis macha), and the clam  are landed in smaller quantities [Eleginops maclovinus) and sea silverside (Odontesthes regia) have been fished by the Kawésqar people for centuries and this traditional fishery persists today at a small scale in the Almirante Montt Gulf area, close to Puerto Natales [According to the Chilean Fisheries Service, many of the demersal fisheries in southern Chile are overexploited . The maj Natales , 75.Merluccius australis) constituted the main target species of the mixed demersal fisheries in the KNR with annual landings between 1,300 and 3,300 t [Genypterus blacodes) and yellownose skate (Zearaja chilensis) with the main fishing areas in Nelson Strait, close to Islas Lobos, and the Strait of Magellan, close to Carlos III [From 2000–2010, the artisanal fishery for southern hake , identification and protection of VMEs, establishment of marine protected areas, monitoring of ecosystem health and human use, and support of indigenous management and traditional ecological knowledge. Our scientific findings are consistent with indigenous knowledge of the region and highlight the importance of incorporating this knowledge in research and management of this inimitable region.S1 Table(DOCX)Click here for additional data file.S2 TableFeed = feeding groups: 1 = passive suspension feeders, 2 = active suspension feeders, 3 = herbivorous browsers, 4 = carnivores, 5 = omnivores, 6 = deposit feeders.(DOCX)Click here for additional data file.S3 Table-2, with one standard deviation of the mean in parentheses. Family names in bold.Pisc = piscivore; Inv = invertivore. Mean total length (TL) in cm are from quantitative underwater transects. Values are mean number of individuals.m(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 TableFrequency of occurrence (%) and MaxN .(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.