Flagyl online canada

The U.S flagyl online canada. Department of Health and Human Services (HHS), through the Health Resources and Services Administration (HRSA), today announced $103 million in awards to improve the retention of health care workers and help respond to the nation’s critical staffing needs by reducing burnout and promoting mental health and wellness among the health care workforce. These awards will fund evidence-informed programs, flagyl online canada practices and training, with a specific focus on providers in underserved and rural communities.

The funds, secured through the Biden-Harris Administration’s American Rescue Plan, will be disbursed to 45 grantees.“I have traveled to many health centers across the country and know that the buy antibiotics flagyl has intensified issues that have long been a source of stress for frontline health care workers — from increased patient volumes to long working hours,” said Health and Human Services Secretary Xavier Becerra. €œThis funding reflects the Biden-Harris Administration’s commitment to ensuring we flagyl online canada have enough critical frontline workers by supporting health care providers now and beyond as they face burnout and mental health challenges. We will continue to promote the well-being of those who have made so many sacrifices to keep others well.” buy antibiotics has compounded rates of depression and anxiety among health care workers.

The relentless physical and emotional demands of treating patients during a flagyl have exacerbated longstanding barriers to workplace well-being. While the challenge is complex, these multi-year awards will support flagyl online canada proven strategies for health care providers, academic institutions, and other recipients to reduce burnout and build resiliency. These strategies will include the creation of partnerships and utilization of local resources to directly support health professionals’ response to workplace stressors, and provide training to help individuals manage the constantly changing, high-stress environment of health care.

€œNow more than ever, it is critical to support the well-being of our health care workforce, who are working every day to protect each of us,” said HRSA Administrator Carole flagyl online canada Johnson. €œToday’s awards will provide new tools to help support our health professionals’ resilience as they continue to face the stress and challenges of responding to buy antibiotics and other health care needs and provide high quality care.” HRSA is making these awards through three programs. Promoting Resilience and Mental Health Among Health Professional Workforce – HRSA is awarding $28.6 million to 10 grantees to help health care organizations establish, improve, or expand evidence-informed programs and practices to promote mental health and well-being among the health workforce, including flagyl online canada their employees.

Health and Public Safety Workforce Resiliency Training Program – HRSA is awarding $68.2 million to 34 grantees to support tailored evidence-informed training development within health profession and nursing training activities. This curriculum will help reduce burnout and promote resilience among health care students, residents, health care professionals, paraprofessionals, trainees and public safety officers, such as firefighters, law enforcement officers, and ambulance crew members. Health and Public Safety Workforce Resiliency Technical Assistance Center – HRSA is awarding $6 million to George Washington University to provide tailored training and technical assistance flagyl online canada to today’s awardees.See a list of the award recipients here.

Https://bhw.hrsa.gov/funding/health-workforce-resiliency-awards Learn more about HRSA's funding opportunities.Start Preamble Health Resources and Services Administration (HRSA), Department of Health and Human Services (HHS). Notice. Effective December 30, 2021, HRSA accepted recommended updates to the Bright Futures Periodicity Schedule, a HRSA-supported guideline for infants, children and adolescents for purposes of ensuring that non-grandfathered group and individual health insurance issuers provide coverage without cost sharing under the Public Health Service Act.

The updates to the Bright Futures Periodicity Schedule are. A new category for sudden cardiac arrest and sudden cardiac death risk assessment, a new category for hepatitis B flagyl risk assessment, addition of suicide risk as an element of universal depression screening for children ages 12-21, and updated category title from “Psychosocial/Behavioral Assessment” to “Behavioral/Social/Emotional Screening,” with no revision to the ages in which the screening occurs (newborn to 21 years). Finally, two clarifying references related to dental fluoride varnish and fluoride supplementation have been added, with no associated recommended changes to clinical practice or health insurance coverage.

Please see https://mchb.hrsa.gov/​maternal-child-health-topics/​child-health/​bright-futures.html for additional information. Start Further Info Savannah Kidd, M.S. MFT, HRSA/Maternal and Child Health Bureau by calling 301-287-2601 or by emailing at SKidd@hrsa.gov.

End Further Info End Preamble Start Supplemental Information The Bright Futures program has been funded by HRSA since 1990. A primary focus of this program is for the funding recipient to maintain and recommend updates to the Bright Futures Guidelines for Health Supervision of Infants, Children and Adolescents, a set of materials and tools that provide theory-based and evidence-driven guidance for all preventive care screenings and well-child visits. One component of these tools is the Bright Futures Periodicity Schedule, a chart that identifies the recommended screenings, assessments, physical examinations, and procedures to be delivered within preventive checkups at each age milestone.

Over the program's existence, the Bright Futures Periodicity Schedule has become the accepted schedule within the United States for preventive health services through the course of a child's development. Section 2713 of the Public Health Service Act (42 U.S.C. 300gg-13), added by the Patient Protection and Affordable Care Act (Pub.

L. 111-148), requires that non-grandfathered group health plans and health insurance issuers offering Start Printed Page 1763 group or individual health insurance coverage provide coverage without cost-sharing for certain preventive health services. Section 2713(a)(3) describes such services for infants, children, and adolescents as “evidence-informed preventive care and screenings provided for in the comprehensive guidelines supported by the Health Resources and Services Administration.” HHS, along with the Departments of Treasury and Labor, issued an Interim Final Rule on July 19, 2010 (75 FR 41726-41760) that identified two specific resources as the comprehensive guidelines supported by HRSA for infants, children, and adolescents to be covered by insurance without cost sharing by non-grandfathered group health plans and health insurance issuers.

(1) The Bright Futures Periodicity Schedule and (2) the Recommended Uniform Screening Panel of the Advisory Committee on Heritable Disorders in Newborns and Children. The Interim Final Rule provided that a future change to these comprehensive guidelines is considered to be issued for purposes of Section 2713 on the date on which it is accepted by the HRSA Administrator or, if applicable, adopted by the Secretary of HHS. A public comment period was announced and occurred from September 13, 2021, through October 13, 2021 (86 FR 50894, September 13, 2021),[] to allow public comment on the proposed recommended updates affecting clinical practice and health insurance coverage requirements.

A total of 27 respondents gave 57 comments during the public comment period. The Bright Futures grantee, the American Academy of Pediatrics, received and considered the public comments. The annual report (Tab A) provides a description of the comments, including a detailed tabulation of each comment.

On December 30, 2021, the HRSA Administrator accepted the American Academy of Pediatrics' recommended several updates to the Bright Futures Periodicity Schedule. The Bright Futures recommendations included recommended clinical practice updates, along with revisions to the footnotes on the Bright Futures Periodicity Schedule that do not require changes to clinical practice or health insurance coverage. The updates to the Bright Futures Periodicity Schedule are.

(1) A new category for sudden cardiac arrest and sudden cardiac death risk assessment, (2) a new category for hepatitis B flagyl risk assessment, (3) addition of suicide risk as an element of universal depression screening for children ages 12-21, and (4) updated category title from “Psychosocial/Behavioral Assessment” to “Behavioral/Social/Emotional Screening,” with no revision to the ages in which the screening occurs (newborn to 21 years). Finally, two clarifying references related to dental fluoride varnish and fluoride supplementation have been added with no associated recommended changes to clinical practice. In light of these updates, all non-grandfathered group health plans and health insurance issuers offering group or individual health insurance coverage must cover without cost-sharing the services and screenings listed on the updated Bright Futures Periodicity Schedule for plan years (in the individual market, policy years) that begin in 2023, which can be accessed at the following link.

Https://mchb.hrsa.gov/​maternal-child-health-topics/​child-health/​bright-futures.html. Start Signature Diana Espinosa, Acting Administrator. End Signature End Supplemental Information [FR Doc.

2022-00461 Filed 1-11-22. 8:45 am]BILLING CODE 4165-15-P.

Solosec vs flagyl

Latest MedicineNet News By Amy Norton HealthDay ReporterFRIDAY, solosec vs flagyl Feb. 4, 2022 (HealthDay News) You have almost certainly seen the pleas while scrolling through social media. Called crowdfunding, folks try to raise money to pay for their sick loved one's mounting solosec vs flagyl medical bills. But new research shows these grassroots campaigns rarely raise enough money to make a difference. According to GoFundMe, which corners over 90% of the U.S.

Crowdfunding market, more than one-third of solosec vs flagyl its fundraisers are for medical needs. But crowdfunding should be seen as a "symptom" of the U.S. Health care system's failures — not a solution, said Sara Collins, who is vice president of health care coverage and access at the nonprofit Commonwealth Fund, and was not involved with the study. Instead, policymakers should solosec vs flagyl address the reasons that Americans have to resort to online campaigns, she noted. That could include expanding Medicaid — the government health insurance program for low-income Americans — as well as measures to bring down out-of-pocket expenses for people with private insurance.

Under "Obamacare," most U.S. States did expand their Medicaid programs to cover solosec vs flagyl more residents. However, 12 states — largely in the South — have resisted. Crowdfunding for medical bills has often been talked of as an "ad-hoc" safety net — a place for the uninsured or underinsured to turn to in times of need. But the new solosec vs flagyl findings, published Feb.

3 in the American Journal of Public Health, reveal a different reality. People dealing with medical debt are often facing "astronomical" costs, explained lead researcher Nora Kenworthy, an associate professor at the University of Washington, Bothell. So, even a crowdfunding campaign that goes relatively well may still fall far short of solosec vs flagyl getting people out from under medical bills. Nor do crowdfunding sites do what a true safety net would, Kenworthy said. Catch people equally.

For the study, she and her colleague Mark Igra collected data from GoFundMe's website, using its search engine to find solosec vs flagyl campaigns in every U.S. ZIP code. The investigators found over 437,000 fundraisers listed for medical needs between 2016 and 2020. Altogether, those campaigns raised an impressive-sounding solosec vs flagyl $2 billion. But campaigns varied wildly in their success.

The top performer raised $2.4 million, from over 70,000 donors, while 16% of all campaigns raised nothing. When campaigns made money, they typically had modest success, pulling in a median solosec vs flagyl of $1,100 in 2020. ("Median" means half of campaigns made more, and half made less.) And across all study years, almost 90% of campaigns failed to meet their goals. Half reached 25%, while one-third raised half of what they'd hoped. Who was most solosec vs flagyl successful at fundraising?.

The people who already had some advantages. The study found that more campaigns were launched in U.S. States with the highest rates of medical debt and lowest rates of insured residents solosec vs flagyl. Yet, those same campaigns earned the least. A look at the data by income found a similar pattern.

Campaigns in the one-fifth of U.S solosec vs flagyl. ZIP codes with the highest incomes raked in a total of $152 million in 2020. That compared with $70 million in the one-fifth of ZIP codes with the lowest incomes. That disparity is not solosec vs flagyl surprising. "Social networks have a lot to do with it," said Igra, a graduate student in sociology.

"Most campaigns are not reaching a lot of people. They're not going viral." Instead, Igra said, people who try crowdfunding typically reach solosec vs flagyl people they know. And for low-income Americans, that generally means other people facing similar financial struggles. No one is advising people to solosec vs flagyl avoid crowdfunding. Even $1,000 may help with medical needs, Kenworthy said.

But people should be aware, the researchers said, that the big campaigns that spread across social media are not typical. There can also be downsides to crowdfunding, Kenworthy solosec vs flagyl noted, like privacy concerns and earnings being considered income. All agreed that the crowdfunding trend points to underlying systemic issues. Many Americans need better health care coverage and social assistance programs. And ultimately, Collins said, it's the sky-high cost of health care in the United States that needs to be addressed solosec vs flagyl.

"It's not the utilization that's the problem," she said, "it's the prices." More information USA.gov has information on getting government help for medical bills. SOURCES. Nora Kenworthy, solosec vs flagyl PhD, associate professor, nursing and health studies, University of Washington, Bothell. Mark Igra, MA, graduate student, sociology, University of Washington, Bothell. Sara Collins, PhD, vice president, health care coverage and access, Commonwealth Fund, New York City.

American Journal of Public Health, solosec vs flagyl Feb. 3, 2022, online Copyright © 2021 HealthDay. All rights reserved. SLIDESHOW Health Care solosec vs flagyl Reform. Protect Your Health in a Rough Economy See SlideshowLatest antibiotics News FRIDAY, Jan.

4, 2022 (HealthDay News) Medicare will soon provide up to eight free buy antibiotics rapid tests a month to seniors enrolled in the government health insurance program, the Biden administration announced Thursday. The new policy for the over-the-counter tests will take solosec vs flagyl effect in early spring. The at-home tests will be available at pharmacies and other locations for clients with Medicare's "Part B" outpatient benefit, which about 90% of enrollees have. This will be the first time that Medicare has covered an over-the-counter test at no cost to recipients, the Associated Press reported. Access to free buy antibiotics rapid tests solosec vs flagyl will also be available to people with Medicare Advantage, a private insurance option used by 4 in 10 Medicare enrollees.

Those plans can already cover over-the-counter buy antibiotics tests as a supplemental benefit, the AP reported. Medicare will continue to provide free lab-based PCR testing, but those tests have to be ordered by a clinician or an authorized health care professional, White House officials said. Last month, the solosec vs flagyl Biden administration told private insurers to provide up to eight free at-home tests a month for their clients. Thursday's announcement that Medicare will do the same was applauded by the AARP, an advocacy group for older people. €œAARP applauds today's announcement that will guarantee access to at-home over-the-counter buy antibiotics tests at no cost for Medicare's 64 million beneficiaries...

Expanded access to no-cost testing will help protect seniors who have been hit hardest by the flagyl and ensure they can remain connected with their loved solosec vs flagyl ones and community," Nancy LeaMond, an AARP executive vice president and its chief advocacy and engagement officer, said in a statement. "Every American should have an easy way to get at-home buy antibiotics tests. We know that people 65 and older are at much greater risk of serious illness and death from this disease -- they need equal access to tools that can help keep them safe," she added. "The cost of paying for tests and the time needed to find free testing options are barriers that could discourage Medicare beneficiaries from getting tested, leading to greater social isolation and continued spread of the flagyl." Before Thursday's announcement, Medicare enrollees could get free at-home tests by requesting four free tests for home delivery through buy antibioticstests.gov or picking up free tests up from community locations such as libraries solosec vs flagyl or senior centers that distribute them. Those options will remain available while the new policy is going into effect, the AP reported.

More information Visit the U.S. Centers for Disease Control solosec vs flagyl and Prevention for more on buy antibiotics tests. SOURCE. Associated Press Robert Preidt and Robin Foster Copyright © 2021 HealthDay. All rights reserved.

SLIDESHOW Health Care Reform. Protect Your Health in a Rough Economy See Slideshow.

Latest MedicineNet News flagyl online canada By Amy Norton HealthDay ReporterFRIDAY, Feb. 4, 2022 (HealthDay News) You have almost certainly seen the pleas while scrolling through social media. Called crowdfunding, folks try to raise money to pay for their sick loved one's mounting medical bills flagyl online canada. But new research shows these grassroots campaigns rarely raise enough money to make a difference. According to GoFundMe, which corners over 90% of the U.S.

Crowdfunding market, more than one-third of its fundraisers are for medical flagyl online canada needs. But crowdfunding should be seen as a "symptom" of the U.S. Health care system's failures — not a solution, said Sara Collins, who is vice president of health care coverage and access at the nonprofit Commonwealth Fund, and was not involved with the study. Instead, policymakers should address the reasons that Americans have to resort flagyl online canada to online campaigns, she noted. That could include expanding Medicaid — the government health insurance program for low-income Americans — as well as measures to bring down out-of-pocket expenses for people with private insurance.

Under "Obamacare," most U.S. States did expand their flagyl online canada Medicaid programs to cover more residents. However, 12 states — largely in the South — have resisted. Crowdfunding for medical bills has often been talked of as an "ad-hoc" safety net — a place for the uninsured or underinsured to turn to in times of need. But the flagyl online canada new findings, published Feb.

3 in the American Journal of Public Health, reveal a different reality. People dealing with medical debt are often facing "astronomical" costs, explained lead researcher Nora Kenworthy, an associate professor at the University of Washington, Bothell. So, even a crowdfunding flagyl online canada campaign that goes relatively well may still fall far short of getting people out from under medical bills. Nor do crowdfunding sites do what a true safety net would, Kenworthy said. Catch people equally.

For the study, she and her colleague Mark flagyl online canada Igra collected data from GoFundMe's website, using its search engine to find campaigns in every U.S. ZIP code. The investigators found over 437,000 fundraisers listed for medical needs between 2016 and 2020. Altogether, those campaigns flagyl online canada raised an impressive-sounding $2 billion. But campaigns varied wildly in their success.

The top performer raised $2.4 million, from over 70,000 donors, while 16% of all campaigns raised nothing. When campaigns flagyl online canada made money, they typically had modest success, pulling in a median of $1,100 in 2020. ("Median" means half of campaigns made more, and half made less.) And across all study years, almost 90% of campaigns failed to meet their goals. Half reached 25%, while one-third raised half of what they'd hoped. Who was flagyl online canada most successful at fundraising?.

The people who already had some advantages. The study found that more campaigns were launched in U.S. States with flagyl online canada the highest rates of medical debt and lowest rates of insured residents. Yet, those same campaigns earned the least. A look at the data by income found a similar pattern.

Campaigns in the one-fifth of flagyl online canada U.S. ZIP codes with the highest incomes raked in a total of $152 million in 2020. That compared with $70 million in the one-fifth of ZIP codes with the lowest incomes. That disparity flagyl online canada is not surprising. "Social networks have a lot to do with it," said Igra, a graduate student in sociology.

"Most campaigns are not reaching a lot of people. They're not going flagyl online canada viral." Instead, Igra said, people who try crowdfunding typically reach people they know. And for low-income Americans, that generally means other people facing similar financial struggles. No one flagyl online canada is advising people to avoid crowdfunding. Even $1,000 may help with medical needs, Kenworthy said.

But people should be aware, the researchers said, that the big campaigns that spread across social media are not typical. There can also be downsides to crowdfunding, Kenworthy noted, like privacy concerns and earnings being considered flagyl online canada income. All agreed that the crowdfunding trend points to underlying systemic issues. Many Americans need better health care coverage and social assistance programs. And ultimately, Collins said, it's the sky-high cost of health care in the United States that flagyl online canada needs to be addressed.

"It's not the utilization that's the problem," she said, "it's the prices." More information USA.gov has information on getting government help for medical bills. SOURCES. Nora Kenworthy, PhD, associate professor, nursing and flagyl online canada health studies, University of Washington, Bothell. Mark Igra, MA, graduate student, sociology, University of Washington, Bothell. Sara Collins, PhD, vice president, health care coverage and access, Commonwealth Fund, New York City.

American Journal flagyl online canada of Public Health, Feb. 3, 2022, online Copyright © 2021 HealthDay. All rights reserved. SLIDESHOW Health flagyl online canada Care Reform. Protect Your Health in a Rough Economy See SlideshowLatest antibiotics News FRIDAY, Jan.

4, 2022 (HealthDay News) Medicare will soon provide up to eight free buy antibiotics rapid tests a month to seniors enrolled in the government health insurance program, the Biden administration announced Thursday. The new policy for flagyl online canada the over-the-counter tests will take effect in early spring. The at-home tests will be available at pharmacies and other locations for clients with Medicare's "Part B" outpatient benefit, which about 90% of enrollees have. This will be the first time that Medicare has covered an over-the-counter test at no cost to recipients, the Associated Press reported. Access to free buy antibiotics rapid tests will also be available to flagyl online canada people with Medicare Advantage, a private insurance option used by 4 in 10 Medicare enrollees.

