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When explaining the inadequacy of cialis best buy the words “Cheer him up” to describe the purpose of offering a drink to a murderer, TS Elliot’s Sweeney remarks,Well here again that don’t applyBut I’ve gotta use words when I talk to you.1The importance of words (or concepts) to medical ethics cannot be denied. While a narrow view of conceptual analysis is not conducive to good medical ethics,2 the adequacy and clarity of the words we use continues to be the foundation for all ethical analysis. While some key ideas such as ‘paternalism’3 or ‘coercion’4 are well theorised and tend to be used in a consistent way that most understand, other words that are important to cialis best buy ethics, often are not.Our ability to notice, perceive and understand ethical issues is the starting point for all ethical inquiry.

The ethical words we choose structure and give content to our ethical perception. As Kant observed, ‘Thoughts without content are empty, intuitions without concepts are blind.’5 While many do not agree with aspects of Kant’s moral philosophy, his views about what’s required to have an ‘intuition’ or an ‘ethical perception’ seem correct.The ethical words we choose enable us cialis best buy to understand and articulate different ethical features of a situation and words that come from other cultures can enrich our perception of what matters and what we should do. For example, the Māori concept Whakawhanaungatanga refers to the process of establishing meaningful relationships between people via culturally appropriate processes.

In a situation where the decision making capacity of a patient is in question and an important decision needs to be made, this is a process whereby a healthcare practitioner can build a meaningful relationship with that patient and their whānau (family).6 While that’s cialis best buy a process that’s particularly important for Māori patients and whānau, the emphasis on creating and valuing relationships is something that’s ethically important for this kind of decision more generally and arguably not highlighted if we view this kind of situation via the lenses of autonomy or beneficence.The JME invites those interested in writing a short discussion piece that explains an ethical concept or word that is useful and should be more widely understood to contact the journal’s Editor in Chief. €˜Words’ columns will be up to a thousand words, have no more than five references and explain a clinically relevant ethical concept that would benefit from being explained and is preferably from a non-western ethical tradition.This issue of the JME includes a number of papers that demonstrate the importance of explaining words that will then bear weight in argument. Jecker and Atuire analyse arguments relevant to waiving intellectual property rights over erectile dysfunction treatments.7 ‘Intellectual property’ is a concept that would appear to be easily grasped cialis best buy.

It implies an ownership right over knowledge of some kind that has been created. Yet if we are to understand that concept with the depth needed to mount an ethical analysis, we must delve into the rules that currently apply to cialis best buy intellectual property. As Jecker and Atuire explain, the 1995 TRIPS agreement created the means for the stricter protection of IP, including pharmaceuticals.

The agreement itself and those who have argued in support of it, emphasised reasons such as the need to nurture innovation and this, as Jecker and Atuire show, opens the door for a critical ethical analysis of whether IP should be waived for erectile dysfunction treatments.‘Emotional support animals’ can be very helpful to people with mental illnesses and cialis best buy that raises the question of whether those who depend on an ESA have a right to remain connected to their ESA that is similar to someone who has a prosthetic body part. As Kolmes observes, ESA’s ‘…supplement or entirely replace vital functions that their handlers are not able to perform on their own.8’ This description of ESAs characterises them in a way that distinguishes them from other animals with which we might have a strong emotional bond and helps to explain how they can be considered analogous to a prosthetic body part.We’re grateful to the JME authors and reviewers who made it possible for the journal to cover the issues raised by the erectile dysfunction treatment cialis in depth. The cialis continues, but many of the issues have been well explored now and cialis best buy there is less need for a special section in the journal.

The JME has always published topical high quality analysis of health care ethics and we look forward to publishing articles that deepen and broaden scholarship in ethics. Our new Words column is intended to help broaden the scope of scholarship in ethics and we hope to hear from those who would like to explain an important ethical concept.John McMillanEditor cialis best buy in ChiefEthics statementsPatient consent for publicationNot required.AbstractUnlike its friendly cousin the placebo effect, the nocebo effect (the effect of expecting a negative outcome) has been almost ignored. Epistemic and ethical confusions related to its existence have gone all but unnoticed.

