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Funding will redirect people who use drugs from the criminal justice system August 26, 2020 - Peterborough, Ontario - Health Canada Problematic substance use has generic lasix online for sale devastating impacts on people, families and communities across Canada. Tragically, the hypertension medications outbreak has worsened the situation for many Canadians struggling with substance use. The Government of Canada continues to address this serious public health issue by focusing on increasing access to generic lasix online for sale quality treatment and harm reduction services nationwide.

Today, on behalf of the Honourable Patty Hajdu, Minister of Health, the Honourable Maryam Monsef, Minister for Women and Gender Equality and Rural Economic Development, announced more than $1.9 million in funding over the next three years to the Peterborough Police Service. Through this funding, people who use drugs and experience mental health issues will be connected to newly-created community-based generic lasix online for sale outreach and support services. As part of this project, the Peterborough Police Service is working with local partners to create a community-based outreach team to increase the capacity for front-line community services to help people at risk who are referred by police.

With the help of this new team, people who use drugs generic lasix online for sale or experience mental health issues will be redirected from the criminal justice system to harm reduction, peer support, health and social services. Additionally, this initiative will increase access to culturally appropriate services for Indigenous Peoples, LGBTQ2+ populations, youth, women, and those living with HIV through partnerships with other organizations such as Nogojiwanong Friendship Centre and Peterborough AIDS Research Network. The Government of Canada is committed to working with partners, peer generic lasix online for sale workers, people with lived and living experience and other stakeholders to ensure Canadians receive the support they need to reduce the harms related to substance use.From.

Health Canada Media advisory Government of Canada to announce funding for community-based, multi-sector outreach and support services in Peterborough PETERBOROUGH, August 25, 2020 — On behalf of the Federal Minister of Health, Patty Hajdu, the Honourable Maryam Monsef, Minister for Women and Gender Equality and Rural Economic Development, will announce federal funding to help connect people at risk of experiencing opioid-related overdoses to community-based outreach and support services in Peterborough.There will be a media availability immediately following the announcement.DateWednesday, August 26, 2020Time10:00 AM (EDT)LocationThe media availability will be held on Zoom.Zoom link. Https://us02web.zoom.us/j/89698543218Meeting ID generic lasix online for sale. 896 9854 3218 Contacts Media Inquiries:Cole DavidsonOffice of the Honourable Patty HajduMinister of Health613-957-0200Media RelationsHealth Canada613-957-2983hc.media.sc@canada.ca.

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Former Editor-in-Chief of the Postgraduate Medical Journal Dr Barry Ian Hoffbrand died suddenly on April 24, 2020 at the age of 86.A prominent member of a generation of Ventolin evohaler for sale very bright young doctors at University College Hospital (UCH) in London who went buy cheap lasix on to distinguished careers, he was much admired for his keen intellect, clinical perception and skills, gentle good humour and kindly nature, combined with a wonderfully sharp intelligence. Professor Dame Jane Dacre remembered him as ‘a kind, witty, clever man, and a great physician’.He was born in Bradford, West Yorkshire, to Philip Hoffbrand, a bespoke tailor, and Minnie (née Freedman), both from Jewish families from Eastern Europe. After Bradford buy cheap lasix Grammar School, he went up to read medicine from 1952 to 1956 at The Queen’s College, Oxford, where he was a keen member of the college cricket team—the Quondams. He was pleased to feature in the 1950s on the silver Quondams Cup.

Clinical training on a Goldsmid scholarship followed from 1956 to 1958 at UCH Medical School, London, where he was awarded prizes in clinical pathology and haematology. His postgraduate buy cheap lasix medical training was mainly at UCH, where he was house physician to Max (later Lord) Rosenheim, after an initial 6 months at St Luke’s Hospital, Bradford. He also spent a year as senior research fellow from 1967 to 1968 at the Cardiovascular Research Institute, at the University of California Medical Center in San Francisco. Barry’s research on cardiovascular physiology lead to a DM in 1971 from Oxford University.Barry was appointed in 1970 as a consultant physician at the Whittington Hospital and honorary senior clinical lecturer at UCH Medical School, with interests in general and …INTRODUCTIONAs cardiac arrest occurs in around 20% of the patients with severe hypertension medications, a large number of them will require immediate resuscitative efforts.1 Cardiopulmonary resuscitation (CPR) in hypertension medications lasix has become a source of speculation and debate worldwide.

Healthcare professionals buy cheap lasix (HCPs) resuscitating this subset of patients are subject to fears and enormous mental stress pertaining to risk of transmission, breach in personal protective equipment (PPE), unsure effectiveness of PPE and nevertheless bleak positive outcomes in patients despite best resuscitative measures.2 CPR, which is conventionally deemed to be life-saving for patients, appears as an aerosol-generating procedure risking lives of HCPs caring for patients with hypertension medications. Protected code blue algorithm has been formulated to address both performer and patient safety.3POCUS-INTEGRATED CPR. WHY THE NEED buy cheap lasix IN hypertension medications?. Danilo Buonsenso and colleagues have described hypertension medications era as demanding less stethoscope and more ultrasound usage in clinical practice.4 PPE is now an essential measure for HCP protection, and goggles used as a part of PPE are associated with fogging and poor visibility.

This coupled with the inability to confirm endotracheal tube position with stethoscope due to poor accessibility in PPE, increases the risk of oesophageal intubation, re-intubation attempts, aerosol generation and thus HCP exposure. Bedside ultrasound could act as visual stethoscope in the buy cheap lasix described scenario. Sono-CPR in hypertension medications can help intervene quickly in treatable cases and reduce the time spent by HCP in futile resuscitative efforts. Reduced time spent equates to reduced duration of aerosol exposure and thus reduced risk of transmission.

Various algorithms are described for sono-cardiopulmonary resuscitation (sono-CPR) during cardiac arrest, but buy cheap lasix none are discussed to address patients with hypertension medications.5 It would hence be wise to integrate bedside point-of-care ultrasound (POCUS) in the code blue algorithm.HOW THE BEDSIDE TOOL HELPS?. Hypoxemia and respiratory failure attribute over 80% aetiology of cardiac arrest in patients with hypertension medications.1 Prioritising oxygenation and ventilation using definitive airway and use of high-efficiency particulate air filters reduces airborne transmission, thereby making early intubation the dictum of resuscitation.3 Considering poor visualisation due to fogging with the goggles and face shield, inability to use stethoscope and lack of availability of end-tidal CO2 (EtCO2) in resource constraint settings, ultrasound-guided real-time intubation by trained HCP or endotracheal tube (ETT) placement confirmation post intubation could prove beneficial. Confirming ETT placement and direct visualisation of oesophagal lumen buy cheap lasix can be done using a linear ultrasound probe.6 In cases of oesophageal intubation, tissue-air hyperechoic lines are visualised in both trachea and oesophagus, referred to as ‘double-track sign’.State of hypercoagulability and myocardial dysfunction exist in patients with hypertension medications, hence increasing the likelihood of myocardial infarction or pulmonary thromboembolism as aetiologies of cardiac arrest.7 Regional wall motion abnormality, dilated right atrium or right ventricle, plethoric inferior vena cava are easily identified by goal-directed echocardiography. Pneumothorax has been reported in patients with hypertension medications, and ultrasound can identify absence of lung sliding, helping in quick needle thoracocentesis in arrest and peri-arrest cases.

Few cases of cardiac tamponade owing to myopericarditis have also been reported and bedside ultrasound can help diagnose and perform pericardiocentesis in such patients.Literature suggests that the chances of Return Of Spontaneous Circulation (ROSC) and survival to hospital admission at 24 hours is better in patients with baseline cardiac activity rather than no baseline cardiac activity. In patients with no baseline cardiac activity on buy cheap lasix arrival, one can withhold CPR, thereby protecting the HCP in this resource-intensive, aerosol-generating futile resuscitative effort.8 Asystole could be the disguised entity of fine ventricular fibrillation, which can be confirmed by fibrillatory cardiac activity on transthoracic echocardiography and can be defibrillated, thereby increasing the chances of earlier ROSC.9POCUS-INTEGRATED CPR. THE PROPOSED ALGORITHMCPR is a chaotic scenario, and to prevent added chaos, there is a need for a well-trained ultrasound performer placed in an appropriate area (figure 1). Intubating room needs to consist of minimal necessary number of HCPs, and all of them should be equipped with full PPE.

Ultrasound device could be a potential fomite facilitating cross-transmission and requires adequate protection of machine and its components with buy cheap lasix a transparent cover, sheet or bag. When unavailable, low-level disinfectant solution should be used between each patient.Proposed algorithm for integration of POCUS during CPR in patients with hypertension medications with team dynamics. The illustration is original work buy cheap lasix of the authors Dr Brunda RL and colleagues. CPR, cardiopulmonary resuscitation.

HCP, healthcare professional. POCUS, point-of-care ultrasound buy cheap lasix. PPE, personal protective equipment. RA, right atrium.

RV, right ventricle buy cheap lasix. VF, ventricular fibrillation. USG, ultrasonography." data-icon-position data-hide-link-title="0">Figure 1 Proposed algorithm for integration of POCUS during CPR in patients with hypertension medications with team dynamics. The illustration is original work of the authors Dr Brunda buy cheap lasix RL and colleagues.

CPR, cardiopulmonary resuscitation. HCP, healthcare professional buy cheap lasix. POCUS, point-of-care ultrasound. PPE, personal protective equipment.

RA, right atrium buy cheap lasix. RV, right ventricle. VF, ventricular fibrillation. USG, ultrasonography.When a patient experiences cardiac arrest, there is a need for HCPs with full buy cheap lasix PPE to check pulse and begin CPR as per standard guidelines.

After 2 min of CPR, if there is no ROSC, during the 10 second pause for rhythm assessment, a trained HCP can perform POCUS in a stepwise manner. Each step needs buy cheap lasix to be performed individually during 10 second pause without prolonging delay between chest compressions and compromising the quality of CPR. Any treatable aetiology identified during the algorithm requires immediate intervention.Step 1. Assess cardiac activity—Sub-xiphoid view can be procured and cardiac activity assessed.

If absent, consider termination of efforts, and if present, resuscitative efforts can be continued.After repeating 2 min cycle of CPR, if there buy cheap lasix has been no ROSC, consider hypoxic aetiology as the cause of arrest in patients with hypertension medications and intubate without delay. Withholding chest compressions during intubation is recommended.3Step 2. Assess ETT placement—At the level of thyroid gland, above the suprasternal notch, place ultrasound probe transversely and visualise the oesophagus.10 If the posterior wall of oesophagus is obscured by a dark acoustic shadow or if there is ‘double-track’ sign, consider failed endotracheal intubation and perform immediate re-intubation.Step 3. Assess lung for pneumothorax—Assess lung sliding, and if absent look for ‘stratosphere sign’ in M-mode of ultrasound.10 If detected, perform immediate needle thoracocentesis.Step buy cheap lasix 4.

Assess for Cardiac etiology of arrest—Obtain sub-xiphoid window preferably, and look for the presence of cardiac tamponade, chamber dilatation or collapse, regional wall motion abnormality and cardiac contractility.Availability of trained personnel and smaller portable ultrasound devices makes its use during cardiac arrest plausible.CPR with the help of POCUS could thus prove to improve chances of ROSC and also reduced transmission to HCP by early identification, treatment of reversible causes and avoidance of prolonged efforts. Sono-CPR appears to be more HCP-friendly than prolonged blind CPR and necessitates its utility in the era of hypertension medications addressing performer safety as well as patient safety..

Former Editor-in-Chief of the Postgraduate Medical Journal Dr Barry Ian Hoffbrand died suddenly on April 24, 2020 at the age of 86.A prominent member of a generation of very bright young doctors at University College Hospital (UCH) generic lasix online for sale in London who went on to distinguished careers, he was much admired for his keen intellect, clinical perception and skills, gentle good humour and kindly nature, combined with a wonderfully sharp intelligence. Professor Dame Jane Dacre remembered him as ‘a kind, witty, clever man, and a great physician’.He was born in Bradford, West Yorkshire, to Philip Hoffbrand, a bespoke tailor, and Minnie (née Freedman), both from Jewish families from Eastern Europe. After Bradford Grammar School, he went up to read medicine from 1952 to 1956 at The Queen’s College, Oxford, generic lasix online for sale where he was a keen member of the college cricket team—the Quondams. He was pleased to feature in the 1950s on the silver Quondams Cup. Clinical training on a Goldsmid scholarship followed from 1956 to 1958 at UCH Medical School, London, where he was awarded prizes in clinical pathology and haematology.

His postgraduate medical training was mainly at UCH, where generic lasix online for sale he was house physician to Max (later Lord) Rosenheim, after an initial 6 months at St Luke’s Hospital, Bradford. He also spent a year as senior research fellow from 1967 to 1968 at the Cardiovascular Research Institute, at the University of California Medical Center in San Francisco. Barry’s research on cardiovascular physiology lead to a DM in 1971 from Oxford University.Barry was appointed in 1970 as a consultant physician at the Whittington Hospital and honorary senior clinical lecturer at UCH Medical School, with interests in general and …INTRODUCTIONAs cardiac arrest occurs in around 20% of the patients with severe hypertension medications, a large number of them will require immediate resuscitative efforts.1 Cardiopulmonary resuscitation (CPR) in hypertension medications lasix has become a source of speculation and debate worldwide. Healthcare professionals (HCPs) resuscitating this subset of patients are subject to fears and enormous mental stress pertaining to risk of transmission, breach in personal protective equipment (PPE), unsure effectiveness of PPE and nevertheless bleak positive outcomes in patients generic lasix online for sale despite best resuscitative measures.2 CPR, which is conventionally deemed to be life-saving for patients, appears as an aerosol-generating procedure risking lives of HCPs caring for patients with hypertension medications. Protected code blue algorithm has been formulated to address both performer and patient safety.3POCUS-INTEGRATED CPR.