Those plans can already cover over-the-counter buy antibiotics tests as a supplemental benefit, the AP reported. Medicare will continue to provide free lab-based PCR testing, but those tests have to be ordered by a clinician or an authorized health care professional, White House officials said. Last month, the Biden administration told private insurers to provide up to eight free at-home tests a month for their clients. Thursday's announcement that Medicare will do the same was applauded by the AARP, an advocacy group for older people. €œAARP applauds today's announcement that will guarantee access to at-home over-the-counter buy antibiotics tests at no cost for Medicare's 64 million beneficiaries...

Expanded access to no-cost testing will help protect seniors who have been hit hardest by the flagyl and ensure they can remain connected with their loved ones and community," Nancy LeaMond, an AARP executive vice president and its chief advocacy and engagement officer, said in a statement. "Every American should have an easy way to get at-home buy antibiotics tests. We know that people 65 and older are at much greater risk of serious illness and death from this disease -- they need equal access to tools that can help keep them safe," she added. "The cost of paying for tests and the time needed to find free testing options are barriers that could discourage Medicare beneficiaries from getting tested, leading to greater social isolation and continued spread of the flagyl." Before Thursday's announcement, Medicare enrollees could get free at-home tests by requesting four free tests for home delivery through buy antibioticstests.gov or picking up free tests up from community locations such as libraries or senior centers that distribute them. Those options will remain available while the new policy is going into effect, the AP reported.

More information Visit the U.S. Centers for Disease Control and Prevention for more on buy antibiotics tests. SOURCE. Associated Press Robert Preidt and Robin Foster Copyright © 2021 HealthDay. All rights reserved.

SLIDESHOW Health Care Reform. Protect Your Health in a Rough Economy See Slideshow.

What may interact with Flagyl?

Do not take Flagyl with any of the following:

• alcohol or any product that contains alcohol
• amprenavir oral solution
• disulfiram
• paclitaxel injection
• ritonavir oral solution
• sertraline oral solution
• sulfamethoxazole-trimethoprim injection

Flagyl may also interact with the following:

• cimetidine
• lithium
• phenobarbital
• phenytoin
• warfarin

This list may not describe all possible interactions. Give your health care providers a list of all the medicines, herbs, non-prescription drugs, or dietary supplements you use. Also tell them if you smoke, drink alcohol, or use illegal drugs. Some items may interact with your medicine.

Flagyl cream

IntroductionSOX10 belongs to the SOX family Buy cheap seroquel of transcription factors, of which the members are defined based on the presence of a 79 amino acid DNA-binding domain with homology to flagyl cream the high mobility group (HMG) box of SRY (sex-determining region Y. Hence SOX, Sry bOX). These factors are involved in multiple developmental processes, such as male differentiation, skeletogenesis, neurogenesis and neural crest (NC) development, where they control stemness, cell fate and differentiation.1–4 The growing number of developmental disorders associated with mutations in SOX genes underscores their importance during development.5 The SOX10 transcription factor is a flagyl cream characteristic marker for migratory multipotent NC progenitors as well as for various NC derivatives.The NC is a specific population of cells in vertebrates that arise at the edge between the neural and non-neural ectoderm, delaminate from the dorsal aspect of the neural tube, and migrate through several routes to reach target tissues and give rise to neurons and glia of the peripheral nervous system (PNS), including sensory, autonomous and enteric ganglia, Schwann cells and olfactory ensheathing cells, melanocyte pigment cells, skeletal structures and mesenchyme of the head, face and neck, outflow tract of the heart, and smooth muscle cells of the great arteries.6 7Over the years, heterozygous SOX10 mutations have been associated with various phenotypes that extend beyond Waardenburg syndrome (WS. Depigmentation features and deafness) and Hirschsprung disease (HSCR. Intestinal aganglionosis) initial diagnosis flagyl cream.

Here, we present an up-to-date overview of these various clinical manifestations, along with our current understanding of how they are explained by SOX10 dysfunction in several NC derivatives and extra-NC tissues (inner ear and oligodendrocytes), and of the origin of phenotypic variability.SOX10. Structure and regulation of the gene, protein domains and post-transcriptional modificationsThe human SOX10 and mouse Sox10 genes encode an open reading flagyl cream frame of 466 amino acids that share 92% nucleotidic and 98% amino acid sequence identities.8 The absence of a complete description of the human gene 5’ non-coding exon(s) has given rise to two coexisting exon numbering systems. Historically, exons 1 and 2 are non-coding, the initiation codon is found in exon 3, and the stop codon in exon 5.8 The second system is based on the reference transcript NM_006941, with only one non-coding exon in the 5’UTR (untranslated transcribed region) and a total of four exons. A major transcript of ~3 kb is detected in most tissues tested, consistent with the predicted SOX10 mRNA sequence.9 10The protein’s structure is schematised in flagyl cream figure 1. As for all other members of the SOX family, the previously mentioned HMG domain forms an L-shaped module composed of three alpha helices that bind to DNA sequences in the minor groove (matching or resembling C[A/T]TTG[A/T][A/T]), bending the DNA molecule and interacting with other proteins to establish stable and active transcriptional complexes3 4 (the most recent model can be found in Haseeb and Lefebvre11).

This domain also harbours two nuclear import (nuclear localisation signal) and one export (nuclear export signal) signals.12 1310 samples in various tissues (including the brain, gut, nerves (tibial), breast and salivary glands)/total flagyl cream number of A-to-I modifications reported is. AluY. 67/224. AluSx. 25/87.

AluSx. 31/92. And AluSq. 20/73. DIM, dimerisation domain.

ERK, extracellular signal-regulated kinase. HMG, high mobility group domain. NES, nuclear export signal. NLS, nuclear localisation signal. TA/TAC, transactivation domain in C-terminal.

TAM, transactivation domain in the middle of the protein." class="highwire-fragment fragment-images colorbox-load" rel="gallery-fragment-images-1605886105" data-figure-caption="Schematic of the SOX10 protein and post-translational modifications. Domains of human SOX10. The numbers refer to amino acid residues. The pink lines above the HMG domain represent the NLS sequences, one at each end of the HMG domain, and the light pink line the NES sequence. Note that although nucleocytoplasmic shuttling of the protein has been well documented for several SOX factors,12 13 in vivo regulation of SOX10 through nuclear translocation is yet to be clarified.

Black arrowheads represent the localisation of junctions between exons. Post-transcriptional modifications, including acetylation (Ac), phosphorylation (P) and sumoylation (Su), are indicated, along with the position of modified amino acids. Note that a putative acetylation site was identified in SOX2 and is conserved in SOX10.17 Sumoylation by Ubc9 occurs at lysines K55, K246 and K357 with consequences on cell fate decision.19 20 Mechanistically, sumoylated SOXE proteins fail to interact with the coactivator CBP (CREB-binding protein)/p300 and instead recruit the GRG4 corepressor (Groucho-related protein 4/TLE4, transducing-like enhancer of split 4), leading to strong inhibition of SOXE target genes.106 Among the identified phosphorylation sites, note that ERK phosphorylates T240 and T244, inhibiting the sumoylation of SOX10 at K55 and transcriptional activity.107 Additional phosphorylation sites have been described from large-scale proteomic screens in melanoma, breast tumours and mouse neuroblastoma (serine S8, S13, S17, S24, S27, S30, S40, S45, S221, S224 and S23216). The relevance of most has not been functionally assessed. SOX10 phosphorylation sites are localised in two distinct clusters, one at the amino terminus, 5’ of the dimerisation domain, and the other at the centre of the protein.

FBXW7-mediated ubiquitination of SOX10 has also been shown to control protein stability. The region involves aa 235–244 of the human protein. A search of the public REDIportal (http://srv00.recas.ba.infn.it/atlas/) revealed various A-to-I editing sites located in AluY, AluSx or AluSq sequences embedded in the last SOX10 intron. In each Alu sequence schematised from left to right, the number of A-to-I sites identified in >10 samples in various tissues (including the brain, gut, nerves (tibial), breast and salivary glands)/total number of A-to-I modifications reported is. AluY.

And AluSq. 20/73. DIM, dimerisation domain. ERK, extracellular signal-regulated kinase. HMG, high mobility group domain.

NES, nuclear export signal. NLS, nuclear localisation signal. TA/TAC, transactivation domain in C-terminal. TAM, transactivation domain in the middle of the protein." data-icon-position data-hide-link-title="0">Figure 1 Schematic of the SOX10 protein and post-translational modifications. Domains of human SOX10.

The numbers refer to amino acid residues. The pink lines above the HMG domain represent the NLS sequences, one at each end of the HMG domain, and the light pink line the NES sequence. Note that although nucleocytoplasmic shuttling of the protein has been well documented for several SOX factors,12 13 in vivo regulation of SOX10 through nuclear translocation is yet to be clarified. Black arrowheads represent the localisation of junctions between exons. Post-transcriptional modifications, including acetylation (Ac), phosphorylation (P) and sumoylation (Su), are indicated, along with the position of modified amino acids.

Note that a putative acetylation site was identified in SOX2 and is conserved in SOX10.17 Sumoylation by Ubc9 occurs at lysines K55, K246 and K357 with consequences on cell fate decision.19 20 Mechanistically, sumoylated SOXE proteins fail to interact with the coactivator CBP (CREB-binding protein)/p300 and instead recruit the GRG4 corepressor (Groucho-related protein 4/TLE4, transducing-like enhancer of split 4), leading to strong inhibition of SOXE target genes.106 Among the identified phosphorylation sites, note that ERK phosphorylates T240 and T244, inhibiting the sumoylation of SOX10 at K55 and transcriptional activity.107 Additional phosphorylation sites have been described from large-scale proteomic screens in melanoma, breast tumours and mouse neuroblastoma (serine S8, S13, S17, S24, S27, S30, S40, S45, S221, S224 and S23216). The relevance of most has not been functionally assessed. SOX10 phosphorylation sites are localised in two distinct clusters, one at the amino terminus, 5’ of the dimerisation domain, and the other at the centre of the protein. FBXW7-mediated ubiquitination of SOX10 has also been shown to control protein stability. The region involves aa 235–244 of the human protein.

A search of the public REDIportal (http://srv00.recas.ba.infn.it/atlas/) revealed various A-to-I editing sites located in AluY, AluSx or AluSq sequences embedded in the last SOX10 intron. In each Alu sequence schematised from left to right, the number of A-to-I sites identified in >10 samples in various tissues (including the brain, gut, nerves (tibial), breast and salivary glands)/total number of A-to-I modifications reported is. AluY. 67/224. AluSx.

DIM, dimerisation domain. ERK, extracellular signal-regulated kinase. HMG, high mobility group domain. NES, nuclear export signal. NLS, nuclear localisation signal.

TA/TAC, transactivation domain in C-terminal. TAM, transactivation domain in the middle of the protein.SOX10 shares additional domains with SOX8 and SOX9, all three forming the SOX_E group (SOX factors have been subdivided in several groups based on the amino acid identity within their HMG domain) (figure 1). Among them, the dimeric domain (DIM) confers preferential binding of SOX_E members to target sites containing two inverted SOX motifs separated by three to four nucleotides and promotes homodimerisation or heterodimerisation through DIM:HMG interactions.14Within its C-terminus, SOX10 contains a potent transactivation domain called the TA or TAC (transactivation domain in C-terminal).4 Another weaker and context-dependent transactivation domain has been identified in the middle of SOX10, the so-called K2 domain or TAM (transactivation domain in the middle of the protein), and was recently shown to synergise with TA/TAC in all SOX_E members.11 15Various post-transcriptional and post-translational modifications modulate the activity, stability and intracellular localisation of SOX1016 (figure 1). Several of these modifications are inferred from those occurring in other SOX factors, as for the lysine K136 acetylation site.16–18 Others, including phosphorylation sites, were mainly found from large-scale proteomic screens performed in cancer cells. SOX10 sumoylation by UBC9 (sumo-conjugating enzyme UBC9) is the best described one.

Occurring at lysines K55, K246 and K357,19 it inhibits NC development and promotes development of non-sensory cranial placodes in vivo.20 Absence of A-to-I RNA modification mediated by the ADAR (adenosine deaminase RNA-specific) enzyme family was recently reported to alter melanocyte and Schwann cell development.21 Examination of the public REDIportal shows that SOX10 is under such regulation in humans (but not mice).Finally, several regulatory regions likely involved in driving SOX10/Sox10 expression have been identified using various cell lines and zebrafish or mice models (ref 22 and references therein). Methylation of the Sox10 promoter by DNA methyansferase 3 has also been shown to arrest NC generation in chicks.23Involvement of SOX10 in WS. Role in melanocytes and enteric nervous systemThe identification of Sox10 as the gene mutated in the spontaneous Dom mutant mouse (Dominant megacolon. Intestinal aganglionosis, white belly spot and white paws) first shed light on the essential function of this transcription factor in NC development. In this strain, a Sox10 frameshift mutation results in alteration of binding to some DNA target sequences in vitro, of transactivation capacity and synergistic action with several cofactors.9 24–27 This observation immediately led to test SOX10 involvement in Waardenburg-Hirschsprung disease.8 Also known as WS type 4 (WS4) or Waardenburg-Shah syndrome, Waardenburg-Hirschsprung encompasses symptoms of WS and HSCR (Mendelian inheritance in man, MIM) #613266).28–30HSCR is the most common enteric neuropathy, occurring in 1 of 5000 neonates, and is characterised by the absence of enteric ganglia from a varying length of the distal gut, leading to intestinal obstruction in neonates or severe constipation in adults (MIM #142623).29 30WS is a genetic disorder characterised by sensorineural hearing loss (SNHL) and pigmentation anomalies, including depigmented patches of skin and hair and vivid blue eyes or iris heterochromia (MIM #193500).

Four types of WS are clinically defined, based on additional features due to defects in structures mostly arising from NC derivatives. WS1 is further characterised by dystopia canthorum, WS3 by musculoskeletal abnormalities of the limbs, WS4 by HSCR, whereas WS2 has no further significant features. In addition to SOX10, four main genes are involved in WS thus far. MITF (melanocyte inducing transcription factor) in WS2, PAX3 (transcription factor paired Box 3) in WS1 and WS3, EDN3 (endothelin 3) in WS4, and EDNRB (endothelin receptor type B) in WS4 and WS2.28 31 32 SOX10 has been shown to regulate and interact with several of these genes.28 33SOX10 screening in WS4 cases led to the identification of the first heterozygous mutations in 1998.8 In 2007, SOX10 mutations were shown to be also responsible for approximately 15% of WS2 cases.34 By contrast, SOX10 involvement in isolated HSCR is very limited. For example, screening of 229 isolated HSCR cases led to the identification of only one frameshift mutation inherited from an asymptomatic mother (germline mosaicism has been proposed).35Certain patients with WS4 or PCWH (see later) present with hypoganglionosis or chronic intestinal pseudo-obstruction (CIPO) instead of HSCR.28 36–39 Given the role of SOX10 in enteric nervous system (ENS) development, CIPO is probably neurogenic.

Aganglionosis is therefore not the only mechanism underlying the intestinal dysfunction in patients with SOX10 mutations.Each of the clinical manifestations described above can be explained by dysregulation of SOX10 during melanocyte and ENS development. WS accounts for a developmental defect in both skin melanocytes and a melanocyte-derived cell lineage of the inner ear, called intermediate cells of the stria vascularis, necessary to the inner ear homeostasis.40 In melanocytes, SOX10 controls proliferation, survival and differentiation by directly and sequentially activating a number of downstream target genes.4 41–43 From the NC, a SOX10–PAX3 pair activates the expression of Mitf/MITF, which then acts as a SOX10 partner to activate the expression of Dct (dopachrome tautomerase) and Tyr (tyrosinase), both involved in melanocyte differentiation and melanin synthesis.27 32 42 44 45 In 2015, an extensive genome-wide catalogue of SOX10 targets was obtained.46 For the first time, integrated chromatin occupancy and transcriptome analysis suggested a role of SOX10 in both transcriptional activation and repression. SOX10 was also shown to cooperate with MITF to facilitate BRG1 (Brahma-related gene 1/SMARCA4, SWI/SNF related, matrix associated, actin-dependent regulator of chromatin) binding to distal enhancers of melanocyte-specific genes, promoting differentiation.47In the developing gut, SOX10 is expressed in all NC-derived ENS progenitors.22 24 48–50 Later, SOX10 is maintained in enteric glia but downregulated in cells that are committed to neurons (see refs 25 50 for examples). Most publications suggest a role of SOX10 in the maintenance of enteric progenitors,22 49 and overexpression of SOX10 inhibits enteric neuron differentiation, without altering commitment to the neurogenic lineage.25 51 These cellular functions rely on the capacity of SOX10 to regulate (along with several cofactors) various target genes, including Ret (RET proto-oncogene. A receptor tyrosine kinase involved in ENS development and the main HSCR-related gene), Ednrb and Sox10 itself.22 33 52 53 As an example SOX10 and ZEB2 (zinc-finger E-box binding homeobox 2.

A negative regulator of NC differentiation) both bind to Ednrb promoter-specific regions, highlighting the role of this ‘triade’ in controlling the maintenance of multi-potential enteric progenitors and their differentiation process.33Hearing loss associated with SOX10 mutations. Beyond melanocytes, SOX10 expression in inner ear and related deficitsSNHL due to SOX10 mutations, as for the other WS genes, is typically prelingual, non-evolutive, profound and bilateral. However, it can also be moderate and asymmetric or unilateral.Aside from the intermediate-cell alterations mentioned above, inner ear malformations have been noted in some patients with WS long ago.54 It now appears that only patients with a SOX10 mutation present with these abnormalities. Hypoplasia/dysplasia or agenesis of the semicircular canals and enlarged vestibules are very frequent, while agenesis of the vestibulo-cochlear nerve and cochlear deformities have also been reported.55–57 Consequently, temporal CT scan or MRI is of particular interest in diagnosis. In our experience, this feature is highly penetrant when interpreted by a specialised radiologist.

However, recent papers reported the absence of imaging abnormalities in the inner ear of a few patients with SOX10 mutations. A complete exploration of the vestibular function has yet to be performed.These observations are consistent with an expression profile of Sox10 in the ear. Sox10 is expressed in the placode-derived otic vesicle from E9.5 onward and then in the developing epithelium of the cochlea and vestibule, before being restricted to supporting cells of the neurosensory epithelium. Sox10/SOX10 promotes the survival of cochlear progenitors during formation of the otocyst and the organ of Corti, plays a role in glial development of the cochleovestibular ganglia, and its NC-targeted deletion leads to improper neuronal migration and projection.58–60 The resulting inner ear malformations differ depending on the animal model.58 61 62 RNA-seq studies of inner ear development in a pig model showed dysregulation of WNT1 (Wnt family member 1. A regulator of cell fate and patterning), KCNQ4 (potassium voltage-gated channel subfamily Q), STRC (stereocilin.

