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A former cipro cialis aide to can you get cipro without a prescription New York Gov. Andrew Cuomo can you get cipro without a prescription has been the first to file a criminal complaint with law enforcement after she was allegedly sexually harassed and groped in his office. Do You Think Gov. Andrew Cuomo can you get cipro without a prescription Resign?.

Yes .67%No.26%Undecided.5%Don't care.2%Back to VoteSubscribe to the resultsThe victim, identified in the Attorney General’s report as “Executive Assistant #1,” filed the complaint with the Albany County Sheriff's Department this week, making her the first of Cuomo’s alleged victims to seek criminal charges.Her name has not been released.“Since approximately late 2019, the governor engaged in a pattern of inappropriate conduct with Executive Assistant #1,” the report states.Among the claims made in the can you get cipro without a prescription complaint include intimate hugs, kisses, unwanted touching and grabbing of her privates, as well as inappropriate and suggestive remarks allegedly made by the governor.
“I mean it was … he was like cupping my breast. He cupped my breast,” the woman told investigators on AG Letitia James’ team. €œI have to tell you it was … at the moment I was in such shock that I could just tell you that I just remember looking down seeing his hand, seeing the top of my bra and I remember it was like a little even the cup—the kind of bra that I had to the point I could tell you doesn’t really fit me properly, it was a little loose, I just remember seeing exactly that.” The criminal complaint comes as the governor fends of calls for him to resign from as high up as President Joe Biden, as well as top elected officials on both sides of the aisle and prominent Democrats in New York, many of whom were put in their positions by Cuomo.During can you get cipro without a prescription a lengthy briefing on Friday, Aug. 6, lawyers for Cuomo held a public briefing where they sought to pick apart his complainants’ claims while discounting their accounts and alleging the independent investigators failed to question some of his top aides regarding some of the allegations.Despite calls for his head and his job, Rita Glavin, Cuomo’s attorney, discredited some of the claims made against the governor, while saying that they have not received all of the necessary transcripts and documents while alluding to the fact that the independent investigation was pre-determined.Glavin said that she received a call from another lawyer months back who was questioning the investigators’ integrity and claiming that “the manner of questioning alarmed this lawyer.” “I was being warned that minds were made up and that questions pushed back on evidence that as favorable to the governor and particular information the witness had to provide about credibility,” she said.

€œThe governor deserves to be treated fairly, and can you get cipro without a prescription he must be. That did not happen can you get cipro without a prescription here. This was one-sided, and he was ambushed. There’s a rule of law in this can you get cipro without a prescription country and I believe in it.

Give us the opportunity to have the evidence and we’re going to give our response.” Click here to sign up for Daily Voice's free daily emails and news alerts.Some regions in New York are seeing some buy antibiotics relief, but the Hudson Valley isn't one of them as it saw another rise in the buy antibiotics rate as a two-week trend continues as variants of the cipro continue to rapidly spread.In the past week, according to the Department of Health, the seven-day average positive buy antibiotics rate of those tested in can you get cipro without a prescription the Hudson Valley has dramatically jumped from under 2.30 percent to 2.84 percent as of Thursday, Aug. 5, down up from 2.75 percent the previous day.Statewide, the positive rate jumped from 2.46 percent to 2.79 percent in that same time frame.No new cipro-related fatalities were reported in the Hudson Valley, though deaths were reported in the Bronx, Kings, Queens, Saratoga, and Warren counties.Average seven-day rates in the state’s 10 regions on Aug. 5, according can you get cipro without a prescription to the state Department of Health:Capital Region. 3.94 percent (up .11 percent);Central New York.

3.56 percent (up .23 can you get cipro without a prescription percent);Long Island. 3.36 percent (down can you get cipro without a prescription .01 percent);Finger Lakes. 3.10 percent (up .27 percent);Western New York. 3.07 percent can you get cipro without a prescription (down .10 percent);Mohawk Valley.

3.05 percent (up .02 percent);Hudson Valley. 2.84 percent (up .09 can you get cipro without a prescription percent);New York City. 2.52 percent can you get cipro without a prescription (up .03 percent);North Country. 2.52 percent up .03 percent);Southern Tier.