WHY THE NEED generic lasix online for sale IN hypertension medications?. Danilo Buonsenso and colleagues have described hypertension medications era as demanding less stethoscope and more ultrasound usage in clinical practice.4 PPE is now an essential measure for HCP protection, and goggles used as a part of PPE are associated with fogging and poor visibility. This coupled with the inability to confirm endotracheal tube position with stethoscope due to poor accessibility in PPE, increases the risk of oesophageal intubation, re-intubation attempts, aerosol generation and thus HCP exposure. Bedside ultrasound could act as visual stethoscope in the generic lasix online for sale described scenario. Sono-CPR in hypertension medications can help intervene quickly in treatable cases and reduce the time spent by HCP in futile resuscitative efforts.

Reduced time spent equates to reduced duration of aerosol exposure and thus reduced risk of transmission. Various algorithms are described for sono-cardiopulmonary resuscitation (sono-CPR) during cardiac arrest, but none are discussed to address patients with generic lasix online for sale hypertension medications.5 It would hence be wise to integrate bedside point-of-care ultrasound (POCUS) in the code blue algorithm.HOW THE BEDSIDE TOOL HELPS?. Hypoxemia and respiratory failure attribute over 80% aetiology of cardiac arrest in patients with hypertension medications.1 Prioritising oxygenation and ventilation using definitive airway and use of high-efficiency particulate air filters reduces airborne transmission, thereby making early intubation the dictum of resuscitation.3 Considering poor visualisation due to fogging with the goggles and face shield, inability to use stethoscope and lack of availability of end-tidal CO2 (EtCO2) in resource constraint settings, ultrasound-guided real-time intubation by trained HCP or endotracheal tube (ETT) placement confirmation post intubation could prove beneficial. Confirming ETT placement and direct visualisation of oesophagal lumen can be done using a linear ultrasound probe.6 In cases of oesophageal intubation, tissue-air hyperechoic lines are visualised in both trachea and generic lasix online for sale oesophagus, referred to as ‘double-track sign’.State of hypercoagulability and myocardial dysfunction exist in patients with hypertension medications, hence increasing the likelihood of myocardial infarction or pulmonary thromboembolism as aetiologies of cardiac arrest.7 Regional wall motion abnormality, dilated right atrium or right ventricle, plethoric inferior vena cava are easily identified by goal-directed echocardiography. Pneumothorax has been reported in patients with hypertension medications, and ultrasound can identify absence of lung sliding, helping in quick needle thoracocentesis in arrest and peri-arrest cases.

Few cases of cardiac tamponade owing to myopericarditis have also been reported and bedside ultrasound can help diagnose and perform pericardiocentesis in such patients.Literature suggests that the chances of Return Of Spontaneous Circulation (ROSC) and survival to hospital admission at 24 hours is better in patients with baseline cardiac activity rather than no baseline cardiac activity. In patients with no baseline cardiac activity on arrival, one can withhold CPR, thereby generic lasix online for sale protecting the HCP in this resource-intensive, aerosol-generating futile resuscitative effort.8 Asystole could be the disguised entity of fine ventricular fibrillation, which can be confirmed by fibrillatory cardiac activity on transthoracic echocardiography and can be defibrillated, thereby increasing the chances of earlier ROSC.9POCUS-INTEGRATED CPR. THE PROPOSED ALGORITHMCPR is a chaotic scenario, and to prevent added chaos, there is a need for a well-trained ultrasound performer placed in an appropriate area (figure 1). Intubating room needs to consist of minimal necessary number of HCPs, and all of them should be equipped with full PPE. Ultrasound device generic lasix online for sale could be a potential fomite facilitating cross-transmission and requires adequate protection of machine and its components with a transparent cover, sheet or bag.

When unavailable, low-level disinfectant solution should be used between each patient.Proposed algorithm for integration of POCUS during CPR in patients with hypertension medications with team dynamics. The illustration is original work of the generic lasix online for sale authors Dr Brunda RL and colleagues. CPR, cardiopulmonary resuscitation. HCP, healthcare professional. POCUS, point-of-care ultrasound generic lasix online for sale.

PPE, personal protective equipment. RA, right atrium. RV, right ventricle generic lasix online for sale. VF, ventricular fibrillation. USG, ultrasonography." data-icon-position data-hide-link-title="0">Figure 1 Proposed algorithm for integration of POCUS during CPR in patients with hypertension medications with team dynamics.

The illustration is original work of the authors Dr Brunda RL and colleagues generic lasix online for sale. CPR, cardiopulmonary resuscitation. HCP, healthcare generic lasix online for sale professional. POCUS, point-of-care ultrasound. PPE, personal protective equipment.

RA, right generic lasix online for sale atrium. RV, right ventricle. VF, ventricular fibrillation. USG, ultrasonography.When generic lasix online for sale a patient experiences cardiac arrest, there is a need for HCPs with full PPE to check pulse and begin CPR as per standard guidelines. After 2 min of CPR, if there is no ROSC, during the 10 second pause for rhythm assessment, a trained HCP can perform POCUS in a stepwise manner.

Each step needs to be performed individually during 10 second pause without prolonging delay between chest compressions and generic lasix online for sale compromising the quality of CPR. Any treatable aetiology identified during the algorithm requires immediate intervention.Step 1. Assess cardiac activity—Sub-xiphoid view can be procured and cardiac activity assessed. If absent, consider termination of efforts, and if present, resuscitative efforts can be continued.After repeating 2 min cycle of generic lasix online for sale CPR, if there has been no ROSC, consider hypoxic aetiology as the cause of arrest in patients with hypertension medications and intubate without delay. Withholding chest compressions during intubation is recommended.3Step 2.

Assess ETT placement—At the level of thyroid gland, above the suprasternal notch, place ultrasound probe transversely and visualise the oesophagus.10 If the posterior wall of oesophagus is obscured by a dark acoustic shadow or if there is ‘double-track’ sign, consider failed endotracheal intubation and perform immediate re-intubation.Step 3. Assess lung for pneumothorax—Assess lung sliding, and if absent look for ‘stratosphere sign’ in generic lasix online for sale M-mode of ultrasound.10 If detected, perform immediate needle thoracocentesis.Step 4. Assess for Cardiac etiology of arrest—Obtain sub-xiphoid window preferably, and look for the presence of cardiac tamponade, chamber dilatation or collapse, regional wall motion abnormality and cardiac contractility.Availability of trained personnel and smaller portable ultrasound devices makes its use during cardiac arrest plausible.CPR with the help of POCUS could thus prove to improve chances of ROSC and also reduced transmission to HCP by early identification, treatment of reversible causes and avoidance of prolonged efforts. Sono-CPR appears to be more HCP-friendly than prolonged blind CPR and necessitates its utility in the era of hypertension medications addressing performer safety as well as patient safety..

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Lasix abuse

In a lasix abuse historic move, the US government has announced that it supports waiving patent protections for hypertension medications treatments, a measure aimed at boosting supplies so that people around helpful resources the world can get the shots. €œThe extraordinary circumstances of the hypertension medications lasix call for extraordinary measures,” said US trade representative Katherine Tai in a statement. The move came on 5 May, the first of a two-day meeting of the general council of the World Trade Organization, based in Geneva, Switzerland. Until now, lasix abuse the United States, the European Union, the United Kingdom and Japan have blocked efforts brought by India and South Africa to make it legal to manufacture generic versions of hypertension medications treatments.

Former US presidents from both the Republican and the Democratic parties have staunchly defended intellectual-property rights, so the move by the administration of President Joe Biden has shocked people on both sides of the debate. €œThis marks a major shift in US policy in a pro-public-health way,” says Matthew Kavanagh, a global-health researcher at Georgetown University in Washington DC. Kavanagh is part of the growing chorus of health-policy and global-health researchers advocating patent waivers, as the gap between vaccination rates in rich and poor nations grows larger lasix abuse every day. Fewer than 1% of people in low-income countries have received hypertension medications treatments.

The researchers are quick to note, however, that a waiver on patents covering all aspects of hypertension medications treatments would be just the first step in ramping up treatment supply. First step of three “It’s a one-two-three,” explains Rachel Cohen, US director for the non-profit Drugs and Neglected Diseases initiative in New York City lasix abuse. €œFirst we need to remove patent obstacles, second we need to transfer the knowledge on how to make them, and step three is a massive investment in manufacturing capacity.” And at the moment, step one is far from complete. The World Trade Organization will not negotiate the details of which patents to adjust until all its member countries agree on some sort of waiver.

Health-policy analysts speculate that other countries will follow in the footsteps of the United States, although the European Union might hold out beyond the lasix abuse end of the meeting. South Africa and India have proposed waivers on patents, not only on treatments, but also on hypertension medications-related medical devices, drugs and diagnostic technologies. So far, Tai’s statement mentions only treatments. Drugmakers and others who oppose the measure say that waivers sabotage companies’ enormous investments in drug and treatment development, which are compensated by their ability to lasix abuse set the price on products that they exclusively own.

Normally, patents reward pharmaceutical companies by protecting their inventions from competition by generics for a limited time—US patents on drugs typically last for 20 years. Pharmaceutical-industry backlash Drug companies aren’t the only opponents of the measure. In a 25 April interview with Sky News, global health philanthropist and Microsoft co-founder Bill Gates argued against intellectual-property waivers, saying that manufacturers of generics couldn’t ramp up production quickly, and that treatment quality could be lasix abuse compromised. After the US government's waiver announcement, the industry group Pharmaceutical Research and Manufacturers of America released a statement echoing these points, saying.

€œThe Biden Administration has taken an unprecedented step that will undermine our global response to the lasix and compromise safety.” Proponents of the waiver disagree, pointing out that generics manufacturers have been supplying the world with high-quality treatments and medicines for years. They point out that taxpayers helped to foot the bill for the development of several hypertension medications treatments, and say that the claim that pharmaceutical companies must recoup all the costs is lasix abuse therefore unfair—especially during a crisis. Several other obstacles must be addressed, however, such as making sure distribution is equitable. Cohen says.

€œThese treatments are an unparalleled triumph for science, but if only 20% or 30% of the world winds up benefiting, what is the point of the innovation?.

€œThe extraordinary circumstances generic lasix online for sale of the where can you get lasix hypertension medications lasix call for extraordinary measures,” said US trade representative Katherine Tai in a statement. The move came on 5 May, the first of a two-day meeting of the general council of the World Trade Organization, based in Geneva, Switzerland. Until now, the United States, the European Union, the United Kingdom and Japan have blocked efforts brought by India and South Africa to make it legal to manufacture generic versions of hypertension medications treatments. Former US presidents from both the Republican and the Democratic parties have staunchly defended intellectual-property rights, so the move by the administration of President Joe Biden has shocked people on both sides generic lasix online for sale of the debate. €œThis marks a major shift in US policy in a pro-public-health way,” says Matthew Kavanagh, a global-health researcher at Georgetown University in Washington DC.

Kavanagh is part of the growing chorus of health-policy and global-health researchers advocating patent waivers, as the gap between vaccination rates in rich and poor nations grows larger every day. Fewer than 1% of people generic lasix online for sale in low-income countries have received hypertension medications treatments. The researchers are quick to note, however, that a waiver on patents covering all aspects of hypertension medications treatments would be just the first step in ramping up treatment supply. First step of three “It’s a one-two-three,” explains Rachel Cohen, US director for the non-profit Drugs and Neglected Diseases initiative in New York City. €œFirst we need to remove patent generic lasix online for sale obstacles, second we need to transfer the knowledge on how to make them, and step three is a massive investment in manufacturing capacity.” And at the moment, step one is far from complete.

The World Trade Organization will not negotiate the details of which patents to adjust until all its member countries agree on some sort of waiver. Health-policy analysts speculate that other countries will follow in the footsteps of the United States, although the European Union might hold out beyond the end of the meeting. South Africa and India have generic lasix online for sale proposed waivers on patents, not only on treatments, but also on hypertension medications-related medical devices, drugs and diagnostic technologies. So far, Tai’s statement mentions only treatments. Drugmakers and others who oppose the measure say that waivers sabotage companies’ enormous investments in drug and treatment development, which are compensated by their ability to set the price on products that they exclusively own.

Normally, patents reward pharmaceutical companies by protecting their generic lasix online for sale inventions from competition by generics for a limited time—US patents on drugs typically last for 20 years. Pharmaceutical-industry backlash Drug companies aren’t the only opponents of the measure. In a 25 April interview with Sky News, global health philanthropist and Microsoft co-founder Bill Gates argued against intellectual-property waivers, saying that manufacturers of generics couldn’t ramp up production quickly, and that treatment quality could be compromised. After the US government's waiver announcement, the industry group Pharmaceutical Research and Manufacturers of America generic lasix online for sale released a statement echoing these points, saying. €œThe Biden Administration has taken an unprecedented step that will undermine our global response to the lasix and compromise safety.” Proponents of the waiver disagree, pointing out that generics manufacturers have been supplying the world with high-quality treatments and medicines for years.

They point out that taxpayers helped to foot the bill for the development of several hypertension medications treatments, and say that the claim that pharmaceutical companies must recoup all the costs is therefore unfair—especially during a crisis. Several other obstacles must be addressed, generic lasix online for sale however, such as making sure distribution is equitable. Cohen says. €œThese treatments are an unparalleled triumph for science, but if only 20% or 30% of the world winds up benefiting, what is the point of the innovation?. € This article is reproduced with permission and was first published on May 6 2021..

