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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)..

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The NSW Government is investing an additional $4 million to fast-track the redevelopment of Shoalhaven District Memorial Hospital to begin in 2020-21.Minister for cipro calf pain Health Brad Hazzard said the funding boost will bring the total spend for the project to $438 million, which will also support the acquisition order cipro online of nearby Nowra Park.“The NSW Government is committed to investing in regional hospitals to ensure patients receive high-quality healthcare closer to home,” Mr Hazzard said.“The land acquisition of Nowra Park is necessary to provide for the expansion of clincial services at Shoalhaven Hospital.”The existing hospital site with expansion into the adjacent Nowra Park has been identified as the best solution for the redeveloped hospital.Clinical services planning is already well underway to identify the range of health services the Illawarra Shoalhaven community will require into the future. The additional funding will allow planning activities to progress including:Detailed cipro calf pain site investigations, including in-ground investigations. Enabling works, including services diversion cipro calf pain and potential in-ground works. And Design cipro calf pain http://bretmwebb.com/?p=67 works for the redevelopment, including clinical design.

Member for the South Coast Shelley Hancock released new artist impressions and said residents will benefit from the hospital expansion, with new and upgraded health facilities to be delivered sooner.“Additionally, as we can see in these stunning images, the completed hospital will return green space back to the community, with an inclusive playground a key component of the park,” Mrs Hancock said.Member for Kiama Gareth Ward said he’s pleased work can get underway on the expanded hospital as soon as possible.“With the ongoing investments we have already put into the Shoalhaven District Memorial Hospital, this is the next big step after the completion of the $11.8 million hospital car park project this year,” Mr Ward said.Construction will start on the redeveloped hospital in this term of Government, prior to March 2023The SDMH redevelopment is one of 29 health projects announced before the 2019 election and is a part of the NSW Government’s record $10.7 billion investment in health infrastructure over the next 4 years.In the Illawarra Shoalhaven, other health projects include $700 million for a new Shellharbour Hospital, cipro calf pain $37.1 million towards the Bulli Hospital and Aged Care Centre, and the Dapto and Ulladulla HealthOne projects, delivered as part of the $100 million HealthOne program.Artist impressions are available.Minister for Health Brad Hazzard, Member for Kiama Gareth Ward MP and Member for South Coast Shelley Hancock MP have today announced the Illawarra community is a step closer to having a new world-class $700 million Shellharbour Hospital, with the search now on for the ideal site.Minister Hazzard said the NSW Government is calling for proposals from landowners of suitable greenfield sites in the Shellharbour region.“The NSW Government is fulfilling its promise to deliver the $700 million state-of-the-art Shellharbour Hospital on a greenfield site, together with new networked health services, to meet the communities’ healthcare needs,” Mr Hazzard said.“We’re launching a thorough site selection process to secure a hospital site that is convenient, accessible and best placed to provide future health services to communities across the entire Illawarra region.”The public site selection process is now open, inviting landowners to nominate potentially suitable sites for consideration.Submissions will close on Friday 4 December.Member for Kiama Gareth Ward said finding the right site was key to unlocking the future health growth of the Shellharbour region.“Building Shellharbour Hospital on a new site will enable the expansion of health services which will ease waiting list pressures across the region,” Mr Ward said.“It will also allow for a contemporary new mental health facility, better transport links and opportunities for further expansion in the future.”Member for South Coast Shelley Hancock said local clinicians, staff and the broader community all have a vital role to play in planning for the new hospital.“The community’s input will help shape the future of healthcare in our region, ensuring the new hospital is an enormous asset to our local community in providing the best possible health services and creating jobs well into the future,” Mrs Hancock said.“Following further planning and consultation, the new hospital is expected to include acute medical and surgical services, medical imaging, an emergency department, mental health services, outpatient and ambulatory care and a multistorey car park.”The NSW Government has invested $10 billion to deliver more than 130 new and enhanced health facilities statewide since 2011, including $37.1 million towards the Bulli Hospital and Aged Care Centre. In addition, as part of the $100 million HealthOne Program, two new HealthOne projects have been developed in the Illawarra, at Dapto and Ulladulla.The NSW Government is also investing $10.7 billion more over the next four cipro calf pain years, including $900 million for new and upgraded regional hospitals and health facilities for rural and regional areas in 2020-21.To suggest a site visit Colliers websiteTo learn more about the project visit Shellharbour Hospital Redevelopmentor email ISLHD-SHH-Redevelopment@health.nsw.gov.au.

The NSW Government is investing an additional $4 million to fast-track the redevelopment of Shoalhaven District Memorial Hospital to begin in 2020-21.Minister for Health Brad Hazzard said the funding boost will bring the total spend for the project to $438 million, which will also support the acquisition of nearby Nowra Park.“The NSW Government is committed to investing in regional hospitals to ensure patients receive high-quality healthcare closer to home,” Mr Hazzard said.“The land acquisition of Nowra Park is necessary to provide for the expansion of clincial services at Shoalhaven Hospital.”The existing hospital site with expansion into the adjacent Nowra Park has buy cipro over the counter been identified as the best solution for the redeveloped hospital.Clinical services planning is already well underway to identify the range of health services the Illawarra Shoalhaven community will require into the future. The additional funding will allow planning activities to progress including:Detailed site investigations, buy cipro over the counter including in-ground investigations. Enabling works, including services diversion and potential buy cipro over the counter in-ground works.

And Design works for the redevelopment, including clinical design buy cipro over the counter. Member for the South Coast Shelley Hancock released new artist impressions and said residents will benefit from the hospital expansion, with new and upgraded health facilities to be delivered sooner.“Additionally, as we can see in these stunning images, the completed hospital will return green space back to the community, with an inclusive playground a key component of the park,” Mrs Hancock said.Member for Kiama Gareth Ward said he’s pleased work can get underway on the expanded hospital as soon as possible.“With the ongoing investments we have already put into the Shoalhaven District Memorial Hospital, this is the next big step after the completion of the $11.8 million hospital car park project this year,” Mr Ward said.Construction will start on the redeveloped hospital in this term of Government, prior to March 2023The SDMH redevelopment is one of 29 health projects announced before the 2019 election and is a part of the NSW Government’s record $10.7 billion investment in health infrastructure over the next 4 years.In the Illawarra Shoalhaven, other health projects include $700 million for a new Shellharbour Hospital, $37.1 million towards the Bulli Hospital and Aged Care Centre, and the Dapto and Ulladulla HealthOne projects, delivered as part of the $100 million HealthOne program.Artist impressions are available.Minister for Health Brad Hazzard, Member for Kiama Gareth Ward MP and Member for South Coast Shelley Hancock MP have today announced the Illawarra community is a step closer to having a new world-class $700 million Shellharbour Hospital, with the search now on for the ideal site.Minister Hazzard said the NSW Government is calling for proposals from landowners of suitable greenfield sites in the Shellharbour region.“The NSW Government is fulfilling its promise to deliver the $700 million state-of-the-art Shellharbour Hospital on a greenfield site, together with new networked health services, to meet the communities’ buy cipro over the counter healthcare needs,” Mr Hazzard said.“We’re launching a thorough site selection process to secure a hospital site that is convenient, accessible and best placed to provide future health services to communities across the entire Illawarra region.”The public site selection process is now open, inviting landowners to nominate potentially suitable sites for consideration.Submissions will close on Friday 4 December.Member for Kiama Gareth Ward said finding the right site was key to unlocking the future health growth of the Shellharbour region.“Building Shellharbour Hospital on a new site will enable the expansion of health services which will ease waiting list pressures across the region,” Mr Ward said.“It will also allow for a contemporary new mental health facility, better transport links and opportunities for further expansion in the future.”Member for South Coast Shelley Hancock said local clinicians, staff and the broader community all have a vital role to play in planning for the new hospital.“The community’s input will help shape the future of healthcare in our region, ensuring the new hospital is an enormous asset to our local community in providing the best possible health services and creating jobs well into the future,” Mrs Hancock said.“Following further planning and consultation, the new hospital is expected to include acute medical and surgical services, medical imaging, an emergency department, mental health services, outpatient and ambulatory care and a multistorey car park.”The NSW Government has invested $10 billion to deliver more than 130 new and enhanced health facilities statewide since 2011, including $37.1 million towards the Bulli Hospital and Aged Care Centre. In addition, as part of the $100 million HealthOne Program, two new HealthOne projects have been developed in the Illawarra, at Dapto and Ulladulla.The NSW Government is also investing $10.7 billion more over the next four years, including $900 million for new and upgraded regional hospitals and health facilities for rural and regional areas in 2020-21.To suggest a site visit buy cipro over the counter Colliers websiteTo learn more about the project visit Shellharbour Hospital Redevelopmentor email ISLHD-SHH-Redevelopment@health.nsw.gov.au.

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Aug. 29, 2020 -- Chadwick Boseman, the star of the 2018 Marvel Studios megahit Black Panther, died of colon cancer Friday. He was 43. Boseman, who was diagnosed 4 years ago, had kept his condition a secret. He filmed his recent movies ''during and between countless surgeries and chemotherapy," according to a statement issued on his Twitter account.

When the actor was diagnosed in 2016, the cancer was at stage III -- meaning it had already grown through the colon wall -- but then progressed to the more lethal stage IV, meaning it had spread beyond his colon. Messages of condolences and the hashtag #Wakandaforever, referring to the fictional African nation in the Black Panther film, flooded social media Friday evening. Oprah tweeted. "What a gentle gifted SOUL. Showing us all that Greatness in between surgeries and chemo.

The courage, the strength, the Power it takes to do that. This is what Dignity looks like. " Marvel Studios tweeted. "Your legacy will live on forever." Boseman was also known for his role as Jackie Robinson in the movie 42. Coincidentally, Friday was Major League Baseball's Jackie Robinson Day, where every player on every team wears Robinson's number 42 on their jerseys.