2.27 percent (up .02 percent).Each of the 10 regions has seen a marked spike in can you get cipro without a prescription rates in the past several weeks as the cipro continues spreading.At the beginning of last month, no single region had an rate above 1 percent.New buy antibiotics cases in the Hudson Valley, according to the Department of Health on Aug. 5, by can you get cipro without a prescription county:Westchester. 148 new (132,327 since the cipro began);Orange County. 91 (49,438);Rockland can you get cipro without a prescription.

88 (47,809);Dutchess. 71 (30,175);Ulster can you get cipro without a prescription. 30 (14,259);Putnam can you get cipro without a prescription. 19 (10,827);Sullivan.

16 (6,857).A breakdown of buy antibiotics deaths in can you get cipro without a prescription the Hudson Valley as of Thursday, Aug. 5:Westchester. 2,297;Rockland. 758;Orange County.

725;Dutchess. 447;Ulster. 258;Putnam. 93;Sullivan.

76.There were 134,202 buy antibiotics tests administered in New York on Aug. 5, according to the Department of Health, resulting in 3,700 newly confirmed s for a 2.76 percent daily rate, nearly identical to the previous day.Seventy-two new buy antibiotics patients were admitted to New York hospitals with the cipro as the number rose to 1,050 being treated statewide, up more than 200 from a week ago.A total of 76 percent of New Yorkers over the age of 18 have received at least one dose of the buy antibiotics treatment, while 69 percent are fully vaccinated. Officials said that 63.7 percent of all New Yorkers have received at least one dose, with 57.5 percent completing the vaccination process.As of Aug. 6, 1,302,982 (3,284 new) first doses have been administered to Hudson Valley residents, while 1,172,713 (1,820 new) have completed the process, both among the highest rates in the state."According to the numbers, New Yorkers are listening to the science and getting their vaccinations," New York Gov.

Andrew Cuomo said. "New Yorkers are doing the right thing to protect their communities and keeping vaccination rates up is crucial as we near back to school season. If you haven't already, get your vaccination as soon as you can." Click here to sign up for Daily Voice's free daily emails and news alerts.Guess who’s back. Former President Donald Trump was at his namesake golf club in Northern Westchester this week, where he took the opportunity to take pop shots at embattled New York Gov.

Andrew Cuomo, who is under fire himself for allegedly sexually harassing 11 women.Cuomo, facing an impending impeachment inquiry from the State Assembly Judiciary Committee, has been under the microscope following the release of the findings of independent investigators’ probe into allegations made against the longtime governor.“Cuomo’s got real problems,” the former president reportedly said at the Republican rally held at the Trump Golf Course in Briarcliff, according to The New York Post. €œIt's going to be a hot couple of weeks in Albany. Senator (Robert) Ortt, (the Senate Republican Minority Leader), is going to have a great time.“Cuomo has found himself in an interesting situation.” According to reports, the fundraiser was attended by approximately 500 guests, who raised upwards of $1 million for the state’s Republican Committee.Other topics touched on by Trump during his speech reportedly included buy antibiotics, Cuomo’s handling of the cipro, as well as shoutouts for gubernatorial candidates Lee Zeldin and Andrew Giuliani, son of former White House aide and New York City Mayor Rudy Giuliani, as well as Republican Elise Stefanik, the Republican Party Policy chairwoman. Congresswoman Elise Stefanik got a shout out from former President Donald Trump in Northern Westchester.Twitter/@EliseStefanikTrump also allegedly targeted New York City Mayor Bill de Blasio while touting that New York is “going to have a Republican governor” when Cuomo’s term is up in 2022.The former president played coy when asked if he planned to attempt to reclaim his seat in the Oval Office in 2024, saying simply that “I’ll make an announcement very soon.

I’ll let you guys know.” Click here to sign up for Daily Voice's free daily emails and news alerts.State Police have released the identities of two Hudson Valley residents who were killed during a two-vehicle crash that also injured six others. Orange County residents Nelson Vivarcampoverde, age 48, and passenger Maria Abrigo, age 36, both from Middletown, were killed around 7 p.m., Wednesday, Aug. 4, following the crash on Route 17 in Wallkill, said Trooper Steven Nevel.The crash involved a 2005 Ford Escape operated by Orange County resident Anthony K. Jones, age 24, of Walden, and a 2004 Toyota Sienna, operated by Vivarcampoverde.Jones was traveling alone while Vivarcampoverde’s vehicle was occupied by seven additional passengers, Nevel said.The Toyota Sienna was traveling in the passing lane of Route 17 parallel to the Ford Escape that was in the driving lane when they collided causing the Ford Escape to leave the roadway, striking an earth embankment, and overturning, said Nevel.The Toyota entered the center median, struck the guide rail, and overturned before coming to rest on the westbound median, Nevel said.Vivarcampoverde and Abrigo were ejected from the vehicle and pronounced dead at the scene, according to Nevel.Two additional passengers were flown from the scene to Westchester Medical Center due to the nature of their injuries, Nevel said.The remaining four passengers were transported by ambulance to Garnet Hospital with serious injuries.