Does lasix make horses faster

This edition does lasix make horses faster of the Emergency Medicine Journal has ‘something for buy lasix water pills online everyone’ (as always), and at least one article that will be of interest to everyone (I think). The two main themes in this edition are ‘the difficult airway’ and Paediatric Emergency Medicine. However, we begin this Primary Survey by talking about gender.Gender differences in Emergency MedicineTwo articles look at gender disparity in Emergency does lasix make horses faster Medicine (EM). A short report by Partiali et al looks at the proportion of female speakers, and the length of time these speakers are given to deliver their talks, at a major EM academic conference.

Although both does lasix make horses faster proportion and ‘speaking-time’ are increasing over the period reviewed, there remains a large gender difference. In the paper by Parsons et al, the worldwide difference in academic representation between the genders is discussed, and is especially interesting given the fact that more females matriculate from medical school in both the USA (since 2017) and the UK (since 1996–7). The authors then go on to look at gender differences in medical leadership in EM does lasix make horses faster in the USA. The discrepancy revealed in this paper will, unfortunately, be unsurprising.Whilst writing this ‘Primary Survey’ my bedtime reading is a novel by the late-Victorian writer George Gissing, who in many of his novels explored the position of women in the late nineteenth century.

One of does lasix make horses faster the characters opines “Woman is still enslaved, though men nowadays think it necessary to disguise it.” Having read these two articles it may be that the medical profession has evolved little in this regard over the last 150 years.The difficult airwayThree papers in this edition look at difficult airways and their management. In a paper from Japan by Takahashi et al, there is a review of a database from a large observational study on emergency airway management. This has revealed an increase does lasix make horses faster in major (but not minor) adverse events in the older population undergoing emergency intubation, largely due to post-intubation hypotension. From the Helicopter Emergency Medical Service in London, there is a 20 year review of emergency cricothyroidotomy which reveals a very low rate of requirement for surgical airways in the pre-hospital environmentWhen performed, it is often due to blood in the airway preventing laryngoscopy.

Gaffar et al have looked at trauma CT scans and calculated does lasix make horses faster the average cricothyroid membrane depth, and factors associated with a greater depth. Some of these factors might be surprising, however these ought to be considered by those preparing to perform an emergency surgical airway.Paediatric Emergency MedicineThere are several papers looking at issues in Paediatric Emergency Medicine. The results from a Paediatric Emergency registry in Nicaragua (reviewed in Bressan et al) is sobering, and the use lasix water pills online of point-of-care EEG in an ED (described by Simma et al) in intriguing." data-icon-position data-hide-link-title="0">Two further papers particularly catch the eye. The Editors Choice this month is a paper looking at the likely cervical spine imaging in a Paediatric population, when using three different clinical decision rules does lasix make horses faster (CDRs) (Philips et al).

There were large differences between cervical spine injury rates and imaging rates. However the use of does lasix make horses faster CDRs would have increased the rate of imaging. The second paper is the short report by Cameron et al, highlighting the dangers of travel cups to children. Of interest to all of those who use them.Other articles of interestThe problem of pre-hospital ‘missed stroke’ is considered in the systematic review by Jones et al, and reading this paper reveals the challenges faced by does lasix make horses faster clinicians ‘in the field’.

The clinical impact of this, and the potential for improving sensitivity of tools to identify stroke pre-hospital is discussed.Two original research papers relating to hypertension medications are of interest. Lyall and Lone look at the effect on non-hypertension medications admissions during the first lockdown in Scotland, while Bertaina et does lasix make horses faster al look at non-invasive ventilation in acute respiratory failure due to hypertension medications.And finally…And the article I think will be of interest to everyone?. This is the Best Evidence topic report on homemade or cloth facemasks as a preventative measure for respiratory lasix transmission- an evidence review on a topic that, is affecting all our lives.‘Tis a lesson you should heedTry, try again.If at first you don’t succeed,Try, try again.— Thomas H Palmer Teacher’s ManualPaediatric cervical spine injuries are rare events, particularly in young children. An individual emergency provider may see less than a handful in her entire career, even does lasix make horses faster as she is continuously presented with patients considered at risk for injury.

In the same career, each provider will likely expose thousands of children to significant doses of radiation with an indeterminate but finite risk of inducing a downstream malignancy. Thus, with the increasing awareness of the cumulative risks associated with radiation exposure, the decision as to which patient should be radiographically studied and at what threshold often becomes an uncomfortable one.Useful clinical decision rules (CDRs) for identifying cervical spine injuries have been derived, validated and are broadly embraced for adult does lasix make horses faster patients. The National Emergency X-Radiography Utilization Study (NEXUS) from the US and the Canadian C-Spine Rules (CCR).1 2 No comparable, validated paediatric decision-making tools have been created and medical providers have been largely left to extrapolate the findings of adult studies to their paediatric patients whose injuries and risks differ mechanistically and physiologically from their future selves. In an effort to provide better guidance to emergency providers, the investigators of the NEXUS trial analysed a paediatric subset with a very limited sample size (n=3065 with 30 cervical spine injuries), while the Pediatric Emergency Care Applied Research Network (PECARN) attempted to tackle the problem differently through a case-controlled methodology.3 4 Both of these paediatric efforts suffer significant limitations compared with the afore-mentioned large prospective observational studies.In a side-by-side comparison of these three decision tools, ….

This edition of the Emergency Medicine Journal has ‘something for everyone’ (as always), generic lasix online for sale and at least one article that will be of http://www.ec-paul-bert-schiltigheim.ac-strasbourg.fr/lelementaire/nos-projets/ce1-basket-ball-a-lecole-paul-bert/ interest to everyone (I think). The two main themes in this edition are ‘the difficult airway’ and Paediatric Emergency Medicine. However, we begin this Primary Survey by talking generic lasix online for sale about gender.Gender differences in Emergency MedicineTwo articles look at gender disparity in Emergency Medicine (EM).

A short report by Partiali et al looks at the proportion of female speakers, and the length of time these speakers are given to deliver their talks, at a major EM academic conference. Although both proportion and ‘speaking-time’ are increasing over the period reviewed, there remains a generic lasix online for sale large gender difference. In the paper by Parsons et al, the worldwide difference in academic representation between the genders is discussed, and is especially interesting given the fact that more females matriculate from medical school in both the USA (since 2017) and the UK (since 1996–7).

The authors then go on generic lasix online for sale to look at gender differences in medical leadership in EM in the USA. The discrepancy revealed in this paper will, unfortunately, be unsurprising.Whilst writing this ‘Primary Survey’ my bedtime reading is a novel by the late-Victorian writer George Gissing, who in many of his novels explored the position of women in the late nineteenth century. One of the characters opines “Woman is still enslaved, though men nowadays think generic lasix online for sale it necessary to disguise it.” Having read these two articles it may be that the medical profession has evolved little in this regard over the last 150 years.The difficult airwayThree papers in this edition look at difficult airways and their management.

In a paper from Japan by Takahashi et al, there is a review of a database from a large observational study on emergency airway management. This has revealed an increase in major (but not minor) adverse events in the older generic lasix online for sale population undergoing emergency intubation, largely due to post-intubation hypotension. From the Helicopter Emergency Medical Service in London, there is a 20 year review of emergency cricothyroidotomy which reveals a very low rate of requirement for surgical airways in the pre-hospital environmentWhen performed, it is often due to blood in the airway preventing laryngoscopy.

Gaffar et al have looked at trauma CT scans and calculated the average cricothyroid membrane generic lasix online for sale depth, and factors associated with a greater depth. Some of these factors might be surprising, however these ought to be considered by those preparing to perform an emergency surgical airway.Paediatric Emergency MedicineThere are several papers looking at issues in Paediatric Emergency Medicine. The results from a Paediatric Emergency registry in Nicaragua (reviewed in Bressan et al) is sobering, and the use of point-of-care EEG in an ED (described by Simma et al) in intriguing." data-icon-position data-hide-link-title="0">Two further papers particularly catch the eye.

The Editors Choice this month is a paper looking generic lasix online for sale at the likely cervical spine imaging in a Paediatric population, when using three different clinical decision rules (CDRs) (Philips et al). There were large differences between cervical spine injury rates and imaging rates. However the generic lasix online for sale use of CDRs would have increased the rate of imaging.

The second paper is the short report by Cameron et al, highlighting the dangers of travel cups to children. Of interest to all of generic lasix online for sale those who use them.Other articles of interestThe problem of pre-hospital ‘missed stroke’ is considered in the systematic review by Jones et al, and reading this paper reveals the challenges faced by clinicians ‘in the field’. The clinical impact of this, and the potential for improving sensitivity of tools to identify stroke pre-hospital is discussed.Two original research papers relating to hypertension medications are of interest.

Lyall and Lone look at the effect on non-hypertension medications admissions during the first generic lasix online for sale lockdown in Scotland, while Bertaina et al look at non-invasive ventilation in acute respiratory failure due to hypertension medications.And finally…And the article I think will be of interest to everyone?. This is the Best Evidence topic report on homemade or cloth facemasks as a preventative measure for respiratory lasix transmission- an evidence review on a topic that, is affecting all our lives.‘Tis a lesson you should heedTry, try again.If at first you don’t succeed,Try, try again.— Thomas H Palmer Teacher’s ManualPaediatric cervical spine injuries are rare events, particularly in young children. An individual emergency provider may generic lasix online for sale see less than a handful in her entire career, even as she is continuously presented with patients considered at risk for injury.

In the same career, each provider will likely expose thousands of children to significant doses of radiation with an indeterminate but finite risk of inducing a downstream malignancy. Thus, with the increasing awareness of the cumulative risks associated with radiation exposure, the decision as to which patient should be generic lasix online for sale radiographically studied and at what threshold often becomes an uncomfortable one.Useful clinical decision rules (CDRs) for identifying cervical spine injuries have been derived, validated and are broadly embraced for adult patients. The National Emergency X-Radiography Utilization Study (NEXUS) from the US and the Canadian C-Spine Rules (CCR).1 2 No comparable, validated paediatric decision-making tools have been created and medical providers have been largely left to extrapolate the findings of adult studies to their paediatric patients whose injuries and risks differ mechanistically and physiologically from their future selves.

In an effort to provide better guidance to emergency providers, the investigators of the NEXUS trial analysed a paediatric subset with a very limited sample size (n=3065 with 30 cervical spine injuries), while the Pediatric Emergency Care Applied Research Network (PECARN) attempted to tackle the problem differently through a case-controlled methodology.3 4 Both of these paediatric efforts suffer significant limitations compared with the afore-mentioned large prospective observational studies.In a side-by-side comparison of these three decision tools, ….

Does lasix cause hypokalemia

On 26 May, US President Joe Biden tasked the US Intelligence Community to see this join efforts to find hypertension’s origins, whatever they might be, and report back in 90 does lasix cause hypokalemia days. Australia, the European Union and Japan have also called for a robust investigation into hypertension’s origins in China. The WHO has yet to reveal the next phase of its investigation. But China has asked does lasix cause hypokalemia that the probe examine other countries. Such reticence, and the fact that China has withheld information in the past, has fuelled suspicions of a ‘lab leak’.

For instance, Chinese government officials suppressed crucial public-health data at the start of the hypertension medications lasix, and during the 2002–04 severe acute respiratory syndrome (SARS) epidemic, according to high-level reports. At the assembly, Mike Ryan, director of health emergencies at the WHO, asked for less politicization of calls for an origin investigation, does lasix cause hypokalemia which have, in many ways, devolved into accusations. €œOver the last number of days, we have seen more and more and more discourse in the media, with terribly little actual news, or evidence, or new material,” said Ryan. €œThis is disturbing.” Nature looks at the key arguments that support a lab leak, and the extent to which research has answers. There’s not does lasix cause hypokalemia yet any substantial evidence for a lab leak.

Why are scientists still considering it?. Scientists don’t have enough evidence about the origins of hypertension to rule out the lab-leak hypothesis, or to prove the alternative—that the lasix has a natural origin. Many infectious-disease researchers agree does lasix cause hypokalemia that the most probable scenario is that the lasix evolved naturally and spread from a bat either directly to a person or through an intermediate animal. Most emerging infectious diseases begin with a spillover from nature, as was seen with HIV, influenza epidemics, Ebola outbreaks and the hypertensiones that caused the SARS epidemic beginning in 2002 and the Middle East respiratory syndrome (MERS) outbreak beginning in 2012. Researchers have some leads that support a natural origin.

Bats are known carriers of hypertensiones, and scientists have does lasix cause hypokalemia determined that the genome of hypertension is most similar to that of RATG13, a hypertension that was first found in a horseshoe bat (Rhinolophus affinis) in the southern Chinese province of Yunnan in 2013. But RATG13’s genome is only 96% identical to hypertension’s, suggesting that a closer relative of the lasix—the one passed to humans—remains unknown. Still, the possibility remains that hypertension escaped from a lab. Although lab leaks have does lasix cause hypokalemia never caused an epidemic, they have resulted in small outbreaks involving well-documented lasixes. A relevant example happened in 2004, when two researchers were independently infected by the lasix that causes SARS at a virology lab in Beijing that studied the disease.

They spread the to seven others before the outbreak was contained. What are does lasix cause hypokalemia the key arguments for a lab leak?. In theory, hypertension medications could have come from a lab in a few ways. Researchers might have collected hypertension from an animal and maintained it in their lab to study, or they might have created it by engineering hypertension genomes. In these scenarios, a person in the lab might have does lasix cause hypokalemia then been accidentally or deliberately infected by the lasix, and then spread it to others—sparking the lasix.