Boseman's other starring roles include portraying James Brown in Get on Up and U.S. Supreme Court Justice Thurgood Marshall in Marshall. But his role as King T'Challa in Black Panther, the super hero protagonist, made him an icon and an inspiration. About Colon Cancer Boseman's death reflects a troubling recent trend, says Mark Hanna, MD, a colorectal surgeon at City of Hope, a comprehensive cancer center near Los Angeles. "We have noticed an increasing incidence of colorectal cancer in young adults," says Hanna, who did not treat Boseman.

"I've seen patients as young as their early 20s." About 104,000 cases of colon cancer will be diagnosed this year, according to American Cancer Society estimates, and another 43,000 cases of rectal cancer will be diagnosed. About 12% of those, or 18,000 cases, will be in people under age 50. As the rates have declined in older adults due to screening, rates in young adults have steadily risen. Younger patients are often diagnosed at a later stage than older adults, Hanna says, because patients and even their doctors don't think about the possibility of colon cancer. Because it is considered a cancer affecting older adults, many younger people may brush off the symptoms or delay getting medical attention, Hanna says.

In a survey of 885 colorectal cancer patients conducted by Colorectal Cancer Alliance earlier this year, 75% said they visited two or more doctors before getting their diagnosis, and 11% went to 10 or more before finding out. If found early, colon cancer is curable, Hanna says. About 50% of those with colon cancer will be diagnosed at stage I or II, which is considered localized disease, he says. "The majority have a very good prognosis." The 5-year survival rate is about 90% for both stage I and II. But when it progresses to stage III, the cancer has begun to grow into surrounding tissues and the lymph nodes, Hanna says, and the survival rate for 5 years drops to 75%.

About 25% of patients are diagnosed at stage III, he says. If the diagnosis is made at stage IV, the 5-year survival rate drops to about 10% or 15%, he says. Experts have been trying to figure out why more young adults are getting colon cancer and why some do so poorly. "Traditionally we thought that patients who are older would have a worse outlook," Hanna says, partly because they tend to have other medical conditions too. Some experts say that younger patients might have more ''genetically aggressive disease," Hanna says.

"Our understanding of colorectal cancer is becoming more nuanced, and we know that not all forms are the same." For instance, he says, testing is done for specific genetic mutations that have been tied to colon cancer. "It's not just about finding the mutations, but finding the drug that targets [that form] best." Paying Attention to Red Flags "If you have any of what we call the red flag signs, do not ignore your symptoms no matter what your age is," Hanna says. Those are. In 2018, the American Cancer Society changed its guidelines for screening, recommending those at average risk start at age 45, not 50. The screening can be stool-based testing, such as a fecal occult blood test, or visual, such as a colonoscopy.

Hanna says he orders a colonoscopy if the symptoms suggest colon cancer, regardless of a patient's age. Family history of colorectal cancer is a risk factor, as are being obese or overweight, being sedentary, and eating lots of red meat. Sources Mark Hanna, MD, colorectal surgeon and assistant clinical professor of surgery, City of Hope, Los Angeles. American Cancer Society. "Key Statistics for Colorectal Cancer." Twitter statement.

Chadwick Boseman. American Cancer Society. "Colorectal Cancer Risk Factors." American Cancer Society. '"Colorectal Cancer Rates Rise in Younger Adults." American Society of Clinical Oncology annual meeting, May 29-31, 2020. American Cancer Society "Survival Rates for Colorectal Cancer." American Cancer Society.

"Colorectal Cancer Facts &. Figures. 2017-2019." © 2020 WebMD, LLC. All rights reserved.FRIDAY, Aug. 28, 2020 (HealthDay News) -- As many as 20% of Americans don't believe in treatments, a new study finds.

Misinformed treatment beliefs drive opposition to public treatment policies even more than politics, education, religion or other factors, researchers say. The findings are based on a survey of nearly 2,000 U.S. Adults done in 2019, during the largest measles outbreak in 25 years. The researchers, from the Annenberg Public Policy Center (APPC) of the University of Pennsylvania, found that negative misperceptions about vaccinations. reduced the likelihood of supporting mandatory childhood treatments by 70%, reduced the likelihood of opposing religious exemptions by 66%, reduced the likelihood of opposing personal belief exemptions by 79%.

"There are real implications here for a treatment for buy antibiotics," lead author Dominik Stecula said in an APPC news release. He conducted the research while at APPC and is now an assistant professor of political science at Colorado State University. "The negative treatment beliefs we examined aren't limited only to the measles, mumps and rubella [MMR] treatment, but are general attitudes about vaccination." Stecula called for an education campaign by public health professionals and journalists, among others, to preemptively correct misinformation and prepare the public to accept a buy antibiotics treatment. Overall, there was strong support for vaccination policies. 72% strongly or somewhat supported mandatory childhood vaccination, 60% strongly or somewhat opposed religious exemptions, 66% strongly or somewhat opposed treatment exemptions based on personal beliefs.

"On the one hand, these are big majorities. Well above 50% of Americans support mandatory childhood vaccinations and oppose religious and personal belief exemptions to vaccination," said co-author Ozan Kuru, a former APPC researcher, now an assistant professor of communications at the National University of Singapore. "Still, we need a stronger consensus in the public to bolster pro-treatment attitudes and legislation and thus achieve community immunity," he added in the release. A previous study from the 2018-2019 measles outbreak found that people who rely on social media were more likely to be misinformed about treatments. And a more recent one found that people who got information from social media or conservative news outlets at the start of the buy antibiotics cipro were more likely to be misinformed about how to prevent and hold conspiracy theories about it.

With the antibiotics cipro still raging, the number of Americans needed to be vaccinated to achieve community-wide immunity is not known, the researchers said. The findings were recently published online in the American Journal of Public Health.By Robert Preidt HealthDay Reporter FRIDAY, Aug. 28, 2020 (HealthDay News) -- Breastfeeding mothers are unlikely to transmit the new antibiotics to their babies via their milk, researchers say. No cases of an infant contracting buy antibiotics from breast milk have been documented, but questions about the potential risk remain. Researchers examined 64 samples of breast milk collected from 18 women across the United States who were infected with the new antibiotics (antibiotics) that causes buy antibiotics.

One sample tested positive for antibiotics RNA, but follow-up tests showed that the cipro couldn't replicate and therefore, couldn't infect the breastfed infant, according to the study recently published online in the Journal of the American Medical Association. "Detection of viral RNA does not equate to . It has to grow and multiply in order to be infectious and we did not find that in any of our samples," said study author Christina Chambers, a professor of pediatrics at the University of California, San Diego. She is also director of the Mommy's Milk Human Milk Research Biorepository. "Our findings suggest breast milk itself is not likely a source of for the infant," Chambers said in a UCSD news release.

To prevent transmission of the cipro while breastfeeding, wearing a mask, hand-washing and sterilizing pumping equipment after each use are recommended. "We hope our results and future studies will give women the reassurance needed for them to breastfeed. Human milk provides invaluable benefits to mom and baby," said co-author Dr. Grace Aldrovandi, chief of the Division of Infectious Diseases at UCLA Mattel Children's Hospital in Los Angeles. WebMD News from HealthDay Sources SOURCE.

University of California, San Diego, news release, Aug. 19, 2020 Copyright © 2013-2020 HealthDay. All rights reserved.Nursing home staff will have to be tested regularly for buy antibiotics, and facilities that fail to do so will face fines, the Trump administration said Tuesday. Even though they account for less than 1% of the nation's population, long-term care facilities account for 42% of buy antibiotics deaths in the United States, the Associated Press reported. There have been more than 70,000 deaths in U.S.

Nursing homes, according to the buy antibiotics Tracking Project. It's been months since the White House first urged governors to test all nursing home residents and staff, the AP reported. WebMD News from HealthDay Copyright © 2013-2020 HealthDay. All rights reserved.August 28, 2020 -- Alcohol-based hand sanitizers that are packaged in containers that look like food items or drinks could cause injury or death if ingested, according to a new warning the FDA issued Thursday. Hand sanitizers are being packaged in beer cans, water bottles, juice bottles, vodka bottles and children’s food pouches, the FDA said.

Some sanitizers also contain flavors, such as chocolate or raspberry, which could cause confusion. €œI am increasingly concerned about hand sanitizer being packaged to appear to be consumable products, such as baby food or beverages,” Stephen Hahn, MD, the FDA commissioner, said in a statement. Accidentally drinking hand sanitizer — even a small amount — is potentially lethal to children. €œThese products could confuse consumers into accidentally ingesting a potentially deadly product,” he said. €œIt’s dangerous to add scents with food flavors to hand sanitizers which children could think smells like food, eat and get alcohol poisoning.” For example, the FDA received a report about a consumer who purchased a bottle that looked like drinkable water but was actually hand sanitizer.

In another report, a retailer informed the agency about a hand sanitizer product that was marketed in a pouch that looks like a children’s snack and had cartoons on it. Meanwhile, the FDA's warning list about dangerous hand sanitizers containing methanol continues to grow as some people are drinking the sanitizers to get an alcohol high. Others have believed a rumor, circulated online, that drinking the highly potent and toxic alcohol can disinfect the body, protecting them from buy antibiotics . Earlier this month, the FDA also issued a warning about hand sanitizers contaminated with 1-propanol. Ingesting 1-propanol can cause central nervous system depression, which can be fatal, the agency says.

Symptoms of 1-propanol exposure can include confusion, decreased consciousness, and slowed pulse and breathing. One brand of sanitizer, Harmonic Nature S de RL de MI of Mexico, are labeled to contain ethanol or isopropyl alcohol but have tested positive for 1-propanol contamination. Poison control centers and state health departments have reported an increasing number of adverse events associated with hand sanitizer ingestion, including heart issues, nervous system problems, hospitalizations and deaths, according to the statement. The FDA encouraged consumers and health care professionals to report issues to the MedWatch Adverse Event Reporting program. The agency is working with manufacturers to recall confusing and dangerous products and is encouraging retailers to remove some products from shelves.

The FDA is also updating its list of hand sanitizer products that consumers should avoid. €œManufacturers should be vigilant about packaging and marketing their hand sanitizers in food or drink packages in an effort to mitigate any potential inadvertent use by consumers,” Hahn said..