Contrary to what is often asserted, adverse events following from taking placebo interventions are not necessarily nocebo cialis best buy effects. They could have arisen due to natural history. Meanwhile, ethical informed consent (in clinical trials and clinical practice) has centred almost exclusively on the need to inform patients about intervention risks with cialis best buy patients to preserve their autonomy.

Researchers have failed to consider the harm caused by the way in which the information is conveyed. In this paper, I argue that the magnitude of nocebo cialis best buy effects must be measured using control groups consisting of untreated patients. And, because the nocebo effect can produce harm, the principle of non-maleficence must be taken into account alongside autonomy when obtaining (ethical) informed consent and communicating intervention risks with patients.informed consentautonomyresearch ethicsepidemiologyethicsData availability statementThere are no data in this work..

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9 December buy cialis get free viagra 2021 There are currently vacancies on the cellular pathology, clinical chemistry, cytopathology, transfusion science cialis contra viagra and virology specialist advisory panels Our eight specialist advisory panels are vital in ensuring the success of the Institute’s work. Each panel is made up of around twelve "expert" members that normally includes a Chair, Deputy Chair, Chief Examiner, two Deputy Chief Examiners, a Company Member and around six “ordinary” members. There are cialis contra viagra panels for cellular pathology, clinical chemistry, cytopathology, haematology, immunology, medical microbiology, transfusion science and virology.There are currently vacancies on the cellular pathology, clinical chemistry, cytopathology, transfusion science and virology panels although expressions of interest to join any of the other panels would also be welcome.

The work of the panel members includes. · Organising the scientific programme for their discipline for the IBMS Congress and contributing to other aspects of this major event · Working with the Institute on revisions to the existing, and the development of new, professional qualifications, such as the Specialist Portfolio, Diploma of Expert Practice, Higher Specialist Diploma and Advanced Specialist Diploma · Representing the Institute on local and national groups and committees · Commenting cialis contra viagra on revisions to Institute professional guidance documents · Acting as a source of advice on a range of topics, including patient/service users, emerging technologies and laboratory training · Working with the Institute’s Education and Professional Standards Committee and other working groups To join as an "ordinary" member you need to have. · Institute Member (MIBMS) or Fellow (FIBMS) status · Scientific or managerial experience in your chosen discipline · Evidence of liaison with professional and/or academic contacts · Chartered Scientist (CSci) status is also desirable - non-chartered individuals can initially be co-opted before moving to full membership once chartered “One of the most enjoyable aspects of being a panel member is helping to organise Congress – working with colleagues to find interesting and topical speakers and liaising with panel members on the day to ensure everything runs smoothly.

Another aspect I enjoy is meeting colleagues from across the country, working together to keep the Institute ahead of developments in clinical chemistry and thereby supporting our members and the profession.” Joanna Andrew – IBMS Council Member and former member of Clinical Chemistry Advisory Panel "Being a panel member helps me to stay up to date with the plans and policies affecting pathology services in my discipline, and to contribute to them. It has broadened my network, my understanding of how laboratories around the country deal with issues and challenges and deepened my knowledge of current hospital laboratory practice.” Sarah Pitt – IBMS Council Member and Chief Examiner on Virology Advisory Panel If you are interested, please send a copy of your CV cialis contra viagra (three sides at most) and a supporting letter from your manager to Chris Ward, Head of Examinations (examinations@ibms.org), by Friday 7 January 20229 December 2021 The Institute of Biomedical Science invites applications for a Deputy Editor to assist the Editor in the general day-to-day running of the British Journal of Biomedical Science The Deputy Editor will work with the Editor and two other Deputy Editors to appoint referees and ensure their report within two weeks. The journal publishes original research articles, reviews, Biomedical Science in Brief articles (short summaries of advances in biomedical science) and case reports.