There is currently no clear evidence to back these scenarios, but they aren’t impossible. People have made a number of arguments for a lab origin for hypertension that are currently conjecture. One holds that it’s suspicious that, almost a year and a half into the lasix, hypertension’s closest relative still hasn’t does lasix cause hypokalemia been found in an animal. Another suggests it is no coincidence that hypertension medications was first detected in Wuhan, where a top lab studying hypertensiones, the WIV, is located. Some lab-leak proponents contend that the lasix contains unusual features and genetic sequences signalling that it was engineered by humans.

And some say that hypertension spreads among people so readily that it must have been created with that intention does lasix cause hypokalemia. Another argument suggests that hypertension might have derived from hypertensiones found in an unused mine where WIV researchers collected samples from bats between 2012 and 2015. So what do infectious disease researchers and evolutionary biologists say about these arguments?. Is it suspicious that no animal has been does lasix cause hypokalemia identified as transmitting the lasix to humans?. Outbreak-origin investigations often take years, and some culprits remain unknown.

It took 14 years to nail down the origin of the SARS epidemic, which began with a lasix in bats that spread to humans, most likely through civets. To date, a complete Ebola lasix has never been isolated from does lasix cause hypokalemia an animal in the region where the world’s largest outbreak occurred between 2013 and 2016. Origin investigations are complicated because outbreaks among animals that aren't the main hosts of a particular lasix, such as civets in the case of SARS, are often sporadic. Researchers must find the right animal before it dies or clears the . And, even if does lasix cause hypokalemia the animal tests positive, lasixes found in saliva, faeces or blood are often degraded, making it difficult to sequence the pathogen’s whole genome.

Scientists have made some progress since the lasix began, however. For example, a report, posted to the preprint server bioRxiv on 27 May, suggests that RmYN02, a hypertension in bats in southern China, might be more closely related to hypertension than RATG13 is. As for finding an does lasix cause hypokalemia intermediate host animal, researchers in China have tested more than 80,000 wild and domesticated animals. None have been positive for hypertension. But this number is a tiny fraction of the animals in the country.

To narrow the does lasix cause hypokalemia search down, researchers say, more strategic testing is needed to isolate animals that are most susceptible to and those that come in close contact with people. They also suggest using antibody tests to identify animals that have previously been infected with the lasix. Is it suspicious that the WIV is in Wuhan?. Virology labs tend to specialize in the lasixes around them, says Vincent Munster, a virologist at the does lasix cause hypokalemia Rocky Mountain Laboratories, a division of the National Institutes of Health, in Hamilton, Montana. The WIV specializes in hypertensiones because many have been found in and around http://www.ec-schloessel-ostwald.ac-strasbourg.fr/?p=1496 China.

Munster names other labs that focus on endemic viral diseases. Influenza labs in Asia, haemorrhagic fever labs in Africa and dengue-fever labs in Latin America, for does lasix cause hypokalemia example. €œNine out of ten times, when there’s a new outbreak, you’ll find a lab that will be working on these kinds of lasixes nearby,” says Munster. Researchers note that a hypertension outbreak in Wuhan isn’t surprising, because it’s a city of 11 million people in a broader region where hypertensiones have been found. It contains an airport, train stations and markets selling goods and wildlife does lasix cause hypokalemia transported there from around the region — meaning a lasix could enter the city and spread rapidly.

Does the lasix have features that suggest it was created in a lab?. Several researchers have looked into whether features of hypertension signal that it was bioengineered. One of the first teams to do so, led by Kristian does lasix cause hypokalemia Andersen, a virologist at Scripps Research in La Jolla, California, determined that this was “improbable” for a few reasons, including a lack of signatures of genetic manipulation. Since then, others have asked whether the lasix’s furin cleavage site—a feature that helps it to enter cells—is evidence of engineering, because hypertension has these sites but its closest relatives don’t. The furin cleavage site is important because it's in the lasix's spike protein, and cleavage of the protein at that site is necessary for the lasix to infect cells.

But many other hypertensiones have furin cleavage sites, such does lasix cause hypokalemia as hypertensiones that cause colds. Because lasixes containing the site are scattered across the hypertension family tree, rather than confined to a group of closely related lasixes, Stephen Goldstein, a virologist at the University of Utah in Salt Lake City, says the site probably evolved multiple times because it provides an evolutionary advantage. Convergent evolution—the process by which organisms that aren’t closely related independently evolve similar traits as a result of adapting to similar environments—is incredibly common. Another feature of hypertension that has drawn attention is a combination of nucleotides that does lasix cause hypokalemia underlie a segment of the furin cleavage site. CGG (these encode the amino acid arginine).

A Medium article that speculates on a lab origin for hypertension quotes David Baltimore, a Nobel laureate and professor emeritus at the California Institute of Technology in Pasadena, as saying that lasixes don’t usually have that particular code for arginine, but humans often do—a “smoking gun”, hinting that researchers might have tampered with hypertension’s genome. Andersen says that Baltimore was does lasix cause hypokalemia incorrect about that detail, however. In hypertension, about 3% of the nucleotides encoding arginine are CGG, he says. And he points out that around 5% of those encoding arginine in the lasix that caused the original SARS epidemic are CGG, too. In an e-mail to Nature, Baltimore says Andersen could be correct that evolution produced hypertension, but adds that does lasix cause hypokalemia “there are other possibilities and they need careful consideration, which is all I meant to be saying”.

Is it true that hypertension must have been engineered, because it's perfect for causing a lasix?. Many scientists say no. Just because the lasix spreads among humans doesn't does lasix cause hypokalemia mean it was designed to do so. It also flourishes among mink and infects a host of carnivorous mammals. And it wasn’t optimally transmissible among humans for the better part of last year.

Rather, new, more efficient variants have does lasix cause hypokalemia evolved around the world. To name one example, the highly transmissible variant of hypertension first reported in India (B.1.617.2, or Delta) has mutations in the nucleotides encoding its furin cleavage site that appear to make the lasix better at infecting cells. €œThis was not some supremely adapted pathogen,” says Joel Wertheim, a molecular epidemiologist at the University of California San Diego. Did researchers collect hypertension does lasix cause hypokalemia from a mine?. Researchers from the WIV collected hundreds of samples from bats roosting in a mine between 2012 and 2015, after several miners working there had gotten sick with an unknown respiratory disease.

(Last year, researchers reported that blood samples taken from the miners tested negative for antibodies against hypertension, meaning that the sickness was probably not hypertension medications.) Back at the lab, WIV researchers detected nearly 300 hypertensiones in the bat samples, but they were able to get whole or partial genomic sequences from fewer than a dozen , and none of those that were reported were hypertension. During the WHO-led origins probe earlier this year, WIV researchers told investigators that they cultured only does lasix cause hypokalemia three hypertensiones at the lab, and none were closely related to hypertension. Although the investigators didn’t sift through freezers at the WIV to confirm this information, the low number of genomes and cultures doesn’t surprise virologists. Munster says it’s exceedingly difficult to extract intact hypertensiones from bat samples. lasix levels tend to be low in the animals, and lasixes are often degraded in faeces, does lasix cause hypokalemia saliva and droplets of blood.

Additionally, when researchers want to study or genetically alter lasixes, they need to keep them (or synthetic mimics of them) alive, by finding the appropriate live animal cells for the lasixes to inhabit in the lab, which can be a challenge. So, for hypertension to have come from this mine in China, WIV researchers would have had to overcome some serious technical challenges—and they would have kept the information secret for a number of years and misled investigators on the WHO-led mission, scientists point out. There's no evidence of this, but it can't be does lasix cause hypokalemia ruled out. What’s next for lab-leak investigations?. Biden asked the US Intelligence Community to report back to him in 90 days.

Perhaps this investigation will shed light on undisclosed US intel reported by The Wall Street Journal suggesting that three staff members at the WIV were sick in November 2019, before the first cases of hypertension medications were reported does lasix cause hypokalemia in China. The article claims that US officials have different opinions on the quality of that intel. And researchers at the WIV have maintained that staff at the institute tested negative for antibodies that would indicate hypertension prior to January 2020. Last week, Anthony Fauci, Biden’s chief does lasix cause hypokalemia medical adviser, asked Chinese officials to release the hospital records of WIV staff members. Others have asked for blood samples from WIV staff members, and access to WIV bat and lasix samples, laboratory notebooks and hard drives.

But it’s unclear what such asks will yield because China has not conceded to demands for a full lab investigation. A spokesperson for the Ministry of Foreign Affairs of the People's Republic of China, Zhao Lijian, said that US labs should instead be investigated, and that some people in the United States “don't care about facts or truth and have zero does lasix cause hypokalemia interest in a serious science-based study of origins”. As Biden's investigation commences and the WHO considers the next phase in its origin study, lasix experts are bracing themselves for a long road ahead. €œWe want an answer,” says Jason Kindrachuk, a virologist at the University of Manitoba in Winnipeg, Canada.

However, a lab leak has not been ruled out, and many are calling for a deeper investigation into the hypothesis that the lasix emerged from the Wuhan Institute https://eingrext.at/shop/ of Virology (WIV), located in the Chinese city where generic lasix online for sale the first hypertension medications cases were reported. On 26 May, US President Joe Biden tasked the US Intelligence Community to join efforts to find hypertension’s origins, whatever they might be, and report back in 90 days. Australia, the European Union and Japan have also called for a robust investigation into hypertension’s origins in China. The WHO has generic lasix online for sale yet to reveal the next phase of its investigation.

But China has asked that the probe examine other countries. Such reticence, and the fact that China has withheld information in the past, has fuelled suspicions of a ‘lab leak’. For instance, Chinese government officials suppressed crucial public-health data at the start generic lasix online for sale of the hypertension medications lasix, and during the 2002–04 severe acute respiratory syndrome (SARS) epidemic, according to high-level reports. At the assembly, Mike Ryan, director of health emergencies at the WHO, asked for less politicization of calls for an origin investigation, which have, in many ways, devolved into accusations.

€œOver the last number of days, we have seen more and more and more discourse in the media, with terribly little actual news, or evidence, or new material,” said Ryan. €œThis is disturbing.” Nature looks at the generic lasix online for sale key arguments that support a lab leak, and the extent to which research has answers. There’s not yet any substantial evidence for a lab leak. Why are scientists still considering it?.

Scientists don’t have enough evidence about the origins of hypertension to rule out the lab-leak hypothesis, or to prove the alternative—that the lasix has a generic lasix online for sale natural origin. Many infectious-disease researchers agree that the most probable scenario is that the lasix evolved naturally and spread from a bat either directly to a person or through an intermediate animal. Most emerging infectious diseases begin with a spillover from nature, as was seen with HIV, influenza epidemics, Ebola outbreaks and the hypertensiones that caused the SARS epidemic beginning in 2002 and the Middle East respiratory syndrome (MERS) outbreak beginning in 2012. Researchers have some leads that support a generic lasix online for sale natural origin.

Bats are known carriers of hypertensiones, and scientists have determined that the genome of hypertension is most similar to that of RATG13, a hypertension that was first found in a horseshoe bat (Rhinolophus affinis) in the southern Chinese province of Yunnan in 2013. But RATG13’s genome is only 96% identical to hypertension’s, suggesting that a closer relative of the lasix—the one passed to humans—remains unknown. Still, the possibility remains that hypertension escaped from a lab generic lasix online for sale. Although lab leaks have never caused an epidemic, they have resulted in small outbreaks involving well-documented lasixes.

A relevant example happened in 2004, when two researchers were independently infected by the lasix that causes SARS at a virology lab in Beijing that studied the disease. They spread the generic lasix online for sale to seven others before the outbreak was contained. What are the key arguments for a lab leak?. In theory, hypertension medications could have come from a lab in a few ways.

Researchers might have collected hypertension from an animal and maintained it in their lab to study, or they might have created it by engineering hypertension genomes generic lasix online for sale. In these scenarios, a person in the lab might have then been accidentally or deliberately infected by the lasix, and then spread it to others—sparking the lasix. There is currently no clear evidence to back these scenarios, but they aren’t impossible. People have made a number of generic lasix online for sale arguments for a lab origin for hypertension that are currently conjecture.

One holds that it’s suspicious that, almost a year and a half into the lasix, hypertension’s closest relative still hasn’t been found in an animal. Another suggests it is no coincidence that hypertension medications was first detected in Wuhan, where a top lab studying hypertensiones, the WIV, is located. Some lab-leak proponents contend that the lasix contains unusual features and genetic sequences signalling that it was engineered by generic lasix online for sale humans. And some say that hypertension spreads among people so readily that it must have been created with that intention.

Another argument suggests that hypertension might have derived from hypertensiones found in an unused mine where WIV researchers collected samples from bats between 2012 and 2015. So what do infectious disease researchers and evolutionary biologists generic lasix online for sale say about these arguments?. Is it suspicious that no animal has been identified as transmitting the lasix to humans?. Outbreak-origin investigations often take years, and some culprits remain unknown.

It took 14 years to nail down the origin of the SARS epidemic, which began with generic lasix online for sale a lasix in bats that spread to humans, most likely through civets. To date, a complete Ebola lasix has never been isolated from an animal in the region where the world’s largest outbreak occurred between 2013 and 2016. Origin investigations are complicated because outbreaks among animals that aren't the main hosts of a particular lasix, such as civets in the case of SARS, are often sporadic. Researchers must find the right animal before it dies or clears the generic lasix online for sale.

And, even if the animal tests positive, lasixes found in saliva, faeces or blood are often degraded, making it difficult to sequence the pathogen’s whole genome. Scientists have made some progress since the lasix began, however. For example, a report, posted to the preprint server bioRxiv on generic lasix online for sale 27 May, suggests that RmYN02, a hypertension in bats in southern China, might be more closely related to hypertension than RATG13 is. As for finding an intermediate host animal, researchers in China have tested more than 80,000 wild and domesticated animals.