Aug cipro pills online buy cipro over the counter. 29, 2020 -- Chadwick Boseman, the star of the 2018 Marvel Studios megahit Black Panther, died of colon cancer Friday. He was buy cipro over the counter 43. Boseman, who was diagnosed 4 years ago, had kept his condition a secret.

He filmed his recent movies ''during and between countless surgeries and chemotherapy," according to a statement issued on his Twitter account. When the actor was diagnosed in 2016, the cancer was at stage III -- meaning it had already grown through the colon wall -- but then progressed to the more lethal stage buy cipro over the counter IV, meaning it had spread beyond his colon. Messages of condolences and the hashtag #Wakandaforever, referring to the fictional African nation in the Black Panther film, flooded social media Friday evening. Oprah tweeted.

"What a gentle buy cipro over the counter gifted SOUL. Showing us all that Greatness in between surgeries and chemo. The courage, the strength, the Power it takes to do that. This is buy cipro over the counter what Dignity looks like.

" Marvel Studios tweeted. "Your legacy will live on forever." Boseman was also known for his role as Jackie Robinson in the movie 42. Coincidentally, Friday was Major League Baseball's Jackie Robinson Day, where every player on every team wears Robinson's number 42 on their buy cipro over the counter jerseys. Boseman's other starring roles include portraying James Brown in Get on Up and U.S.

Supreme Court Justice Thurgood Marshall in Marshall. But his role as King T'Challa in Black Panther, the super hero protagonist, made buy cipro over the counter him an icon and an inspiration. About Colon Cancer Boseman's death reflects a troubling recent trend, says Mark Hanna, MD, a colorectal surgeon at City of Hope, a comprehensive cancer center near Los Angeles. "We have noticed an increasing incidence of colorectal cancer in young adults," says Hanna, who did not treat Boseman.

"I've seen patients as young as their early 20s." About 104,000 cases of colon cancer will be diagnosed this year, according to American Cancer Society estimates, and another 43,000 cases of rectal cancer will buy cipro over the counter be diagnosed. About 12% of those, or 18,000 cases, will be in people under age 50. As the buy cipro over the counter rates have declined in older adults due to screening, rates in young adults have steadily risen. Younger patients are often diagnosed at a later stage than older adults, Hanna says, because patients and even their doctors don't think about the possibility of colon cancer.

Because it is considered a cancer affecting older adults, many younger people may brush off the symptoms or delay getting medical attention, Hanna says. In a survey of 885 colorectal buy cipro over the counter cancer patients conducted by Colorectal Cancer Alliance earlier this year, 75% said they visited two or more doctors before getting their diagnosis, and 11% went to 10 or more before finding out. If found early, colon cancer is curable, Hanna says. About 50% of those with colon cancer will be diagnosed at stage I or II, which is considered localized disease, he says.

"The majority buy cipro over the counter have a very good prognosis." The 5-year survival rate is about 90% for both stage I and II. But when it progresses to stage III, the cancer has begun to grow into surrounding tissues and the lymph nodes, Hanna says, and the survival rate for 5 years drops to 75%. About 25% of patients are diagnosed at stage III, he says. If the diagnosis is made at stage IV, the 5-year survival buy cipro over the counter rate drops to about 10% or 15%, he says.

Experts have been trying to figure out why more young adults are getting colon cancer and why some do so poorly. "Traditionally we thought that patients who are older would have a worse outlook," Hanna says, partly because they tend to have other medical conditions too. Some experts say that younger patients might buy cipro over the counter have more ''genetically aggressive disease," Hanna says. "Our understanding of colorectal cancer is becoming more nuanced, and we know that not all forms are the same." For instance, he says, testing is done for specific genetic mutations that have been tied to colon cancer.

"It's not just about finding the mutations, but finding the drug that targets [that form] best." Paying Attention to Red Flags "If you have any of what we call the red flag signs, do not ignore your symptoms no matter what your age is," Hanna says. Those are buy cipro over the counter. In 2018, the American Cancer Society changed its guidelines for screening, recommending those at average risk start at age 45, not 50. The screening can be stool-based testing, such as a fecal occult blood test, or visual, such as a colonoscopy.

Hanna says he orders buy cipro over the counter a colonoscopy if the symptoms suggest colon cancer, regardless of a patient's age. Family history of colorectal cancer is a risk factor, as are being obese or overweight, being sedentary, and eating lots of red meat. Sources Mark Hanna, MD, colorectal surgeon and assistant clinical professor of surgery, City of Hope, Los Angeles. American Cancer buy cipro over the counter Society.

"Key Statistics for Colorectal Cancer." Twitter statement. Chadwick Boseman buy cipro over the counter. American Cancer Society. "Colorectal Cancer Risk Factors." American Cancer Society.

'"Colorectal Cancer Rates Rise in Younger Adults." American Society of Clinical buy cipro over the counter Oncology annual meeting, May 29-31, 2020. American Cancer Society "Survival Rates for Colorectal Cancer." American Cancer Society. "Colorectal Cancer Facts &. Figures.

2017-2019." © 2020 WebMD, LLC. All rights reserved.FRIDAY, Aug. 28, 2020 (HealthDay News) -- As many as 20% of Americans don't believe in treatments, a new study finds. Misinformed treatment beliefs drive opposition to public treatment policies even more than politics, education, religion or other factors, researchers say.

The findings are based on a survey of read this article nearly 2,000 U.S. Adults done in 2019, during the largest measles outbreak in 25 years. The researchers, from the Annenberg Public Policy Center (APPC) of the University of Pennsylvania, found that negative misperceptions about vaccinations. reduced the likelihood of supporting mandatory childhood treatments by 70%, reduced the likelihood of opposing religious exemptions by 66%, reduced the likelihood of opposing personal belief exemptions by 79%.

"There are real implications here for a treatment for buy antibiotics," lead author Dominik Stecula said in an APPC news release. He conducted the research while at APPC and is now an assistant professor of political science at Colorado State University. "The negative treatment beliefs we examined aren't limited only to the measles, mumps and rubella [MMR] treatment, but are general attitudes about vaccination." Stecula called for an education campaign by public health professionals and journalists, among others, to preemptively correct misinformation and prepare the public to accept a buy antibiotics treatment. Overall, there was strong support for vaccination policies.

72% strongly or somewhat supported mandatory childhood vaccination, 60% strongly or somewhat opposed religious exemptions, 66% strongly or somewhat opposed treatment exemptions based on personal beliefs. "On the one hand, these are big majorities. Well above 50% of Americans support mandatory childhood vaccinations and oppose religious and personal belief exemptions to vaccination," said co-author Ozan Kuru, a former APPC researcher, now an assistant professor of communications at the National University of Singapore. "Still, we need a stronger consensus in the public to bolster pro-treatment attitudes and legislation and thus achieve community immunity," he added in the release.

A previous study from the 2018-2019 measles outbreak found that people who rely on social media were more likely to be misinformed about treatments. And a more recent one found that people who got information from social media or conservative news outlets at the start of the buy antibiotics cipro were more likely to be misinformed about how to prevent and hold conspiracy theories about it. With the antibiotics cipro still raging, the number of Americans needed to be vaccinated to achieve community-wide immunity is not known, the researchers said. The findings were recently published online in the American Journal of Public Health.By Robert Preidt HealthDay Reporter FRIDAY, Aug.

28, 2020 (HealthDay News) -- Breastfeeding mothers are unlikely to transmit the new antibiotics to their babies via their milk, researchers say. No cases of an infant contracting buy antibiotics from breast milk have been documented, but questions about the potential risk remain. Researchers examined 64 samples of breast milk collected from 18 women across the United States who were infected with the new antibiotics (antibiotics) that causes buy antibiotics. One sample tested positive for antibiotics RNA, but follow-up tests showed that the cipro couldn't replicate and therefore, couldn't infect the breastfed infant, according to the study recently published online in the Journal of the American Medical Association.

"Detection of viral RNA does not equate to . It has to grow and multiply in order to be infectious and we did not find that in any of our samples," said study author Christina Chambers, a professor of pediatrics at the University of California, San Diego. She is also director of the Mommy's Milk Human Milk Research Biorepository. "Our findings suggest breast milk itself is not likely a source of for the infant," Chambers said in a UCSD news release.

To prevent transmission of the cipro while breastfeeding, wearing a mask, hand-washing and sterilizing pumping equipment after each use are recommended. "We hope our results and future studies will give women the reassurance needed for them to breastfeed. Human milk provides invaluable benefits to mom and baby," said co-author Dr. Grace Aldrovandi, chief of the Division of Infectious Diseases at UCLA Mattel Children's Hospital in Los Angeles.

WebMD News from HealthDay Sources SOURCE. University of California, San Diego, news release, Aug. 19, 2020 Copyright © 2013-2020 HealthDay. All rights reserved.Nursing home staff will have to be tested regularly for buy antibiotics, and facilities that fail to do so will face fines, the Trump administration said Tuesday.

Even though they account for less than 1% of the nation's population, long-term care facilities account for 42% of buy antibiotics deaths in the United States, the Associated Press reported. There have been more than 70,000 deaths in U.S. Nursing homes, according to the buy antibiotics Tracking Project. It's been months since the White House first urged governors to test all nursing home residents and staff, the AP reported.

WebMD News from HealthDay Copyright © 2013-2020 HealthDay. All rights reserved.August 28, 2020 -- Alcohol-based hand sanitizers that are packaged in containers that look like food items or drinks could cause injury or death if ingested, according to a new warning the FDA issued Thursday. Hand sanitizers are being packaged in beer cans, water bottles, juice bottles, vodka bottles and children’s food pouches, the FDA said. Some sanitizers also contain flavors, such as chocolate or raspberry, which could cause confusion.