A protein associated with the hair bundle of the ear sensory cells) and PAX6 (transcription factor Paired Box 6) networks.62In agreement with this broad function, SNHL appears to be the most penetrant sign in cases of SOX10 mutation, leading to the observation that certain patients can present with isolated SNHL until minor signs are revealed on medical reinterview.63PCWH and PCW phenotypes. Important function of SOX10 in Schwann cells and oligodendrocytesBeyond WS2 or WS4, neurological alterations have been identified in the so-called PCWH syndrome (peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, Hirschsprung disease. MIM #609136).28 36 64 Depending on the severity of myelin defects in the PNS and central nervous system (CNS), patients with PCWH exhibit variable symptoms that often include delayed motor and cognitive development, cerebral palsy, ataxia, spasticity, congenital nystagmus, hyporeflexia, distal sensory impairments and distal muscle wasting. This phenotype is recapitulated in a transgenic mouse model with several copies of SOX10 carrying the first PCWH mutation described65 66 and is mostly explained by the role of SOX10 during differentiation of myelinating Schwann cells and oligodendrocytes, both ensuring rapid salutatory conduction along axons.67 68In the PNS, SOX10 controls each differentiation step by inducing stage-restricted transcriptional regulators, which are then recruited as partners to activate specific sets of target genes, allowing progression to the next stage.67–72 For example, in immature Schwann cells, SOX10 induces the expression of OCT6 (POU3F1, POU class 3 homeobox 1). Both factors then cooperatively activate the programme required for progression into the promyelinating stage.

Their target EGR2 (early growth response 2) then associates with SOX10 to activate the myelination programme.In the CNS, analyses of various animal models revealed an essential role of SOX10 in the terminal differentiation of oligodendrocytes in coordination with OLIG1 (OLIGodendrocyte transcription factor 1), MYRF (myelin regulatory factor), TCF4 (transcription factor 4, which has an important role in CNS development) and CHD7 (chromodomain helicase DNA-binding protein 7. The gene involved in CHARGE syndrome (Coloboma, Heart anomaly, choanal Atresia, Retardation, Genital and Ear anomalies)).68 Many genes that are activated during terminal differentiation of oligodendrocytes are direct targets of SOX10, but there are only few known SOX10 targets in oligodendrocyte precursors.68 73 74 Recently, MYRF was identified as a decisive factor that helps SOX10 to switch between its target genes along oligodendrocyte differentiation process.75Of interest, some of the genes directly regulated by SOX10 in PNS and CNS are known to be responsible for hypomyelinating/demyelinating diseases, with some described mutations in these genes that directly result from a loss of regulation by SOX10.76–78Involvement of SOX10 in Kallmann syndrome and its role in olfactory ensheathing cellsSOX10 was considered to be a candidate gene for Kallmann syndrome (KS, hypogonadotropic hypogonadism and anosmia. MIM #308700) based on the unexpected high frequency of olfactory bulb agenesis55 associated with rare clinical reports of hypogonadism or anosmia in patients with WS/PCWH with a SOX10 mutation. The screening of cohorts indeed revealed SOX10 mutations in patients with KS, most of whom also have hearing impairment.79 Since then, many other SOX10 mutations have been characterised in KS or normosmic idiopathic hypogonadotropic hypogonadism (nIHH), although they were usually not functionally characterised and a subset of them appeared unlikely to be pathogenic (see Review of SOX10 variations). Interestingly, KS and WS are not mutually exclusive, and some patients with an initial diagnosis of WS have been further diagnosed with hypogonadism at puberty.80 We believe that anosmia and hypogonadism are still underestimated in patients with WS with a SOX10 mutation, as signs of KS are difficult to diagnose before puberty.

Of note, in the absence of pigmentary disturbances, the association of KS+hearing impairment+abnormalities of the semicircular canals can lead to a differential diagnosis with mild forms of CHARGE syndrome (MIM #214800) (examples in online supplemental table 1).Supplemental materialThe common cause of anosmia and hypogonadism is a defect in a developmental sequence of GnRH (gonadotropin-releasing hormone) neurons migrating along the peripheral olfactory nerve up to and through the olfactory bulb. In the Sox10 knockout mouse, a primary defect of the olfactory ensheathing cells leads to a secondary defect of the olfactory nerve pathway, defasciculation and misrouting of the nerve fibres, impaired migration of GnRH cells along this route, and disorganisation of the olfactory nerve layer of the olfactory bulbs.79 Dysregulation of the frizzled related protein FRZB may contribute to explain the defect in olfactory axon targeting but not GnRH neuron migration.81A summary of the recurrent clinical manifestations due to constitutive SOX10 mutations along with affected cell types is presented in figure 2.Summary of the clinical spectrum due to SOX10 mutations and the corresponding SOX10 function(s). The picture is organised around three clinical poles that correspond to different diagnosis entries. The WS pole is indicated in red, the myelin pole in blue and the KS pole in green. The plain line corresponds to the definition of the disease, while the dotted lines indicate the main clinical extension of these syndromes in case of SOX10 mutation.

Note that the area of the circles is not proportionate to the relative frequency of each syndrome (for an idea about the number of patients, see figure 3B). CIPO, chronic intestinal pseudo-obstruction. CNS, central nervous system. ENS, enteric nervous system. GnRH, gonadotropin-releasing hormone.

HSCR, Hirschsprung disease. KS, Kallmann syndrome. NIHH, normosmic idiopathic hypogonadotropic hypogonadism. PCWH, peripheral demyelinating neuropathy, central dysmyelination, Waardenburg syndrome, with Hirschsprung disease. PNS, peripheral nervous system.

SNHL, sensorineural hearing loss. WS, Waardenburg syndrome. WS2, Waardenburg syndrome type 2. WS4, Waardenburg syndrome type 4." data-icon-position data-hide-link-title="0">Figure 2 Summary of the clinical spectrum due to SOX10 mutations and the corresponding SOX10 function(s). The picture is organised around three clinical poles that correspond to different diagnosis entries.

The WS pole is indicated in red, the myelin pole in blue and the KS pole in green. The plain line corresponds to the definition of the disease, while the dotted lines indicate the main clinical extension of these syndromes in case of SOX10 mutation. Note that the area of the circles is not proportionate to the relative frequency of each syndrome (for an idea about the number of patients, see figure 3B). CIPO, chronic intestinal pseudo-obstruction. CNS, central nervous system.

ENS, enteric nervous system. GnRH, gonadotropin-releasing hormone. HSCR, Hirschsprung disease. KS, Kallmann syndrome. NIHH, normosmic idiopathic hypogonadotropic hypogonadism.

PCWH, peripheral demyelinating neuropathy, central dysmyelination, Waardenburg syndrome, with Hirschsprung disease. PNS, peripheral nervous system. SNHL, sensorineural hearing loss. WS, Waardenburg syndrome. WS2, Waardenburg syndrome type 2.

WS4, Waardenburg syndrome type 4.Involvement in cancer, sex reversal, associations and reports of the first biallelic mutationsBeyond congenital disorders, a role of SOX10 in cancer progression has been reported. SOX10 protein is highly expressed in breast, glioma, glioblastoma, salivary adenoid cystic tumours and hepatocellular carcinoma (see The Cancer Genome Atlas). The association of SOX10 with melanoma is the best described, but only a limited number of variants have been reported so far.82–84Several reports of duplications in the 22q13.1 region have been published that may include one or several signs of WS/PCWH and sex reversal in a number of cases.85 Sex reversal has been suggested to be due to the overexpression of SOX10, consistent with observations in a Sox10 transgenic mouse model.86 However, we found a SOX10 triplication (four doses of SOX10 instead of two) in a 47,XX baby girl without sex reversal (online supplemental table 1), indicating that overexpression of SOX10 alone may not be sufficient, the sign is not fully penetrant or the overexpression of other genes has the opposite effect, depending on the size of the rearrangement.More complex and questionable associations have also been described. For example, increased DNA methylation of SOX10 has been linked to oligodendrocyte dysfunction in patients with schizophrenia.87Two cases of biallelic SOX10 deletion have been characterised and, although not reported in the papers, they appear to represent the first and second pregnancy from the same couple.88 89 Both parents are heterozygous for one of the two SOX10 deletions and present with a classic form of WS. Biallelic SOX10 loss-of-function results in a severe polymalformative fetal phenotype.

Eighteen other genes were included in the maternal deletion and may participate in the phenotype.Finally, the development of large gene panels for diagnosis and whole exome/whole genome sequencing has led to SOX10 mutations being found in unexpected contexts. A number of cases have thus been listed in cohorts of neurodevelopmental defects, hearing impairment and endocrinological problems. Due to the diverse phenotypes related to SOX10 mutations, making sense of such findings can be challenging.Review of SOX10 variationsDuring the first 15 years after their discovery, most SOX10 disease-associated point mutations were shown to result in premature termination codons, with strikingly few exceptions.28 Missense mutations started to be found simultaneously with the finding that SOX10 mutations can cause less severe syndromes than life-threatening WS4 or PCWH.90An up-to-date summary of confident mutations of SOX10 (approximately 300 independent cases, including unpublished ones in online supplemental table 1) is presented in figure 3A. Truncations (stops, frameshifts) are found in slightly more than half of all cases. Approximately one-third of all mutations are non-truncating variations, either missense or small inframe insertions/deletions, the rest being either complete or partial copy number variations of the gene (approximately 10%) and rare mutational mechanisms (splice mutations, mutation of the initiation codon or non-stop mutations (five cases to date)).

Truncating mutations can be located anywhere, except in the very extreme C-terminus. On the contrary, missense mutations are tightly clustered in the DNA-binding domain (HMG), a frequent finding for transcription factors. We have thus far found no specific link between SOX10 missense mutations and residues involved in post-translational modifications.Review of SOX10 mutations. (A) Representation of SOX10 truncating and non-truncation mutations along the SOX10 protein. We made a list of all published SOX10 mutations, starting with the LOVD database that we curated up to 2015 (https://databases.lovd.nl/shared/genes/SOX10), updated with the literature and finally completed using the HGMD (Human Gene Mutation Database) professional database (https://digitalinsights.qiagen.com/products-overview/clinical-insights-portfolio/human-gene-mutation-database) for a few mutations that were reported in cohorts of unspecific diagnosis and would have been missed by common keywords.

We prioritised the strength of the data to create this figure, as our goal was not to include all cases but to provide a reliable picture of the SOX10 mutational spectrum. We retained papers for which the data allowed curation and removed neutral variants and variants of unknown significance, duplicated patients or publications, and publications with inconsistent findings. Finally, we added our unpublished cases (listed in online supplemental table 1). €˜M1?. €™ indicates a mutation of the initiation codon (p.Met1?.

). (B) Proportion (in percentage) of the different types of mutations for each syndrome. €˜n’ indicates the number of independent cases included in each group. (C) Localisation of the truncating (stop and frameshift) mutations along the SOX10 protein associated with each phenotype. Note that (1) the phenotypic description was sometimes too incomplete for inclusion in B and C.

(2) among the familial cases showing intrafamilial differences in phenotype, we considered the phenotype of the index case. (3) KS/nIHH is given regardless of the presence of WS signs or not, and anosmia without hypogonadism was not considered. And (4) because presence or absence of a demyelination is frequently unreported or not evaluated, we conserved all the patients with neurological features in the PCW/PCWH group when the data seemed consistent. DIM, dimeric domain. HMG, high mobility group.

KS, Kallmann syndrome. LOVD, Leiden OPen Variation Database. NIHH, normosmic idiopathic hypogonadotropic hypogonadism. PCW/PCWH, peripheral demyelinating neuropathy, central demyelination, Waardenburg syndrome, with or without Hirschsprung disease. TA, transactivation domain in C-terminal.

TAM, transactivation domain in the middle of the protein. WS, Waardenburg syndrome. WS2, Waardenburg syndrome type 2. WS4, Waardenburg syndrome type 4." data-icon-position data-hide-link-title="0">Figure 3 Review of SOX10 mutations. (A) Representation of SOX10 truncating and non-truncation mutations along the SOX10 protein.

We made a list of all published SOX10 mutations, starting with the LOVD database that we curated up to 2015 (https://databases.lovd.nl/shared/genes/SOX10), updated with the literature and finally completed using the HGMD (Human Gene Mutation Database) professional database (https://digitalinsights.qiagen.com/products-overview/clinical-insights-portfolio/human-gene-mutation-database) for a few mutations that were reported in cohorts of unspecific diagnosis and would have been missed by common keywords. We prioritised the strength of the data to create this figure, as our goal was not to include all cases but to provide a reliable picture of the SOX10 mutational spectrum. We retained papers for which the data allowed curation and removed neutral variants and variants of unknown significance, duplicated patients or publications, and publications with inconsistent findings. Finally, we added our unpublished cases (listed in online supplemental table 1). €˜M1?.

€™ indicates a mutation of the initiation codon (p.Met1?. ). (B) Proportion (in percentage) of the different types of mutations for each syndrome. €˜n’ indicates the number of independent cases included in each group. (C) Localisation of the truncating (stop and frameshift) mutations along the SOX10 protein associated with each phenotype.

Note that (1) the phenotypic description was sometimes too incomplete for inclusion in B and C. (2) among the familial cases showing intrafamilial differences in phenotype, we considered the phenotype of the index case. (3) KS/nIHH is given regardless of the presence of WS signs or not, and anosmia without hypogonadism was not considered. And (4) because presence or absence of a demyelination is frequently unreported or not evaluated, we conserved all the patients with neurological features in the PCW/PCWH group when the data seemed consistent. DIM, dimeric domain.

HMG, high mobility group. KS, Kallmann syndrome. LOVD, Leiden OPen Variation Database. NIHH, normosmic idiopathic hypogonadotropic hypogonadism. PCW/PCWH, peripheral demyelinating neuropathy, central demyelination, Waardenburg syndrome, with or without Hirschsprung disease.

TA, transactivation domain in C-terminal. TAM, transactivation domain in the middle of the protein. WS, Waardenburg syndrome. WS2, Waardenburg syndrome type 2. WS4, Waardenburg syndrome type 4.Of course, rarity in control populations is not sufficient to confer pathogenicity and the prediction of pathogenicity by dedicated tools is of indicative value only.

Among the published SOX10 missense variations that are located outside of the HMG domain, most should be considered variants of unknown significance. From our experience and bibliography review, it appears that extremely rare or new missense variations have a high probability of being truly pathogenic if located in the HMG domain, whereas missense mutations located outside of this domain, even if rare and ‘predicted pathogenic’ by in silico tools, are less likely to be pathogenic and should be considered cautiously. We have worked on SOX10 since its characterisation, both in the research and clinical context, and have only once found an exception to each of these rules. With the increasing number of mutations described, it appears that there may be a second, rare spot of mutations in the dimerisation domain (three variations reported in four independent cases, creating or removing valines at residues 76, 79 and 80), although functional tests are required to reach a definitive conclusion91 92 (online supplemental table 1).Due to the well-documented incomplete penetrance and digenism in KS and nIHH, there is a tendency in the literature to overevaluate the pathogenic probability of rare variants. Certain SOX10 variations have been considered to be pathogenic or likely pathogenic without many arguments (low pathogenicity scores, no functional tests, proven not pathogenic in another paper, inherited from healthy parents or without segregation study, and/or associated with an obvious causative mutation in another KS/nIHH gene).

On careful review, we consider several of these rare missense variants to more likely be neutral (although some still may be hypomorphic variants exerting their effect on a multigenic background, but this has thus far not been proven) and did not include them in figure 3.In early studies, most SOX10 variants were found to be de novo, which was thought to be due to the severity of HSCR in WS4. Given the cumulative number of WS2 cases now described, the life-threatening hypothesis cannot completely explain the proportion of de novo mutations. The possibility of hypogonadism in patients probably also contributes to this observation. However, the proportion of familial cases has tended to increase over the years and now represents approximately 20% of cases. These cases revealed an important intrafamilial phenotypic variability.

Several mutations have now been found in independent cases and also show interfamilial phenotypic variability. Parental mosaicism is found in approximately 3%–4% of cases, but a recent study reported a higher proportion in a small series using more sensitive methods.93The proportion of mutations relating to phenotype is summarised in figure 3B. There is a large proportion of truncating mutations in WS4 and PCWH. The proportion of missense increases in WS2 and even more strongly in KS. Thus, not all missense mutations may be null mutations.The location of truncating mutations along the gene (figure 3C) confirms the correlation between PCWH and the escape from non-sense-mediated RNA decay (NMD) (mutations located in the last coding exon and last 45 nt of the penultimate exon).64 Of note, some of the cases that appear not to respect the NMD rule may be misclassified (whether demyelination is proven or not is not always reported).

The severity of PCWH was shown to be linked to the location of the mutation within the last exon. The earlier the truncation, the more severe the phenotype.64 This tendency is visible in the graphs (compare the truncating mutations of WS vs PCWH in the last exon). A few cases escape the rule, with no clear explanation so far.Finally, heterozygous deletions/duplications can be intragenic or lead to complete gene loss and be as large as several Mb, encompassing other genes and leading to more complex phenotypes.Functional consequences of SOX10 mutations and the origin of phenotypic variabilityMost in vitro functional tests found in the literature rely on the ability of SOX10 to activate its target genes, alone or in combination with its cofactors. The construct most frequently used is a luciferase reporter under the control of the MITF-M (melanocyte-specific isoform) promoter. Additional targets, immunohistochemistry and assessment of the contribution of the DNA-binding capabilities have sometimes enriched such studies.Functional analysis of the first SOX10 missense mutation suggested that differential tissue-specific gene regulation could account for the phenotype observed in patients.94–96 Since, many SOX10 missense mutations associated with a variety of phenotypes, ranging from WS2 to WS4 and PCWH, have been tested, but no clear correlation between in vitro results and the phenotypes could be established.79 90 97The development of in vivo tests is therefore required to facilitate the establishment of genotype–phenotype correlations.

The only model currently published is in ovo chick electroporation in the developing neural tube.26 However, the effect of most of the mutations on early NC development precludes the analysis of their role in later developmental processes. Use of an inducible model would be of interest. Alternatively, zebrafish or the use of induced pluripotent stem cells differentiated towards NC derivatives of interest could be future models of choice.As mentioned earlier, the presence or absence of a neurological phenotype that characterises PCWH or PCW was proposed to be related to NMD.64 The proposed mechanism is that mutant proteins that have escaped degradation via the NMD pathway result in dominant-negative activity that impairs the function of the wildtype SOX10 allele and lead to PCWH, while those located in the first coding exons activate the NMD RNA surveillance pathway, leading to degradation, haploinsufficiency and a classic WS phenotype.However, several non-stop mutations have also been described to be associated with a PCW/PCWH phenotype. This is thought to be due to the generation of a specific inframe new C-terminus generated by the loss of normal termination. Functional studies of an equivalent mouse mutant allele showed that the additional 82 amino acids contain a deleterious (tryptophan-arginine (WR) domain, supporting a toxic gain-of-function.98 This is consistent with the recent report of a frameshift mutation that also elongates the protein, but in a different reading frame does not lead to PCWH (p.Tyr460Leufs*42).99 The observation of another, transgenic mouse model carrying different copy number variations of the first described SOX10 non-stop mutation suggested PCWH is due to a dominant-additive, rather than dominant-negative, effect.66 Finally, duplication of the 22q13.1 region, including SOX10, can also induce PCWH,85 supporting the hypothesis that it is promoted by a gain-of-function rather than a dominant-negative effect.