None have been positive for hypertension. But this number is a tiny generic lasix online for sale fraction of the animals in the country. To narrow the search down, researchers say, more strategic testing is needed to isolate animals that are most susceptible to and those that come in close contact with people. They also suggest using antibody tests to identify animals that have previously been infected with the lasix.

Is it generic lasix online for sale suspicious that the WIV is in Wuhan?. Virology labs tend to specialize in the lasixes around them, says Vincent Munster, a virologist at the Rocky Mountain Laboratories, a division of the National Institutes of Health, in Hamilton, Montana. The WIV specializes in hypertensiones because many have been found in and around China. Munster names other labs that focus on endemic generic lasix online for sale viral diseases.

Influenza labs in Asia, haemorrhagic fever labs in Africa and dengue-fever labs in Latin America, for example. €œNine out of ten times, when there’s a new outbreak, you’ll find a lab that will be working on these kinds of lasixes nearby,” says Munster. Researchers note that a hypertension outbreak in Wuhan isn’t surprising, because it’s a city of 11 million people in a generic lasix online for sale broader region where hypertensiones have been found. It contains an airport, train stations and markets selling goods and wildlife transported there from around the region — meaning a lasix could enter the city and spread rapidly.

Does the lasix have features that suggest it was created in a lab?. Several researchers have looked into whether features generic lasix online for sale of hypertension signal that it was bioengineered. One of the first teams to do so, led by Kristian Andersen, a virologist at Scripps Research in La Jolla, California, determined that this was “improbable” for a few reasons, including a lack of signatures of genetic manipulation. Since then, others have asked whether the lasix’s furin cleavage site—a feature that helps it to enter cells—is evidence of engineering, because hypertension has these sites but its closest relatives don’t.

The furin cleavage generic lasix online for sale site is important because it's in the lasix's spike protein, and cleavage of the protein at that site is necessary for the lasix to infect cells. But many other hypertensiones have furin cleavage sites, such as hypertensiones that cause colds. Because lasixes containing the site are scattered across the hypertension family tree, rather than confined to a group of closely related lasixes, Stephen Goldstein, a virologist at the University of Utah in Salt Lake City, says the site probably evolved multiple times because it provides an evolutionary advantage. Convergent evolution—the process by which organisms that aren’t closely related independently evolve similar traits as a result of generic lasix online for sale adapting to similar environments—is incredibly common.

Another feature of hypertension that has drawn attention is a combination of nucleotides that underlie a segment of the furin cleavage site. CGG (these encode the amino acid arginine). A Medium article that speculates on a generic lasix online for sale lab origin for hypertension quotes David Baltimore, a Nobel laureate and professor emeritus at the California Institute of Technology in Pasadena, as saying that lasixes don’t usually have that particular code for arginine, but humans often do—a “smoking gun”, hinting that researchers might have tampered with hypertension’s genome. Andersen says that Baltimore was incorrect about that detail, however.

In hypertension, about 3% of the nucleotides encoding arginine are CGG, he says. And he points out that around 5% of those encoding arginine in the lasix that caused the original SARS epidemic are CGG, too generic lasix online for sale. In an e-mail to Nature, Baltimore says Andersen could be correct that evolution produced hypertension, but adds that “there are other possibilities and they need careful consideration, which is all I meant to be saying”. Is it true that hypertension must have been engineered, because it's perfect for causing a lasix?.

Many scientists say no generic lasix online for sale. Just because the lasix spreads among humans doesn't mean it was designed to do so. It also flourishes among mink and infects a host of carnivorous mammals. And it wasn’t optimally transmissible among humans for the better generic lasix online for sale part of last year.

Rather, new, more efficient variants have evolved around the world. To name one example, the highly transmissible variant of hypertension first reported in India (B.1.617.2, or Delta) has mutations in the nucleotides encoding its furin cleavage site that appear to make the lasix better at infecting cells. €œThis was not some supremely adapted pathogen,” says Joel Wertheim, a molecular epidemiologist at the University of California San Diego generic lasix online for sale. Did researchers collect hypertension from a mine?.

Researchers from the WIV collected hundreds of samples from bats roosting in a mine between 2012 and 2015, after several miners working there had gotten sick with an unknown respiratory disease. (Last year, researchers reported that blood samples taken from the miners tested negative for antibodies against hypertension, meaning that the sickness was probably not hypertension medications.) Back at the lab, WIV researchers detected nearly 300 hypertensiones in the bat samples, but they were able to get whole or partial genomic sequences from fewer than a dozen , generic lasix online for sale and none of those that were reported were hypertension. During the WHO-led origins probe earlier this year, WIV researchers told investigators that they cultured only three hypertensiones at the lab, and none were closely related to hypertension. Although the investigators didn’t sift through freezers at the WIV to confirm this information, the low number of genomes and cultures doesn’t surprise virologists.

Munster says it’s generic lasix online for sale exceedingly difficult to extract intact hypertensiones from bat samples. lasix levels tend to be low in the animals, and lasixes are often degraded in faeces, saliva and droplets of blood. Additionally, when researchers want to study or genetically alter lasixes, they need to keep them (or synthetic mimics of them) alive, by finding the appropriate live animal cells for the lasixes to inhabit in the lab, which can be a challenge. So, for hypertension to have come from this generic lasix online for sale mine in China, WIV researchers would have had to overcome some serious technical challenges—and they would have kept the information secret for a number of years and misled investigators on the WHO-led mission, scientists point out.

There's no evidence of this, but it can't be ruled out. What’s next for lab-leak investigations?. Biden asked the US Intelligence Community to report generic lasix online for sale back to him in 90 days. Perhaps this investigation will shed light on undisclosed US intel reported by The Wall Street Journal suggesting that three staff members at the WIV were sick in November 2019, before the first cases of hypertension medications were reported in China.

The article claims that US officials have different opinions on the quality of that intel. And researchers at the WIV have maintained that staff at the institute tested negative for antibodies that would indicate hypertension prior to January 2020. Last week, Anthony Fauci, Biden’s chief medical adviser, asked Chinese officials to release the hospital records of WIV staff members. Others have asked for blood samples from WIV staff members, and access to WIV bat and lasix samples, laboratory notebooks and hard drives.

But it’s unclear what such asks will yield because China has not conceded to demands for a full lab investigation. A spokesperson for the Ministry of Foreign Affairs of the People's Republic of China, Zhao Lijian, said that US labs should instead be investigated, and that some people in the United States “don't care about facts or truth and have zero interest in a serious science-based study of origins”. As Biden's investigation commences and the WHO considers the next phase in its origin study, lasix experts are bracing themselves for a long road ahead.

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AbstractIntroduction http://pamelabarroncobo.com/gallery/retrato/ lasix 40mg furosemida. We report a very rare case of familial breast cancer and diffuse gastric cancer, with germline pathogenic variants in both BRCA1 and CDH1 genes. To the best of our knowledge, this is the first report of such an association.Family description lasix 40mg furosemida. The proband is a woman diagnosed with breast cancer at the age of 52 years. She requested genetic counselling in 2012, at the age of 91 years, because of a history of breast cancer lasix 40mg furosemida in her daughter, her sister, her niece and her paternal grandmother and was therefore concerned about her relatives.

Her sister and maternal aunt also had gastric cancer. She was tested for several genes associated with hereditary breast lasix 40mg furosemida cancer.Results. A large deletion of BRCA1 from exons 1 to 7 and two CDH1 pathogenic cis variants were identified.Conclusion. This complex situation is challenging for genetic counselling and management of at-risk individuals.cancer lasix 40mg furosemida. Breastcancer.

Gastricclinical geneticsgenetic lasix 40mg furosemida screening/counsellingmolecular geneticsIntroductionGLI-Kruppel family member 3 (GLI3) encodes for a zinc finger transcription factor which plays a key role in the sonic hedgehog (SHH) signalling pathway essential in both limb and craniofacial development.1 2 In hand development, SHH is expressed in the zone of polarising activity (ZPA) on the posterior side of the handplate. The ZPA expresses SHH, creating a gradient of SHH from the posterior to the anterior side of the handplate. In the presence of SHH, full length GLI3-protein is produced (GLI3A), whereas absence of SHH causes cleavage of GLI3 into its repressor form (GLI3R).3 4 Abnormal expression of this SHH/GLI3R gradient can cause both preaxial and postaxial polydactyly.2Concordantly, pathogenic DNA variants in the GLI3 gene are known to cause multiple syndromes with craniofacial and limb involvement, lasix 40mg furosemida such as. Acrocallosal syndrome5 (OMIM. 200990), Greig lasix 40mg furosemida cephalopolysyndactyly syndrome6 (OMIM.

175700) and Pallister-Hall syndrome7 (OMIM. 146510). Also, in non-syndromic polydactyly, such as preaxial polydactyly-type 4 (PPD4, OMIM. 174700),8 pathogenic variants in GLI3 have been described. Out of these diseases, Pallister-Hall syndrome is the most distinct entity, defined by the presence of central polydactyly and hypothalamic hamartoma.9 The other GLI3 syndromes are defined by the presence of preaxial and/or postaxial polydactyly of the hand and feet with or without syndactyly (Greig syndrome, PPD4).

Also, various mild craniofacial features such as hypertelorism and macrocephaly can occur. Pallister-Hall syndrome is caused by truncating variants in the middle third of the GLI3 gene.10–12 The truncation of GLI3 causes an overexpression of GLI3R, which is believed to be the key difference between Pallister-Hall and the GLI3-mediated polydactyly syndromes.9 11 Although multiple attempts have been made, the clinical and genetic distinction between the GLI3-mediated polydactyly syndromes is less evident. This has for example led to the introduction of subGreig and the formulation of an Oro-facial-digital overlap syndrome.10 Other authors, suggested that we should not regard these diseases as separate entities, but as a spectrum of GLI3-mediated polydactyly syndromes.13Although phenotype/genotype correlation of the different syndromes has been cumbersome, clinical and animal studies do provide evidence that distinct regions within the gene, could be related to the individual anomalies contributing to these syndromes. First, case studies show isolated preaxial polydactyly is caused by both truncating and non-truncating variants throughout the GLI3 gene, whereas in isolated postaxial polydactyly cases truncating variants at the C-terminal side of the gene are observed.12 14 These results suggest two different groups of variants for preaxial and postaxial polydactyly. Second, recent animal studies suggest that posterior malformations in GLI3-mediated polydactyly syndromes are likely related to a dosage effect of GLI3R rather than due to the influence of an altered GLI3A expression.15Past attempts for phenotype/genotype correlation in GLI3-mediated polydactyly syndromes have directly related the diagnosed syndrome to the observed genotype.10–12 16 Focusing on individual hand phenotypes, such as preaxial and postaxial polydactyly and syndactyly might be more reliable because it prevents misclassification due to inconsistent use of syndrome definition.

Subsequently, latent class analysis (LCA) provides the possibility to relate a group of observed variables to a set of latent, or unmeasured, parameters and thereby identifying different subgroups in the obtained dataset.17 As a result, LCA allows us to group different phenotypes within the GLI3-mediated polydactyly syndromes and relate the most important predictors of the grouped phenotypes to the observed GLI3 variants.The aim of our study was to further investigate the correlation of the individual phenotypes to the genotypes observed in GLI3-mediated polydactyly syndromes, using LCA. Cases were obtained by both literature review and the inclusion of local clinical cases. Subsequently, we identified two subclasses of limb anomalies that relate to the underlying GLI3 variant. We provide evidence for two different phenotypic and genotypic groups with predominantly preaxial and postaxial hand and feet anomalies, and we specify those cases with a higher risk for corpus callosum anomalies.MethodsLiterature reviewThe Human Gene Mutation Database (HGMD Professional 2019) was reviewed to identify known pathogenic variants in GLI3 and corresponding phenotypes.18 All references were obtained and cases were included when they were diagnosed with either Greig or subGreig syndrome or PPD4.10–12 Pallister-Hall syndrome and acrocallosal syndrome were excluded because both are regarded distinct syndromes and rather defined by the presence of the non-hand anomalies, than the presence of preaxial or postaxial polydactyly.13 19 Isolated preaxial or postaxial polydactyly were excluded for two reasons. The phenotype/genotype correlations are better understood and both anomalies can occur sporadically which could introduce falsely assumed pathogenic GLI3 variants in the analysis.

Additionally, cases were excluded when case-specific phenotypic or genotypic information was not reported or if these two could not be related to each other. Families with a combined phenotypic description, not reducible to individual family members, were included as one case in the analysis.Clinical casesThe Sophia Children’s Hospital Database was reviewed for cases with a GLI3 variant. Within this population, the same inclusion criteria for the phenotype were valid. Relatives of the index patients were also contacted for participation in this study, when they showed comparable hand, foot, or craniofacial malformations or when a GLI3 variant was identified. Phenotypes of the hand, foot and craniofacial anomalies of the patients treated in the Sophia Children's Hospital were collected using patient documentation.

Family members were identified and if possible, clinically verified. Alternatively, family members were contacted to verify their phenotypes. If no verification was possible, cases were excluded.PhenotypesThe phenotypes of both literature cases and local cases were extracted in a similar fashion. The most frequently reported limb and craniofacial phenotypes were dichotomised. The dichotomised hand and foot phenotypes were preaxial polydactyly, postaxial polydactyly and syndactyly.