The driver of the Ford Escape was also transported to Garnet Hospital and treated for minor injuries. The cause of the crash remains under investigation. Click here to sign up for Daily Voice's free daily emails and news alerts.There's a new police chief in Rockland County, a man who has served as a police officer in the Hudson Valley for more than 34 years.Interim Chief Jeffrey Wanamaker was selected to serve as the next chief of the town of Clarkstown following a vote by Clarkstown Supervisor George Hoehmann and the Town Board on Thursday, Aug. 5.In addition to the chief's position, the board also voted to elected Lt.

Glenn Cummings to serve as Captain and promoted Sgt. Joseph Dwyer to the rank of Lieutenant for the Town of Clarkstown Police Department.“I am truly honored to serve as your Chief of Police,” said Wanamaker. €œI could not be prouder of this department, our officers and support staff’s professionalism and compassion never wavered in providing superior service to the Clarkstown community, and we will continue to provide the town with exceptional service.”Wanamaker has served with the Clarkstown Police Department for over 34 years. Since joining the department he has been assigned as a patrol officer, detective, patrol sergeant, special operations sergeant, patrol lieutenant, administrative lieutenant, captain, member of both the Honor Guard and Critical Incident Response Team.A graduate of Nyack Schools, Wanamaker received a bachelor of arts from SUNY Oneonta and a master's degree from Seton Hall University.

He attended the FBI National Academy and has received numerous awards throughout his career.Cummings has served with the Clarkstown Police Department for 23 years. He previously held the ranks of patrol officer, patrol sergeant, patrol lieutenant, and administrative lieutenant.Dwyer has served with the Clarkstown Police Department for over 24 years. He has previously held the rank of patrol officer, patrol sergeant and is a member of the Accident Investigation Team. Click here to sign up for Daily Voice's free daily emails and news alerts..

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Start Preamble Office of the Secretary, Health and Human how is cipro taken Service, a fantastic read HHS. Notice and request for comments. Office of the Assistant Secretary for Public Affairs is requesting Start Printed Page 70511 OMB approval for a new father Generic how is cipro taken Clearance. In compliance with the requirement of the Paperwork Reduction Act of 1995, the Office of the Secretary (OS), Department of Health and Human Services, is publishing the following summary of a proposed collection for public comment.

Comments on the ICR must be received on or before January 10, 2022. Submit your how is cipro taken comments to OIRA_submission@omb.eop.gov or via facsimile to (202) 395-5806. Start Further Info Sherrette Funn, Sherrette.Funn@hhs.gov or (202) 795-7714. When requesting information, please include the how is cipro taken document identifier 0990-New-30D and project title for reference.

End Further Info End Preamble Start Supplemental Information Interested persons are invited to send comments regarding this burden estimate or any other aspect of this collection of information, including any of the following subjects. (1) The necessity and utility of the proposed information collection for the proper performance of the agency's functions. (2) the accuracy how is cipro taken of the estimated burden. (3) ways to enhance the quality, utility, and clarity of the information to be collected.

And (4) the use of automated collection techniques or other forms of information technology to minimize the information collection burden. Title of the how is cipro taken Collection. The National Hypertension Control Initiative. Type how is cipro taken of Collection.

Father Generic. OMB No.. 0990-NEW—within OS/Office of how is cipro taken Minority and Health. Abstract.