From January cialis contra viagra 2022 the BJBS is moving to open access with Frontiers. BJBS’s impact factor grew from 2.712 in 2019 to 3.829 in 2020 and now ranks 6th out of 29 you can try this out journals in the Medical Laboratory Technology JCR category. The Deputy Editor will have access to cialis contra viagra all members of the Institute Advisory Panels who are a rich source of referees and may invite each other to referee.

She/He will of course be free to appoint referees not on the Panels, i.e. Those of their personal network, or members of the Institute who are not yet Fellows. Other duties (writing a monthly report for The Biomedical Scientist and writing a JBL from articles within an issue (if possible) may be undertaken by the DEs in rotation cialis contra viagra.

However, the DEs will together collaborate with the Editor in writing the annual ‘What have we learned’ editorial each Autumn. Interviews will take place cialis contra viagra via MS Teams in January 2022, and full training on the journal software will be given on a subsequent occasion. The position is for a period of three years and a modest honorarium will be provided.

Person specification Essential criteria · Considerable practical experience of biomedical science · Excellent organisational skills · Ability to navigate publishing cialis contra viagra software · FIBMS · PhD/Doctorate in Biomedical Science Desirable criteria · Experience in more than one biomedical science discipline · Experience of in vitro or in vivo mechanistic studies · Prior journal experience preferably as an associate or deputy editor and/or serving on an editorial board · Experience in teaching biomedical science in higher education Role Description · Appoint referee(s) for each manuscript passed on from the Editor. Ensure the referee reports within two weeks · On rotation with the other deputy editors;( i) provide a summary of each issue for ‘The Biomedical Scientist’( ii)generate a Journal Based Learning exercise on each issue (material allowing) · Collaborate in writing the annual ‘What have we learned’ article · Deputise for the Editor as required Applications If you are interested to apply, please send a CV, with an accompanying statement of no more than 500 words saying what you feel you could bring to this role. Applications should be sent to Lynda Rigby, Executive Head of Marketing and Membership by 4 January 2022 via mc@ibms.org.

9 December 2021 There are currently vacancies on the cellular pathology, clinical chemistry, cytopathology, transfusion science and virology specialist advisory panels Our eight specialist advisory panels are vital click for more info in ensuring the success of the cialis best buy Institute’s work. Each panel is made up of around twelve "expert" members that normally includes a Chair, Deputy Chair, Chief Examiner, two Deputy Chief Examiners, a Company Member and around six “ordinary” members. There are panels for cellular pathology, clinical chemistry, cytopathology, haematology, immunology, medical microbiology, transfusion science and virology.There are currently vacancies on the cellular pathology, clinical chemistry, cialis best buy cytopathology, transfusion science and virology panels although expressions of interest to join any of the other panels would also be welcome. The work of the panel members includes.