Regardless of the mechanism, these observations all indicate that NC derivatives are highly sensitive to the dose of SOX10 and its function.SOX10 expression is regulated by numerous enhancers. It is thus possible that certain cases with minor expression could also be due to specific dysregulation of one or a subset of such enhancers. This paradigm is supported by disruption of tissue-specific, long-distance regulatory regions of SOX9 causing endophenotypes.100 101 A large de novo deletion encompassing three SOX10 regulatory elements has been characterised in a patient with typical WS4,102 leading to the hypothesis that variations affecting certain identified regulatory sequences could be the cause of unexplained WS2 or isolated HSCR. Screening for mutations in SOX10 regulatory regions in WS2 turned out to be unfruitful.103 On the contrary, one deletion and two point variations affecting binding sites for known NC transcription factors were identified in 3 of 144 cases of isolated HSCR, both variations being in association with a HSCR-predisposition polymorphism at the RET locus.104 With the implementation of population databases, it now appears that one of these two variants is less rare than expected (22-38016774 G-C. About 1/1000 according to the gnomAD database.

Https://gnomad.broadinstitute.org/), which questions its involvement. These results are yet to be replicated for a pertinent interpretation.In any case, in vitro/in vivo tests will not be able to explain all phenotypes, as phenotypic variability is commonly recognised in patients with SOX10 mutations, even those with the same mutation and even within the same family. This suggests that the genetic background is influential, as has often been suggested for HSCR.53 105 Because the identification of modifier genes has been hampered by the small number of available patients, most modifier gene studies have relied on Sox10 mouse models.22Despite such variability, certain specificities have been reported for a few peculiar missense mutations. Here, we want to discuss the case of the Gln174/Pro175 missense mutations. The observation that certain SOX10 missense mutants accumulate in nuclear foci in transfected cells, where they colocalise with p54NRB (nucleo ribo binding protein, 54 kDa/NONO, non-Pou domain containing octamer binding.

A multifunctional protein known to be a marker of ‘paraspeckles’), leads to characterise missense mutations of amino acids 174 and 175 as associated with a peculiar phenotype (refs 79 97 and unpublished data (S. Marlin, N. Bondurand and V. Pingault, 2016)). Remarkably, the cotransfection of foci-forming mutant with wildtype constructs led to the sequestration of wildtype SOX10 in these ‘foci’ and altered the synergistic activity of SOX10 and p54NRB.

A dominant-negative effect was therefore proposed to contribute to or be at the origin of the progressive central and peripheral neurological phenotypes observed in patients carrying these specific missense mutations and may thus be the basis of a hitherto unexplored molecular mechanism for genotype–phenotype correlations. These data need to be confirmed in more physiological models.The phenotype variability finally leads to question the risk of a more severe phenotype in cases of recurrence in a family. The risk of the PCWH phenotype after a first non-PCWH case is considered to be low. On the contrary, there is a risk of WS4 after a first, milder case of WS2. This situation has been reported several times.

However, a bias in the representation of these cases in the literature can be expected, as a second mildly affected member is less likely to result in a visit of the family to the geneticist’s office, molecular analysis and ultimately publication. As a result, the true risk is difficult to quantify.Conclusion, with a few tips to help in variant classificationDuring twenty years of cases and cohorts reporting, SOX10 variants have been involved in WS2/WS4/PCWH/Kallmann syndrome/pseudo-isolated hearing loss/HSCR or CIPO and any combination. This is correlated with the known developmental functions of SOX10. All these phenotypes should be considered as a clinical continuum with variable expression, rather than as independent diseases, conferring a mild to life-threatening syndrome. Observation of the familial cases and of a few recurrent variations documented a high phenotypic variability, even within a single family.Most mutations predict a truncation of the protein, but the proportion of missense variations has increased with time.

Missense variations (or small in-frame insertions/deletions) outside of the HMG domain should be considered with caution, even with good in silico pathogenicity scores. The fact that the variation is already published can be used as a supporting argument only if the strength of the published data has been verified (also a general recommendation of the American College of Medical Genetic).The most (almost fully) penetrant sign observed in patients is hearing impairment. Pigmentation defects are not always present. Confirmed incomplete penetrance appears to be very rare, but a targeted clinical reevaluation may be necessary to assess mild signs. Searching for inner ear-specific malformations by imaging is highly informative.

The absence of olfactory bulbs could be investigated at the same time by MRI. The only strong phenotype-genotype correlation usable in phenotype prediction, thus far, is the link between NMD escape and PCW/PCWH.Ethics statementsPatient consent for publicationNot required.Ethics approvalEthics approval is not applicable. This study does not involve human participants in a research study. Only mutations found on a diagnosis basis are reviewed in a retrospective manner (list of mutations along with scarce clinical information).AcknowledgmentsWe apologise to all whose contributions were not cited due to space limitations.IntroductionIn recent years, a large increase in the use of multigene panel tests for breast cancer associated pathogenic variants (PVs) has expanded the number of potentially actionable PVs beyond BRCA1 and BRCA2.1–9 These studies have shown an almost equal rate of BRCA1/2 PVs to all additional potentially actionable gene PVs combined. In addition, much of the increased detection is due to variants in less actionable moderate-risk genes,10ATM and CHEK2, with higher background population prevalence.

The only other actionable breast cancer gene variants consistently identified at substantial rates is PALB2, which is now also considered to be a high-risk susceptibility gene.11Although higher frequencies of actionable gene variants are reported in those at particularly young ages (<40 years) particularly for TP53, the PV rates of ATM and CHEK2 do not appear to be strongly related if at all to age-at-onset, although a small effect was seen for CHEK2 in two studies.1 2 Very few studies have concentrated testing on women with very early onset breast cancer. We previously reported a high rate of BRCA1, BRCA2 and TP53 PVs in a population based series of breast cancer in women ≤30 years of age at diagnosis.12 13 Fewer than 1 in 1000 women develop breast cancer by age 30 years and UK statistics showed that only 222 of 54 450 (0.41%) of breast cancers occurred in women aged <30 years14 (0.59% if ~100 breast cancers in women aged 30 years are included).14 Although this is a small group of patients with breast cancer, the prognosis of breast cancer diagnosed in this young age group is poor.12 13 15 16 BRCA1 and BRCA2 PVs have been reported in small numbers of women diagnosed aged ≤30 years. However, the studies reporting these individuals include many women with breast cancer diagnosed at older ages and do not specify the detection rates within the ≤30 years age group.15 16 The Prospective study of Outcomes in Sporadic vs Hereditary breast cancer (POSH) reported a 12% rate of BRCA1/2 PVs in 338 of 2733 women diagnosed aged ≤40 years, but only 316 of a total 3095 women in POSH were aged ≤30 years and no separate analysis was presented.15 16 In another study, the rate of TP53 PVs was reported as 6% in an unselected subset within 333 women with breast cancer aged ≤30 years.17 The Myriad study is the only large study that has assessed the detection rate of PVs in other breast cancer genes in women with breast cancer aged <30 years. In this study, 783 (2.2%) of 35 409 women were aged <30 years;6 however, it is likely that there was considerable pretesting in this series for BRCA1/2 and TP53 PVs as acknowledged by the authors and evidenced by the low detection rates among Ashkenazi Jews.We present analysis of BRCA1/2 and TP53 testing in 379 patients with breast cancer aged ≤30 years, and of extended testing of a panel of additional breast cancer genes in 184 patients, expanding our previous population-based study of 115 women.12 13MethodsIndividuals with a confirmed breast cancer diagnosis aged ≤30 years were eligible for the study. Affected women came from two sources.

The first was a population-based study of 288 women with breast cancer presenting between January 1980 and December 1997 from the Manchester region (population=4.5M) of North-West England identified from the regional cancer registry.12 13 From this, 175 women were alive and potentially available for genetic testing.12 Fifty (28.6%) of these did not provide a DNA sample (it was either not appropriate to recontact or the individual did not wish to participate or could not be traced). This increases by 10 the number with available DNA samples from our previous report to 125.13 Only 39 currently living patients have not consented to the study. An additional 256 women were referred to the Manchester Centre for Genomic Medicine (MCGM) between 1990 and 2019. All women gave clinical consent for testing of breast cancer genes. Samples were initially screened for point mutations and copy number variants in BRCA1, BRCA2, TP53 and for the CHEK2 c.1100delC PV.13 When a PV was identified, no further testing was carried out.

Samples testing negative were selected for next generation sequencing panels which included, as a minimum, the additional breast cancer associated genes. PALB2, CHEK2, ATM, CDH1, PTEN, RAD50, RAD51D and NBN. In addition, 1567 population control samples without breast cancer at entry aged 47–73 years from the PROCAS study18 were tested as part of the Breast Cancer Risk after Diagnostic Gene Sequencing (BRIDGES) programme.19PV frequencies in the Manchester early onset cohort were compared with PV frequencies observed in women aged ≤30 years who took part in the prospectively ascertained POSH study (01/2000–01/2008).15 16Tumour pathology information was obtained through hospital record and cancer registries. The pathology adjusted Manchester Scoring System was used to assess likelihoods of BRCA1/2 PVs.20 Pathology-adjusted Manchester score (MS) of 15–19 is equivalent to a 10% probability of a BRCA1/2 PV and a 20–24 point score is equivalent to a 20% probability.The type and number of PVs were determined in the full cohort as well as in different age groups, specific tumour pathology characteristics and MS.ResultsA total of 381 women with breast cancer diagnosed ≤30 years were included. Two women met diagnostic criteria for neurofibromatosis type 1, explaining their early onset of breast cancer.

The remaining 379 were screened for variants in BRCA1, BRCA2, TP53 and the CHEK2 c.1100delC variant. This strategy detected 134 PVs. BRCA1=75 (19.79%), BRCA2=35 (9.23%), TP53=22 (5.80%), CHEK2 c.1100delC=2 (0.53%). One woman harboured both a BRCA1 and BRCA2 PV. Of those testing negative, 184 (74.8%) underwent extended genetic testing.

Sixty-two women did not undergo further testing due to poor quality, or insufficient, DNA. The detection rate was 4.35% (n=8) for actionable breast cancer PVs (ATM=2, PALB2=4, CHEK2=1, PTEN=1, online supplemental table 1). Single PVs were identified in other genes associated with breast cancer risk, BRIP1 (c.2392C>T. P.Arg798Ter), RECQL (c.1667_1667+3delAGTA. P.?.

) and RAD50 (c.1300_ 1306del. P.Asp434LysfsTer7).Supplemental materialRisk associations for each gene were determined using the population controls from the PROCAS study (table 1). Significant associations with a more than twofold increased risk were found for BRCA1. OR=193.10 (95% CI 51.58 to 804.8), BRCA2. OR=17.61 (95% CI 8.59 to 36.53), TP53.

OR=308.10 (95% CI 51.20 to 3202) and PALB2. OR=11.59 (95% CI 3.08 to 46.15). PV rates in the POSH study were established among the 287 women with invasive breast cancer at the age of ≤30 years. A total of 56 (19.5%) PVs were identified in BRCA1 (32 PVs, 11.1%), BRCA2 (17 PVs 5.9%), TP53 (5 PVs, 1.7%) and CHEK2 c.1100delC (3 PVs, 1.1%) (table 1).View this table:Table 1 Association of pathogenic variants with early onset of breast cancerDetection rate of pathogenic variants in different age groupsSurprisingly, the youngest age group (<26 years) showed a lower rate of BRCA1/2 PVs. Only 9/61 (14.75%) compared with 101/318 (31.76%) for those aged 26–30 years (p=0.0083) (table 2).

TP53 showed the reverse trend with 7/61 (11.48%) aged <26 years compared with 4.72% (15/318) in those aged 26–30 years (p=0.0649). Thus, only 12.93% (15/116) PVs in BRCA1/2/TP53 in those aged 26–30 years were in TP53 compared with 43.8% (7/16) in those <26 years (p=0.0060). The lower rates in the younger age group for BRCA1/2 PVs were similar to the rates in the POSH cohort ≤30 years potentially reflecting ascertainment differences. The higher rate of TP53 PVs (5.8%) compared with 1.7% in POSH likely reflects that the POSH study specifically excluded women with only DCIS and no invasive tumour component.View this table:Table 2 Rates of pathogenic variants by age group, pathology and Manchester Scoring SystemThe CHEK2 c.1100delC PV was identified in only 2/379 (0.53%) compared with 1.7% (55/3177) in women with breast cancer aged >30 years (p=0.0835) seen at the MCGM and 2.3% in the POSH study aged ≤40% and 1% in POSH cases≤30 years (table 1).Manchester scoreThe detection of PVs in BRCA1 and BRCA2 was, as expected, strongly correlated with breast cancer pathology and family history. The MS accurately predicted the likelihood of a BRCA1/BRCA2 variant at both the 10% (15–19 points) and 20% (20–24 points) thresholds (table 2).

By including PVs in TP53, 100% of women with a MS ≥40 had a PV in BRCA1/2 or TP53.Tumour characteristicsWe identified 61 (48.8%) PVs in BRCA1/2/TP53 in 125 women with triple-negative breast cancer (TNBC) (table 3). Unexpectedly, a similar rate of BRCA1/2/TP53 PVs was detected in cases of pure DCIS (11/26 [42.3%]), although TP53 accounted for 54.5% (6/11) of these. Eight were comedo DCIS of which four had a TP53 PV. The majority of DCIS were high grade (18/26) and 8/18 harboured a PV (2 in BRCA1, 1 in BRCA2 and 5 in TP53) (table 3). None of the cases of pure DCIS were detected on screening for familial risk.View this table:Table 3 Rates of pathogenic variants found in patients with DCISHER2+ breast cancer showed a similar predominance of TP53 PVs (8/43 (18.6%)), but BRCA1/2 PVs were uncommon (3/43 (6.9%)).Presence of cancer in both breasts was also predictive of PVs, with 36/63 (57.1%) cases with BRCA1/2/TP53 PVs (including 10/22 TP53 PVs) having bilateral breast cancer.Sporadic breast cancerOf 147 women without a family history of breast or ovarian cancer at original diagnosis, 24 (16.3%) had a PV.

Only 10 (6.8%) had BRCA1/2 PVs (BRCA1=7. BRCA2=4. 1 woman had both BRCA1 and BRCA2 PVs), 12 women had a TP53 PV and the remaining 2 women had a PALB2 or a CHEK2 PV. All BRCA1 PVs were detected in women with sporadic TNBC 7/59 (11.9%). There were six other PVs identified in sporadic TNBC in BRCA2=3, TP53=2 and PALB2=1.

Of 26 people with HER2+ sporadic breast cancers, 7 (26.9%) had PVs. (TP53=6. BRCA2=1). Outside of these confirmed pathologies 5/62 (8.1%) had PVs (TP53=4, CHEK2=1), but receptor status was unknown in 43 cases, including 13 with DCIS, two of whom had a TP53 PV.TP53 carriersAmong TP53 carriers, 10/22 (45.5%) had a family history of breast cancer at initial diagnosis. Additional relatives in three of these families had Li Fraumeni spectrum tumours (one had none at diagnosis) and one had a personal history of childhood adrenocortical cancer.

Additionally, four families without relatives with breast cancer, had family histories, including the index breast cancer, consistent with classical Li Fraumeni syndrome including at least one sarcoma aged <45 years. One de novo case had an osteosarcoma of the leg aged 19 years. Seven (33%) apparently de novo TP53-associated cases (confirmed after parental testing), with no significant personal or family history of cancer, presented with breast cancer. Thus, 7/144 (4.9%) apparently sporadic breast cancer cases ≤30 years had TP53 de novo variants that would not have been expected from personal or family history.One of the TP53 PVs was identified at a variant allele frequency of 22% suggesting mosaicism (online supplemental table 1). The PV was found in the tumour (20%-neoplastic content) at 15% and 11% in normal breast excluding clonal haematopoiesis (in a woman with Paget’s/DCIS who had not undergone radiotherapy/chemotherapy).Assessment of population level of testingThere were 135 women diagnosed with breast cancer in the Manchester region aged ≤30 years between 01/01/1990 and 31/12/1997 (since cancer genetic testing was introduced in Manchester) within the population study giving an annual rate of 16.9 cases.

During this time, we tested 73/135 (54.1%) of affected women and identified BRCA1=13 (17.8%), BRCA2=8 (11%) and TP53=3 (4.1%) PVs. Of our population based study group of 125 women who underwent genetic testing (presenting with cancer between 1980 and 1997), there were PVs in BRCA1=23 (18.4%), BRCA2=11 (8.8%), TP53=5 (4%) and BRIP1=1,12 13 demonstrating a very similar overall detection rate. In the cohort referred to MCGM between 01/01/1998 and 3/11/2019, we tested 219 women and identified PVs in BRCA1=46 (21.0%), BRCA2=17 (7.8%) and TP53=16 (7.3%). The combined rate of BRCA1/2 PVs at 27.2% (population-based study) and 28.8% (referrals) are similar, suggesting no substantial testing bias. However, 68/125 (54.4%) in the population study (1980–1997) had no family history, compared with 77/219 (35.2%) in the recent cases (1998–2019) (p=0.0006).

All but 18 of the 219 tested since 1997 had full pathology and ER receptor status available, and only eight ER+ ductal carcinomas had unknown HER2 status.Co-occurrence of actionable breast cancer gene variantsOf 920 breast cancer cases with no prescreening tested at MCGM, no co-occurrence of two actionable breast cancer gene variants was found. Among 4916 non-Jewish breast cancer cases undergoing full BRCA1 and BRCA2 testing, only two co-occurrences of BRCA1 and BRCA2 PVs has occurred including the single case reported in this study.DiscussionWe report here the results of 379 patients with breast cancer ≤30 years initially tested for PVs in BRCA1, BRCA2, TP53 and CHEK2 c.1100delC. Of the patients testing negative for these genes, 184 underwent testing of a panel of breast cancer associated genes. A total of 145 PVs were detected in 144 women, of which the majority (134 PVs) were identified in BRCA1, BRCA2, TP53 and CHEK2 c.1100delC. Only eight actionable PVs were found through extended panel testing.

The rate of PVs in the unselected population series (n=125) was 18.9% in BRCA1, 8.8% in BRCA2 and 4% in TP53. The overall detection rate for TP53 (5.8%) in all samples is similar to the rate (6%) published previously.17 The Myriad study assessed this age group (783 women) and found combined rates of BRCA1/2 PVs of 14% in women aged 25–29 years and 9% in women aged <25 years,6 although this cannot be considered a population study. Our study supports this lower detection rate in the very youngest age group, in contrast to the overall trend to increasing frequency of BRCA1/2 at younger ages seen in population based testing.21 This is similar to the lower rates found in ovarian cancer <30 years.22 The Myriad study6 also showed a similarly increased detection rate for TNBC <30 years. Although there was no breakdown between BRCA1 and BRCA2, it is highly likely that this was BRCA1 driven as in our study. There is no specific figure given for TP53 in this age group, but it is also likely that the increased detection rates for non-BRCA genes from <4% (similar to all other age groups) in the 25–29 age group to ~8% in the <25 group is due to TP53.

In this study, we noted an increased detection rate from 4.8% to 11.7%, due to the inclusion of TP53. Specific data from 287 of the POSH cases diagnosed aged <31 who have been analysed for TP53 and CHEK2 c.1100delC in addition to BRCA1/2 showed overall PV rate was higher in the <26 age group (28.9%) compared with 18.1% in the higher age group (online supplemental table 2). TP53 and BRCA2 PVs were more prevalent in the youngest age groups in the POSH study although numbers were small. Nonetheless, combining the frequencies from both studies the rates of BRCA1 and BRCA2 fell from 17.1% and 7.9% in the 26–30 age group to 10.1% and 7.1% in the <26 age group, respectively, although this was not significant for BRCA1 (p=0.1) and combined BRCA1 and BRCA2 (p=0.09). The increase for TP53 detection remained significant from 3.2% to 9.1% (p=0.01).