€œI am increasingly concerned about hand sanitizer being packaged to appear to be consumable products, such as baby food or beverages,” Stephen Hahn, MD, the FDA commissioner, said in a statement. Accidentally drinking hand sanitizer — even a small amount — is potentially lethal to children. €œThese products could confuse consumers into accidentally ingesting a potentially deadly product,” he said. €œIt’s dangerous to add scents with food flavors to hand sanitizers which children could think smells like food, eat and get alcohol poisoning.” For example, the FDA received a report about a consumer who purchased a bottle that looked like drinkable water but was actually hand sanitizer.

In another report, a retailer informed the agency about a hand sanitizer product that was marketed in a pouch that looks like a children’s snack and had cartoons on it. Meanwhile, the FDA's warning list about dangerous hand sanitizers containing methanol continues to grow as some people are drinking the sanitizers to get an alcohol high. Others have believed a rumor, circulated online, that drinking the highly potent and toxic alcohol can disinfect the body, protecting them from buy antibiotics . Earlier this month, the FDA also issued a warning about hand sanitizers contaminated with 1-propanol.

Ingesting 1-propanol can cause central nervous system depression, which can be fatal, the agency says. Symptoms of 1-propanol exposure can include confusion, decreased consciousness, and slowed pulse and breathing. One brand of sanitizer, Harmonic Nature S de RL de MI of Mexico, are labeled to contain ethanol or isopropyl alcohol but have tested positive for 1-propanol contamination. Poison control centers and state health departments have reported an increasing number of adverse events associated with hand sanitizer ingestion, including heart issues, nervous system problems, hospitalizations and deaths, according to the statement.

The FDA encouraged consumers and health care professionals to report issues to the MedWatch Adverse Event Reporting program. The agency is working with manufacturers to recall confusing and dangerous products and is encouraging retailers to remove some products from shelves. The FDA is also updating its list of hand sanitizer products that consumers should avoid. €œManufacturers should be vigilant about packaging and marketing their hand sanitizers in food or drink packages in an effort to mitigate any potential inadvertent use by consumers,” Hahn said..

Madera de cipres caracteristicas y usos

This report madera de cipres caracteristicas y usos includes demographic information about the provider, their operational status, beneficiary status, and planned resumption of normal operations. This information is provided whether or not a PHE has been declared. We are now developing a streamlined, automated process to standardize submission of this information directly by the provider during emergencies and eliminating the need for SA to provide it.

It will madera de cipres caracteristicas y usos consist of a public facing web form. This information will be used by CMS to receive, triage, respond to and report on requests and/or inquiries for Medicare, Medicaid, and CHIP beneficiaries. This information will be Start Printed Page 66992used to make decisions about approving or denying waiver and flexibility requests and may be used to identify trends that inform CMS Conditions for Coverage or Conditions for Participation policies during public health emergencies, when declared by the President and the HHS Secretary.

Subsequent to madera de cipres caracteristicas y usos the Emergency information collection request, we are revising the package to include a second form, Healthcare Facility Status Workflow, which is for operational status information which will be used to assist providers in delivering critical care to beneficiaries during emergencies. Form Number. CMS-10752 (OMB control number.

Affected Public. Private Sector. Business or other for-profits and Not-for-profit institutions and State, Local or Tribal Governments.

Number of Respondents. 3,730. Total Annual Responses.

(For policy questions regarding this collection, contact Adriane Saunders at 404-562-7484.) 2. Type of Information Collection Request. Revision of a currently approved collection.

Title of Information Collection. Solicitation for Applications for Medicare Prescription Drug Plan 2022 Contracts. Use.

Coverage for the prescription drug benefit is provided through contracted prescription drug plans (PDPs) or through Medicare Advantage (MA) plans that offer integrated prescription drug and health care coverage (MA-PD plans). Cost Plans that are regulated under Section 1876 of the Social Security Act, and Employer Group Waiver Plans (EGWP) may also provide a Part D benefit. Organizations wishing to provide services under the Prescription Drug Benefit Program must complete an application, negotiate rates, and receive final approval from CMS.

Existing Part D Sponsors may also expand their contracted service area by completing the Service Area Expansion (SAE) application. Collection of this information is mandated in Part D of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (MMA) in Subpart 3. The application requirements are codified in Subpart K of 42 CFR 423 entitled “Application Procedures and Contracts with PDP Sponsors.” The information will be collected under the solicitation of proposals from PDP, MA-PD, Cost Plan, Program of All Inclusive Care for the Elderly (PACE), and EGWP applicants.

The collected information will be used by CMS to. (1) Ensure that applicants meet CMS requirements for offering Part D plans (including network adequacy, contracting requirements, and compliance program requirements, as described in the application), (2) support the determination of contract awards. Form Number.

CMS-10137 (OMB control number. 0938-0936). Frequency.

Business or other for-profits and Not-for-profit institutions and State, Local or Tribal Governments. Number of Respondents. 658.

Total Annual Responses. 331. Total Annual Hours.

1,550. (For policy questions regarding this collection, contact Arianne Spaccarelli at 410-786-5715.) 3. Type of Information Collection Request.

Revision of a currently approved collection. Title of Information Collection. CMS Plan Benefit Package (PBP) and Formulary CY 2022.

Use. Under the Medicare Modernization Act (MMA), Medicare Advantage (MA) and Prescription Drug Plan (PDP) organizations are required to submit plan benefit packages for all Medicare beneficiaries residing in their service area. The plan benefit package submission consists of the Plan Benefit Package (PBP) software, formulary file, and supporting documentation, as necessary.

MA and PDP organizations use the PBP software to describe their organization's plan benefit packages, including information on premiums, cost sharing, authorization rules, and supplemental benefits. They also generate a formulary to describe their list of drugs, including information on prior authorization, step therapy, tiering, and quantity limits. CMS requires that MA and PDP organizations submit a completed PBP and formulary as part of the annual bidding process.

During this process, organizations prepare their proposed plan benefit packages for the upcoming contract year and submit them to CMS for review and approval. CMS uses this data to review and approve the benefit packages that the plans will offer to Medicare beneficiaries. This allows CMS to review the benefit packages in a consistent way across all submitted bids during with incredibly tight timeframes.

This data is also used to populate data on Medicare Plan Finder, which allows beneficiaries to access and compare Medicare Advantage and Prescription Drug plans. Form Number. CMS-R-262 (OMB control number.

Affected Public. Private Sector. Business or other for-profits and Not-for-profit institutions and State, Local or Tribal Governments.

Number of Respondents. 753. Total Annual Responses.

(For policy questions regarding this collection, contact Kristy Holtje at 410-786-2209.) 4. Type of Information Collection Request. Revision of a currently approved collection.

Title of Information Collection. Generic Clearance. Questionnaire Testing and Methodological Research for the Medicare Current Beneficiary Survey (MCBS).

Use. The current generic clearance for MCBS Questionnaire Testing and Methodological Research encompasses development and testing of MCBS questionnaires, instrumentation, and data collection protocols, as well as a mechanism for conducting methodological experiments. The current clearance includes conducting field tests and experiments, including split ballot experiments, within the MCBS production environment, and conducting usability tests.

The purpose of this OMB clearance package is to revise the current clearance to expand the methods to allow for field tests outside of MCBS production Field tests conducted within production do not incur any additional burden on respondents whereas tests conducted outside production must account for additional respondent burden. The MCBS is a continuous, multipurpose survey of a nationally representative sample of aged, disabled, and institutionalized Medicare beneficiaries. The MCBS, which is sponsored by the Centers for Medicare &.

Medicaid Services (CMS), is the only comprehensive source of information on the health status, health care use and expenditures, health insurance coverage, and socioeconomic and demographic characteristics of the entire spectrum of Medicare beneficiaries. The core of the MCBS is a series of interviews with a stratified random sample of the Medicare population, including aged and disabled enrollees, residing in the community or in institutions. Questions are asked about enrollees' patterns of health care use, charges, insurance coverage, and payments over time.

Respondents are asked about their sources of health care coverage and payment, their demographic characteristics, their health and work history, and their family living circumstances. In addition to collecting information through the core questionnaire, the MCBS collects information on special topics. Form Number.

CMS-10549 (OMB control number. 0938-1275). Frequency.

Occasionally. Affected Public. Individuals or Households.

Number of Respondents. 11,655. Total Annual Responses.

11,655. Total Annual Hours. Start Printed Page 669933,947.

(For policy questions regarding this collection, contact William Long at 410-786-7927.) Start Signature Dated. October 16, 2020. William N.

Parham, III, Director, Paperwork Reduction Staff, Office of Strategic Operations and Regulatory Affairs. End Signature End Supplemental Information [FR Doc. 2020-23335 Filed 10-20-20.

8:45 am]BILLING CODE 4120-01-PStart Preamble Start Printed Page 66989 Centers for Medicare &. Medicaid Services (CMS), HHS. Final notice.

This final notice announces our decision to approve The Joint Commission for continued recognition as a national accrediting organization for Ambulatory Surgical Centers that wish to participate in the Medicare or Medicaid programs. The decision announced in this notice is effective on December 20, 2020 through December 20, 2024. Joy Webb (410) 786-1667.

Erin Imhoff (410) 786-2337. I. Background Ambulatory Surgical Centers (ASCs) are distinct entities that operate exclusively for the purpose of furnishing outpatient surgical services to patients.

Under the Medicare program, eligible beneficiaries may receive covered services from an ASC provided certain requirements are met. Section 1832(a)(2)(F)(i) of the Social Security Act (the Act) establishes distinct criteria for a facility seeking designation as an ASC. Regulations concerning provider agreements are at 42 CFR part 489 and those pertaining to activities relating to the survey and certification of facilities are at 42 CFR part 488.

The regulations at 42 CFR part 416 specify the conditions that an ASC must meet in order to participate in the Medicare program, the scope of covered services, and the conditions for Medicare payment for ASCs. Generally, to enter into an agreement, an ASC must first be certified by a State survey agency (SA) as complying with the conditions or requirements set forth in part 416 of our Medicare regulations. Thereafter, the ASC is subject to regular surveys by an SA to determine whether it continues to meet these requirements.