· Organising cialis best buy the scientific programme for their discipline for the IBMS Congress and contributing to other aspects of this major event · Working with the Institute on revisions to the existing, and the development of new, professional qualifications, such as the Specialist Portfolio, Diploma of Expert Practice, Higher Specialist Diploma and Advanced Specialist Diploma · Representing the Institute on local and national groups and committees · Commenting on revisions to Institute professional guidance documents · Acting as a source of advice on a range of topics, including patient/service users, emerging technologies and laboratory training · Working with the Institute’s Education and Professional Standards Committee and other working groups To join as an "ordinary" member you need to have. · Institute Member (MIBMS) or Fellow (FIBMS) status · Scientific or managerial experience in your chosen discipline · Evidence of liaison with professional and/or academic contacts · Chartered Scientist (CSci) status is also desirable - non-chartered individuals can initially be co-opted before moving to full membership once chartered “One of the most enjoyable aspects of being a panel member is helping to organise Congress – working with colleagues to find interesting and topical speakers and liaising with panel members on the day to ensure everything runs smoothly. Another aspect I enjoy is meeting colleagues from across the country, working together to keep the Institute ahead of developments in clinical chemistry and thereby supporting our members and the profession.” Joanna Andrew – IBMS Council Member and former member of Clinical Chemistry Advisory Panel "Being a panel member helps me to stay up to date with the plans and policies affecting pathology services in my discipline, and to contribute to them. It has broadened my network, my understanding of how laboratories around the country deal with issues and challenges and deepened my knowledge of current hospital laboratory practice.” Sarah Pitt – IBMS Council Member and Chief Examiner on Virology Advisory Panel If you are interested, please send a copy of your CV (three sides at most) and a supporting letter from your manager to Chris Ward, Head of Examinations (examinations@ibms.org), by Friday 7 cialis best buy January 20229 December 2021 The Institute of Biomedical Science invites applications for a Deputy Editor to assist the Editor in the general day-to-day running of the British Journal of Biomedical Science The Deputy Editor will work with the Editor and two other Deputy Editors to appoint referees and ensure their report within two weeks.

The journal publishes original research articles, reviews, Biomedical Science in Brief articles (short summaries of advances in biomedical science) and case reports. From January 2022 the BJBS is moving to open access with Frontiers cialis best buy. BJBS’s impact factor grew from 2.712 in 2019 to 3.829 in 2020 and now ranks 6th out of 29 journals in the Medical Laboratory Technology JCR category. The Deputy Editor will have access to all members of cialis best buy the Institute Advisory Panels who are a rich source of referees and may invite each other to referee.

She/He will of course be free to appoint referees not on the Panels, i.e. Those of their personal network, or members of the Institute who are not yet Fellows. Other duties (writing a monthly report for The Biomedical Scientist and cialis best buy writing a JBL from articles within an issue (if possible) may be undertaken by the DEs in rotation. However, the DEs will together collaborate with the Editor in writing the annual ‘What have we learned’ editorial each Autumn.

Interviews will take place via MS Teams in January 2022, and full training on the journal software will be given on a cialis best buy subsequent occasion. The position is for a period of three years and a modest honorarium will be provided. Person specification cialis best buy Essential criteria · Considerable practical experience of biomedical science · Excellent organisational skills · Ability to navigate publishing software · FIBMS · PhD/Doctorate in Biomedical Science Desirable criteria · Experience in more than one biomedical science discipline · Experience of in vitro or in vivo mechanistic studies · Prior journal experience preferably as an associate or deputy editor and/or serving on an editorial board · Experience in teaching biomedical science in higher education Role Description · Appoint referee(s) for each manuscript passed on from the Editor. Ensure the referee reports within two weeks · On rotation with the other deputy editors;( i) provide a summary of each issue for ‘The Biomedical Scientist’( ii)generate a Journal Based Learning exercise on each issue (material allowing) · Collaborate in writing the annual ‘What have we learned’ article · Deputise for the Editor as required Applications If you are interested to apply, please send a CV, with an accompanying statement of no more than 500 words saying what you feel you could bring to this role.

Applications should be sent to Lynda Rigby, Executive Head of Marketing and Membership by 4 January 2022 via mc@ibms.org.

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New York's Buy kamagra online uk next day delivery Exchange Portal cialis lilly tablets. A Gateway to Coverage for Immigrants The report includes a new tool -- Immigrant Eligibility Crosswalk -- Eligibility by Immigration Status-- designed to help advocates and policymakers sort through the tangle of immigrant eligibility categories to determine who is eligible for which health care programs in 2014 and beyond. The report was made possible with support from the United Hospital Fund and benefited from the advice and input from many of our national partners in the effort to ensure maximum participation of immigrants in the nation's healthcare system as well as experts from the New York State Department of Health and the Centers for Medicare and Medicaid Services. SEE more about "PRUCOL" immigrant eligibility for Medicaid in this article.