The difference in incidence of PVs between POSH and this study may be due to sampling, certainly excluding cases with no invasive component to the presenting cancer would explain the lower rate of TP53 in the POSH study as well as excluding previous malignancy which jointly made up 12/22 (54%) of TP53 carriers in Manchester.We have also analysed available online data from Ambry genetics commercial testing (https://www.ambrygen.com/providers/resources/prevalence-tool, accessed 29/08/2020).23 While it is not possible to assess the level of pretesting for TP53, and BRCA1/2 or the presence of a Li Fraumeni family history, there is a clear upward trend of prevalence of BRCA1 and BRCA2 PVs with reducing age at breast cancer until 26 years of age (online supplemental table 3). In contrast TP53 detection is increased in the <26 year age group (p=0.03), consistent with our findings.Although the Myriad study is larger than the present study, there is a lack of detail, in particular regarding how much pretesting had been undertaken for PVs in BRCA1/2/TP53. Many women may have been tested for BRCA1/2 years earlier and subsequently taken advantage of extended testing. Similarly, women diagnosed with breast cancer and features of Li Fraumeni syndrome may have undergone clinical bespoke TP53 testing. Nine of 15 (60%) such TP53 cases in the present study triggered clinical testing based on personal or family history.

The lower rates for BRCA1/2/TP53 PVs in the Myriad study probably reflects this level of pretesting and the more likely accurate rates are from the pure population-based series in the present study from 1980 to 1997.16The current study has convincingly shown that PVs in BRCA1 are the biggest contributor to breast cancer in women diagnosed aged ≤30 years. Even in the pure population-based study, this was at least twice the rate of BRCA2. BRCA1 PVs were also twice as prevalent in this age group as BRCA2 PVs in the POSH study. Given the lower population prevalence of BRCA1 PVs, the risk of breast cancer in some women with a BRCA1 PV will be sufficient to recommend MRI screening in BRCA1 PV carriers<30 years. New UK guidance from the National Screening Committee will allow screening in BRCA1/2 PV carriers once their 10 year risk is 8%.24 This level of risk is estimated in BRCA1 PV carriers aged 25 years with a first degree relative diagnosed <40 years in both the Tyrer-Cuzick and BOADICEA models.25 26 Many other countries already offer screening in BRCA1/2 PV carriers from 25 years.

The presence of seven TP53 carriers with breast cancer <26 years of age may well justify MRI screening from age 20 years as is already recommended in a number of guidelines.24The present study has shown limited clinical benefit from testing of genes apart from BRCA1, BRCA2 and TP53 in women with invasive or in situ breast cancer aged ≤30 years. The individual with a PTEN PV had a classical phenotype and had PTEN bespoke testing rather than a panel. The detection rate in other actionable breast cancer genes was only 4.3% (8/184). Even allowing for an increased detection rate from testing the remaining 62 cases, this would have only reached 11/246 cases. Nevertheless, as at least seven TP53 cases would not have been suspected based on personal or family history, TP53 should be included in first-line testing as long as the panel does not reduce sensitivity for BRCA1/2 variant detection.

While a single BRIP1 PV was detected, this gene is not convincingly associated with breast cancer risk and the current evidence does not support actionability for these variants.27 Similarly there has been no clinical validation for RECQL28 29 and RAD50 and the cases in the current series was consistent with population frequencies. We also found no RAD51C or RAD51D variants consistent with their primary association with ovarian cancer susceptibility.30 31All different tumour pathologies had a >9% detection rate for BRCA1/2 and TP53 PVs. A striking finding was that the rate of PVs associated with DCIS (42.3%) was almost as high as that associated with TNBC (48.3%). The previous association with TP53 and high-grade comedo DCIS was noted.13 We also found a rate of 15.4% (4/26) for BRCA1/2 PVs in DCIS cases. The 23.1% rate for TP53 PVs in DCIS in our study reflects the very strong association of DCIS even with invasive cancers with 41 of 45 (91.1%) of all cases containing DCIS in one study of TP53 related breast cancers.32 Currently, many countries in Europe have not instituted extended panel testing for breast cancer and in England testing for a three gene panel of BRCA1, BRCA2 and PALB2 will be provided by the public healthcare system unless a specific request is made for TP53 by a geneticist.

Our study would suggest that TP53 should be discussed and potentially added to all breast cancer gene screens≤30 years unless the woman declines following counselling of the implications of this test. The importance of identifying TP53 variants is shown by the extremely high rate of contralateral breast cancer, nearly 50% in the present study and with annual contralateral rates of ~40%.33 Given the concerns about radiation treatment and new primaries with TP53,34 35 a discussion about mastectomy and even bilateral mastectomy needs to be undertaken as well as instituting proven early detection strategies for other malignancies, including whole body MRI as published in two recent guidelines.34 35This study has some limitations. Not all 379 women underwent full testing of the panel of breast cancer associated genes. However, we have shown that there is a very low likelihood that an individual identified with a PV in BRCA1/2 or TP53 would also carry a PV in another breast cancer gene. It is therefore unlikely that failure to test those with known BRCA1/2 PVs missed PVs in other breast cancer genes.

Unfortunately, full pathology and receptor status was not available on all women. This reflects the chronological, real life data nature of the study. Breast cancer grade was only reported reliably after 1990 and ER receptor status after 1995. HER2 status was not usually reported until 1999, after approval of Herceptin (trastuzumab) for treating HER2+ breast cancer. Nonetheless, there were still a large number of TNBCs available for assessment and since 1997 the majority of women had full pathology available, including HER2 status.

The strengths of this study include. The large number of patients with what is a rare cancer in young women. The well characterised nature of the cohort with extensive family history. A pure population-based cohort with high ascertainment even in the postcohort study period, and the presence of a population control for evaluated genes. The sensitivity of our testing, especially for BRCA1/2 and TP53, is high, indicated by the 100% detection rate of a PV in the 31 women with MS of ≥40.

Although the score was designed for BRCA1/2, it has also clearly captured very early onset highly penetrant TP53 families.In conclusion, we have identified a high rate of actionable PVs in breast cancer genes in women with breast cancer aged ≤30 years. The clear association of TP53 PVs in very young women presenting only with DCIS is noteworthy and adds to the published association of HER2+ invasive disease in young women with TP53 PVs.32 TP53 and BRCA1/2 PVs are of similar frequency in women with breast cancer <26 years but BRCA1/2 PVs predominate in those aged 26–30 years. Overall, there is little additional benefit of testing breast cancer-associated genes apart from BRCA1, BRCA2 and TP53 in this age group.Data availability statementData are available on reasonable request. The datasets analysed during the current study are available from the corresponding author on reasonable request.Ethics statementsPatient consent for publicationNot required.Ethics approvalResearch aspects of this study were approved by the North Manchester research ethics committee (Reference 08/H1006/77)..

IntroductionSOX10 belongs to the SOX family of transcription factors, of which the members are defined based on the presence of a 79 amino acid DNA-binding domain with homology to the high mobility look at this now group flagyl online canada (HMG) box of SRY (sex-determining region Y. Hence SOX, Sry bOX). These factors are involved in multiple developmental processes, such as male differentiation, skeletogenesis, neurogenesis and neural crest (NC) development, where they control stemness, cell fate and differentiation.1–4 The growing number of developmental disorders associated with mutations in SOX genes underscores their importance during development.5 The SOX10 transcription factor is a characteristic marker for migratory multipotent NC progenitors as well as for various NC derivatives.The NC is a specific population of cells in vertebrates that arise at the edge between the neural and non-neural ectoderm, delaminate from the dorsal aspect of the flagyl online canada neural tube, and migrate through several routes to reach target tissues and give rise to neurons and glia of the peripheral nervous system (PNS), including sensory, autonomous and enteric ganglia, Schwann cells and olfactory ensheathing cells, melanocyte pigment cells, skeletal structures and mesenchyme of the head, face and neck, outflow tract of the heart, and smooth muscle cells of the great arteries.6 7Over the years, heterozygous SOX10 mutations have been associated with various phenotypes that extend beyond Waardenburg syndrome (WS.

Depigmentation features and deafness) and Hirschsprung disease (HSCR. Intestinal aganglionosis) initial flagyl online canada diagnosis. Here, we present an up-to-date overview of these various clinical manifestations, along with our current understanding of how they are explained by SOX10 dysfunction in several NC derivatives and extra-NC tissues (inner ear and oligodendrocytes), and of the origin of phenotypic variability.SOX10.

Structure and regulation of the gene, protein domains and post-transcriptional modificationsThe human SOX10 and mouse Sox10 genes encode flagyl online canada an open reading frame of 466 amino acids that share 92% nucleotidic and 98% amino acid sequence identities.8 The absence of a complete description of the human gene 5’ non-coding exon(s) has given rise to two coexisting exon numbering systems. Historically, exons 1 and 2 are non-coding, the initiation codon is found in exon 3, and the stop codon in exon 5.8 The second system is based on the reference transcript NM_006941, with only one non-coding exon in the 5’UTR (untranslated transcribed region) and a total of four exons. A major transcript of ~3 kb is detected in most tissues tested, consistent with the predicted SOX10 mRNA sequence.9 10The protein’s structure flagyl online canada is schematised in figure 1.

As for all other members of the SOX family, the previously mentioned HMG domain forms an L-shaped module composed of three alpha helices that bind to DNA sequences in the minor groove (matching or resembling C[A/T]TTG[A/T][A/T]), bending the DNA molecule and interacting with other proteins to establish stable and active transcriptional complexes3 4 (the most recent model can be found in Haseeb and Lefebvre11). This domain also harbours two nuclear import (nuclear localisation signal) and one export (nuclear export signal) signals.12 1310 samples in various tissues (including the brain, gut, nerves (tibial), flagyl online canada breast and salivary glands)/total number of A-to-I modifications reported is. AluY.

20/73. DIM, dimerisation domain. ERK, extracellular signal-regulated kinase.

HMG, high mobility group domain. NES, nuclear export signal. NLS, nuclear localisation signal.

TA/TAC, transactivation domain in C-terminal. TAM, transactivation domain in the middle of the protein." class="highwire-fragment fragment-images colorbox-load" rel="gallery-fragment-images-1605886105" data-figure-caption="Schematic of the SOX10 protein and post-translational modifications. Domains of human SOX10.

The numbers refer to amino acid residues. The pink lines above the HMG domain represent the NLS sequences, one at each end of the HMG domain, and the light pink line the NES sequence. Note that although nucleocytoplasmic shuttling of the protein has been well documented for several SOX factors,12 13 in vivo regulation of SOX10 through nuclear translocation is yet to be clarified.

Black arrowheads represent the localisation of junctions between exons. Post-transcriptional modifications, including acetylation (Ac), phosphorylation (P) and sumoylation (Su), are indicated, along with the position of modified amino acids. Note that a putative acetylation site was identified in SOX2 and is conserved in SOX10.17 Sumoylation by Ubc9 occurs at lysines K55, K246 and K357 with consequences on cell fate decision.19 20 Mechanistically, sumoylated SOXE proteins fail to interact with the coactivator CBP (CREB-binding protein)/p300 and instead recruit the GRG4 corepressor (Groucho-related protein 4/TLE4, transducing-like enhancer of split 4), leading to strong inhibition of SOXE target genes.106 Among the identified phosphorylation sites, note that ERK phosphorylates T240 and T244, inhibiting the sumoylation of SOX10 at K55 and transcriptional activity.107 Additional phosphorylation sites have been described from large-scale proteomic screens in melanoma, breast tumours and mouse neuroblastoma (serine S8, S13, S17, S24, S27, S30, S40, S45, S221, S224 and S23216).

The relevance of most has not been functionally assessed. SOX10 phosphorylation sites are localised in two distinct clusters, one at the amino terminus, 5’ of the dimerisation domain, and the other at the centre of the protein. FBXW7-mediated ubiquitination of SOX10 has also been shown to control protein stability.

The region involves aa 235–244 of the human protein. A search of the public REDIportal (http://srv00.recas.ba.infn.it/atlas/) revealed various A-to-I editing sites located in AluY, AluSx or AluSq sequences embedded in the last SOX10 intron. In each Alu sequence schematised from left to right, the number of A-to-I sites identified in >10 samples in various tissues (including the brain, gut, nerves (tibial), breast and salivary glands)/total number of A-to-I modifications reported is.

ERK, extracellular signal-regulated kinase. HMG, high mobility group domain. NES, nuclear export signal.

NLS, nuclear localisation signal. TA/TAC, transactivation domain in C-terminal. TAM, transactivation domain in the middle of the protein." data-icon-position data-hide-link-title="0">Figure 1 Schematic of the SOX10 protein and post-translational modifications.

Domains of human SOX10. The numbers refer to amino acid residues. The pink lines above the HMG domain represent the NLS sequences, one at each end of the HMG domain, and the light pink line the NES sequence.

Note that although nucleocytoplasmic shuttling of the protein has been well documented for several SOX factors,12 13 in vivo regulation of SOX10 through nuclear translocation is yet to be clarified. Black arrowheads represent the localisation of junctions between exons. Post-transcriptional modifications, including acetylation (Ac), phosphorylation (P) and sumoylation (Su), are indicated, along with the position of modified amino acids.

Note that a putative acetylation site was identified in SOX2 and is conserved in SOX10.17 Sumoylation by Ubc9 occurs at lysines K55, K246 and K357 with consequences on cell fate decision.19 20 Mechanistically, sumoylated SOXE proteins fail to interact with the coactivator CBP (CREB-binding protein)/p300 and instead recruit the GRG4 corepressor (Groucho-related protein 4/TLE4, transducing-like enhancer of split 4), leading to strong inhibition of SOXE target genes.106 Among the identified phosphorylation sites, note that ERK phosphorylates T240 and T244, inhibiting the sumoylation of SOX10 at K55 and transcriptional activity.107 Additional phosphorylation sites have been described from large-scale proteomic screens in melanoma, breast tumours and mouse neuroblastoma (serine S8, S13, S17, S24, S27, S30, S40, S45, S221, S224 and S23216). The relevance of most has not been functionally assessed. SOX10 phosphorylation sites are localised in two distinct clusters, one at the amino terminus, 5’ of the dimerisation domain, and the other at the centre of the protein.

FBXW7-mediated ubiquitination of SOX10 has also been shown to control protein stability. The region involves aa 235–244 of the human protein. A search of the public REDIportal (http://srv00.recas.ba.infn.it/atlas/) revealed various A-to-I editing sites located in AluY, AluSx or AluSq sequences embedded in the last SOX10 intron.

In each Alu sequence schematised from left to right, the number of A-to-I sites identified in >10 samples in various tissues (including the brain, gut, nerves (tibial), breast and salivary glands)/total number of A-to-I modifications reported is. AluY. 67/224.

DIM, dimerisation domain. ERK, extracellular signal-regulated kinase. HMG, high mobility group domain.

NES, nuclear export signal. NLS, nuclear localisation signal. TA/TAC, transactivation domain in C-terminal.

TAM, transactivation domain in the middle of the protein.SOX10 shares additional domains with SOX8 and SOX9, all three forming the SOX_E group (SOX factors have been subdivided in several groups based on the amino acid identity within their HMG domain) (figure 1). Among them, the dimeric domain (DIM) confers preferential binding of SOX_E members to target sites containing two inverted SOX motifs separated by three to four nucleotides and promotes homodimerisation or heterodimerisation through DIM:HMG interactions.14Within its C-terminus, SOX10 contains a potent transactivation domain called the TA or TAC (transactivation domain in C-terminal).4 Another weaker and context-dependent transactivation domain has been identified in the middle of SOX10, the so-called K2 domain or TAM (transactivation domain in the middle of the protein), and was recently shown to synergise with TA/TAC in all SOX_E members.11 15Various post-transcriptional and post-translational modifications modulate the activity, stability and intracellular localisation of SOX1016 (figure 1). Several of these modifications are inferred from those occurring in other SOX factors, as for the lysine K136 acetylation site.16–18 Others, including phosphorylation sites, were mainly found from large-scale proteomic screens performed in cancer cells.

SOX10 sumoylation by UBC9 (sumo-conjugating enzyme UBC9) is the best described one. Occurring at lysines K55, K246 and K357,19 it inhibits NC development and promotes development of non-sensory cranial placodes in vivo.20 Absence of A-to-I RNA modification mediated by the ADAR (adenosine deaminase RNA-specific) enzyme family was recently reported to alter melanocyte and Schwann cell development.21 Examination of the public REDIportal shows that SOX10 is under such regulation in humans (but not mice).Finally, several regulatory regions likely involved in driving SOX10/Sox10 expression have been identified using various cell lines and zebrafish or mice models (ref 22 and references therein). Methylation of the Sox10 promoter by DNA methyansferase 3 has also been shown to arrest NC generation in chicks.23Involvement of SOX10 in WS.

Role in melanocytes and enteric nervous systemThe identification of Sox10 as the gene mutated in the spontaneous Dom mutant mouse (Dominant megacolon. Intestinal aganglionosis, white belly spot and white paws) first shed light on the essential function of this transcription factor in NC development. In this strain, a Sox10 frameshift mutation results in alteration of binding to some DNA target sequences in vitro, of transactivation capacity and synergistic action with several cofactors.9 24–27 This observation immediately led to test SOX10 involvement in Waardenburg-Hirschsprung disease.8 Also known as WS type 4 (WS4) or Waardenburg-Shah syndrome, Waardenburg-Hirschsprung encompasses symptoms of WS and HSCR (Mendelian inheritance in man, MIM) #613266).28–30HSCR is the most common enteric neuropathy, occurring in 1 of 5000 neonates, and is characterised by the absence of enteric ganglia from a varying length of the distal gut, leading to intestinal obstruction in neonates or severe constipation in adults (MIM #142623).29 30WS is a genetic disorder characterised by sensorineural hearing loss (SNHL) and pigmentation anomalies, including depigmented patches of skin and hair and vivid blue eyes or iris heterochromia (MIM #193500).

Four types of WS are clinically defined, based on additional features due to defects in structures mostly arising from NC derivatives. WS1 is further characterised by dystopia canthorum, WS3 by musculoskeletal abnormalities of the limbs, WS4 by HSCR, whereas WS2 has no further significant features. In addition to SOX10, four main genes are involved in WS thus far.

MITF (melanocyte inducing transcription factor) in WS2, PAX3 (transcription factor paired Box 3) in WS1 and WS3, EDN3 (endothelin 3) in WS4, and EDNRB (endothelin receptor type B) in WS4 and WS2.28 31 32 SOX10 has been shown to regulate and interact with several of these genes.28 33SOX10 screening in WS4 cases led to the identification of the first heterozygous mutations in 1998.8 In 2007, SOX10 mutations were shown to be also responsible for approximately 15% of WS2 cases.34 By contrast, SOX10 involvement in isolated HSCR is very limited. For example, screening of 229 isolated HSCR cases led to the identification of only one frameshift mutation inherited from an asymptomatic mother (germline mosaicism has been proposed).35Certain patients with WS4 or PCWH (see later) present with hypoganglionosis or chronic intestinal pseudo-obstruction (CIPO) instead of HSCR.28 36–39 Given the role of SOX10 in enteric nervous system (ENS) development, CIPO is probably neurogenic. Aganglionosis is therefore not the only mechanism underlying the intestinal dysfunction in patients with SOX10 mutations.Each of the clinical manifestations described above can be explained by dysregulation of SOX10 during melanocyte and ENS development.