Section 1865(a)(1) of the Act provides that, if a provider entity demonstrates through accreditation by a Centers for Medicare &. Medicaid Services (CMS) approved national accrediting organization (AO) that all applicable Medicare conditions are met or exceeded, we may deem that provider entity as having met the requirements. Accreditation by an AO is voluntary and is not required for Medicare participation.

If an AO is recognized by the Secretary of the Department of Health and Human Services as having standards for accreditation that meet or exceed Medicare requirements, any provider entity accredited by the national accrediting body's approved program may be deemed to meet the Medicare conditions. The AO applying for approval of its accreditation program under part 488, subpart A, must provide CMS with reasonable assurance that the AO requires the accredited provider entities to meet requirements that are at least as stringent as the Medicare conditions. Our regulations concerning the approval of AOs are set forth at § 488.5.

The Joint Commission's (TJC's) current term of approval for its ASC program expires December 20, 2020. II. Application Approval Process Section 1865(a)(3)(A) of the Act provides a statutory timetable to ensure that our review of applications for CMS-approval of an accreditation program is conducted in a timely manner.

The Act provides us 210 days after the date of receipt of a complete application, with any documentation necessary to make the determination, to complete our survey activities and application process. Within 60 days after receiving a complete application, we must publish a notice in the Federal Register that identifies the national accrediting body making the request, describes the request, and provides no less than a 30-day public comment period. At the end of the 210-day period, we must publish a notice in the Federal Register approving or denying the application.

III. Provisions of the Proposed Notice On May 26, 2020 we published a proposed notice in the Federal Register (85 FR 31511), announcing TJC's request for continued approval of its Medicare ASC accreditation program. In the May 26, 2020 proposed notice, we detailed our evaluation criteria.

Under section 1865(a)(2) of the Act and in our regulations at § 488.5, we conducted a review of TJC's Medicare ASC accreditation application in accordance with the criteria specified by our regulations, which include, but are not limited to the following. An administrative review of TJC's. (1) Corporate policies.

(2) financial and human resources available to accomplish the proposed surveys. (3) procedures for training, monitoring, and evaluation of its ASC surveyors. (4) ability to investigate and respond appropriately to complaints against accredited ASCs.

And (5) survey review and decision-making process for accreditation. The comparison of TJC's Medicare ASC accreditation program standards to our current Medicare ASC conditions for coverage (CfCs). A documentation review of TJC's survey process to do the following.

++ Determine the composition of the survey team, surveyor qualifications, and TJC's ability to provide continuing surveyor training. ++ Compare TJC's processes to those we require of state survey agencies, including periodic resurvey and the ability to investigate and respond appropriately to complaints against TJC-accredited ASCs. ++ Evaluate TJC's procedures for monitoring accredited ASCs it has found to be out of compliance with TJC's program requirements.

(This pertains only to monitoring procedures when TJC identifies non-compliance. If noncompliance is identified by a SA through a validation survey, the SA monitors corrections as specified at § 488.9(c)). ++ Assess TJC's ability to report deficiencies to the surveyed ASCs and respond to the ASCs' plans of correction in a timely manner.

++ Establish TJC's ability to provide CMS with electronic data and reports necessary for effective validation and assessment of the organization's survey process. ++ Determine the adequacy of TJC's staff and other resources. ++ Confirm TJC's ability to provide adequate funding for performing required surveys.

++ Confirm TJC's policies with respect to surveys being unannounced. ++ Confirm TJC's policies and procedures to avoid conflicts of interest, including the appearance of conflicts of interest, involving individuals who conduct surveys or participate in accreditation decisions. ++ Obtain TJC's agreement to provide CMS with a copy of the most current accreditation survey together with any other information related to the survey as we may require, including corrective action plans.Start Printed Page 66990 IV.

Analysis of and Responses to Public Comments on the Proposed Notice In accordance with section 1865(a)(3)(A) of the Act, the May 26, 2020 proposed notice also solicited public comments regarding whether TJC's requirements met or exceeded the Medicare CfCs for ASCs. No comments were received in response to our proposed notice. V.

Provisions of the Final Notice A. Differences Between TJC's Standards and Requirements for Accreditation and Medicare Conditions and Survey Requirements We compared TJC's ASC accreditation requirements and survey process with the Medicare CfCs of parts 416, and the survey and certification process requirements of parts 488 and 489. Our review and evaluation of TJC's ASC application, which were conducted as described in section III of this final notice, yielded the following areas where, as of the date of this notice, TJC has completed revising its standards and certification processes in order to do all of the following.

Meet the standard's requirements of all of the following regulations. ++ Section 416.2, to include the regulatory definition of an ASC as a comparable TJC standard instead of a glossary definition. ++ Section 416.43(c)(2), to address the broad requirement under the quality improvement program to track adverse patient events.

++ Section 416.44(c), to include reference to the Health Care Facilities Code (HCFC) of the National Fire Protection Association (NFPA) 99 (2012 edition). ++ Section 416.45(a), to include adequate review of credential and personnel files during survey activity. ++ Section 416.48(a), to include policies regarding the administration of drugs be in accordance with acceptable standards of practice.

++ Section 416.50(a), to provide the correct regulatory citation reference to the CMS standard, “Condition for Coverage—Patient Rights. Notice of Rights.” ++ Section 488.5(a)(4)(iv), to include the requirement that all comparable Medicare CfC citations be included in the findings sections of TJC's survey reports. CMS also reviewed TJC's comparable survey processes, which were conducted as described in section III.

Of this final notice, and yielded the following areas where, as of the date of this notice, TJC has completed revising its survey processes in order to demonstrate that it uses survey processes that are comparable to state survey agency processes by. ++ Modifying TJC's accreditation award letter to facilities to remove the term “lengthen” to eliminate potential conflict as it relates to survey cycle length not to exceed 36 months, as survey cycles for deeming purposes do not exceed this timeframe. ++ Adding references to the HCFC of the NFPA 99 (2012 edition).

(NFPA 99) within its Accreditation Process and Surveyor Activity Guide. ++ Providing clarification to its Surveyor Activity Guide indicating that the 2012 edition of the NFPA Life Safety Code and NFPA 99 applies to ASCs, regardless of the number of patients served. ++ Clarifying the process for TJC's performance of on-site Evidence of Standard Compliance (ESC) processes, including what it means to provide coaching and guidance as part of TJC's ESC survey activities.

B. Term of Approval Based on our review described in section III. And section V.

Of this final notice, we approve TJC as a national accreditation organization for ASCs that request participation in the Medicare program. The decision announced in this final notice is effective December 20, 2020 through December 20, 2024. In accordance with § 488.5(e)(2)(i) the term of the approval will not exceed 6 years.

Due to travel restrictions and the reprioritization of survey activities brought on by the 2019 Novel antibiotics Disease (buy antibiotics) Public Health Emergency (PHE), CMS was unable to observe an ASC survey completed by TJC surveyors as part of the application review process, which is one component of the comparability evaluation. Therefore, we are providing TJC with a shorter period of approval. Based on our discussions with TJC and the information provided in its application, we are confident that TJC will continue to ensure that its accredited ASCs will continue to meet or exceed Medicare standards.

While TJC has taken actions based on the findings annotated in section V.A., of this final notice, (Differences Between TJC's Standards and Requirements for Accreditation and Medicare Conditions and Survey Requirements) as authorized under § 488.8, we will continue ongoing review of TJC's ASC survey processes and will conduct a survey observation once the buy antibiotics PHE has expired. In keeping with CMS's initiative to increase AO oversight broadly, and ensure that our requested revisions by TJC are completed, CMS expects more frequent review of TJC's activities in the future. VI.

Collection of Information and Regulatory Impact Statement This document does not impose information collection requirements, that is, reporting, recordkeeping or third party disclosure requirements. Consequently, there is no need for review by the Office of Management and Budget under the authority of the Paperwork Reduction Act of 1995 (44 U.S.C. 3501 et seq.).

The Administrator of the Centers for Medicare &. Medicaid Services (CMS), Seema Verma, having reviewed and approved this document, authorizes Lynette Wilson, who is the Federal Register Liaison, to electronically sign this document for purposes of publication in the Federal Register. Start Signature Dated.

October 8, 2020. Lynette Wilson, Federal Register Liaison, Department of Health and Human Services.

When this occurs, State Survey Agencies (SA) deliver a provider/beneficiary tracking report can i buy cipro regarding the current status of all buy cipro over the counter affected healthcare providers and their beneficiaries. This report includes demographic information about the provider, their operational status, beneficiary status, and planned resumption of normal operations. This information is provided whether or not a PHE has been declared. We are now developing a streamlined, automated process to standardize submission of this information directly by the provider during emergencies and eliminating the need for buy cipro over the counter SA to provide it.

It will consist of a public facing web form. This information will be used by CMS to receive, triage, respond to and report on requests and/or inquiries for Medicare, Medicaid, and CHIP beneficiaries. This information buy cipro over the counter will be Start Printed Page 66992used to make decisions about approving or denying waiver and flexibility requests and may be used to identify trends that inform CMS Conditions for Coverage or Conditions for Participation policies during public health emergencies, when declared by the President and the HHS Secretary. Subsequent to the Emergency information collection request, we are revising the package to include a second form, Healthcare Facility Status Workflow, which is for operational status information which will be used to assist providers in delivering critical care to beneficiaries during emergencies.

Form Number. CMS-10752 (OMB control number buy cipro over the counter. 0938-1384). Frequency.

Occasionally. Affected Public. Private Sector. Business or other for-profits and Not-for-profit institutions and State, Local or Tribal Governments.

Number of Respondents. 3,730. Total Annual Responses. 3,730.

Total Annual Hours. 3,730. (For policy questions regarding this collection, contact Adriane Saunders at 404-562-7484.) 2. Type of Information Collection Request.

Revision of a currently approved collection. Title of Information Collection. Solicitation for Applications for Medicare Prescription Drug Plan 2022 Contracts. Use.