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New York's Buy kamagra online uk next day delivery Exchange Portal cialis best buy. A Gateway to Coverage for Immigrants The report includes a new tool -- Immigrant Eligibility Crosswalk -- Eligibility by Immigration Status-- designed to help advocates and policymakers sort through the tangle of immigrant eligibility categories to determine who is eligible for which health care programs in 2014 and beyond. The report was made possible with support from the United Hospital Fund and benefited from the advice and input from many of our national partners in the effort to ensure maximum participation of immigrants in the nation's healthcare system as well as experts from the New York State Department of Health and the Centers for Medicare and Medicaid Services. SEE more about "PRUCOL" immigrant eligibility for Medicaid in this article. "Undocumented" immigrants are, with some exceptions for pregnant women and Child Health Plus, only eligible for "emergency Medicaid.".

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Just six months after erectile dysfunction treatments were first administered in the United States, more than 60 percent of adults have received cialis 5 at least one dose, including more than 85 percent of those age 65 and older. In recent weeks, this success in vaccination administration, along with mask-wearing, social distancing precautions, and warm weather, has produced a rapid and welcome decline in cases, hospitalizations, and deaths throughout the country. But progress has been uneven across states, with a growing gap as the pace of vaccination slows cialis 5 more in some states than others. Widespread media attention has centered on “treatment hesitancy,” but state-to-state variation in vaccination rates is exhibiting a familiar pattern. States that have invested more to ensure access to health care are pulling ahead while others lag.

States Are Administering New treatment Doses cialis 5 at Different Paces. Health Systems That Offer Better Health Care Access Are Pulling Ahead Early on, all states focused on getting a limited supply of shots in the arms of the people most at risk. The pace of vaccination was fairly consistent across states. All reached 35 percent of adults cialis 5 with at least one dose by around the first week of April. However, as supply and eligibility opened up, differences emerged.

While the pace of vaccination has accelerated in California, Washington, and several Northeast states, it is slowing cialis 5 in others — particularly in Southern states like Louisiana, Mississippi, and Alabama. There is now a nearly 40 percentage-point difference in the share of adults with at least one treatment dose between high- and low-vaccination states. What accounts for the recent changing pace of vaccination?. Several factors are likely at play, but when we compare the patterns of vaccination to the Commonwealth Fund’s annual State Scorecard rankings, it is hard to miss that states that have previously performed well on measures of access and patient engagement are seeing higher rates of treatment administration, cialis 5 while lower-performing states are progressing more slowly. Opportunities in All States to Reach High Vaccination Levels Much of the focus around vaccination rates has centered on the willingness of people to take a treatment.

But recent estimates indicate that adult hesitancy in most states may not be a barrier to reaching target adult vaccination levels of 70 percent to 80 percent. As illustrated in this exhibit, using hesitancy estimates from cialis 5 the federal government and current vaccination rates, we see adult hesitancy rates around 10 percent to 20 percent in most states. This means that even in states with lower vaccination rates, there is a significant target population that would likely take the treatment under the right circumstances. Efforts to educate the public about the dangers of erectile dysfunction treatment and the effectiveness and safety of the treatments will continue to be important. But with such a large proportion of people open to receiving the treatment, there is an opportunity to employ near-term access strategies, particularly for communities of color that cialis 5 face greater access barriers.

The challenge may be more formidable in states that have invested less in ensuring access to care and public health services, including those with largely rural populations. Moving Forward With demand at mass vaccination sites starting to decline, ongoing progress will depend on local approaches that deliver treatments to the places most convenient cialis 5 and comfortable for communities. Most important will be ensuring that a broad variety of providers — particularly ambulatory clinics, primary care doctors, and pediatricians — have the treatment available for their patients. Allowing people to get the treatment in a familiar setting will improve their comfort level and increase their options. Wisconsin has been putting cialis 5 treatments in community provider offices since early in the vaccination campaign with success.