WS accounts for a developmental defect in both skin melanocytes and a melanocyte-derived cell lineage of the inner ear, called intermediate cells of the stria vascularis, necessary to the inner ear homeostasis.40 In melanocytes, SOX10 controls proliferation, survival and differentiation by directly and sequentially activating a number of downstream target genes.4 41–43 From the NC, a SOX10–PAX3 pair activates the expression of Mitf/MITF, which then acts as a SOX10 partner to activate the expression of Dct (dopachrome tautomerase) and Tyr (tyrosinase), both involved in melanocyte differentiation and melanin synthesis.27 32 42 44 45 In 2015, an extensive genome-wide catalogue of SOX10 targets was obtained.46 For the first time, integrated chromatin occupancy and transcriptome analysis suggested a role of SOX10 in both transcriptional activation and repression. SOX10 was also shown to cooperate with MITF to facilitate BRG1 (Brahma-related gene 1/SMARCA4, SWI/SNF related, matrix associated, actin-dependent regulator of chromatin) binding to distal enhancers of melanocyte-specific genes, promoting differentiation.47In the developing gut, SOX10 is expressed in all NC-derived ENS progenitors.22 24 48–50 Later, SOX10 is maintained in enteric glia but downregulated in cells that are committed to neurons (see refs 25 50 for examples). Most publications suggest a role of SOX10 in the maintenance of enteric progenitors,22 49 and overexpression of SOX10 inhibits enteric neuron differentiation, without altering commitment to the neurogenic lineage.25 51 These cellular functions rely on the capacity of SOX10 to regulate (along with several cofactors) various target genes, including Ret (RET proto-oncogene.

A receptor tyrosine kinase involved in ENS development and the main HSCR-related gene), Ednrb and Sox10 itself.22 33 52 53 As an example SOX10 and ZEB2 (zinc-finger E-box binding homeobox 2. A negative regulator of NC differentiation) both bind to Ednrb promoter-specific regions, highlighting the role of this ‘triade’ in controlling the maintenance of multi-potential enteric progenitors and their differentiation process.33Hearing loss associated with SOX10 mutations. Beyond melanocytes, SOX10 expression in inner ear and related deficitsSNHL due to SOX10 mutations, as for the other WS genes, is typically prelingual, non-evolutive, profound and bilateral.

However, it can also be moderate and asymmetric or unilateral.Aside from the intermediate-cell alterations mentioned above, inner ear malformations have been noted in some patients with WS long ago.54 It now appears that only patients with a SOX10 mutation present with these abnormalities. Hypoplasia/dysplasia or agenesis of the semicircular canals and enlarged vestibules are very frequent, while agenesis of the vestibulo-cochlear nerve and cochlear deformities have also been reported.55–57 Consequently, temporal CT scan or MRI is of particular interest in diagnosis. In our experience, this feature is highly penetrant when interpreted by a specialised radiologist.

However, recent papers reported the absence of imaging abnormalities in the inner ear of a few patients with SOX10 mutations. A complete exploration of the vestibular function has yet to be performed.These observations are consistent with an expression profile of Sox10 in the ear. Sox10 is expressed in the placode-derived otic vesicle from E9.5 onward and then in the developing epithelium of the cochlea and vestibule, before being restricted to supporting cells of the neurosensory epithelium.

Sox10/SOX10 promotes the survival of cochlear progenitors during formation of the otocyst and the organ of Corti, plays a role in glial development of the cochleovestibular ganglia, and its NC-targeted deletion leads to improper neuronal migration and projection.58–60 The resulting inner ear malformations differ depending on the animal model.58 61 62 RNA-seq studies of inner ear development in a pig model showed dysregulation of WNT1 (Wnt family member 1. A regulator of cell fate and patterning), KCNQ4 (potassium voltage-gated channel subfamily Q), STRC (stereocilin. A protein associated with the hair bundle of the ear sensory cells) and PAX6 (transcription factor Paired Box 6) networks.62In agreement with this broad function, SNHL appears to be the most penetrant sign in cases of SOX10 mutation, leading to the observation that certain patients can present with isolated SNHL until minor signs are revealed on medical reinterview.63PCWH and PCW phenotypes.

Important function of SOX10 in Schwann cells and oligodendrocytesBeyond WS2 or WS4, neurological alterations have been identified in the so-called PCWH syndrome (peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, Hirschsprung disease. MIM #609136).28 36 64 Depending on the severity of myelin defects in the PNS and central nervous system (CNS), patients with PCWH exhibit variable symptoms that often include delayed motor and cognitive development, cerebral palsy, ataxia, spasticity, congenital nystagmus, hyporeflexia, distal sensory impairments and distal muscle wasting. This phenotype is recapitulated in a transgenic mouse model with several copies of SOX10 carrying the first PCWH mutation described65 66 and is mostly explained by the role of SOX10 during differentiation of myelinating Schwann cells and oligodendrocytes, both ensuring rapid salutatory conduction along axons.67 68In the PNS, SOX10 controls each differentiation step by inducing stage-restricted transcriptional regulators, which are then recruited as partners to activate specific sets of target genes, allowing progression to the next stage.67–72 For example, in immature Schwann cells, SOX10 induces the expression of OCT6 (POU3F1, POU class 3 homeobox 1).

Both factors then cooperatively activate the programme required for progression into the promyelinating stage. Their target EGR2 (early growth response 2) then associates with SOX10 to activate the myelination programme.In the CNS, analyses of various animal models revealed an essential role of SOX10 in the terminal differentiation of oligodendrocytes in coordination with OLIG1 (OLIGodendrocyte transcription factor 1), MYRF (myelin regulatory factor), TCF4 (transcription factor 4, which has an important role in CNS development) and CHD7 (chromodomain helicase DNA-binding protein 7. The gene involved in CHARGE syndrome (Coloboma, Heart anomaly, choanal Atresia, Retardation, Genital and Ear anomalies)).68 Many genes that are activated during terminal differentiation of oligodendrocytes are direct targets of SOX10, but there are only few known SOX10 targets in oligodendrocyte precursors.68 73 74 Recently, MYRF was identified as a decisive factor that helps SOX10 to switch between its target genes along oligodendrocyte differentiation process.75Of interest, some of the genes directly regulated by SOX10 in PNS and CNS are known to be responsible for hypomyelinating/demyelinating diseases, with some described mutations in these genes that directly result from a loss of regulation by SOX10.76–78Involvement of SOX10 in Kallmann syndrome and its role in olfactory ensheathing cellsSOX10 was considered to be a candidate gene for Kallmann syndrome (KS, hypogonadotropic hypogonadism and anosmia.

MIM #308700) based on the unexpected high frequency of olfactory bulb agenesis55 associated with rare clinical reports of hypogonadism or anosmia in patients with WS/PCWH with a SOX10 mutation. The screening of cohorts indeed revealed SOX10 mutations in patients with KS, most of whom also have hearing impairment.79 Since then, many other SOX10 mutations have been characterised in KS or normosmic idiopathic hypogonadotropic hypogonadism (nIHH), although they were usually not functionally characterised and a subset of them appeared unlikely to be pathogenic (see Review of SOX10 variations). Interestingly, KS and WS are not mutually exclusive, and some patients with an initial diagnosis of WS have been further diagnosed with hypogonadism at puberty.80 We believe that anosmia and hypogonadism are still underestimated in patients with WS with a SOX10 mutation, as signs of KS are difficult to diagnose before puberty.

Of note, in the absence of pigmentary disturbances, the association of KS+hearing impairment+abnormalities of the semicircular canals can lead to a differential diagnosis with mild forms of CHARGE syndrome (MIM #214800) (examples in online supplemental table 1).Supplemental materialThe common cause of anosmia and hypogonadism is a defect in a developmental sequence of GnRH (gonadotropin-releasing hormone) neurons migrating along the peripheral olfactory nerve up to and through the olfactory bulb. In the Sox10 knockout mouse, a primary defect of the olfactory ensheathing cells leads to a secondary defect of the olfactory nerve pathway, defasciculation and misrouting of the nerve fibres, impaired migration of GnRH cells along this route, and disorganisation of the olfactory nerve layer of the olfactory bulbs.79 Dysregulation of the frizzled related protein FRZB may contribute to explain the defect in olfactory axon targeting but not GnRH neuron migration.81A summary of the recurrent clinical manifestations due to constitutive SOX10 mutations along with affected cell types is presented in figure 2.Summary of the clinical spectrum due to SOX10 mutations and the corresponding SOX10 function(s). The picture is organised around three clinical poles that correspond to different diagnosis entries.

The WS pole is indicated in red, the myelin pole in blue and the KS pole in green. The plain line corresponds to the definition of the disease, while the dotted lines indicate the main clinical extension of these syndromes in case of SOX10 mutation. Note that the area of the circles is not proportionate to the relative frequency of each syndrome (for an idea about the number of patients, see figure 3B).

CIPO, chronic intestinal pseudo-obstruction. CNS, central nervous system. ENS, enteric nervous system.

GnRH, gonadotropin-releasing hormone. HSCR, Hirschsprung disease. KS, Kallmann syndrome.

NIHH, normosmic idiopathic hypogonadotropic hypogonadism. PCWH, peripheral demyelinating neuropathy, central dysmyelination, Waardenburg syndrome, with Hirschsprung disease. PNS, peripheral nervous system.

SNHL, sensorineural hearing loss. WS, Waardenburg syndrome. WS2, Waardenburg syndrome type 2.

WS4, Waardenburg syndrome type 4." data-icon-position data-hide-link-title="0">Figure 2 Summary of the clinical spectrum due to SOX10 mutations and the corresponding SOX10 function(s). The picture is organised around three clinical poles that correspond to different diagnosis entries. The WS pole is indicated in red, the myelin pole in blue and the KS pole in green.

The plain line corresponds to the definition of the disease, while the dotted lines indicate the main clinical extension of these syndromes in case of SOX10 mutation. Note that the area of the circles is not proportionate to the relative frequency of each syndrome (for an idea about the number of patients, see figure 3B). CIPO, chronic intestinal pseudo-obstruction.

CNS, central nervous system. ENS, enteric nervous system. GnRH, gonadotropin-releasing hormone.

HSCR, Hirschsprung disease. KS, Kallmann syndrome. NIHH, normosmic idiopathic hypogonadotropic hypogonadism.

PCWH, peripheral demyelinating neuropathy, central dysmyelination, Waardenburg syndrome, with Hirschsprung disease. PNS, peripheral nervous system. SNHL, sensorineural hearing loss.

WS, Waardenburg syndrome. WS2, Waardenburg syndrome type 2. WS4, Waardenburg syndrome type 4.Involvement in cancer, sex reversal, associations and reports of the first biallelic mutationsBeyond congenital disorders, a role of SOX10 in cancer progression has been reported.

SOX10 protein is highly expressed in breast, glioma, glioblastoma, salivary adenoid cystic tumours and hepatocellular carcinoma (see The Cancer Genome Atlas). The association of SOX10 with melanoma is the best described, but only a limited number of variants have been reported so far.82–84Several reports of duplications in the 22q13.1 region have been published that may include one or several signs of WS/PCWH and sex reversal in a number of cases.85 Sex reversal has been suggested to be due to the overexpression of SOX10, consistent with observations in a Sox10 transgenic mouse model.86 However, we found a SOX10 triplication (four doses of SOX10 instead of two) in a 47,XX baby girl without sex reversal (online supplemental table 1), indicating that overexpression of SOX10 alone may not be sufficient, the sign is not fully penetrant or the overexpression of other genes has the opposite effect, depending on the size of the rearrangement.More complex and questionable associations have also been described. For example, increased DNA methylation of SOX10 has been linked to oligodendrocyte dysfunction in patients with schizophrenia.87Two cases of biallelic SOX10 deletion have been characterised and, although not reported in the papers, they appear to represent the first and second pregnancy from the same couple.88 89 Both parents are heterozygous for one of the two SOX10 deletions and present with a classic form of WS.

Biallelic SOX10 loss-of-function results in a severe polymalformative fetal phenotype. Eighteen other genes were included in the maternal deletion and may participate in the phenotype.Finally, the development of large gene panels for diagnosis and whole exome/whole genome sequencing has led to SOX10 mutations being found in unexpected contexts. A number of cases have thus been listed in cohorts of neurodevelopmental defects, hearing impairment and endocrinological problems.

Due to the diverse phenotypes related to SOX10 mutations, making sense of such findings can be challenging.Review of SOX10 variationsDuring the first 15 years after their discovery, most SOX10 disease-associated point mutations were shown to result in premature termination codons, with strikingly few exceptions.28 Missense mutations started to be found simultaneously with the finding that SOX10 mutations can cause less severe syndromes than life-threatening WS4 or PCWH.90An up-to-date summary of confident mutations of SOX10 (approximately 300 independent cases, including unpublished ones in online supplemental table 1) is presented in figure 3A. Truncations (stops, frameshifts) are found in slightly more than half of all cases. Approximately one-third of all mutations are non-truncating variations, either missense or small inframe insertions/deletions, the rest being either complete or partial copy number variations of the gene (approximately 10%) and rare mutational mechanisms (splice mutations, mutation of the initiation codon or non-stop mutations (five cases to date)).

Truncating mutations can be located anywhere, except in the very extreme C-terminus. On the contrary, missense mutations are tightly clustered in the DNA-binding domain (HMG), a frequent finding for transcription factors. We have thus far found no specific link between SOX10 missense mutations and residues involved in post-translational modifications.Review of SOX10 mutations.

(A) Representation of SOX10 truncating and non-truncation mutations along the SOX10 protein. We made a list of all published SOX10 mutations, starting with the LOVD database that we curated up to 2015 (https://databases.lovd.nl/shared/genes/SOX10), updated with the literature and finally completed using the HGMD (Human Gene Mutation Database) professional database (https://digitalinsights.qiagen.com/products-overview/clinical-insights-portfolio/human-gene-mutation-database) for a few mutations that were reported in cohorts of unspecific diagnosis and would have been missed by common keywords. We prioritised the strength of the data to create this figure, as our goal was not to include all cases but to provide a reliable picture of the SOX10 mutational spectrum.

We retained papers for which the data allowed curation and removed neutral variants and variants of unknown significance, duplicated patients or publications, and publications with inconsistent findings. Finally, we added our unpublished cases (listed in online supplemental table 1). €˜M1?.

€™ indicates a mutation of the initiation codon (p.Met1?. ). (B) Proportion (in percentage) of the different types of mutations for each syndrome.

€˜n’ indicates the number of independent cases included in each group. (C) Localisation of the truncating (stop and frameshift) mutations along the SOX10 protein associated with each phenotype. Note that (1) the phenotypic description was sometimes too incomplete for inclusion in B and C.

(2) among the familial cases showing intrafamilial differences in phenotype, we considered the phenotype of the index case. (3) KS/nIHH is given regardless of the presence of WS signs or not, and anosmia without hypogonadism was not considered. And (4) because presence or absence of a demyelination is frequently unreported or not evaluated, we conserved all the patients with neurological features in the PCW/PCWH group when the data seemed consistent.

DIM, dimeric domain. HMG, high mobility group. KS, Kallmann syndrome.

LOVD, Leiden OPen Variation Database. NIHH, normosmic idiopathic hypogonadotropic hypogonadism. PCW/PCWH, peripheral demyelinating neuropathy, central demyelination, Waardenburg syndrome, with or without Hirschsprung disease.

TA, transactivation domain in C-terminal. TAM, transactivation domain in the middle of the protein. WS, Waardenburg syndrome.

WS2, Waardenburg syndrome type 2. WS4, Waardenburg syndrome type 4." data-icon-position data-hide-link-title="0">Figure 3 Review of SOX10 mutations. (A) Representation of SOX10 truncating and non-truncation mutations along the SOX10 protein.

We made a list of all published SOX10 mutations, starting with the LOVD database that we curated up to 2015 (https://databases.lovd.nl/shared/genes/SOX10), updated with the literature and finally completed using the HGMD (Human Gene Mutation Database) professional database (https://digitalinsights.qiagen.com/products-overview/clinical-insights-portfolio/human-gene-mutation-database) for a few mutations that were reported in cohorts of unspecific diagnosis and would have been missed by common keywords. We prioritised the strength of the data to create this figure, as our goal was not to include all cases but to provide a reliable picture of the SOX10 mutational spectrum. We retained papers for which the data allowed curation and removed neutral variants and variants of unknown significance, duplicated patients or publications, and publications with inconsistent findings.

Finally, we added our unpublished cases (listed in online supplemental table 1). €˜M1?. €™ indicates a mutation of the initiation codon (p.Met1?.

). (B) Proportion (in percentage) of the different types of mutations for each syndrome. €˜n’ indicates the number of independent cases included in each group.

(C) Localisation of the truncating (stop and frameshift) mutations along the SOX10 protein associated with each phenotype. Note that (1) the phenotypic description was sometimes too incomplete for inclusion in B and C. (2) among the familial cases showing intrafamilial differences in phenotype, we considered the phenotype of the index case.

(3) KS/nIHH is given regardless of the presence of WS signs or not, and anosmia without hypogonadism was not considered. And (4) because presence or absence of a demyelination is frequently unreported or not evaluated, we conserved all the patients with neurological features in the PCW/PCWH group when the data seemed consistent. DIM, dimeric domain.

HMG, high mobility group. KS, Kallmann syndrome. LOVD, Leiden OPen Variation Database.

NIHH, normosmic idiopathic hypogonadotropic hypogonadism. PCW/PCWH, peripheral demyelinating neuropathy, central demyelination, Waardenburg syndrome, with or without Hirschsprung disease. TA, transactivation domain in C-terminal.

TAM, transactivation domain in the middle of the protein. WS, Waardenburg syndrome. WS2, Waardenburg syndrome type 2.

WS4, Waardenburg syndrome type 4.Of course, rarity in control populations is not sufficient to confer pathogenicity and the prediction of pathogenicity by dedicated tools is of indicative value only. Among the published SOX10 missense variations that are located outside of the HMG domain, most should be considered variants of unknown significance. From our experience and bibliography review, it appears that extremely rare or new missense variations have a high probability of being truly pathogenic if located in the HMG domain, whereas missense mutations located outside of this domain, even if rare and ‘predicted pathogenic’ by in silico tools, are less likely to be pathogenic and should be considered cautiously.

We have worked on SOX10 since its characterisation, both in the research and clinical context, and have only once found an exception to each of these rules. With the increasing number of mutations described, it appears that there may be a second, rare spot of mutations in the dimerisation domain (three variations reported in four independent cases, creating or removing valines at residues 76, 79 and 80), although functional tests are required to reach a definitive conclusion91 92 (online supplemental table 1).Due to the well-documented incomplete penetrance and digenism in KS and nIHH, there is a tendency in the literature to overevaluate the pathogenic probability of rare variants. Certain SOX10 variations have been considered to be pathogenic or likely pathogenic without many arguments (low pathogenicity scores, no functional tests, proven not pathogenic in another paper, inherited from healthy parents or without segregation study, and/or associated with an obvious causative mutation in another KS/nIHH gene).

On careful review, we consider several of these rare missense variants to more likely be neutral (although some still may be hypomorphic variants exerting their effect on a multigenic background, but this has thus far not been proven) and did not include them in figure 3.In early studies, most SOX10 variants were found to be de novo, which was thought to be due to the severity of HSCR in WS4. Given the cumulative number of WS2 cases now described, the life-threatening hypothesis cannot completely explain the proportion of de novo mutations. The possibility of hypogonadism in patients probably also contributes to this observation.