Coverage for the prescription drug benefit is provided through contracted prescription drug plans (PDPs) or through Medicare Advantage (MA) plans that offer integrated prescription drug and health care coverage (MA-PD plans). Cost Plans that are regulated under Section 1876 of the Social Security Act, and Employer Group Waiver Plans (EGWP) may also provide a Part D benefit. Organizations wishing to provide services under the Prescription Drug Benefit Program must complete an application, negotiate rates, and receive final approval from CMS. Existing Part D Sponsors may also expand their contracted service area by completing the Service Area Expansion (SAE) application.

Collection of this information is mandated in Part D of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (MMA) in Subpart 3. The application requirements are codified in Subpart K of 42 CFR 423 entitled “Application Procedures and Contracts with PDP Sponsors.” The information will be collected under the solicitation of proposals from PDP, MA-PD, Cost Plan, Program of All Inclusive Care for the Elderly (PACE), and EGWP applicants. The collected information will be used by CMS to. (1) Ensure that applicants meet CMS requirements for offering Part D plans (including network adequacy, contracting requirements, and compliance program requirements, as described in the application), (2) support the determination of contract awards.

Form Number. CMS-10137 (OMB control number. 0938-0936). Frequency.

Yearly. Affected Public. Private Sector. Business or other for-profits and Not-for-profit institutions and State, Local or Tribal Governments.

Number of Respondents. 658. Total Annual Responses. 331.

Total Annual Hours. 1,550. (For policy questions regarding this collection, contact Arianne Spaccarelli at 410-786-5715.) 3. Type of Information Collection Request.

Revision of a currently approved collection. Title of Information Collection. CMS Plan Benefit Package (PBP) and Formulary CY 2022. Use.

Under the Medicare Modernization Act (MMA), Medicare Advantage (MA) and Prescription Drug Plan (PDP) organizations are required to submit plan benefit packages for all Medicare beneficiaries residing in their service area. The plan benefit package submission consists of the Plan Benefit Package (PBP) software, formulary file, and supporting documentation, as necessary. MA and PDP organizations use the PBP software to describe their organization's plan benefit packages, including information on premiums, cost sharing, authorization rules, and supplemental benefits. They also generate a formulary to describe their list of drugs, including information on prior authorization, step therapy, tiering, and quantity limits.

CMS requires that MA and PDP organizations submit a completed PBP and formulary as part of the annual bidding process. During this process, organizations prepare their proposed plan benefit packages for the upcoming contract year and submit them to CMS for review and approval. CMS uses this data to review and approve the benefit packages that the plans will offer to Medicare beneficiaries. This allows CMS to review the benefit packages in a consistent way across all submitted bids during with incredibly tight timeframes.

This data is also used to populate data on Medicare Plan Finder, which allows beneficiaries to access and compare Medicare Advantage and Prescription Drug plans. Form Number. CMS-R-262 (OMB control number. 0938-0763).

Frequency. Yearly. Affected Public. Private Sector.

Business or other for-profits and Not-for-profit institutions and State, Local or Tribal Governments. Number of Respondents. 753. Total Annual Responses.

8,090. Total Annual Hours. 74,038. (For policy questions regarding this collection, contact Kristy Holtje at 410-786-2209.) 4.

Type of Information Collection Request. Revision of a currently approved collection. Title of Information Collection. Generic Clearance.

Questionnaire Testing and Methodological Research for the Medicare Current Beneficiary Survey (MCBS). Use. The current generic clearance for MCBS Questionnaire Testing and Methodological Research encompasses development and testing of MCBS questionnaires, instrumentation, and data collection protocols, as well as a mechanism for conducting methodological experiments. The current clearance includes conducting field tests and experiments, including split ballot experiments, within the MCBS production environment, and conducting usability tests.

The purpose of this OMB clearance package is to revise the current clearance to expand the methods to allow for field tests outside of MCBS production Field tests conducted within production do not incur any additional burden on respondents whereas tests conducted outside production must account for additional respondent burden. The MCBS is a continuous, multipurpose survey of a nationally representative sample of aged, disabled, and institutionalized Medicare beneficiaries. The MCBS, which is sponsored by the Centers for Medicare &. Medicaid Services (CMS), is the only comprehensive source of information on the health status, health care use and expenditures, health insurance coverage, and socioeconomic and demographic characteristics of the entire spectrum of Medicare beneficiaries.

The core of the MCBS is a series of interviews with a stratified random sample of the Medicare population, including aged and disabled enrollees, residing in the community or in institutions. Questions are asked about enrollees' patterns of health care use, charges, insurance coverage, and payments over time. Respondents are asked about their sources of health care coverage and payment, their demographic characteristics, their health and work history, and their family living circumstances. In addition to collecting information through the core questionnaire, the MCBS collects information on special topics.

Form Number. CMS-10549 (OMB control number. 0938-1275). Frequency.

Occasionally. Affected Public. Individuals or Households. Number of Respondents http://txresearchanalyst.com/2014/08/231/.

11,655. Total Annual Responses. 11,655. Total Annual Hours.

Start Printed Page 669933,947. (For policy questions regarding this collection, contact William Long at 410-786-7927.) Start Signature Dated. October 16, 2020. William N.

Parham, III, Director, Paperwork Reduction Staff, Office of Strategic Operations and Regulatory Affairs. End Signature End Supplemental Information [FR Doc. 2020-23335 Filed 10-20-20. 8:45 am]BILLING CODE 4120-01-PStart Preamble Start Printed Page 66989 Centers for Medicare &.

Medicaid Services (CMS), HHS. Final notice. This final notice announces our decision to approve The Joint Commission for continued recognition as a national accrediting organization for Ambulatory Surgical Centers that wish to participate in the Medicare or Medicaid programs. The decision announced in this notice is effective on December 20, 2020 through December 20, 2024.

Joy Webb (410) 786-1667. Erin Imhoff (410) 786-2337. I. Background Ambulatory Surgical Centers (ASCs) are distinct entities that operate exclusively for the purpose of furnishing outpatient surgical services to patients.

Under the Medicare program, eligible beneficiaries may receive covered services from an ASC provided certain requirements are met. Section 1832(a)(2)(F)(i) of the Social Security Act (the Act) establishes distinct criteria for a facility seeking designation as an ASC. Regulations concerning provider agreements are at 42 CFR part 489 and those pertaining to activities relating to the survey and certification of facilities are at 42 CFR part 488. The regulations at 42 CFR part 416 specify the conditions that an ASC must meet in order to participate in the Medicare program, the scope of covered services, and the conditions for Medicare payment for ASCs.

Generally, to enter into an agreement, an ASC must first be certified by a State survey agency (SA) as complying with the conditions or requirements set forth in part 416 of our Medicare regulations. Thereafter, the ASC is subject to regular surveys by an SA to determine whether it continues to meet these requirements. Section 1865(a)(1) of the Act provides that, if a provider entity demonstrates through accreditation by a Centers for Medicare &. Medicaid Services (CMS) approved national accrediting organization (AO) that all applicable Medicare conditions are met or exceeded, we may deem that provider entity as having met the requirements.

Accreditation by an AO is voluntary and is not required for Medicare participation. If an AO is recognized by the Secretary of the Department of Health and Human Services as having standards for accreditation that meet or exceed Medicare requirements, any provider entity accredited by the national accrediting body's approved program may be deemed to meet the Medicare conditions. The AO applying for approval of its accreditation program under part 488, subpart A, must provide CMS with reasonable assurance that the AO requires the accredited provider entities to meet requirements that are at least as stringent as the Medicare conditions. Our regulations concerning the approval of AOs are set forth at § 488.5.

The Joint Commission's (TJC's) current term of approval for its ASC program expires December 20, 2020. II. Application Approval Process Section 1865(a)(3)(A) of the Act provides a statutory timetable to ensure that our review of applications for CMS-approval of an accreditation program is conducted in a timely manner. The Act provides us 210 days after the date of receipt of a complete application, with any documentation necessary to make the determination, to complete our survey activities and application process.

Within 60 days after receiving a complete application, we must publish a notice in the Federal Register that identifies the national accrediting body making the request, describes the request, and provides no less than a 30-day public comment period. At the end of the 210-day period, we must publish a notice in the Federal Register approving or denying the application. III. Provisions of the Proposed Notice On May 26, 2020 we published a proposed notice in the Federal Register (85 FR 31511), announcing TJC's request for continued approval of its Medicare ASC accreditation program.

In the May 26, 2020 proposed notice, we detailed our evaluation criteria. Under section 1865(a)(2) of the Act and in our regulations at § 488.5, we conducted a review of TJC's Medicare ASC accreditation application in accordance with the criteria specified by our regulations, which include, but are not limited to the following. An administrative review of TJC's. (1) Corporate policies.

(2) financial and human resources available to accomplish the proposed surveys. (3) procedures for training, monitoring, and evaluation of its ASC surveyors. (4) ability to investigate and respond appropriately to complaints against accredited ASCs. And (5) survey review and decision-making process for accreditation.

The comparison of TJC's Medicare ASC accreditation program standards to our current Medicare ASC conditions for coverage (CfCs). A documentation review of TJC's survey process to do the following. ++ Determine the composition of the survey team, surveyor qualifications, and TJC's ability to provide continuing surveyor training. ++ Compare TJC's processes to those we require of state survey agencies, including periodic resurvey and the ability to investigate and respond appropriately to complaints against TJC-accredited ASCs.

++ Evaluate TJC's procedures for monitoring accredited ASCs it has found to be out of compliance with TJC's program requirements. (This pertains only to monitoring procedures when TJC identifies non-compliance. If noncompliance is identified by a SA through a validation survey, the SA monitors corrections as specified at § 488.9(c)). ++ Assess TJC's ability to report deficiencies to the surveyed ASCs and respond to the ASCs' plans of correction in a timely manner.

++ Establish TJC's ability to provide CMS with electronic data and reports necessary for effective validation and assessment of the organization's survey process. ++ Determine the adequacy of TJC's staff and other resources. ++ Confirm TJC's ability to provide adequate funding for performing required surveys. ++ Confirm TJC's policies with respect to surveys being unannounced.