North Dakota, which has smaller, more rural facilities that need less supply, has set up ua-cold freezer storage that allows the state to distribute smaller treatment allocations to providers. States also have shorter-term options, like mobile vaccination sites or pop-up clinics, as Massachusetts and Vermont have done, or ensuring walk-in appointments, like New York has. In addition to enhancing access in traditional settings, many states are employing creative campaigns cialis 5 that allow residents to get their shot at a baseball game, or enjoy a free beer, a $100 savings bond or exclusive lottery ticket entry with proof of vaccination. Combined with more explicit equitable distribution strategies like those employed in California that reserve supply for high-need neighborhoods, these access improvements could help close remaining disparities and bump all states to a vaccination level that will help the U.S. Ward off variants and cialis 5 future outbreaks.

Still, the vaccination rollout is showing once again that states that do more to enable access to health care services can leverage that investment in a crisis, whether it is a cialis or natural disaster. erectile dysfunction treatment highlights the importance of sustaining primary care and public health infrastructure over time so all Americans can receive the services they need not just in times of crisis, but in their day-to-day lives.Explore full-page map While the number of new erectile dysfunction treatment s in rural America continued to decline last week, the number of erectile dysfunction treatment-related deaths grew for the second week in row. New s in rural America dropped by 18% last week, marking the sixth consecutive week of declining cases in the nation’s nonmetropolitan cialis 5 counties. Cases fell from 25,876 two weeks ago to 21,179 last week, according to a Daily Yonder analysis of data provided by USA Facts. During the same period, new erectile dysfunction treatment-related deaths in rural counties grew by about 25%, from 681 two weeks ago to 849 last week.

Deaths have grown by 40% over the past three weeks in rural counties, cialis 5 while climbing only 4% in metropolitan counties. Oklahoma had the largest number of erectile dysfunction treatment-related deaths in rural counties last week, with 152. The next greatest numbers of rural deaths were in New Mexico, 90. Michigan, 53 cialis 5. And Georgia, 51.

Maryland, where only about cialis 5 3% of the population lives in a rural county, had the highest rate of rural erectile dysfunction treatment-related deaths last week. The 30 rural deaths there equaled a rate of 19.9 deaths per 100,000 residents. The next highest rural death rates were in New Mexico, 13.1 per 100,000. And Oklahoma, cialis 5 11.4 per 100,000. Like this story?.

Sign up for our newsletter. Nationally, the rural erectile dysfunction treatment death rate last week was 38% higher than the metropolitan death rate (1.8 per 100,000 residents vs, 1.3 per 100,000 residents) The Daily cialis 5 Yonder’s analysis of erectile dysfunction treatment in rural America covers May 23-29, 2021. New cases in rural counties were at their lowest point in nearly a year and were down 91% from the peak, which occurred in January.Rural deaths, while increasing for the past two weeks, were still about 80% below the peak of 4,127 deaths set in mid-January.The number of rural counties on the red-zone list dropped by about a quarter last week, from 259 to 197. Red-zone counties are defined as cialis 5 having 100 or more new cases per 100,000 residents in a one-week period. The White House erectile dysfunction treatment team says that counties on the red-zone list should take additional measures to contain the erectile dysfunction.Montana added the largest number of rural counties to the red-zone list last week, with seven.

Missouri added six, and Washington added 5.Michigan, a recent hotspot for the cialis, dropped 20 rural counties from its red-zone list. Only 20% cialis 5 of the state’s 57 nonmetropolitan counties were on the red-zone list last week. Just a month ago, all but three of the state’s rural counties were on the red-zone list.Washington became the new rural red-zone leader last week, with more than half of its 18 nonmetropolitan counties on the list.West Virginia had the highest rate of new s last week, at 113 per 100,000 residents. Washington was a close second at 108 per 100,000. (Washington had the highest metropolitan new- rate, at 108 per 100,000).Half of cialis 5 the nations’ 1,976 rural counties saw decreases in s last week.