However, the proportion of familial cases has tended to increase over the years and now represents approximately 20% of cases. These cases revealed an important intrafamilial phenotypic variability. Several mutations have now been found in independent cases and also show interfamilial phenotypic variability.

Parental mosaicism is found in approximately 3%–4% of cases, but a recent study reported a higher proportion in a small series using more sensitive methods.93The proportion of mutations relating to phenotype is summarised in figure 3B. There is a large proportion of truncating mutations in WS4 and PCWH. The proportion of missense increases in WS2 and even more strongly in KS.

Thus, not all missense mutations may be null mutations.The location of truncating mutations along the gene (figure 3C) confirms the correlation between PCWH and the escape from non-sense-mediated RNA decay (NMD) (mutations located in the last coding exon and last 45 nt of the penultimate exon).64 Of note, some of the cases that appear not to respect the NMD rule may be misclassified (whether demyelination is proven or not is not always reported). The severity of PCWH was shown to be linked to the location of the mutation within the last exon. The earlier the truncation, the more severe the phenotype.64 This tendency is visible in the graphs (compare the truncating mutations of WS vs PCWH in the last exon).

A few cases escape the rule, with no clear explanation so far.Finally, heterozygous deletions/duplications can be intragenic or lead to complete gene loss and be as large as several Mb, encompassing other genes and leading to more complex phenotypes.Functional consequences of SOX10 mutations and the origin of phenotypic variabilityMost in vitro functional tests found in the literature rely on the ability of SOX10 to activate its target genes, alone or in combination with its cofactors. The construct most frequently used is a luciferase reporter under the control of the MITF-M (melanocyte-specific isoform) promoter. Additional targets, immunohistochemistry and assessment of the contribution of the DNA-binding capabilities have sometimes enriched such studies.Functional analysis of the first SOX10 missense mutation suggested that differential tissue-specific gene regulation could account for the phenotype observed in patients.94–96 Since, many SOX10 missense mutations associated with a variety of phenotypes, ranging from WS2 to WS4 and PCWH, have been tested, but no clear correlation between in vitro results and the phenotypes could be established.79 90 97The development of in vivo tests is therefore required to facilitate the establishment of genotype–phenotype correlations.

The only model currently published is in ovo chick electroporation in the developing neural tube.26 However, the effect of most of the mutations on early NC development precludes the analysis of their role in later developmental processes. Use of an inducible model would be of interest. Alternatively, zebrafish or the use of induced pluripotent stem cells differentiated towards NC derivatives of interest could be future models of choice.As mentioned earlier, the presence or absence of a neurological phenotype that characterises PCWH or PCW was proposed to be related to NMD.64 The proposed mechanism is that mutant proteins that have escaped degradation via the NMD pathway result in dominant-negative activity that impairs the function of the wildtype SOX10 allele and lead to PCWH, while those located in the first coding exons activate the NMD RNA surveillance pathway, leading to degradation, haploinsufficiency and a classic WS phenotype.However, several non-stop mutations have also been described to be associated with a PCW/PCWH phenotype.

This is thought to be due to the generation of a specific inframe new C-terminus generated by the loss of normal termination. Functional studies of an equivalent mouse mutant allele showed that the additional 82 amino acids contain a deleterious (tryptophan-arginine (WR) domain, supporting a toxic gain-of-function.98 This is consistent with the recent report of a frameshift mutation that also elongates the protein, but in a different reading frame does not lead to PCWH (p.Tyr460Leufs*42).99 The observation of another, transgenic mouse model carrying different copy number variations of the first described SOX10 non-stop mutation suggested PCWH is due to a dominant-additive, rather than dominant-negative, effect.66 Finally, duplication of the 22q13.1 region, including SOX10, can also induce PCWH,85 supporting the hypothesis that it is promoted by a gain-of-function rather than a dominant-negative effect. Regardless of the mechanism, these observations all indicate that NC derivatives are highly sensitive to the dose of SOX10 and its function.SOX10 expression is regulated by numerous enhancers.

It is thus possible that certain cases with minor expression could also be due to specific dysregulation of one or a subset of such enhancers. This paradigm is supported by disruption of tissue-specific, long-distance regulatory regions of SOX9 causing endophenotypes.100 101 A large de novo deletion encompassing three SOX10 regulatory elements has been characterised in a patient with typical WS4,102 leading to the hypothesis that variations affecting certain identified regulatory sequences could be the cause of unexplained WS2 or isolated HSCR. Screening for mutations in SOX10 regulatory regions in WS2 turned out to be unfruitful.103 On the contrary, one deletion and two point variations affecting binding sites for known NC transcription factors were identified in 3 of 144 cases of isolated HSCR, both variations being in association with a HSCR-predisposition polymorphism at the RET locus.104 With the implementation of population databases, it now appears that one of these two variants is less rare than expected (22-38016774 G-C.

About 1/1000 according to the gnomAD database. Https://gnomad.broadinstitute.org/), which questions its involvement. These results are yet to be replicated for a pertinent interpretation.In any case, in vitro/in vivo tests will not be able to explain all phenotypes, as phenotypic variability is commonly recognised in patients with SOX10 mutations, even those with the same mutation and even within the same family.

This suggests that the genetic background is influential, as has often been suggested for HSCR.53 105 Because the identification of modifier genes has been hampered by the small number of available patients, most modifier gene studies have relied on Sox10 mouse models.22Despite such variability, certain specificities have been reported for a few peculiar missense mutations. Here, we want to discuss the case of the Gln174/Pro175 missense mutations. The observation that certain SOX10 missense mutants accumulate in nuclear foci in transfected cells, where they colocalise with p54NRB (nucleo ribo binding protein, 54 kDa/NONO, non-Pou domain containing octamer binding.

A multifunctional protein known to be a marker of ‘paraspeckles’), leads to characterise missense mutations of amino acids 174 and 175 as associated with a peculiar phenotype (refs 79 97 and unpublished data (S. Marlin, N. Bondurand and V.

Pingault, 2016)). Remarkably, the cotransfection of foci-forming mutant with wildtype constructs led to the sequestration of wildtype SOX10 in these ‘foci’ and altered the synergistic activity of SOX10 and p54NRB. A dominant-negative effect was therefore proposed to contribute to or be at the origin of the progressive central and peripheral neurological phenotypes observed in patients carrying these specific missense mutations and may thus be the basis of a hitherto unexplored molecular mechanism for genotype–phenotype correlations.

These data need to be confirmed in more physiological models.The phenotype variability finally leads to question the risk of a more severe phenotype in cases of recurrence in a family. The risk of the PCWH phenotype after a first non-PCWH case is considered to be low. On the contrary, there is a risk of WS4 after a first, milder case of WS2.

This situation has been reported several times. However, a bias in the representation of these cases in the literature can be expected, as a second mildly affected member is less likely to result in a visit of the family to the geneticist’s office, molecular analysis and ultimately publication. As a result, the true risk is difficult to quantify.Conclusion, with a few tips to help in variant classificationDuring twenty years of cases and cohorts reporting, SOX10 variants have been involved in WS2/WS4/PCWH/Kallmann syndrome/pseudo-isolated hearing loss/HSCR or CIPO and any combination.

This is correlated with the known developmental functions of SOX10. All these phenotypes should be considered as a clinical continuum with variable expression, rather than as independent diseases, conferring a mild to life-threatening syndrome. Observation of the familial cases and of a few recurrent variations documented a high phenotypic variability, even within a single family.Most mutations predict a truncation of the protein, but the proportion of missense variations has increased with time.

Missense variations (or small in-frame insertions/deletions) outside of the HMG domain should be considered with caution, even with good in silico pathogenicity scores. The fact that the variation is already published can be used as a supporting argument only if the strength of the published data has been verified (also a general recommendation of the American College of Medical Genetic).The most (almost fully) penetrant sign observed in patients is hearing impairment. Pigmentation defects are not always present.

Confirmed incomplete penetrance appears to be very rare, but a targeted clinical reevaluation may be necessary to assess mild signs. Searching for inner ear-specific malformations by imaging is highly informative. The absence of olfactory bulbs could be investigated at the same time by MRI.

The only strong phenotype-genotype correlation usable in phenotype prediction, thus far, is the link between NMD escape and PCW/PCWH.Ethics statementsPatient consent for publicationNot required.Ethics approvalEthics approval is not applicable. This study does not involve human participants in a research study. Only mutations found on a diagnosis basis are reviewed in a retrospective manner (list of mutations along with scarce clinical information).AcknowledgmentsWe apologise to all whose contributions were not cited due to space limitations.IntroductionIn recent years, a large increase in the use of multigene panel tests for breast cancer associated pathogenic variants (PVs) has expanded the number of potentially actionable PVs beyond BRCA1 and BRCA2.1–9 These studies have shown an almost equal rate of BRCA1/2 PVs to all additional potentially actionable gene PVs combined.

In addition, much of the increased detection is due to variants in less actionable moderate-risk genes,10ATM and CHEK2, with higher background population prevalence. The only other actionable breast cancer gene variants consistently identified at substantial rates is PALB2, which is now also considered to be a high-risk susceptibility gene.11Although higher frequencies of actionable gene variants are reported in those at particularly young ages (<40 years) particularly for TP53, the PV rates of ATM and CHEK2 do not appear to be strongly related if at all to age-at-onset, although a small effect was seen for CHEK2 in two studies.1 2 Very few studies have concentrated testing on women with very early onset breast cancer. We previously reported a high rate of BRCA1, BRCA2 and TP53 PVs in a population based series of breast cancer in women ≤30 years of age at diagnosis.12 13 Fewer than 1 in 1000 women develop breast cancer by age 30 years and UK statistics showed that only 222 of 54 450 (0.41%) of breast cancers occurred in women aged <30 years14 (0.59% if ~100 breast cancers in women aged 30 years are included).14 Although this is a small group of patients with breast cancer, the prognosis of breast cancer diagnosed in this young age group is poor.12 13 15 16 BRCA1 and BRCA2 PVs have been reported in small numbers of women diagnosed aged ≤30 years.

However, the studies reporting these individuals include many women with breast cancer diagnosed at older ages and do not specify the detection rates within the ≤30 years age group.15 16 The Prospective study of Outcomes in Sporadic vs Hereditary breast cancer (POSH) reported a 12% rate of BRCA1/2 PVs in 338 of 2733 women diagnosed aged ≤40 years, but only 316 of a total 3095 women in POSH were aged ≤30 years and no separate analysis was presented.15 16 In another study, the rate of TP53 PVs was reported as 6% in an unselected subset within 333 women with breast cancer aged ≤30 years.17 The Myriad study is the only large study that has assessed the detection rate of PVs in other breast cancer genes in women with breast cancer aged <30 years. In this study, 783 (2.2%) of 35 409 women were aged <30 years;6 however, it is likely that there was considerable pretesting in this series for BRCA1/2 and TP53 PVs as acknowledged by the authors and evidenced by the low detection rates among Ashkenazi Jews.We present analysis of BRCA1/2 and TP53 testing in 379 patients with breast cancer aged ≤30 years, and of extended testing of a panel of additional breast cancer genes in 184 patients, expanding our previous population-based study of 115 women.12 13MethodsIndividuals with a confirmed breast cancer diagnosis aged ≤30 years were eligible for the study. Affected women came from two sources.

The first was a population-based study of 288 women with breast cancer presenting between January 1980 and December 1997 from the Manchester region (population=4.5M) of North-West England identified from the regional cancer registry.12 13 From this, 175 women were alive and potentially available for genetic testing.12 Fifty (28.6%) of these did not provide a DNA sample (it was either not appropriate to recontact or the individual did not wish to participate or could not be traced). This increases by 10 the number with available DNA samples from our previous report to 125.13 Only 39 currently living patients have not consented to the study. An additional 256 women were referred to the Manchester Centre for Genomic Medicine (MCGM) between 1990 and 2019.

All women gave clinical consent for testing of breast cancer genes. Samples were initially screened for point mutations and copy number variants in BRCA1, BRCA2, TP53 and for the CHEK2 c.1100delC PV.13 When a PV was identified, no further testing was carried out. Samples testing negative were selected for next generation sequencing panels which included, as a minimum, the additional breast cancer associated genes.

PALB2, CHEK2, ATM, CDH1, PTEN, RAD50, RAD51D and NBN. In addition, 1567 population control samples without breast cancer at entry aged 47–73 years from the PROCAS study18 were tested as part of the Breast Cancer Risk after Diagnostic Gene Sequencing (BRIDGES) programme.19PV frequencies in the Manchester early onset cohort were compared with PV frequencies observed in women aged ≤30 years who took part in the prospectively ascertained POSH study (01/2000–01/2008).15 16Tumour pathology information was obtained through hospital record and cancer registries. The pathology adjusted Manchester Scoring System was used to assess likelihoods of BRCA1/2 PVs.20 Pathology-adjusted Manchester score (MS) of 15–19 is equivalent to a 10% probability of a BRCA1/2 PV and a 20–24 point score is equivalent to a 20% probability.The type and number of PVs were determined in the full cohort as well as in different age groups, specific tumour pathology characteristics and MS.ResultsA total of 381 women with breast cancer diagnosed ≤30 years were included.

Two women met diagnostic criteria for neurofibromatosis type 1, explaining their early onset of breast cancer. The remaining 379 were screened for variants in BRCA1, BRCA2, TP53 and the CHEK2 c.1100delC variant. This strategy detected 134 PVs.

BRCA1=75 (19.79%), BRCA2=35 (9.23%), TP53=22 (5.80%), CHEK2 c.1100delC=2 (0.53%). One woman harboured both a BRCA1 and BRCA2 PV. Of those testing negative, 184 (74.8%) underwent extended genetic testing.

Sixty-two women did not undergo further testing due to poor quality, or insufficient, DNA. The detection rate was 4.35% (n=8) for actionable breast cancer PVs (ATM=2, PALB2=4, CHEK2=1, PTEN=1, online supplemental table 1). Single PVs were identified in other genes associated with breast cancer risk, BRIP1 (c.2392C>T.

P.Arg798Ter), RECQL (c.1667_1667+3delAGTA. P.?. ) and RAD50 (c.1300_ 1306del.

P.Asp434LysfsTer7).Supplemental materialRisk associations for each gene were determined using the population controls from the PROCAS study (table 1). Significant associations with a more than twofold increased risk were found for BRCA1. OR=193.10 (95% CI 51.58 to 804.8), BRCA2.

OR=17.61 (95% CI 8.59 to 36.53), TP53. OR=308.10 (95% CI 51.20 to 3202) and PALB2. OR=11.59 (95% CI 3.08 to 46.15).

PV rates in the POSH study were established among the 287 women with invasive breast cancer at the age of ≤30 years. A total of 56 (19.5%) PVs were identified in BRCA1 (32 PVs, 11.1%), BRCA2 (17 PVs 5.9%), TP53 (5 PVs, 1.7%) and CHEK2 c.1100delC (3 PVs, 1.1%) (table 1).View this table:Table 1 Association of pathogenic variants with early onset of breast cancerDetection rate of pathogenic variants in different age groupsSurprisingly, the youngest age group (<26 years) showed a lower rate of BRCA1/2 PVs. Only 9/61 (14.75%) compared with 101/318 (31.76%) for those aged 26–30 years (p=0.0083) (table 2).

TP53 showed the reverse trend with 7/61 (11.48%) aged <26 years compared with 4.72% (15/318) in those aged 26–30 years (p=0.0649). Thus, only 12.93% (15/116) PVs in BRCA1/2/TP53 in those aged 26–30 years were in TP53 compared with 43.8% (7/16) in those <26 years (p=0.0060). The lower rates in the younger age group for BRCA1/2 PVs were similar to the rates in the POSH cohort ≤30 years potentially reflecting ascertainment differences.

The higher rate of TP53 PVs (5.8%) compared with 1.7% in POSH likely reflects that the POSH study specifically excluded women with only DCIS and no invasive tumour component.View this table:Table 2 Rates of pathogenic variants by age group, pathology and Manchester Scoring SystemThe CHEK2 c.1100delC PV was identified in only 2/379 (0.53%) compared with 1.7% (55/3177) in women with breast cancer aged >30 years (p=0.0835) seen at the MCGM and 2.3% in the POSH study aged ≤40% and 1% in POSH cases≤30 years (table 1).Manchester scoreThe detection of PVs in BRCA1 and BRCA2 was, as expected, strongly correlated with breast cancer pathology and family history. The MS accurately predicted the likelihood of a BRCA1/BRCA2 variant at both the 10% (15–19 points) and 20% (20–24 points) thresholds (table 2). By including PVs in TP53, 100% of women with a MS ≥40 had a PV in BRCA1/2 or TP53.Tumour characteristicsWe identified 61 (48.8%) PVs in BRCA1/2/TP53 in 125 women with triple-negative breast cancer (TNBC) (table 3).

Unexpectedly, a similar rate of BRCA1/2/TP53 PVs was detected in cases of pure DCIS (11/26 [42.3%]), although TP53 accounted for 54.5% (6/11) of these. Eight were comedo DCIS of which four had a TP53 PV. The majority of DCIS were high grade (18/26) and 8/18 harboured a PV (2 in BRCA1, 1 in BRCA2 and 5 in TP53) (table 3).

None of the cases of pure DCIS were detected on screening for familial risk.View this table:Table 3 Rates of pathogenic variants found in patients with DCISHER2+ breast cancer showed a similar predominance of TP53 PVs (8/43 (18.6%)), but BRCA1/2 PVs were uncommon (3/43 (6.9%)).Presence of cancer in both breasts was also predictive of PVs, with 36/63 (57.1%) cases with BRCA1/2/TP53 PVs (including 10/22 TP53 PVs) having bilateral breast cancer.Sporadic breast cancerOf 147 women without a family history of breast or ovarian cancer at original diagnosis, 24 (16.3%) had a PV. Only 10 (6.8%) had BRCA1/2 PVs (BRCA1=7. BRCA2=4.

1 woman had both BRCA1 and BRCA2 PVs), 12 women had a TP53 PV and the remaining 2 women had a PALB2 or a CHEK2 PV. All BRCA1 PVs were detected in women with sporadic TNBC 7/59 (11.9%). There were six other PVs identified in sporadic TNBC in BRCA2=3, TP53=2 and PALB2=1.

Of 26 people with HER2+ sporadic breast cancers, 7 (26.9%) had PVs. (TP53=6. BRCA2=1).

Outside of these confirmed pathologies 5/62 (8.1%) had PVs (TP53=4, CHEK2=1), but receptor status was unknown in 43 cases, including 13 with DCIS, two of whom had a TP53 PV.TP53 carriersAmong TP53 carriers, 10/22 (45.5%) had a family history of breast cancer at initial diagnosis. Additional relatives in three of these families had Li Fraumeni spectrum tumours (one had none at diagnosis) and one had a personal history of childhood adrenocortical cancer. Additionally, four families without relatives with breast cancer, had family histories, including the index breast cancer, consistent with classical Li Fraumeni syndrome including at least one sarcoma aged <45 years.

One de novo case had an osteosarcoma of the leg aged 19 years. Seven (33%) apparently de novo TP53-associated cases (confirmed after parental testing), with no significant personal or family history of cancer, presented with breast cancer. Thus, 7/144 (4.9%) apparently sporadic breast cancer cases ≤30 years had TP53 de novo variants that would not have been expected from personal or family history.One of the TP53 PVs was identified at a variant allele frequency of 22% suggesting mosaicism (online supplemental table 1).