As part of the federal response to buy antibiotics, the U.S. Department of Health and Human Services (HHS) has funded a new initiative involving two cooperative agreements with the American Heart how is cipro taken Association (AHA) to improve buy antibiotics-related health outcomes by addressing hypertension (high blood pressure) among racial and ethnic minority populations. The $32 million project from the HHS Office of Minority Health (OMH) and the Health Resources and Services Administration (HRSA) Bureau of Primary Health Care will support the implementation of the National Hypertension Control Initiative (NHCI), a national initiative to improve blood pressure control among the most at-risk populations, including racial and ethnic minorities. The NHCI will support 350 participating HRSA-funded health centers by providing patient and provider education and training for effective hypertension control as well as integration of remote blood pressure monitoring technology into the treatment of hypertension how is cipro taken for patients served by participating health centers.

The project will also utilize the American Heart Association's targeted media campaigns and existing partnerships with community-based organizations (CBOs) to help reach Black, Latino, and other impacted communities with (i) culturally and linguistically appropriate messages, (ii) access to blood pressure screenings, and (iii) connection to health centers to encourage proper treatment and management of hypertension of screened individuals. This initiative serves to increase the number of adult patients with controlled hypertension and reduce the potential risk of buy antibiotics-related health outcomes. AHA aims to conduct an evaluation to assess the feasibility of the implementation of each of the three how is cipro taken NHCI strategies. The findings of this evaluation will inform the improvement and tailoring of AHA's communication approaches about the importance of and techniques for improving blood pressure control, including the benefits of accurately measuring, rapidly acting, and having a patient-focused approach to blood pressure control.

Methodology The evaluation of the NHCI project will use a mixed methods design, integrating both quantitative and qualitative data collection and analyses. Three main goals of how is cipro taken data collection will be to. (1) Track and monitor systems change implementation process information from Community Health Centers (CHCs) on a quarterly basis, (2) assess the capacity of NHCI partners to implement the NHCI project, their needs, the strengths and weaknesses of the systems change approach, and the feasibility of the implementation of the NHCI in their organizations and communities, and (3) assess the reach and success of NHCI project strategies implemented by partners. Estimated Annualized Burden TableType of respondentNumber of respondentsNumber responses per respondentAverage burden per response (in hours)Total burden hoursCommunity and Social Service Occupations (CBO quarterly data entry into MERD)53430/60106Consumers (ETS health lesson learning questionnaires)63,600110/6010,600Health care professionals (quarterly data entry in MERD)35041.52,100Health care how is cipro taken professionals (annual focus group)1611.524Community and Social Service Occupations (annual focus group)1611.524Total64,03512,854 Start Signature Sherrette A.

Funn, Paperwork Reduction Act Reports Clearance Officer, Office of the Secretary. End Signature End Supplemental Information [FR Doc. 2021-26805 Filed how is cipro taken 12-9-21. 8:45 am]BILLING CODE 4150-29-PExplore the full-page map New buy antibiotics s in rural America shot up by nearly 50% last week, hitting numbers we haven’t seen since early fall.

Part of the increase may be attributable to a long tail from Thanksgiving interruptions in data reporting. But regional patterns (such as worsening how is cipro taken conditions on the perimeter of the South and continued spread in the Northeast) indicate that the spike is based on conditions on the ground. Three different data sets confirmed the increase, according to a Daily Yonder analysis. Cases were on the rise in data from how is cipro taken USA Facts (the primary source for the Daily Yonder’s analysis), the New York Times, and the Centers for Disease Control and Prevention.

New cases of buy antibiotics in rural counties climbed to about 148,000 last week, an increase of 48,000 (or 48%) from two weeks ago. buy antibiotics-related deaths in rural counties also grew by roughly half last week, climbing from about 1,400 two weeks ago to 2,200 last week. Since the start how is cipro taken of the cipro, 135,339 rural Americans have died from buy antibiotics. Last week, the rural death rate was 130% higher than the metropolitan death rate.

The rural death rate has been roughly twice as high as the metropolitan rate since late August. Metropolitan counties how is cipro taken also had a similar percentage increase in new s and deaths last week. Like this story?. Sign up for how is cipro taken our newsletter.

Data for the Daily Yonder’s analysis of buy antibiotics in rural America comes from the nonprofit USA Facts. This week we supplemented USA Facts data with information from the Centers for Disease Control and Prevention for the state of Florida, which did not include information on deaths through USA Facts. Highs and Lows New Hampshire had the highest rural rate in the how is cipro taken U.S. Last week, at 755 new cases per 100,000 rural residents over the seven-day period.Michigan had the second-highest rural rate at 696 new cases per 100,000 for the week.Vermont and Indiana were third- and fourth-highest respectively, both with rural rates over 600 per 100,000 residents for the week.Three Deep South states had the best rural rates.