About a quarter of rural counties had an increase in cases, while another quarter had little change. You Might Also Like.

Just six months after erectile dysfunction treatment cialis best buy treatments were first administered in the United States, more than 60 percent of adults have received at least one dose, including more than 85 percent of those age 65 and older. In recent weeks, this success in vaccination administration, along with mask-wearing, social distancing precautions, and warm weather, has produced a rapid and welcome decline in cases, hospitalizations, and deaths throughout the country. But progress has been uneven across states, with a growing gap as the pace of vaccination slows more in some states than others cialis best buy. Widespread media attention has centered on “treatment hesitancy,” but state-to-state variation in vaccination rates is exhibiting a familiar pattern.

States that have invested more to ensure access to health care are pulling ahead while others lag. States Are Administering cialis best buy New treatment Doses at Different Paces. Health Systems That Offer Better Health Care Access Are Pulling Ahead Early on, all states focused on getting a limited supply of shots in the arms of the people most at risk. The pace of vaccination was fairly consistent across states.

All reached 35 percent of adults with at least cialis best buy one dose by around the first week of April. However, as supply and eligibility opened up, differences emerged. While the pace of vaccination has accelerated in California, Washington, and several Northeast states, cialis best buy it is slowing in others — particularly in Southern states like Louisiana, Mississippi, and Alabama. There is now a nearly 40 percentage-point difference in the share of adults with at least one treatment dose between high- and low-vaccination states.

What accounts for the recent changing pace of vaccination?. Several factors are likely at play, but when we compare the patterns of vaccination to the Commonwealth Fund’s annual State Scorecard rankings, it is hard to miss that states that have previously performed cialis best buy well on measures of access and patient engagement are seeing higher rates of treatment administration, while lower-performing states are progressing more slowly. Opportunities in All States to Reach High Vaccination Levels Much of the focus around vaccination rates has centered on the willingness of people to take a treatment. But recent estimates indicate that adult hesitancy in most states may not be a barrier to reaching target adult vaccination levels of 70 percent to 80 percent.

As illustrated in this exhibit, using hesitancy estimates from the federal government and current vaccination rates, we see adult hesitancy rates around 10 percent to cialis best buy 20 percent in most states. This means that even in states with lower vaccination rates, there is a significant target population that would likely take the treatment under the right circumstances. Efforts to educate the public about the dangers of erectile dysfunction treatment and the effectiveness and safety of the treatments will continue to be important. But with such a large proportion of people open to receiving the treatment, there is an opportunity to employ cialis best buy near-term access strategies, particularly for communities of color that face greater access barriers.

The challenge may be more formidable in states that have invested less in ensuring access to care and public health services, including those with largely rural populations. Moving Forward With demand at mass vaccination sites starting to decline, ongoing progress will depend on local approaches that deliver treatments to the places most convenient and cialis best buy comfortable for communities. Most important will be ensuring that a broad variety of providers — particularly ambulatory clinics, primary care doctors, and pediatricians — have the treatment available for their patients. Allowing people to get the treatment in a familiar setting will improve their comfort level and increase their options.

Wisconsin has been putting treatments in community provider offices since early in the vaccination campaign cialis best buy with success. North Dakota, which has smaller, more rural facilities that need less supply, has set up ua-cold freezer storage that allows the state to distribute smaller treatment allocations to providers. States also have shorter-term options, like mobile vaccination sites or pop-up clinics, as Massachusetts and Vermont have done, or ensuring walk-in appointments, like New York has. In addition to cialis best buy enhancing access in traditional settings, many states are employing creative campaigns that allow residents to get their shot at a baseball game, or enjoy a free beer, a $100 savings bond or exclusive lottery ticket entry with proof of vaccination.