Broad halluces or thumbs were commonly reported by authors and were dichotomised as a presentation of preaxial polydactyly. The extracted dichotomised craniofacial phenotypes were hypertelorism, macrocephaly and corpus callosum agenesis. All other phenotypes were registered, but not dichotomised.Pathogenic GLI3 variantsAll GLI3 variants were extracted and checked using Alamut Visual V.2.14. If indicated, variants were renamed according to standard Human Genome Variation Society nomenclature.20 Variants were grouped in either missense, frameshift, nonsense or splice site variants. In the group of frameshift variants, a subgroup with possible splice site effect were identified for subgroup analysis when indicated.

Similarly, nonsense variants prone for nonsense mediated decay (NMD) and nonsense variants with experimentally confirmed NMD were identified.21 Deletions of multiple exons, CNVs and translocations were excluded for analysis. A full list of included mutations is available in the online supplementary materials.Supplemental materialThe location of the variant was compared with five known structural domains of the GLI3 gene. (1) repressor domain, (2) zinc finger domain, (3) cleavage site, (4) activator domain, which we defined as a concatenation of the separately identified transactivation zones, the CBP binding domain and the mediator binding domain (MBD) and (5) the MID1 interaction region domain.1 6 22–24 The boundaries of each of the domains were based on available literature (figure 1, exact locations available in the online supplementary materials). The boundaries used by different authors did vary, therefore a consensus was made.In this figure the posterior probability of an anterior phenotype is plotted against the location of the variant, stratified for the type of mutation that was observed. For better overview, only variants with a location effect were displayed.

The full figure, including all variant types, can be found in the online supplementary figure 1. Each mutation is depicted as a dot, the size of the dot represents the number of observations for that variant. If multiple observations were made, the mean posterior odds and IQR are plotted. For the nonsense variants, variants that were predicted to produce nonsense mediated decay, are depicted using a triangle. Again, the size indicates the number of observations." data-icon-position data-hide-link-title="0">Figure 1 In this figure the posterior probability of an anterior phenotype is plotted against the location of the variant, stratified for the type of mutation that was observed.

For better overview, only variants with a location effect were displayed. The full figure, including all variant types, can be found in the online supplementary figure 1. Each mutation is depicted as a dot, the size of the dot represents the number of observations for that variant. If multiple observations were made, the mean posterior odds and IQR are plotted. For the nonsense variants, variants that were predicted to produce nonsense mediated decay, are depicted using a triangle.

Again, the size indicates the number of observations.Supplemental materialLatent class analysisTo cluster phenotypes and relate those to the genotypes of the patients, an explorative analysis was done using LCA in R (R V.3.6.1 for Mac. Polytomous variable LCA, poLCA V.1.4.1.). We used our LCA to detect the number of phenotypic subgroups in the dataset and subsequently predict a class membership for each case in the dataset based on the posterior probabilities.In order to make a reliable prediction, only phenotypes that were sufficiently reported and/or ruled out were feasible for LCA, limiting the analysis to preaxial polydactyly, postaxial polydactyly and syndactyly of the hands and feet. Only full cases were included. To determine the optimal number of classes, we fitted a series of models ranging from a one-class to a six-class model.

The optimal number of classes was based on the conditional Akaike information criterion (cAIC), the non adjusted and the sample-size adjusted Bayesian information criterion (BIC and aBIC) and the obtained entropy.25 The explorative LCA produces both posterior probabilities per case for both classes and predicted class membership. Using the predicted class membership, the phenotypic features per class were determined in a univariate analysis (χ2, SPSS V.25). Using the posterior probabilities on latent class (LC) membership, a scatter plot was created using the location of the variant on the x-axis and the probability of class membership on the y-axis for each of the types of variants (Tibco Spotfire V.7.14). Using these scatter plots, variants that give similar phenotypes were clustered.Genotype/phenotype correlationBecause an LC has no clinical value, the correlation between genotypes and phenotypes was investigated using the predictor phenotypes and the clustered phenotypes. First, those phenotypes that contribute most to LC membership were identified.

Second those phenotypes were directly related to the different types of variants (missense, nonsense, frameshift, splice site) and their clustered locations. Quantification of the relation was performed using a univariate analysis using a χ2 test. Because of our selection criteria, meaning patients at least have two phenotypes, a multivariate using a logistic regression analysis was used to detect the most significant predictors in the overall phenotype (SPSS V.25). Finally, we explored the relation of the clustered genotypes to the presence of corpus callosum agenesis, a rare malformation in GLI3-mediated polydactyly syndromes which cannot be readily diagnosed without additional imaging.ResultsWe included 251 patients from the literature and 46 local patients,10–12 16 21 26–43 in total 297 patients from 155 different families with 127 different GLI3 variants, 32 of which were large deletions, CNVs or translocations. In six local cases, the exact variant could not be retrieved by status research.The distribution of the most frequently observed phenotypes and variants are presented in table 1.

Other recurring phenotypes included developmental delay (n=22), broad nasal root (n=23), frontal bossing or prominent forehead (n=16) and craniosynostosis (n=13), camptodactyly (n=8) and a broad first interdigital webspace of the foot (n=6).View this table:Table 1 Baseline phenotypes and genotypes of selected populationThe LCA model was fitted using the six defined hand/foot phenotypes. Model fit indices for the LCA are displayed in table 2. Based on the BIC, a two-class model has the best fit for our data. The four-class model does show a gain in http://www.hamburg-zeigt-kunst.de/5d7413265167d5c264f4d1e90170c21f/ entropy, however with a higher BIC and loss of df. Therefore, based on the majority of performance statistics and the interpretability of the model, a two-class model was chosen.

Table 3 displays the distribution of phenotypes and genotypes over the two classes.View this table:Table 2 Model fit indices for the one-class through six-class model evaluated in our LCAView this table:Table 3 Distribution of phenotypes and genotypes in the two latent classes (LC)Table 1 depicts the baseline phenotypes and genotypes in the obtained population. Note incomplete data especially in the cranium phenotypes. In total 259 valid genotypes were present. In total, 289 cases had complete data for all hand and foot phenotypes (preaxial polydactyly, postaxial polydactyly and syndactyly) and thus were available for LCA. Combined, for phenotype/genotype correlation 258 cases were available with complete genotypes and complete hand and foot phenotypes.Table 2 depicts the model fit indices for all models that have been fitted to our data.Table 3 depicts the distribution of phenotypes and genotypes over the two assigned LCs.

Hand and foot phenotypes were used as input for the LCA, thus are all complete cases. Malformation of the cranium and genotypes do have missing cases. Note that for the LCA, full case description was required, resulting in eight cases due to incomplete phenotypes. Out of these eight, one also had a genotype that thus needed to be excluded. Missingness of genotypic data was higher in LC2, mostly due to CNVs (table 1).In 54/60 cases, a missense variant produced a posterior phenotype.

Likewise, splice site variants show the same phenotype in 23/24 cases (table 3). For both frameshift and nonsense variants, this relation is not significant (52 anterior vs 54 posterior and 26 anterior vs 42 posterior, respectively). Therefore, only for nonsense and frameshift variants the location of the variant was plotted against the probability for LC2 membership in figure 1. A full scatterplot of all variants is available in online supplementary figure 1.Figure 1 reveals a pattern for these nonsense and frameshift variants that reveals that variants at the C-terminal of the gene predict anterior phenotypes. When relating the domains of the GLI3 protein to the observed phenotype, we observe that the majority of patients with a nonsense or frameshift variant in the repressor domain, the zinc finger domain or the cleavage site had a high probability of an LC2/anterior phenotype.

This group contains all variants that are either experimentally determined to be subject to NMD (triangle marker in figure 1) or predicted to be subject to NMD (diamond marker in figure 1). Frameshift and nonsense variants in the activator domain result in high probability for an LC1/posterior phenotype. These variants will be further referred to as truncating variants in the activator domain.The univariate relation of the individual phenotypes to these two groups of variants are estimated and presented in table 4. In our multivariate analysis, postaxial polydactyly of the foot and hand are the strongest predictors (Beta. 2.548, p<0001 and Beta.

1.47, p=0.013, respectively) for patients to have a truncating variant in the activator domain. Moreover, the effect sizes of preaxial polydactyly of the hand and feet (Beta. ˆ’0.797, p=0123 and −1.772, p=0.001) reveals that especially postaxial polydactyly of the foot is the dominant predictor for the genetic substrate of the observed anomalies.View this table:Table 4 Univariate and multivariate analysis of the phenotype/genotype correlationTable 4 shows exploration of the individual phenotypes on the genotype, both univariate and multivariate. The multivariate analysis corrects for the presence of multiple phenotypes in the underlying population.Although the craniofacial anomalies could not be included in the LCA, the relation between the observed anomalies and the identified genetic substrates can be studied. The prevalence of hypertelorism was equally distributed over the two groups of variants (47/135 vs 21/47 respectively, p<0.229).

However for corpus callosum agenesis and macrocephaly, there was a higher prevalence in patients with a truncating variant in the activator domain (3/75 vs 11/41, p<0.001. OR. 8.8, p<0.001) and 42/123 vs 24/48, p<0.05). Noteworthy is the fact that 11/14 cases with corpus callosum agenesis in the dataset had a truncating variant in the activator domain.DiscussionIn this report, we present new insights into the correlation between the phenotype and the genotype in patients with GLI3-mediated polydactyly syndromes. We illustrate that there are two LCs of patients, best predicted by postaxial polydactyly of the hand and foot for LC1, and the preaxial polydactyly of the hand and foot and syndactyly of the foot for LC2.

Patients with postaxial phenotypes have a higher risk of having a truncating variant in the activator domain of the GLI3 gene which is also related to a higher risk of corpus callosum agenesis. These results suggest a functional difference between truncating variants on the N-terminal and the C-terminal side of the GLI3 cleavage site.Previous attempts of phenotype to genotype correlation have not yet provided the clinical confirmation of these assumed mechanisms in the pathophysiology of GLI3-mediated polydactyly syndromes. Johnston et al have successfully determined the Pallister-Hall region in which truncating variants produce a Pallister-Hall phenotype rather than Greig syndrome.11 However, in their latest population study, subtypes of both syndromes were included to explain the full spectrum of observed malformations. In 2015, Demurger et al reported the higher incidence of corpus callosum agenesis in the Greig syndrome population with truncating mutations in the activator domain.12 Al-Qattan in his review summarises the concept of a spectrum of anomalies dependent on haplo-insufficiency (through different mechanisms) and repressor overexpression.13 However, he bases this theory mainly on reviewed experimental data. Our report is the first to provide an extensive clinical review of cases that substantiate the phenotypic difference between the two groups that could fit the suggested mechanisms.

We agree with Al-Qattan et al that a variation of anomalies can be observed given any pathogenic variant in the GLI3 gene, but overall two dominant phenotypes are present. A population with predominantly preaxial anomalies and one with postaxial anomalies. The presence of preaxial or postaxial polydactyly and syndactyly is not mutually exclusive for one of these two subclasses. Meaning that preaxial polydactyly can co-occur with postaxial polydactyly. However, truncating mutations in the activator domain produce a postaxial phenotype, as can be derived from the risk in table 4.

The higher risk of corpus callosum agenesis in this population shows that differentiating between a preaxial phenotype and a postaxial phenotype, instead of between the different GLI3-mediated polydactyly syndromes, might be more relevant regarding diagnostics for corpus callosum agenesis.We chose to use LCA as an exploratory tool only in our population for two reasons. First of all, LCA can be useful to identify subgroups, but there is no ‘true’ model or number of subgroups you can detect. The best fitting model can only be estimated based on the available measures and approximates the true subgroups that might be present. Second, LC membership assignment is a statistical procedure based on the posterior probability, with concordant errors of the estimation, rather than a clinical value that can be measured or evaluated. Therefore, we decided to use our LCA only in an exploratory tool, and perform our statistics using the actual phenotypes that predict LC membership and the associated genotypes.

Overall, this method worked well to differentiate the two subgroups present in our dataset. However, outliers were observed. A qualitative analysis of these outliers is available in the online supplementary data.The genetic substrate for the two phenotypic clusters can be discussed based on multiple experiments. Overall, we hypothesise two genetic clusters. One that is due to haploinsufficiency and one that is due to abnormal truncation of the activator.

The hypothesised cluster of variants that produce haploinsufficiency is mainly based on the experimental data that confirms NMD in two variants and the NMD prediction of other nonsense variants in Alamut. For the frameshift variants, it is also likely that the cleavage of the zinc finger domain results in functional haploinsufficiency either because of a lack of signalling domains or similarly due to NMD. Missense variants could cause haploinsufficiency through the suggested mechanism by Krauss et al who have illustrated that missense variants in the MID1 domain hamper the functional interaction with the MID1-α4-PP2A complex, leading to a subcellular location of GLI3.24 The observed missense variants in our study exceed the region to which Krauss et al have limited the MID-1 interaction domain. An alternative theory is suggested by Zhou et al who have shown that missense variants in the MBD can cause deficiency in the signalling of GLI3A, functionally implicating a relative overexpression of GLI3R.22 However, GLI3R overexpression would likely produce a posterior phenotype, as determined by Hill et al in their fixed homo and hemizygous GLI3R models.15 Therefore, our hypothesis is that all included missense variants have a similar pathogenesis which is more likely in concordance with the mechanism introduced by Krauss et al. To our knowledge, no splice site variants have been functionally described in literature.

However, it is noted that the 15 and last exon encompasses the entire activator domain, thus any splice site mutation is by definition located on the 5′ side of the activator. Based on the phenotype, we would suggest that these variants fail to produce a functional protein. We hypothesise that the truncating variants of the activator domain lead to overexpression of GLI3R in SHH rich areas. In normal development, the presence of SHH prevents the processing of full length GLI34 into GLI3R, thus producing the full length activator. In patients with a truncating variant of the activator domain of GLI3, thus these variants likely have the largest effect in SHH rich areas, such as the ZPA located at the posterior side of the hand/footplate.