These were Georgia, Florida and Alabama.Seven of the 10 states with the lowest rural rates were in the South.The South’s rate cooled after the early stages of the Delta-variant surge this summer and early fall. But the perimeter how is cipro taken of the South saw increased cases last week. Trouble Spots and Red Zones The Northeast and Upper Midwest remained the trouble spots for this phase of the cipro. All eight states that border the Great Lakes how is cipro taken had above-average rates.

Collectively, those states accounted for just under half of all rural s last week. Every county in the Great Lakes states is in the red zone, meaning they have rates of at least 100 new cases per 100,000 for a one-week period. The White House has advised localities in the red zone to take additional measures to contain the cipro.Texas saw a dramatic increase in the number of how is cipro taken rural red-zone counties last week. The state jumped from 21 rural red-zone counties two weeks ago to 86 last week, an increase of 65, or more than 200%.Tennessee’s rural red-zone county list grew by nearly 300%, from 12 two weeks ago to 46 last week.Half of the states had 90% or more of their rural counties in the red zone last week.Only three states (Alabama, Missouri, Oklahoma) had fewer rural red-zone counties last week than they did two weeks ago.Rural rates were higher than metropolitan rates in 36 states last week.

Start Preamble Buy cipro no prescription Office of can you get cipro without a prescription the Secretary, Health and Human Service, HHS. Notice and request for comments. Office of the Assistant Secretary for Public Affairs is requesting Start Printed Page 70511 OMB approval for a new father Generic Clearance can you get cipro without a prescription.

In compliance with the requirement of the Paperwork Reduction Act of 1995, the Office of the Secretary (OS), Department of Health and Human Services, is publishing the following summary of a proposed collection for public comment. Comments on the ICR must be received on or before January 10, 2022. Submit your comments to OIRA_submission@omb.eop.gov or can you get cipro without a prescription via facsimile to (202) 395-5806.

Start Further Info Sherrette Funn, Sherrette.Funn@hhs.gov or (202) 795-7714. When requesting information, please include the document identifier 0990-New-30D can you get cipro without a prescription and project title for reference. End Further Info End Preamble Start Supplemental Information Interested persons are invited to send comments regarding this burden estimate or any other aspect of this collection of information, including any of the following subjects.

(1) The necessity and utility of the proposed information collection for the proper performance of the agency's functions. (2) the can you get cipro without a prescription accuracy of the estimated burden. (3) ways to enhance the quality, utility, and clarity of the information to be collected.

And (4) the use of automated collection techniques or other forms of information technology to minimize the information collection burden. Title of the can you get cipro without a prescription Collection. The National Hypertension Control Initiative.

Type of Collection can you get cipro without a prescription. Father Generic. OMB No..

0990-NEW—within OS/Office of can you get cipro without a prescription Minority and Health. Abstract. As part of the federal response to buy antibiotics, the U.S.

Department of Health and Human Services (HHS) has funded a new initiative involving two cooperative agreements with the American Heart Association (AHA) to improve buy antibiotics-related health outcomes by addressing hypertension (high blood pressure) among racial and ethnic minority populations can you get cipro without a prescription. The $32 million project from the HHS Office of Minority Health (OMH) and the Health Resources and Services Administration (HRSA) Bureau of Primary Health Care will support the implementation of the National Hypertension Control Initiative (NHCI), a national initiative to improve blood pressure control among the most at-risk populations, including racial and ethnic minorities. The NHCI will support 350 participating HRSA-funded health centers by providing patient and provider education and training for effective hypertension control as well as integration of remote blood pressure monitoring technology into the treatment can you get cipro without a prescription of hypertension for patients served by participating health centers.

The project will also utilize the American Heart Association's targeted media campaigns and existing partnerships with community-based organizations (CBOs) to help reach Black, Latino, and other impacted communities with (i) culturally and linguistically appropriate messages, (ii) access to blood pressure screenings, and (iii) connection to health centers to encourage proper treatment and management of hypertension of screened individuals. This initiative serves to increase the number of adult patients with controlled hypertension and reduce the potential risk of buy antibiotics-related health outcomes. AHA aims to conduct an evaluation can you get cipro without a prescription to assess the feasibility of the implementation of each of the three NHCI strategies.