Combined with more explicit equitable distribution strategies like those employed in California that reserve supply for high-need neighborhoods, these access improvements could help close remaining disparities and bump all states to a vaccination level that will help the U.S. Ward off cialis best buy variants and future outbreaks. Still, the vaccination rollout is showing once again that states that do more to enable access to health care services can leverage that investment in a crisis, whether it is a cialis or natural disaster. erectile dysfunction treatment highlights the importance of sustaining primary care and public health infrastructure over time so all Americans can receive the services they need not just in times of crisis, but in their day-to-day lives.Explore full-page map While the number of new erectile dysfunction treatment s in rural America continued to decline last week, the number of erectile dysfunction treatment-related deaths grew for the second week in row.

New s in rural America dropped by 18% last week, cialis best buy marking the sixth consecutive week of declining cases in the nation’s nonmetropolitan counties. Cases fell from 25,876 two weeks ago to 21,179 last week, according to a Daily Yonder analysis of data provided by USA Facts. During the same period, new erectile dysfunction treatment-related deaths in rural counties grew by about 25%, from 681 two weeks ago to 849 last week. Deaths have grown cialis best buy by 40% over the past three weeks in rural counties, while climbing only 4% in metropolitan counties.

Oklahoma had the largest number of erectile dysfunction treatment-related deaths in rural counties last week, with 152. The next greatest numbers of rural deaths were in New Mexico, 90. Michigan, 53 cialis best buy. And Georgia, 51.

Maryland, where only about 3% of the population lives in a cialis best buy rural county, had the highest rate of rural erectile dysfunction treatment-related deaths last week. The 30 rural deaths there equaled a rate of 19.9 deaths per 100,000 residents. The next highest rural death rates were in New Mexico, 13.1 per 100,000. And Oklahoma, 11.4 per 100,000 cialis best buy.

Like this story?. Sign up for our newsletter. Nationally, the rural erectile dysfunction treatment death rate last week was 38% higher than cialis best buy the metropolitan death rate (1.8 per 100,000 residents vs, 1.3 per 100,000 residents) The Daily Yonder’s analysis of erectile dysfunction treatment in rural America covers May 23-29, 2021. New cases in rural counties were at their lowest point in nearly a year and were down 91% from the peak, which occurred in January.Rural deaths, while increasing for the past two weeks, were still about 80% below the peak of 4,127 deaths set in mid-January.The number of rural counties on the red-zone list dropped by about a quarter last week, from 259 to 197.

Red-zone counties cialis best buy are defined as having 100 or more new cases per 100,000 residents in a one-week period. The White House erectile dysfunction treatment team says that counties on the red-zone list should take additional measures to contain the erectile dysfunction.Montana added the largest number of rural counties to the red-zone list last week, with seven. Missouri added six, and Washington added 5.Michigan, a recent hotspot for the cialis, dropped 20 rural counties from its red-zone list. Only 20% of the state’s 57 nonmetropolitan counties were on the red-zone list last cialis best buy week.

Just a month ago, all but three of the state’s rural counties were on the red-zone list.Washington became the new rural red-zone leader last week, with more than half of its 18 nonmetropolitan counties on the list.West Virginia had the highest rate of new s last week, at 113 per 100,000 residents. Washington was a close second at 108 per 100,000. (Washington had the highest metropolitan new- rate, at 108 per 100,000).Half of the nations’ 1,976 cialis best buy rural counties saw decreases in s last week. About a quarter of rural counties had an increase in cases, while another quarter had little change.

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AbstractIntroduction http://abelvettes.com/?page_id=6 can you take viagra and cialis at the same time. 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 can you take viagra and cialis at the same time 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 can you take viagra and cialis at the same time and her paternal grandmother and was therefore concerned about her relatives. Her sister and maternal aunt also had gastric cancer.

She was can you take viagra and cialis at the same time 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 can you take viagra and cialis at the same time 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 can you take viagra and cialis at the same time 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 you take viagra and cialis at the same time 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 can you take viagra and cialis at the same time (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 low price cialis 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)..

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)..