Moreover, the lack of posterior anomalies in the GLI3∆699/- mouse model (hemizygous fixed repressor model) compared with the GLI3∆699/∆699 mouse model (homozygous fixed repressor model), suggesting a dosage effect of GLI3R to be responsible for posterior hand anomalies.15 These findings are supported by Lewandowski et al, who show that the majority of the target genes in GLI signalling are regulated by GLI3R rather than GLI3A.44 Together, these findings suggest a role for the location and type of variant in GLI3-mediated syndromes.Interestingly, the difference between Pallister-Hall syndrome and GLI3-mediated polydactyly syndromes has also been attributed to the GLI3R overexpression. However, the difference in phenotype observed in the cases with a truncating variant in the activator domain and Pallister-Hall syndrome suggest different functional consequences. When studying figure 1, it is noted that the included truncating variants on the 3′ side of the cleavage site seldomly affect the CBP binding region, which could provide an explanation for the observed differences. This binding region is included in the Pallister-Hall region as defined by Johnston et al and is necessary for the downstream signalling with GLI1.10 11 23 45 Interestingly, recent reports show that pathogenic variants in GLI1 can produce phenotypes concordant with Ellis von Krefeld syndrome, which includes overlapping features with Pallister-Hall syndrome.46 The four truncating variants observed in this study that do affect the CBP but did not result in a Pallister-Hall phenotype are conflicting with this theory. Krauss et al postulate an alternative hypothesis, they state that the MID1-α4-PP2A complex, which is essential for GLI3A signalling, could also be the reason for overlapping features of Opitz syndrome, caused by variants in MID1, and Pallister-Hall syndrome.

Further analysis is required to fully appreciate the functional differences between truncating mutations that cause Pallister-Hall syndrome and those that result in GLI3-mediated polydactyly syndromes.For the clinical evaluation of patients with GLI3-mediated polydactyly syndromes, intracranial anomalies are likely the most important to predict based on the variant. Unfortunately, the presence of corpus callosum agenesis was not routinely investigated or reported thus this feature could not be used as an indicator phenotype for LC membership. Interestingly when using only hand and foot phenotypes, we did notice a higher prevalence of corpus callosum agenesis in patients with posterior phenotypes. The suggested relation between truncating mutations in the activator domain causing these posterior phenotypes and corpus callosum agenesis was statistically confirmed (OR. 8.8, p<0.001).

Functionally this relation could be caused by the GLI3-MED12 interaction at the MBD. Pathogenic DNA variants in MED12 can cause Opitz-Kaveggia syndrome, a syndrome in which presentation includes corpus callosum agenesis, broad halluces and thumbs.47In conclusion, there are two distinct phenotypes within the GLI3-mediated polydactyly population. Patients with more posteriorly and more anteriorly oriented hand anomalies. Furthermore, this difference is related to the observed variant in GLI3. We hypothesise that variants that cause haploinsufficiency produce anterior anomalies of the hand, whereas variants with abnormal truncation of the activator domain have more posterior anomalies.

Furthermore, patients that have a variant that produces abnormal truncation of the activator domain, have a greater risk for corpus callosum agenesis. Thus, we advocate to differentiate preaxial or postaxial oriented GLI3 phenotypes to explain the pathophysiology as well as to get a risk assessment for corpus callosum agenesis.Data availability statementData are available upon reasonable request.Ethics statementsPatient consent for publicationNot required.Ethics approvalThe research protocol was approved by the local ethics board of the Erasmus MC University Medical Center (MEC 2015-679)..

AbstractIntroduction. We report a very rare case of familial breast cancer and diffuse gastric cancer, with germline pathogenic variants in both BRCA1 and CDH1 genes. To the best of our knowledge, this is the first report of such an association.Family description.

The proband is a woman diagnosed with breast cancer at the age of 52 years. She requested genetic counselling in 2012, at the age of 91 years, because of a history of breast cancer in her daughter, her sister, her niece and her paternal grandmother and was therefore concerned about her relatives. Her sister and maternal aunt also had gastric cancer.

She was tested for several genes associated with hereditary breast cancer.Results. A large deletion of BRCA1 from exons 1 to 7 and two CDH1 pathogenic cis variants were identified.Conclusion. This complex situation is challenging for genetic counselling and management of at-risk individuals.cancer.

Breastcancer. Gastricclinical geneticsgenetic screening/counsellingmolecular geneticsIntroductionGLI-Kruppel family member 3 (GLI3) encodes for a zinc finger transcription factor which plays a key role in the sonic hedgehog (SHH) signalling pathway essential in both limb and craniofacial development.1 2 In hand development, SHH is expressed in the zone of polarising activity (ZPA) on the posterior side of the handplate. The ZPA expresses SHH, creating a gradient of SHH from the posterior to the anterior side of the handplate.

In the presence of SHH, full length GLI3-protein is produced (GLI3A), whereas absence of SHH causes cleavage of GLI3 into its repressor form (GLI3R).3 4 Abnormal expression of this SHH/GLI3R gradient can cause both preaxial and postaxial polydactyly.2Concordantly, pathogenic DNA variants in the GLI3 gene are known to cause multiple syndromes with craniofacial and limb involvement, such as. Acrocallosal syndrome5 (OMIM. 200990), Greig cephalopolysyndactyly syndrome6 (OMIM.

175700) and Pallister-Hall syndrome7 (OMIM. 146510). Also, in non-syndromic polydactyly, such as preaxial polydactyly-type 4 (PPD4, OMIM.

174700),8 pathogenic variants in GLI3 have been described. Out of these diseases, Pallister-Hall syndrome is the most distinct entity, defined by the presence of central polydactyly and hypothalamic hamartoma.9 The other GLI3 syndromes are defined by the presence of preaxial and/or postaxial polydactyly of the hand and feet with or without syndactyly (Greig syndrome, PPD4). Also, various mild craniofacial features such as hypertelorism and macrocephaly can occur.

Pallister-Hall syndrome is caused by truncating variants in the middle third of the GLI3 gene.10–12 The truncation of GLI3 causes an overexpression of GLI3R, which is believed to be the key difference between Pallister-Hall and the GLI3-mediated polydactyly syndromes.9 11 Although multiple attempts have been made, the clinical and genetic distinction between the GLI3-mediated polydactyly syndromes is less evident. This has for example led to the introduction of subGreig and the formulation of an Oro-facial-digital overlap syndrome.10 Other authors, suggested that we should not regard these diseases as separate entities, but as a spectrum of GLI3-mediated polydactyly syndromes.13Although phenotype/genotype correlation of the different syndromes has been cumbersome, clinical and animal studies do provide evidence that distinct regions within the gene, could be related to the individual anomalies contributing to these syndromes. First, case studies show isolated preaxial polydactyly is caused by both truncating and non-truncating variants throughout the GLI3 gene, whereas in isolated postaxial polydactyly cases truncating variants at the C-terminal side of the gene are observed.12 14 These results suggest two different groups of variants for preaxial and postaxial polydactyly.

Second, recent animal studies suggest that posterior malformations in GLI3-mediated polydactyly syndromes are likely related to a dosage effect of GLI3R rather than due to the influence of an altered GLI3A expression.15Past attempts for phenotype/genotype correlation in GLI3-mediated polydactyly syndromes have directly related the diagnosed syndrome to the observed genotype.10–12 16 Focusing on individual hand phenotypes, such as preaxial and postaxial polydactyly and syndactyly might be more reliable because it prevents misclassification due to inconsistent use of syndrome definition. Subsequently, latent class analysis (LCA) provides the possibility to relate a group of observed variables to a set of latent, or unmeasured, parameters and thereby identifying different subgroups in the obtained dataset.17 As a result, LCA allows us to group different phenotypes within the GLI3-mediated polydactyly syndromes and relate the most important predictors of the grouped phenotypes to the observed GLI3 variants.The aim of our study was to further investigate the correlation of the individual phenotypes to the genotypes observed in GLI3-mediated polydactyly syndromes, using LCA. Cases were obtained by both literature review and the inclusion of local clinical cases.

Subsequently, we identified two subclasses of limb anomalies that relate to the underlying GLI3 variant. We provide evidence for two different phenotypic and genotypic groups with predominantly preaxial and postaxial hand and feet anomalies, and we specify those cases with a higher risk for corpus callosum anomalies.MethodsLiterature reviewThe Human Gene Mutation Database (HGMD Professional 2019) was reviewed to identify known pathogenic variants in GLI3 and corresponding phenotypes.18 All references were obtained and cases were included when they were diagnosed with either Greig or subGreig syndrome or PPD4.10–12 Pallister-Hall syndrome and acrocallosal syndrome were excluded because both are regarded distinct syndromes and rather defined by the presence of the non-hand anomalies, than the presence of preaxial or postaxial polydactyly.13 19 Isolated preaxial or postaxial polydactyly were excluded for two reasons. The phenotype/genotype correlations are better understood and both anomalies can occur sporadically which could introduce falsely assumed pathogenic GLI3 variants in the analysis.

Additionally, cases were excluded when case-specific phenotypic or genotypic information was not reported or if these two could not be related to each other. Families with a combined phenotypic description, not reducible to individual family members, were included as one case in the analysis.Clinical casesThe Sophia Children’s Hospital Database was reviewed for cases with a GLI3 variant. Within this population, the same inclusion criteria for the phenotype were valid.

Relatives of the index patients were also contacted for participation in this study, when they showed comparable hand, foot, or craniofacial malformations or when a GLI3 variant was identified. Phenotypes of the hand, foot and craniofacial anomalies of the patients treated in the Sophia Children's Hospital were collected using patient documentation. Family members were identified and if possible, clinically verified.

Alternatively, family members were contacted to verify their phenotypes. If no verification was possible, cases were excluded.PhenotypesThe phenotypes of both literature cases and local cases were extracted in a similar fashion. The most frequently reported limb and craniofacial phenotypes were dichotomised.

The dichotomised hand and foot phenotypes were preaxial polydactyly, postaxial polydactyly and syndactyly. Broad halluces or thumbs were commonly reported by authors and were dichotomised as a presentation of preaxial polydactyly. The extracted dichotomised craniofacial phenotypes were hypertelorism, macrocephaly and corpus callosum agenesis.

All other phenotypes were registered, but not dichotomised.Pathogenic GLI3 variantsAll GLI3 variants were extracted and checked using Alamut Visual V.2.14. If indicated, variants were renamed according to standard Human Genome Variation Society nomenclature.20 Variants were grouped in either missense, frameshift, nonsense or splice site variants. In the group of frameshift variants, a subgroup with possible splice site effect were identified for subgroup analysis when indicated.

Similarly, nonsense variants prone for nonsense mediated decay (NMD) and nonsense variants with experimentally confirmed NMD were identified.21 Deletions of multiple exons, CNVs and translocations were excluded for analysis. A full list of included mutations is available in the online supplementary materials.Supplemental materialThe location of the variant was compared with five known structural domains of the GLI3 gene. (1) repressor domain, (2) zinc finger domain, (3) cleavage site, (4) activator domain, which we defined as a concatenation of the separately identified transactivation zones, the CBP binding domain and the mediator binding domain (MBD) and (5) the MID1 interaction region domain.1 6 22–24 The boundaries of each of the domains were based on available literature (figure 1, exact locations available in the online supplementary materials).

The boundaries used by different authors did vary, therefore a consensus was made.In this figure the posterior probability of an anterior phenotype is plotted against the location of the variant, stratified for the type of mutation that was observed. For better overview, only variants with a location effect were displayed. The full figure, including all variant types, can be found in the online supplementary figure 1.

Each mutation is depicted as a dot, the size of the dot represents the number of observations for that variant. If multiple observations were made, the mean posterior odds and IQR are plotted. For the nonsense variants, variants that were predicted to produce nonsense mediated decay, are depicted using a triangle.

Again, the size indicates the number of observations." data-icon-position data-hide-link-title="0">Figure 1 In this figure the posterior probability of an anterior phenotype is plotted against the location of the variant, stratified for the type of mutation that was observed. For better overview, only variants with a location effect were displayed. The full figure, including all variant types, can be found in the online supplementary figure 1.

Each mutation is depicted as a dot, the size of the dot represents the number of observations for that variant. If multiple observations were made, the mean posterior odds and IQR are plotted. For the nonsense variants, variants that were predicted to produce nonsense mediated decay, are depicted using a triangle.

Again, the size indicates the number of observations.Supplemental materialLatent class analysisTo cluster phenotypes and relate those to the genotypes of the patients, an explorative analysis was done using LCA in R (R V.3.6.1 for Mac. Polytomous variable LCA, poLCA V.1.4.1.). We used our LCA to detect the number of phenotypic subgroups in the dataset and subsequently predict a class membership for each case in the dataset based on the posterior probabilities.In order to make a reliable prediction, only phenotypes that were sufficiently reported and/or ruled out were feasible for LCA, limiting the analysis to preaxial polydactyly, postaxial polydactyly and syndactyly of the hands and feet.

Only full cases were included. To determine the optimal number of classes, we fitted a series of models ranging from a one-class to a six-class model. The optimal number of classes was based on the conditional Akaike information criterion (cAIC), the non adjusted and the sample-size adjusted Bayesian information criterion (BIC and aBIC) and the obtained entropy.25 The explorative LCA produces both posterior probabilities per case for both classes and predicted class membership.

Using the predicted class membership, the phenotypic features per class were determined in a univariate analysis (χ2, SPSS V.25). Using the posterior probabilities on latent class (LC) membership, a scatter plot was created using the location of the variant on the x-axis and the probability of class membership on the y-axis for each of the types of variants (Tibco Spotfire V.7.14). Using these scatter plots, variants that give similar phenotypes were clustered.Genotype/phenotype correlationBecause an LC has no clinical value, the correlation between genotypes and phenotypes was investigated using the predictor phenotypes and the clustered phenotypes.