The findings of this evaluation will inform the improvement and tailoring of AHA's communication approaches about the importance of and techniques for improving blood pressure control, including the benefits of accurately measuring, rapidly acting, and having a patient-focused approach to blood pressure control. Methodology The evaluation of the NHCI project will use a mixed methods design, integrating both quantitative and qualitative data collection and analyses. Three main goals of data can you get cipro without a prescription collection will be to.

(1) Track and monitor systems change implementation process information from Community Health Centers (CHCs) on a quarterly basis, (2) assess the capacity of NHCI partners to implement the NHCI project, their needs, the strengths and weaknesses of the systems change approach, and the feasibility of the implementation of the NHCI in their organizations and communities, and (3) assess the reach and success of NHCI project strategies implemented by partners. Estimated Annualized Burden TableType of respondentNumber of respondentsNumber responses per respondentAverage burden per response (in hours)Total burden hoursCommunity and Social Service Occupations (CBO quarterly data entry can you get cipro without a prescription into MERD)53430/60106Consumers (ETS health lesson learning questionnaires)63,600110/6010,600Health care professionals (quarterly data entry in MERD)35041.52,100Health care professionals (annual focus group)1611.524Community and Social Service Occupations (annual focus group)1611.524Total64,03512,854 Start Signature Sherrette A. Funn, Paperwork Reduction Act Reports Clearance Officer, Office of the Secretary.

End Signature End Supplemental Information [FR Doc. 2021-26805 Filed can you get cipro without a prescription 12-9-21. 8:45 am]BILLING CODE 4150-29-PExplore the full-page map New buy antibiotics s in rural America shot up by nearly 50% last week, hitting numbers we haven’t seen since early fall.

Part of the increase may be attributable to a long tail from Thanksgiving interruptions in data reporting. But regional patterns (such as worsening conditions on the perimeter of the South and continued spread in the Northeast) indicate that the spike is can you get cipro without a prescription based on conditions on the ground. Three different data sets confirmed the increase, according to a Daily Yonder analysis.

Cases were on the rise in data from USA Facts (the primary source for the Daily Yonder’s analysis), the New York can you get cipro without a prescription Times, and the Centers for Disease Control and Prevention. New cases of buy antibiotics in rural counties climbed to about 148,000 last week, an increase of 48,000 (or 48%) from two weeks ago. buy antibiotics-related deaths in rural counties also grew by roughly half last week, climbing from about 1,400 two weeks ago to 2,200 last week.

Since the start of the cipro, can you get cipro without a prescription 135,339 rural Americans have died from buy antibiotics. Last week, the rural death rate was 130% higher than the metropolitan death rate. The rural death rate has been roughly twice as high as the metropolitan rate since late August.

Metropolitan counties also had a similar percentage can you get cipro without a prescription increase in new s and deaths last week. Like this story?. Sign up for our newsletter can you get cipro without a prescription.

Data for the Daily Yonder’s analysis of buy antibiotics in rural America comes from the nonprofit USA Facts. This week we supplemented USA Facts data with information from the Centers for Disease Control and Prevention for the state of Florida, which did not include information on deaths through USA Facts. Highs and Lows New Hampshire had the highest rural rate in the can you get cipro without a prescription U.S.

Last week, at 755 new cases per 100,000 rural residents over the seven-day period.Michigan had the second-highest rural rate at 696 new cases per 100,000 for the week.Vermont and Indiana were third- and fourth-highest respectively, both with rural rates over 600 per 100,000 residents for the week.Three Deep South states had the best rural rates. These were Georgia, Florida and Alabama.Seven of the 10 states with the lowest rural rates were in the South.The South’s rate cooled after the early stages of the Delta-variant surge this summer and early fall. But the perimeter of the can you get cipro without a prescription South saw increased cases last week.

Trouble Spots and Red Zones The Northeast and Upper Midwest remained the trouble spots for this phase of the cipro. All eight states that border the Great Lakes had above-average rates. Collectively, those states accounted for just under half of all rural s last week.