++ Confirm TJC's policies and procedures to avoid conflicts of interest, including the appearance of conflicts of interest, involving individuals who conduct surveys or participate in accreditation decisions. ++ Obtain TJC's agreement to provide CMS with a copy of the most current accreditation survey together with any other information related to the survey as we may require, including corrective action plans.Start Printed Page 66990 IV. Analysis of and Responses to Public Comments on the Proposed Notice In accordance with section 1865(a)(3)(A) of the Act, the May 26, 2020 proposed notice also solicited public comments regarding whether TJC's requirements met or exceeded the Medicare CfCs for ASCs. No comments were received in response to our proposed notice.

V. Provisions of the Final Notice A. Differences Between TJC's Standards and Requirements for Accreditation and Medicare Conditions and Survey Requirements We compared TJC's ASC accreditation requirements and survey process with the Medicare CfCs of parts 416, and the survey and certification process requirements of parts 488 and 489. Our review and evaluation of TJC's ASC application, which were conducted as described in section III of this final notice, yielded the following areas where, as of the date of this notice, TJC has completed revising its standards and certification processes in order to do all of the following.

Meet the standard's requirements of all of the following regulations. ++ Section 416.2, to include the regulatory definition of an ASC as a comparable TJC standard instead of a glossary definition. ++ Section 416.43(c)(2), to address the broad requirement under the quality improvement program to track adverse patient events. ++ Section 416.44(c), to include reference to the Health Care Facilities Code (HCFC) of the National Fire Protection Association (NFPA) 99 (2012 edition).

++ Section 416.45(a), to include adequate review of credential and personnel files during survey activity. ++ Section 416.48(a), to include policies regarding the administration of drugs be in accordance with acceptable standards of practice. ++ Section 416.50(a), to provide the correct regulatory citation reference to the CMS standard, “Condition for Coverage—Patient Rights. Notice of Rights.” ++ Section 488.5(a)(4)(iv), to include the requirement that all comparable Medicare CfC citations be included in the findings sections of TJC's survey reports.

CMS also reviewed TJC's comparable survey processes, which were conducted as described in section III. Of this final notice, and yielded the following areas where, as of the date of this notice, TJC has completed revising its survey processes in order to demonstrate that it uses survey processes that are comparable to state survey agency processes by. ++ Modifying TJC's accreditation award letter to facilities to remove the term “lengthen” to eliminate potential conflict as it relates to survey cycle length not to exceed 36 months, as survey cycles for deeming purposes do not exceed this timeframe. ++ Adding references to the HCFC of the NFPA 99 (2012 edition).

(NFPA 99) within its Accreditation Process and Surveyor Activity Guide. ++ Providing clarification to its Surveyor Activity Guide indicating that the 2012 edition of the NFPA Life Safety Code and NFPA 99 applies to ASCs, regardless of the number of patients served. ++ Clarifying the process for TJC's performance of on-site Evidence of Standard Compliance (ESC) processes, including what it means to provide coaching and guidance as part of TJC's ESC survey activities. B.

Term of Approval Based on our review described in section III. And section V. Of this final notice, we approve TJC as a national accreditation organization for ASCs that request participation in the Medicare program. The decision announced in this final notice is effective December 20, 2020 through December 20, 2024.

In accordance with § 488.5(e)(2)(i) the term of the approval will not exceed 6 years. Due to travel restrictions and the reprioritization of survey activities brought on by the 2019 Novel antibiotics Disease (buy antibiotics) Public Health Emergency (PHE), CMS was unable to observe an ASC survey completed by TJC surveyors as part of the application review process, which is one component of the comparability evaluation. Therefore, we are providing TJC with a shorter period of approval. Based on our discussions with TJC and the information provided in its application, we are confident that TJC will continue to ensure that its accredited ASCs will continue to meet or exceed Medicare standards.

While TJC has taken actions based on the findings annotated in section V.A., of this final notice, (Differences Between TJC's Standards and Requirements for Accreditation and Medicare Conditions and Survey Requirements) as authorized under § 488.8, we will continue ongoing review of TJC's ASC survey processes and will conduct a survey observation once the buy antibiotics PHE has expired. In keeping with CMS's initiative to increase AO oversight broadly, and ensure that our requested revisions by TJC are completed, CMS expects more frequent review of TJC's activities in the future. VI. Collection of Information and Regulatory Impact Statement This document does not impose information collection requirements, that is, reporting, recordkeeping or third party disclosure requirements.

Consequently, there is no need for review by the Office of Management and Budget under the authority of the Paperwork Reduction Act of 1995 (44 U.S.C. 3501 et seq.). The Administrator of the Centers for Medicare &. Medicaid Services (CMS), Seema Verma, having reviewed and approved this document, authorizes Lynette Wilson, who is the Federal Register Liaison, to electronically sign this document for purposes of publication in the Federal Register.

Cipro and alcohol

Having trouble cipro and alcohol seeing things clearly?. Is it difficult to watch TV, scroll on your phone, look at your computer, read a book or complete close-up tasks such as cross-stitching?. Does it cipro and alcohol make you dizzy when you try to do any of these things?. Have you been told you have a lazy eye?.

Does it seem like cipro and alcohol your glasses aren’t working well?. Has your eye doctor told you there’s nothing wrong with your current glasses prescription?. If you’ve answered yes to any of these questions, you may have a problem with your visual or vestibular system, such as a condition of convergence insufficiency or unilateral or bilateral vestibular hypofunction. The visual sensory system consists of the receptors in our eyes that detect light and the colors of objects, and the ability to cipro and alcohol have fine discrimination and visual acuity through the pupil.

In other words, it helps us see things clearly. The oculomotor system cipro and alcohol is a motor component of the eye function. It helps bring targets onto the fovea, found in the pupil, and keeps the targets on the fovea. This system uses six muscles that we have in each eye to move the eye in cipro and alcohol all positions.

The eye movements perform two functions. First, it holds the image on the retina, and second, it allows the gaze or focus to be shifted. One of the cipro and alcohol two functions of the eye movements is that it holds the images in the retina. There are three ways that oculomotor control works with eye movements to hold images onto the retina.

Visual Fixation cipro and alcohol. Where the retina holds the image of a stationary object on the fovea while your head is stationary, for example, reading a posted sign while standing to look at it.Vestibular Ocular Reflex. Where images of the seen world are held steady on the retina during brief head rotations, for example, following a cipro and alcohol flying insect or animal.Optokinetic Reflex. Where images of what we see in the world are held steady on the retina during sustained low frequency head rotation, such as driving and looking out the window at passing objects or reading.

The second function of the eye movement allows your gaze to be shifted. There are cipro and alcohol three types of gaze shifting. Smooth pursuit. Holds the image of a moving target on our eyesSaccades cipro and alcohol.

Where there is rapid movements of the eyes, for example, when we’re watching a tennis matchVergence. Moving the eyes in opposite directions to provide depth perception such as cipro and alcohol when reading a book or looking at your computer or phone. Vestibular hypofunction occurs when the vestibular nerve is not responding to movement at an accurate rate. The person experiencing this cipro and alcohol can feel “off,” dizzy, unbalanced and may have trouble focusing.

Vestibular therapy and exercises can help with these conditions. Occupational Therapist Dawn Wylie, O.T.R.L., is a vestibular and balance specialist and part of MidMichigan Health’s Rehabilitation Services team. She sees patients in Alma.There are four key phases for a wound to heal successfully:[click image to enlarge] Specialized Wound Treatment Centers have better outcomes because they bring together many disciplines to not only treat wounds, but also to address the underlying barriers to healing.Hemostasis – clotting to control bleeding.Inflammation – swelling occurs as helpful materials are transported to the wound site and invasive microbes are pushed out.Proliferation – a protective layer of tissue is formed.Remodeling – rebuilding of tissue and revascularization and reorganization of the new tissue cipro and alcohol to function like the surrounding tissue.Any factors that interfere with one or more of these phases can prevent wounds from healing. Some of the most common factors include:Poor Circulation – Oxygen and materials needed for healing can’t get to the wound site.

Dead cells cipro and alcohol and harmful materials can’t be carried away.Diabetes – Diabetes interferes with healing in many ways, including lower oxygen levels, weaker immunity and decreased ability to form new skin cells and blood vessels. Diabetic nerve damage can also make it harder to sense a wound and seek treatment. – Harmful bacteria can prolong inflammation and prevent newNutrition Deficits – Wounds need energy, protein and other vital nutrients to heal.Repeat Trauma – Wounds on feet, moving joints and any body parts that may easily get bumped, rubbed or pressured are more susceptible to reopening. Other factors that can interfere with healing include age, sex hormones, stress, obesity, some medications, alcoholism and smoking.Specialized Wound Treatment Addresses Root Causes Specialized Wound Treatment Centers have better outcomes because they bring together many disciplines to not only treat the wound, but to also address these underlying issues that may be barriers to healing. The Wound Treatment Centers at MidMichigan Health have a cross functional team with specialists in these cipro and alcohol and other areas:infectious disease managementcardiologydiabetes educationnutrition managementphysical therapypain managementlab and imagingdebridementhyperbaric oxygen therapyMidMichigan’s specialized Wound Treatment Centers in Alma, Alpena, Clare, Midland and West Branch have a median time to heal of 28 days and 94 percent patient satisfaction.

These outcomes places us among the top 21 percent of nearly 800 Healogics centers nationwide. Healogics is the nation’s leading wound care management company.Take Action cipro and alcohol. Seek Specialized Treatment.If you or someone you love is living with a non-healing wound, don’t wait – seek specialized treatment. Even if you have tried other treatments, but your wound isn’t healing, a multi-disciplinary Wound Treatment Center can identify cipro and alcohol and address the underlying reasons that the wound did not heal.

Call MidMichigan’s Wound Treatment Centers toll free at (877) 683-0800 or visit www.midmichigan.org/woundcenter.Source. Www.ncbi.nlm.nih.gov/pmc/articles/PMC2903966/.