First, those phenotypes that contribute most to LC membership were identified. Second those phenotypes were directly related to the different types of variants (missense, nonsense, frameshift, splice site) and their clustered locations. Quantification of the relation was performed using a univariate analysis using a χ2 test.

Because of our selection criteria, meaning patients at least have two phenotypes, a multivariate using a logistic regression analysis was used to detect the most significant predictors in the overall phenotype (SPSS V.25). Finally, we explored the relation of the clustered genotypes to the presence of corpus callosum agenesis, a rare malformation in GLI3-mediated polydactyly syndromes which cannot be readily diagnosed without additional imaging.ResultsWe included 251 patients from the literature and 46 local patients,10–12 16 21 26–43 in total 297 patients from 155 different families with 127 different GLI3 variants, 32 of which were large deletions, CNVs or translocations. In six local cases, the exact variant could not be retrieved by status research.The distribution of the most frequently observed phenotypes and variants are presented in table 1.

Other recurring phenotypes included developmental delay (n=22), broad nasal root (n=23), frontal bossing or prominent forehead (n=16) and craniosynostosis (n=13), camptodactyly (n=8) and a broad first interdigital webspace of the foot (n=6).View this table:Table 1 Baseline phenotypes and genotypes of selected populationThe LCA model was fitted using the six defined hand/foot phenotypes. Model fit indices for the LCA are displayed in table 2. Based on the BIC, a two-class model has the best fit for our data.

The four-class model does show a gain in entropy, however with a higher BIC and loss of df. Therefore, based on the majority of performance statistics and the interpretability of the model, a two-class model was chosen. Table 3 displays the distribution of phenotypes and genotypes over the two classes.View this table:Table 2 Model fit indices for the one-class through six-class model evaluated in our LCAView this table:Table 3 Distribution of phenotypes and genotypes in the two latent classes (LC)Table 1 depicts the baseline phenotypes and genotypes in the obtained population.

Note incomplete data especially in the cranium phenotypes. In total 259 valid genotypes were present. In total, 289 cases had complete data for all hand and foot phenotypes (preaxial polydactyly, postaxial polydactyly and syndactyly) and thus were available for LCA.

Combined, for phenotype/genotype correlation 258 cases were available with complete genotypes and complete hand and foot phenotypes.Table 2 depicts the model fit indices for all models that have been fitted to our data.Table 3 depicts the distribution of phenotypes and genotypes over the two assigned LCs. Hand and foot phenotypes were used as input for the LCA, thus are all complete cases. Malformation of the cranium and genotypes do have missing cases.

Note that for the LCA, full case description was required, resulting in eight cases due to incomplete phenotypes. Out of these eight, one also had a genotype that thus needed to be excluded. Missingness of genotypic data was higher in LC2, mostly due to CNVs (table 1).In 54/60 cases, a missense variant produced a posterior phenotype.

Likewise, splice site variants show the same phenotype in 23/24 cases (table 3). For both frameshift and nonsense variants, this relation is not significant (52 anterior vs 54 posterior and 26 anterior vs 42 posterior, respectively). Therefore, only for nonsense and frameshift variants the location of the variant was plotted against the probability for LC2 membership in figure 1.

A full scatterplot of all variants is available in online supplementary figure 1.Figure 1 reveals a pattern for these nonsense and frameshift variants that reveals that variants at the C-terminal of the gene predict anterior phenotypes. When relating the domains of the GLI3 protein to the observed phenotype, we observe that the majority of patients with a nonsense or frameshift variant in the repressor domain, the zinc finger domain or the cleavage site had a high probability of an LC2/anterior phenotype. This group contains all variants that are either experimentally determined to be subject to NMD (triangle marker in figure 1) or predicted to be subject to NMD (diamond marker in figure 1).

Frameshift and nonsense variants in the activator domain result in high probability for an LC1/posterior phenotype. These variants will be further referred to as truncating variants in the activator domain.The univariate relation of the individual phenotypes to these two groups of variants are estimated and presented in table 4. In our multivariate analysis, postaxial polydactyly of the foot and hand are the strongest predictors (Beta.

2.548, p<0001 and Beta. 1.47, p=0.013, respectively) for patients to have a truncating variant in the activator domain. Moreover, the effect sizes of preaxial polydactyly of the hand and feet (Beta.

ˆ’0.797, p=0123 and −1.772, p=0.001) reveals that especially postaxial polydactyly of the foot is the dominant predictor for the genetic substrate of the observed anomalies.View this table:Table 4 Univariate and multivariate analysis of the phenotype/genotype correlationTable 4 shows exploration of the individual phenotypes on the genotype, both univariate and multivariate. The multivariate analysis corrects for the presence of multiple phenotypes in the underlying population.Although the craniofacial anomalies could not be included in the LCA, the relation between the observed anomalies and the identified genetic substrates can be studied. The prevalence of hypertelorism was equally distributed over the two groups of variants (47/135 vs 21/47 respectively, p<0.229).

However for corpus callosum agenesis and macrocephaly, there was a higher prevalence in patients with a truncating variant in the activator domain (3/75 vs 11/41, p<0.001. OR. 8.8, p<0.001) and 42/123 vs 24/48, p<0.05).

Noteworthy is the fact that 11/14 cases with corpus callosum agenesis in the dataset had a truncating variant in the activator domain.DiscussionIn this report, we present new insights into the correlation between the phenotype and the genotype in patients with GLI3-mediated polydactyly syndromes. We illustrate that there are two LCs of patients, best predicted by postaxial polydactyly of the hand and foot for LC1, and the preaxial polydactyly of the hand and foot and syndactyly of the foot for LC2. Patients with postaxial phenotypes have a higher risk of having a truncating variant in the activator domain of the GLI3 gene which is also related to a higher risk of corpus callosum agenesis.

These results suggest a functional difference between truncating variants on the N-terminal and the C-terminal side of the GLI3 cleavage site.Previous attempts of phenotype to genotype correlation have not yet provided the clinical confirmation of these assumed mechanisms in the pathophysiology of GLI3-mediated polydactyly syndromes. Johnston et al have successfully determined the Pallister-Hall region in which truncating variants produce a Pallister-Hall phenotype rather than Greig syndrome.11 However, in their latest population study, subtypes of both syndromes were included to explain the full spectrum of observed malformations. In 2015, Demurger et al reported the higher incidence of corpus callosum agenesis in the Greig syndrome population with truncating mutations in the activator domain.12 Al-Qattan in his review summarises the concept of a spectrum of anomalies dependent on haplo-insufficiency (through different mechanisms) and repressor overexpression.13 However, he bases this theory mainly on reviewed experimental data.

Our report is the first to provide an extensive clinical review of cases that substantiate the phenotypic difference between the two groups that could fit the suggested mechanisms. We agree with Al-Qattan et al that a variation of anomalies can be observed given any pathogenic variant in the GLI3 gene, but overall two dominant phenotypes are present. A population with predominantly preaxial anomalies and one with postaxial anomalies.

The presence of preaxial or postaxial polydactyly and syndactyly is not mutually exclusive for one of these two subclasses. Meaning that preaxial polydactyly can co-occur with postaxial polydactyly. However, truncating mutations in the activator domain produce a postaxial phenotype, as can be derived from the risk in table 4.

The higher risk of corpus callosum agenesis in this population shows that differentiating between a preaxial phenotype and a postaxial phenotype, instead of between the different GLI3-mediated polydactyly syndromes, might be more relevant regarding diagnostics for corpus callosum agenesis.We chose to use LCA as an exploratory tool only in our population for two reasons. First of all, LCA can be useful to identify subgroups, but there is no ‘true’ model or number of subgroups you can detect. The best fitting model can only be estimated based on the available measures and approximates the true subgroups that might be present.

Second, LC membership assignment is a statistical procedure based on the posterior probability, with concordant errors of the estimation, rather than a clinical value that can be measured or evaluated. Therefore, we decided to use our LCA only in an exploratory tool, and perform our statistics using the actual phenotypes that predict LC membership and the associated genotypes. Overall, this method worked well to differentiate the two subgroups present in our dataset.

However, outliers were observed. A qualitative analysis of these outliers is available in the online supplementary data.The genetic substrate for the two phenotypic clusters can be discussed based on multiple experiments. Overall, we hypothesise two genetic clusters.

One that is due to haploinsufficiency and one that is due to abnormal truncation of the activator. The hypothesised cluster of variants that produce haploinsufficiency is mainly based on the experimental data that confirms NMD in two variants and the NMD prediction of other nonsense variants in Alamut. For the frameshift variants, it is also likely that the cleavage of the zinc finger domain results in functional haploinsufficiency either because of a lack of signalling domains or similarly due to NMD.

Missense variants could cause haploinsufficiency through the suggested mechanism by Krauss et al who have illustrated that missense variants in the MID1 domain hamper the functional interaction with the MID1-α4-PP2A complex, leading to a subcellular location of GLI3.24 The observed missense variants in our study exceed the region to which Krauss et al have limited the MID-1 interaction domain. An alternative theory is suggested by Zhou et al who have shown that missense variants in the MBD can cause deficiency in the signalling of GLI3A, functionally implicating a relative overexpression of GLI3R.22 However, GLI3R overexpression would likely produce a posterior phenotype, as determined by Hill et al in their fixed homo and hemizygous GLI3R models.15 Therefore, our hypothesis is that all included missense variants have a similar pathogenesis which is more likely in concordance with the mechanism introduced by Krauss et al. To our knowledge, no splice site variants have been functionally described in literature.

However, it is noted that the 15 and last exon encompasses the entire activator domain, thus any splice site mutation is by definition located on the 5′ side of the activator. Based on the phenotype, we would suggest that these variants fail to produce a functional protein. We hypothesise that the truncating variants of the activator domain lead to overexpression of GLI3R in SHH rich areas.

In normal development, the presence of SHH prevents the processing of full length GLI34 into GLI3R, thus producing the full length activator. In patients with a truncating variant of the activator domain of GLI3, thus these variants likely have the largest effect in SHH rich areas, such as the ZPA located at the posterior side of the hand/footplate. Moreover, the lack of posterior anomalies in the GLI3∆699/- mouse model (hemizygous fixed repressor model) compared with the GLI3∆699/∆699 mouse model (homozygous fixed repressor model), suggesting a dosage effect of GLI3R to be responsible for posterior hand anomalies.15 These findings are supported by Lewandowski et al, who show that the majority of the target genes in GLI signalling are regulated by GLI3R rather than GLI3A.44 Together, these findings suggest a role for the location and type of variant in GLI3-mediated syndromes.Interestingly, the difference between Pallister-Hall syndrome and GLI3-mediated polydactyly syndromes has also been attributed to the GLI3R overexpression.

However, the difference in phenotype observed in the cases with a truncating variant in the activator domain and Pallister-Hall syndrome suggest different functional consequences. When studying figure 1, it is noted that the included truncating variants on the 3′ side of the cleavage site seldomly affect the CBP binding region, which could provide an explanation for the observed differences. This binding region is included in the Pallister-Hall region as defined by Johnston et al and is necessary for the downstream signalling with GLI1.10 11 23 45 Interestingly, recent reports show that pathogenic variants in GLI1 can produce phenotypes concordant with Ellis von Krefeld syndrome, which includes overlapping features with Pallister-Hall syndrome.46 The four truncating variants observed in this study that do affect the CBP but did not result in a Pallister-Hall phenotype are conflicting with this theory.

Krauss et al postulate an alternative hypothesis, they state that the MID1-α4-PP2A complex, which is essential for GLI3A signalling, could also be the reason for overlapping features of Opitz syndrome, caused by variants in MID1, and Pallister-Hall syndrome. Further analysis is required to fully appreciate the functional differences between truncating mutations that cause Pallister-Hall syndrome and those that result in GLI3-mediated polydactyly syndromes.For the clinical evaluation of patients with GLI3-mediated polydactyly syndromes, intracranial anomalies are likely the most important to predict based on the variant. Unfortunately, the presence of corpus callosum agenesis was not routinely investigated or reported thus this feature could not be used as an indicator phenotype for LC membership.

Interestingly when using only hand and foot phenotypes, we did notice a higher prevalence of corpus callosum agenesis in patients with posterior phenotypes. The suggested relation between truncating mutations in the activator domain causing these posterior phenotypes and corpus callosum agenesis was statistically confirmed (OR. 8.8, p<0.001).

Functionally this relation could be caused by the GLI3-MED12 interaction at the MBD. Pathogenic DNA variants in MED12 can cause Opitz-Kaveggia syndrome, a syndrome in which presentation includes corpus callosum agenesis, broad halluces and thumbs.47In conclusion, there are two distinct phenotypes within the GLI3-mediated polydactyly population. Patients with more posteriorly and more anteriorly oriented hand anomalies.

Furthermore, this difference is related to the observed variant in GLI3. We hypothesise that variants that cause haploinsufficiency produce anterior anomalies of the hand, whereas variants with abnormal truncation of the activator domain have more posterior anomalies. Furthermore, patients that have a variant that produces abnormal truncation of the activator domain, have a greater risk for corpus callosum agenesis.

Thus, we advocate to differentiate preaxial or postaxial oriented GLI3 phenotypes to explain the pathophysiology as well as to get a risk assessment for corpus callosum agenesis.Data availability statementData are available upon reasonable request.Ethics statementsPatient consent for publicationNot required.Ethics approvalThe research protocol was approved by the local ethics board of the Erasmus MC University Medical Center (MEC 2015-679)..