Every county in the Great Lakes states is in the red zone, meaning they have rates of at least 100 new cases per 100,000 for a one-week period. The White House has advised localities in the red zone to take additional measures to contain the cipro.Texas saw a dramatic increase in the number of rural red-zone counties last week. The state jumped from 21 rural red-zone counties two weeks ago to 86 last week, an increase of 65, or more than 200%.Tennessee’s rural red-zone county list grew by nearly 300%, from 12 two weeks ago to 46 last week.Half of the states had 90% or more of their rural counties in the red zone last week.Only three states (Alabama, Missouri, Oklahoma) had fewer rural red-zone counties last week than they did two weeks ago.Rural rates were higher than metropolitan rates in 36 states last week.

What should I watch for while taking Cipro?

Tell your doctor or health care professional if your symptoms do not improve.

Do not treat diarrhea with over the counter products. Contact your doctor if you have diarrhea that lasts more than 2 days or if it is severe and watery.

You may get drowsy or dizzy. Do not drive, use machinery, or do anything that needs mental alertness until you know how Cipro affects you. Do not stand or sit up quickly, especially if you are an older patient. This reduces the risk of dizzy or fainting spells.

Cipro can make you more sensitive to the sun. Keep out of the sun. If you cannot avoid being in the sun, wear protective clothing and use sunscreen. Do not use sun lamps or tanning beds/booths.

Avoid antacids, aluminum, calcium, iron, magnesium, and zinc products for 6 hours before and 2 hours after taking a dose of Cipro.

Will cipro work for strep throat

Many health experts say creating a small, select group of friends or family to socialise with in-person could be key will cipro work for strep throat to a more sustainable lockdown period, should the need Kamagra online pharmacy uk arise. While plans for reopening Victoria remain unclear, the Andrews government is considering the concept of a social 'bubble'.The antibiotics cipro has shown that even the most introverted people need physical human will cipro work for strep throat contact from time to time. While digital happy hours, pub trivia, and children's playdates have sated the immediate urge for connection, kids are still missing their friends, families are missing their relatives, and will cipro work for strep throat couples are missing, well, each other.But while it's important to keep everyone sane and mentally healthy, we also need to do it safely."We do know that this is particularly challenging for those who live on their own, and we're going to try to support them, and indeed every Victorian," Premier Daniel Andrews said during a press conference, though he has yet to reveal his reopening plan which is scheduled to be unveiled on Sunday.The 'bubble' conceptWith numbers dropping, but still presenting a risk to Victorians, the state government is considering the concept of a quarantine 'pod' or 'bubble'—the idea of nominating a small group of people or households, within your town or city, with whom you can socialise in-person.Like what you see?.

Sign up to our bodyandsoul.com.au newsletter to read more stories like this.The idea of the bubble is that it takes a more compassionate approach, a will cipro work for strep throat route New Zealand, Canada, and the UK took, is ultimately more sustainable. It can allow children will cipro work for strep throat to continue their social development, while for those living on their own, it aims to combat loneliness and the mental health issues that can arise.Exposure is easily contained though it's still not completely without risk and caveats.How would it work?. Medical experts agree the smaller the group, the better.

The larger the group, the higher the risk of transmission, and they recommend always paying attention to local health guidelines on the recommended maximum number of people.Who you choose would have to be exclusive and finite, Dr Mary-Louise McLaws, Professor of Epidemiology at the University of New South Wales, wrote for The Conversation."If you were 'bubbling' with one household, you could not just decide to change to a different one whenever you please," she said."If you did need to alter your bubble, there would have to be a 14-day gap between one set of people leaving and another group joining, to reduce the risk of transmission between bubbles."Set the guidelinesTransparency and communication are key to establishing a will cipro work for strep throat successful bubble. You're entering will cipro work for strep throat into a social contract, after all. Communicating with each other about what activities are OK and what is off-limits is crucial to forming a successful pod."The more communication you have upfront about the various scenarios and how they might play out, the easier it’ll be to navigate those situations as they arise," Dr Julia Marcus, an infectious disease epidemiologist at Harvard Medical School, told the New York Times.It's will cipro work for strep throat also important to pick a family/friends that are as cautious as you are.

Those who are staying home and wearing masks when they go will cipro work for strep throat out. And if group will cipro work for strep throat members break the house rules, you need to be prepared to have those conversations, difficult as they may be."Establish routine check-ins. Set some dates to re-evaluate whether the bubble is working," Carolyn Cannuscio, director of research at the Center for Public Health Initiatives at the University of Pennsylvania told ABC.And, Dr Tara Kirk Sell, a professor at Johns Hopkins Bloomberg School of Public Health, if you do make a mistake and break the rules yourself, own up to it."Don't be a jerk," she said.Never a truer word spoken..