Having trouble my response seeing things buy cipro over the counter clearly?. Is it difficult to watch TV, scroll on your phone, look at your computer, read a book or complete close-up tasks such as cross-stitching?. Does it make you dizzy when you try to do any of these buy cipro over the counter things?.

Have you been told you have a lazy eye?. Does it seem like your glasses aren’t working buy cipro over the counter well?. Has your eye doctor told you there’s nothing wrong with your current glasses prescription?.

If you’ve answered yes to any of these questions, you may have a problem with your visual or vestibular system, such as a condition of convergence insufficiency or unilateral or bilateral vestibular hypofunction. The visual buy cipro over the counter sensory system consists of the receptors in our eyes that detect light and the colors of objects, and the ability to have fine discrimination and visual acuity through the pupil. In other words, it helps us see things clearly.

The oculomotor system is a motor component of buy cipro over the counter the eye function. It helps bring targets onto the fovea, found in the pupil, and keeps the targets on the fovea. This system uses six buy cipro over the counter muscles that we have in each eye to move the eye in all positions.

The eye movements perform two functions. First, it holds the image on the retina, and second, it allows the gaze or focus to be shifted. One of the two buy cipro over the counter functions of the eye movements is that it holds the images in the retina.

There are three ways that oculomotor control works with eye movements to hold images onto the retina. Visual Fixation buy cipro over the counter. Where the retina holds the image of a stationary object on the fovea while your head is stationary, for example, reading a posted sign while standing to look at it.Vestibular Ocular Reflex.

Where images of the seen world are held steady on buy cipro over the counter the retina during brief head rotations, for example, following a flying insect or animal.Optokinetic Reflex. Where images of what we see in the world are held steady on the retina during sustained low frequency head rotation, such as driving and looking out the window at passing objects or reading. The second function of the eye movement allows your gaze to be shifted.

There are three types of gaze buy cipro over the counter shifting. Smooth pursuit. Holds the buy cipro over the counter image of a moving target on our eyesSaccades.

Where there is rapid movements of the eyes, for example, when we’re watching a tennis matchVergence. Moving the eyes in opposite directions buy cipro over the counter to provide depth perception such as when reading a book or looking at your computer or phone. Vestibular hypofunction occurs when the vestibular nerve is not responding to movement at an accurate rate.

The person experiencing this can feel “off,” dizzy, unbalanced and may have trouble buy cipro over the counter focusing. Vestibular therapy and exercises can help with these conditions. Occupational Therapist Dawn Wylie, O.T.R.L., is a vestibular and balance specialist and part of MidMichigan Health’s Rehabilitation Services team.

She sees patients in Alma.There are four key phases for a wound to heal successfully:[click image to enlarge] Specialized Wound Treatment Centers have better outcomes because they bring together many disciplines to not only treat wounds, but also to address the underlying barriers to healing.Hemostasis – clotting to control bleeding.Inflammation – swelling occurs as helpful materials are transported to the wound site and invasive microbes are pushed out.Proliferation – a protective layer of tissue is formed.Remodeling – rebuilding of tissue and revascularization and reorganization of the new tissue to function like the surrounding tissue.Any factors that interfere with one or more of these phases buy cipro over the counter can prevent wounds from healing. Some of the most common factors include:Poor Circulation – Oxygen and materials needed for healing can’t get to the wound site. Dead cells and harmful materials can’t be carried away.Diabetes – Diabetes interferes with healing in many ways, including lower buy cipro over the counter oxygen levels, weaker immunity and decreased ability to form new skin cells and blood vessels.

Diabetic nerve damage can also make it harder to sense a wound and seek treatment. – Harmful bacteria can prolong inflammation and prevent newNutrition Deficits – Wounds need energy, protein and other vital nutrients to heal.Repeat Trauma – Wounds on feet, moving joints and any body parts that may easily get bumped, rubbed or pressured are more susceptible to reopening. Other factors that can interfere with healing include age, sex hormones, stress, obesity, some medications, alcoholism and smoking.Specialized Wound Treatment Addresses Root Causes Specialized Wound Treatment Centers have better outcomes because they bring together many disciplines to not only treat the wound, but to also address these underlying issues that may be barriers to healing. The Wound Treatment Centers at MidMichigan Health have a cross functional team with specialists in these and buy cipro over the counter other areas:infectious disease managementcardiologydiabetes educationnutrition managementphysical therapypain managementlab and imagingdebridementhyperbaric oxygen therapyMidMichigan’s specialized Wound Treatment Centers in Alma, Alpena, Clare, Midland and West Branch have a median time to heal of 28 days and 94 percent patient satisfaction.

These outcomes places us among the top 21 percent of nearly 800 Healogics centers nationwide. Healogics is the nation’s leading wound care management buy cipro over the counter company.Take Action. Seek Specialized Treatment.If you or someone you love is living with a non-healing wound, don’t wait – seek specialized treatment.

Even if you have tried other buy cipro over the counter treatments, but your wound isn’t healing, a multi-disciplinary Wound Treatment Center can identify and address the underlying reasons that the wound did not heal. Call MidMichigan’s Wound Treatment Centers toll free at (877) 683-0800 or visit www.midmichigan.org/woundcenter.Source. Www.ncbi.nlm.nih.gov/pmc/articles/PMC2903966/.

How long can cipro side effects last

IntroductionGLI-Kruppel family member 3 (GLI3) encodes for a how long can cipro side effects last zinc important site finger transcription factor which plays a key role in the sonic hedgehog (SHH) signalling pathway essential in both limb and craniofacial development.1 2 In hand development, SHH is expressed in the zone of polarising activity (ZPA) on the posterior side of the handplate. The ZPA expresses SHH, creating a gradient of SHH from the posterior to the anterior side of the handplate. In the presence of SHH, full length GLI3-protein is produced (GLI3A), whereas absence of SHH causes cleavage of GLI3 into its repressor form (GLI3R).3 4 Abnormal expression of this SHH/GLI3R gradient can cause both preaxial and postaxial polydactyly.2Concordantly, pathogenic DNA variants in the GLI3 gene are known to cause multiple syndromes with craniofacial and how long can cipro side effects last limb involvement, such as. Acrocallosal syndrome5 (OMIM. 200990), Greig how long can cipro side effects last 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, https://essenceflowyoga.co.uk/yin-yoga/ 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)..

IntroductionGLI-Kruppel family member 3 (GLI3) encodes for a zinc buy cipro over the counter 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 buy cipro over the counter length GLI3-protein is produced (GLI3A), whereas absence of SHH causes cleavage of GLI3 into its repressor form (GLI3R).3 4 Abnormal expression of this SHH/GLI3R gradient can cause both preaxial and postaxial polydactyly.2Concordantly, pathogenic DNA variants in the GLI3 gene are known to cause multiple syndromes with craniofacial and limb involvement, such as.

Acrocallosal syndrome5 (OMIM. 200990), Greig cephalopolysyndactyly syndrome6 (OMIM buy cipro over the counter. 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)..

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Cipro calf pain

Cipro calf pain

Last week, without any real pomp, I brewed a couple beers for that thing in the desert. Turns out they were my 100th and 101st batches of homebrew. Yay! They’re both finished – or at least they’d better be, since I’m kegging them today. I had to use Wyeast 1056 (courtesy of DBC) for the […]

Cipro calf pain

Obviously I haven’t updated in a long time. For the most part, that’s because my brewing equipment is packed up in expectation of moving somewhere or other. Pretty much all I’m doing these days is running in the mornings and trying to avoid heat in the afternoons.

Anyway, I ran 10 km this morning. Probably […]

Cipro calf pain

It’s only been spring here for about a month, but I’m starting to get back into a groove. I’m sure I’m positively dogging it by most people’s standards, but it’s gratifying to be seeing improvement almost daily.

Name: Track 096 Date: Jun 5, 2013 9:41 am Map: View on Map Distance: 1.51 miles Elapsed Time: […]

Cipro calf pain

Brewing test batches isn’t necessarily a whole lot of fun, but it does lend itself to some potentially useful experimentation. Throughout my (home) brewing career, I’ve bounced more or less randomly from one Belgian strain to another, in the process collecting most of the common strains, but without really settling on a “house” yeast. For […]

Cipro calf pain

It is exactly as dangerous as it looks.

Heat sticks are becoming popular among home brewers, and for good reason. Having two heated vessels really streamlines a brew day, and makes double brew days significantly less painful. And the economics of electric heat are compelling (in fact, that’s the way I’ve decided to […]

Cipro calf pain

Shaved Parmesan doesn’t work quite as well as shredded.

A recipe that doesn’t involve beer?! I know, I’m in danger of becoming a well-rounded person. These are delicious, though, and very easy to make, and quickly becoming my go-to appetizer for guests. If you have access to Trader Joe’s, they sell a can of […]

Cipro calf pain

Just a quick note. While I was doing some calculations for Two Mile, I decided to expand on a year-old post on draft system balancing, primarily just to include the relevant results for longer draft systems. Enjoy.

Or not. It doesn’t really affect me either way.

[…]

Cipro calf pain

I haven’t posted in… let’s see… six months. Yikes. Here’s a quartet of beer recipes, though, so that’s basically the same as posting almost once per month.

10.2 Mk2: I’m still struggling to get the attenuation I need out of my Belgian-style “Blond” (I use quotation marks because BJCP-wise, it would be a Belgian Specialty […]

Cipro calf pain

I’m not wild about the idea of driving somewhere for the sole purpose of running somewhere else, but I suppose allowances can be made.

Name: Track 023 Date: Apr 26, 2012 11:35 am Map: View on Map Distance: 3.01 miles Elapsed Time: 29:41.2 Avg Speed: 6.1 mph Max Speed: 8.3 mph Avg Pace: 9′ […]

Cipro calf pain

Well, maybe “hate”‘s a strong word. I’ve just never had a wine that I’d prefer over a good beer. I’ll keep trying though. You know, for science.

What I do hate is the wine industry. Bunch of namby-pamby grape gropers whose bottles collect dust and who spit instead of swallow. Which is why my interest […]