Many health experts say creating a small, select group of can you get cipro without a prescription friends or family to socialise with in-person could be key to a more sustainable lockdown period, should the need arise. While plans for reopening Victoria remain unclear, the Andrews government is considering the concept of a social 'bubble'.The can you get cipro without a prescription antibiotics cipro has shown that even the most introverted people need physical human contact from time to time. While digital happy hours, pub trivia, and children's playdates have sated the immediate urge for connection, kids are still missing their friends, families are missing their relatives, and couples are missing, well, each other.But while it's important to keep everyone sane and mentally healthy, we also need to do it safely."We do know that this is particularly challenging can you get cipro without a prescription for those who live on their own, and we're going to try to support them, and indeed every Victorian," Premier Daniel Andrews said during a press conference, though he has yet to reveal his reopening plan which is scheduled to be unveiled on Sunday.The 'bubble' conceptWith numbers dropping, but still presenting a risk to Victorians, the state government is considering the concept of a quarantine 'pod' or 'bubble'—the idea of nominating a small group of people or households, within your town or city, with whom you can socialise in-person.Like what you see?. Sign can you get cipro without a prescription up to our bodyandsoul.com.au newsletter to read more stories like this.The idea of the bubble is that it takes a more compassionate approach, a route New Zealand, Canada, and the UK took, is ultimately more sustainable.

It can allow children to continue their social development, while for those living on can you get cipro without a prescription their own, it aims to combat loneliness and the mental health issues that can arise.Exposure is easily contained though it's still not completely without risk and caveats.How would it work?. Medical experts agree the smaller the group, the better. The larger the group, the higher the risk of transmission, and they recommend always paying attention to local health guidelines on the recommended maximum number of people.Who you choose would have to be exclusive and finite, Dr Mary-Louise McLaws, Professor of Epidemiology at the University of New South Wales, wrote for The Conversation."If you were can you get cipro without a prescription 'bubbling' with one household, you could not just decide to change to a different one whenever you please," she said."If you did need to alter your bubble, there would have to be a 14-day gap between one set of people leaving and another group joining, to reduce the risk of transmission between bubbles."Set the guidelinesTransparency and communication are key to establishing a successful bubble. You're entering into a social contract, after can you get cipro without a prescription all.

Communicating with each other about what activities are OK and what is off-limits is crucial to forming a successful pod."The more communication you have upfront about the various scenarios and how they might play out, the easier it’ll be to navigate those situations as they arise," Dr Julia Marcus, an infectious disease epidemiologist at Harvard Medical School, told the New York Times.It's also important to pick a family/friends that are can you get cipro without a prescription as cautious as you are. Those who are staying can you get cipro without a prescription home and wearing masks when they go out. And if group members break the house rules, you need to be prepared to have those conversations, difficult as they may be."Establish routine check-ins can you get cipro without a prescription. Set some dates to re-evaluate whether the bubble is working," Carolyn Cannuscio, director of research at the Center for Public Health Initiatives at the University of Pennsylvania told ABC.And, Dr Tara Kirk Sell, a professor at Johns Hopkins Bloomberg School of Public Health, if you do make a mistake and break the rules yourself, own up to it."Don't be a jerk," she said.Never a truer word spoken..

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AbstractIntroduction go right here cipro 1000mg for uti. 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 cipro 1000mg for uti 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 cipro 1000mg for uti relatives. Her sister and maternal aunt also had gastric cancer.

She was tested for several genes associated cipro 1000mg for uti 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 cipro 1000mg for uti.

Breastcancer. Gastricclinical geneticsgenetic screening/counsellingmolecular geneticsIntroductionGLI-Kruppel family member 3 cipro 1000mg for uti (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 cipro 1000mg for uti 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 cipro 1000mg for uti 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)..

AbstractIntroduction What do you need to buy ventolin can you get cipro without a prescription. 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 can you get cipro without a prescription. 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 can you get cipro without a prescription her relatives.

Her sister and maternal aunt also had gastric cancer. She was tested for several genes associated can you get cipro without a prescription 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 can you get cipro without a prescription 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 can you get cipro without a prescription 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 can you get cipro without a prescription 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 can you get cipro without a prescription 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)..