The PV was found in the tumour (20%-neoplastic content) at 15% and 11% in normal breast excluding clonal haematopoiesis (in a woman with Paget’s/DCIS who had not undergone radiotherapy/chemotherapy).Assessment of population level of testingThere were 135 women diagnosed with breast cancer in the Manchester region aged ≤30 years between 01/01/1990 and 31/12/1997 (since cancer genetic testing was introduced in Manchester) within the population study giving an annual rate of 16.9 cases. During this time, we tested 73/135 (54.1%) of affected women and identified BRCA1=13 (17.8%), BRCA2=8 (11%) and TP53=3 (4.1%) PVs. Of our population based study group of 125 women who underwent genetic testing (presenting with cancer between 1980 and 1997), there were PVs in BRCA1=23 (18.4%), BRCA2=11 (8.8%), TP53=5 (4%) and BRIP1=1,12 13 demonstrating a very similar overall detection rate.

In the cohort referred to MCGM between 01/01/1998 and 3/11/2019, we tested 219 women and identified PVs in BRCA1=46 (21.0%), BRCA2=17 (7.8%) and TP53=16 (7.3%). The combined rate of BRCA1/2 PVs at 27.2% (population-based study) and 28.8% (referrals) are similar, suggesting no substantial testing bias. However, 68/125 (54.4%) in the population study (1980–1997) had no family history, compared with 77/219 (35.2%) in the recent cases (1998–2019) (p=0.0006).

All but 18 of the 219 tested since 1997 had full pathology and ER receptor status available, and only eight ER+ ductal carcinomas had unknown HER2 status.Co-occurrence of actionable breast cancer gene variantsOf 920 breast cancer cases with no prescreening tested at MCGM, no co-occurrence of two actionable breast cancer gene variants was found. Among 4916 non-Jewish breast cancer cases undergoing full BRCA1 and BRCA2 testing, only two co-occurrences of BRCA1 and BRCA2 PVs has occurred including the single case reported in this study.DiscussionWe report here the results of 379 patients with breast cancer ≤30 years initially tested for PVs in BRCA1, BRCA2, TP53 and CHEK2 c.1100delC. Of the patients testing negative for these genes, 184 underwent testing of a panel of breast cancer associated genes.

A total of 145 PVs were detected in 144 women, of which the majority (134 PVs) were identified in BRCA1, BRCA2, TP53 and CHEK2 c.1100delC. Only eight actionable PVs were found through extended panel testing. The rate of PVs in the unselected population series (n=125) was 18.9% in BRCA1, 8.8% in BRCA2 and 4% in TP53.

The overall detection rate for TP53 (5.8%) in all samples is similar to the rate (6%) published previously.17 The Myriad study assessed this age group (783 women) and found combined rates of BRCA1/2 PVs of 14% in women aged 25–29 years and 9% in women aged <25 years,6 although this cannot be considered a population study. Our study supports this lower detection rate in the very youngest age group, in contrast to the overall trend to increasing frequency of BRCA1/2 at younger ages seen in population based testing.21 This is similar to the lower rates found in ovarian cancer <30 years.22 The Myriad study6 also showed a similarly increased detection rate for TNBC <30 years. Although there was no breakdown between BRCA1 and BRCA2, it is highly likely that this was BRCA1 driven as in our study.

There is no specific figure given for TP53 in this age group, but it is also likely that the increased detection rates for non-BRCA genes from <4% (similar to all other age groups) in the 25–29 age group to ~8% in the <25 group is due to TP53. In this study, we noted an increased detection rate from 4.8% to 11.7%, due to the inclusion of TP53. Specific data from 287 of the POSH cases diagnosed aged <31 who have been analysed for TP53 and CHEK2 c.1100delC in addition to BRCA1/2 showed overall PV rate was higher in the <26 age group (28.9%) compared with 18.1% in the higher age group (online supplemental table 2).

TP53 and BRCA2 PVs were more prevalent in the youngest age groups in the POSH study although numbers were small. Nonetheless, combining the frequencies from both studies the rates of BRCA1 and BRCA2 fell from 17.1% and 7.9% in the 26–30 age group to 10.1% and 7.1% in the <26 age group, respectively, although this was not significant for BRCA1 (p=0.1) and combined BRCA1 and BRCA2 (p=0.09). The increase for TP53 detection remained significant from 3.2% to 9.1% (p=0.01).

The difference in incidence of PVs between POSH and this study may be due to sampling, certainly excluding cases with no invasive component to the presenting cancer would explain the lower rate of TP53 in the POSH study as well as excluding previous malignancy which jointly made up 12/22 (54%) of TP53 carriers in Manchester.We have also analysed available online data from Ambry genetics commercial testing (https://www.ambrygen.com/providers/resources/prevalence-tool, accessed 29/08/2020).23 While it is not possible to assess the level of pretesting for TP53, and BRCA1/2 or the presence of a Li Fraumeni family history, there is a clear upward trend of prevalence of BRCA1 and BRCA2 PVs with reducing age at breast cancer until 26 years of age (online supplemental table 3). In contrast TP53 detection is increased in the <26 year age group (p=0.03), consistent with our findings.Although the Myriad study is larger than the present study, there is a lack of detail, in particular regarding how much pretesting had been undertaken for PVs in BRCA1/2/TP53. Many women may have been tested for BRCA1/2 years earlier and subsequently taken advantage of extended testing.

Similarly, women diagnosed with breast cancer and features of Li Fraumeni syndrome may have undergone clinical bespoke TP53 testing. Nine of 15 (60%) such TP53 cases in the present study triggered clinical testing based on personal or family history. The lower rates for BRCA1/2/TP53 PVs in the Myriad study probably reflects this level of pretesting and the more likely accurate rates are from the pure population-based series in the present study from 1980 to 1997.16The current study has convincingly shown that PVs in BRCA1 are the biggest contributor to breast cancer in women diagnosed aged ≤30 years.

Even in the pure population-based study, this was at least twice the rate of BRCA2. BRCA1 PVs were also twice as prevalent in this age group as BRCA2 PVs in the POSH study. Given the lower population prevalence of BRCA1 PVs, the risk of breast cancer in some women with a BRCA1 PV will be sufficient to recommend MRI screening in BRCA1 PV carriers<30 years.

New UK guidance from the National Screening Committee will allow screening in BRCA1/2 PV carriers once their 10 year risk is 8%.24 This level of risk is estimated in BRCA1 PV carriers aged 25 years with a first degree relative diagnosed <40 years in both the Tyrer-Cuzick and BOADICEA models.25 26 Many other countries already offer screening in BRCA1/2 PV carriers from 25 years. The presence of seven TP53 carriers with breast cancer <26 years of age may well justify MRI screening from age 20 years as is already recommended in a number of guidelines.24The present study has shown limited clinical benefit from testing of genes apart from BRCA1, BRCA2 and TP53 in women with invasive or in situ breast cancer aged ≤30 years. The individual with a PTEN PV had a classical phenotype and had PTEN bespoke testing rather than a panel.

The detection rate in other actionable breast cancer genes was only 4.3% (8/184). Even allowing for an increased detection rate from testing the remaining 62 cases, this would have only reached 11/246 cases. Nevertheless, as at least seven TP53 cases would not have been suspected based on personal or family history, TP53 should be included in first-line testing as long as the panel does not reduce sensitivity for BRCA1/2 variant detection.

While a single BRIP1 PV was detected, this gene is not convincingly associated with breast cancer risk and the current evidence does not support actionability for these variants.27 Similarly there has been no clinical validation for RECQL28 29 and RAD50 and the cases in the current series was consistent with population frequencies. We also found no RAD51C or RAD51D variants consistent with their primary association with ovarian cancer susceptibility.30 31All different tumour pathologies had a >9% detection rate for BRCA1/2 and TP53 PVs. A striking finding was that the rate of PVs associated with DCIS (42.3%) was almost as high as that associated with TNBC (48.3%).

The previous association with TP53 and high-grade comedo DCIS was noted.13 We also found a rate of 15.4% (4/26) for BRCA1/2 PVs in DCIS cases. The 23.1% rate for TP53 PVs in DCIS in our study reflects the very strong association of DCIS even with invasive cancers with 41 of 45 (91.1%) of all cases containing DCIS in one study of TP53 related breast cancers.32 Currently, many countries in Europe have not instituted extended panel testing for breast cancer and in England testing for a three gene panel of BRCA1, BRCA2 and PALB2 will be provided by the public healthcare system unless a specific request is made for TP53 by a geneticist. Our study would suggest that TP53 should be discussed and potentially added to all breast cancer gene screens≤30 years unless the woman declines following counselling of the implications of this test.

The importance of identifying TP53 variants is shown by the extremely high rate of contralateral breast cancer, nearly 50% in the present study and with annual contralateral rates of ~40%.33 Given the concerns about radiation treatment and new primaries with TP53,34 35 a discussion about mastectomy and even bilateral mastectomy needs to be undertaken as well as instituting proven early detection strategies for other malignancies, including whole body MRI as published in two recent guidelines.34 35This study has some limitations. Not all 379 women underwent full testing of the panel of breast cancer associated genes. However, we have shown that there is a very low likelihood that an individual identified with a PV in BRCA1/2 or TP53 would also carry a PV in another breast cancer gene.

It is therefore unlikely that failure to test those with known BRCA1/2 PVs missed PVs in other breast cancer genes. Unfortunately, full pathology and receptor status was not available on all women. This reflects the chronological, real life data nature of the study.

Breast cancer grade was only reported reliably after 1990 and ER receptor status after 1995. HER2 status was not usually reported until 1999, after approval of Herceptin (trastuzumab) for treating HER2+ breast cancer. Nonetheless, there were still a large number of TNBCs available for assessment and since 1997 the majority of women had full pathology available, including HER2 status.

The strengths of this study include. The large number of patients with what is a rare cancer in young women. The well characterised nature of the cohort with extensive family history.

A pure population-based cohort with high ascertainment even in the postcohort study period, and the presence of a population control for evaluated genes. The sensitivity of our testing, especially for BRCA1/2 and TP53, is high, indicated by the 100% detection rate of a PV in the 31 women with MS of ≥40. Although the score was designed for BRCA1/2, it has also clearly captured very early onset highly penetrant TP53 families.In conclusion, we have identified a high rate of actionable PVs in breast cancer genes in women with breast cancer aged ≤30 years.

The clear association of TP53 PVs in very young women presenting only with DCIS is noteworthy and adds to the published association of HER2+ invasive disease in young women with TP53 PVs.32 TP53 and BRCA1/2 PVs are of similar frequency in women with breast cancer <26 years but BRCA1/2 PVs predominate in those aged 26–30 years. Overall, there is little additional benefit of testing breast cancer-associated genes apart from BRCA1, BRCA2 and TP53 in this age group.Data availability statementData are available on reasonable request. The datasets analysed during the current study are available from the corresponding author on reasonable request.Ethics statementsPatient consent for publicationNot required.Ethics approvalResearch aspects of this study were approved by the North Manchester research ethics committee (Reference 08/H1006/77)..

Allergy to flagyl alternative

Control. A brief revisitLet me start by clarifying that I’m not claiming novelty for a concept (the importance of control on life quality) in circulation at least since Aristotle’s halcyon period between 300 and 400 BC. I think, though, emphasising the notion does no harm when it occasionally gets lost in the maelstrom of ‘activity’ inherent to clinical processes. So, in quasi- bullet point terms my argument is along the lines of.

Whichever route individually chosen, we share the need for our lives to ‘have meaning’. One spoke of meaningfulness is the ability to influence one’s own trajectory, in other words, to exert a degree of control and, by extrapolation, autonomy in our/a child’s/a family’s particular circumstances. In broad terms this involves the means to alter the environment and the individual-environmental relationship. So, what’s the environment?.

So broad as to be unanswerable, but arguably, encompasses all exposures from mitochondrial DNA to societal laws to ozone layer protection.Discussion space here is limited but I hope you get the idea. The papers I’ve chosen are very different in terms of content, but are strongly connected by the flavour of sense of control and quality of life.Ethnicity and healthThe complex composite exposures inherent to racism are put under the microscope of Heather Burris (I’m delighted to say, our new advocacy editor) in a blistering, appropriately unsettling, complacency-busting and (excuse the school masterly phraseology) obligatory reading piece.Without divulging too much, the comparable birth outcomes in black women born outside the US and white US born women with a change within a generation, strongly implicate environment. Again, the ‘what is environment’ question rears its head. Yet to be fully decodified, it includes segregation, stress, pollution, and the corrosive psychological effect of chronic discrimination.

See page 212Global child healthZika in the normocephalic childIt was clear from the start of the Zika epidemic that microcephaly after fetal exposure is a harbinger of poor outcome. What is unclear is whether exposure without a measurable abnormality in brain growth (the head circumference) has similar implications or none. Karen Blackmon and colleagues tested this by enrolling seropositive pregnant women and comparing normocephalic Zika-positive children with an unexposed group in Grenada, West Indies. Gratifyingly, there were no differences in neuro-cognitive outcomes, but some subtle ophthalmological discrepancies – grounds then for positivity, but not nonchalance.

See page 244Lead exposureWithout willfully trying to delude myself, I think I’m in a majority in that chronic lead toxicity should feel like the domain of old (1960s) black and white textbooks, complete with grainy bone changes and basophilic stippling on the blood film and the attendant negative cognitive effects, because ‘the environment has improved- right?. €™Not quite. We know, objectively, that isn’t the whole story and Tharwat El Zharman and colleagues in Beirut, Lebanon emphasise this message by revisiting the prevalence and predictors of high serum lead some 18 years after non-leaded petrol legislation became statute in the country. Levels in hospitalised children are now markedly lower than they were during the fuel transition period, but that doesn’t equate with equity.

Smaller houses, time elapsed since last painted and maternal education not including college predicted higher blood levels. See page 251Real lifeTwo oncology manuscripts keep up recent momentum in the area. In the first, Hadeel Hassan and colleagues describe in intricate detail the obstacles encountered in a feasibility study addressing probiotic prophylaxis and mucositis at the start of chemotherapy. In short, recruitment proved very difficult indeed, reasons (from diary records) including unpalatability, to WiFi issues with the study app, to a sense among some, of trial overload.I can’t remember seeing a feasibility study that demonstrates better why feasibility studies are so important.

They reflect real life much more closely than the (often quite blunt) tool that is the actual RCT. We get insights into recruitment, into tolerability and into retention, issues that were they to appear after launching a trial could land a fatal blow. Should feasibility studies simply be part of the CONSORT checklist as much for the children, as researchers as donors?. Looking at post treatment life, Amanda Friend and colleagues assessed provision at UK tertiary cancer centres for fertility preservation, an area one would naturally assume to be equitable.

This isn’t, however, the case. Though sperm and ovarian storage are standard, oocyte preservation is patchy, and many centres are (eye-openingly) reliant on charity rather than central funding. See pages 259 and 265Ambulating or residentEven without the buy antibiotics contribution to the already complex equation, trends in admissions have proved surprisingly resistant to interventions. Smita Dick’s systematic review of RCTs and before and after studies, shows that other than asthma pathways, none of the broad ranging interventions resulted in a reduction in admissions.

My interpretation is that to some extent this mirrors society. Changes in primary, secondary, ED and out of hours care healthcare, the perception of greater vulnerability to criticism. Whatever the underpinning reasons, this isn’t a direction we expected to veer towards when the ambulatory care philosophy garnered momentum and we shouldn’t lose sight of this goal being laudable.I’d be interested in Trousseau’s angle on this, but this will need to wait for another day—or at least the Atoms podcast. See page 234Ethics statementsPatient consent for publicationNot applicable.Ethics approvalThis study does not involve human participants.Whether all children should be offered vaccination against antibiotics has been controversial in children aged 12–15 years old, and remains so for those under 12 years of age, partly because the balance of risk and benefit in this age group is more complex (see figure 1).The risk of severe acute buy antibiotics in healthy children infected with antibiotics is much lower than in adults.1–10 Two longer term consequences of antibiotics might therefore be more of a concern in this age group.

The first is ‘paediatric inflammatory multisystem syndrome-temporally associated with antibiotics (PIMS-TS)’, also known as ‘multisystem inflammatory syndrome in children’, an immune-mediated disease that occurs in a small proportion of children 2–6 weeks after being infected with antibiotics.11–20 The second is long buy antibiotics, the persistence of symptoms following antibiotics , a heterogeneous group of conditions.21Aside from potential long-term consequences, other considerations in deciding on buy antibiotics treatment policy for children include safety (both common reactions and rare serious side effects), population-level factors, such as reducing community transmission, treatment supply, cost of vaccination, the avoidance of quarantine, school closures and other lockdown measures, and the potential impact on routine immunisation programmes.In this review, we do not argue for or against vaccinating children against buy antibiotics but rather outline the points to consider to highlight the complexity of policy decisions on buy antibiotics vaccination in this age group.Benefits and risks of vaccinating children against buy antibioticsThe main question for implementing any treatment is ‘do the benefits of the treatment in preventing the harms of the disease outweigh any known or potential risks associated with vaccination?. €™ To date, two buy antibiotics treatments have been shown to be effective in children aged 12–17 years, and have been authorised for emergency use and subsequently recommended for this age group in many countries.22–26 Both treatments are currently being evaluated in children aged 6 months–12 years and it is likely that emergency authorisation will be sought in this age group soon. Nevertheless, buy antibiotics treatment trials in adolescents so far include less than 4000 participants and appropriately focus on efficacy, immunogenicity and rates of common reactions.25 26 A phase 2/3 trial in children 5–12 years of age recently reported that a messenger RNA (mRNA) treatment was safe, well tolerated and induced robust neutralising antibodies.27 Results from the same trial in children under 5 years of age are expected by the end of 2021. Rare adverse effects are difficult to detect with such sample sizes, and are often seen only after large-scale use.

Outside clinical trials, millions of adolescents between 12 and 18 years of age have been vaccinated, including 13 million in the USA.28 Arguments for and against vaccinating children against buy antibiotics are summarised in table 1.View this table:Table 1 Arguments for and against vaccinating children against buy antibioticsPotential benefits of vaccinating childrenProtection against buy antibioticsbuy antibiotics is generally a mild disease in children with less than 2% of symptomatic children requiring hospital admission.1–10 The rate of intensive care admission of hospitalised children ranges between 2% and 13%.1 7 8 29 30 Higher rates (10%–25%,31 32 up to 33% in some studies33 34) are reported from the USA. However, these numbers often include children who are hospitalised with buy antibiotics and not because of buy antibiotics, and therefore overestimate the severity. In children and adolescents, the risk of death from antibiotics is 0.005%,35 and in those who are hospitalised with buy antibiotics it is 0%–0.7%.1 7 8 29 30 33 34 However, again, these numbers often include children who died with a antibiotics and not because of it (a recent population-based study showed that only 41% of child deaths reported from antibiotics s were from buy antibiotics).35 Therefore, the prevention of antibiotics is not as strong an argument for vaccinating all healthy children as it is for adults. Nevertheless, this might change if new variants emerge which cause more severe disease in otherwise healthy children.There are insufficient data to estimate the risk of myocarditis in children and adolescents with buy antibiotics, although one report from the USA suggested a risk of 876 cases per million.36 Another study reported an adjusted risk ratio for myocarditis from patients with buy antibiotics compared with patients without buy antibiotics of 36.8 in children less than 16 years of age and 7.4 in adolescents 16–24 years of age.37 A third study reported an 8.2-fold increase in myocarditis admissions during the flagyl, but no cases among the 1371 children and adolescents less than 18 years of age.38 Information on the long-term outcome of myocarditis resulting from antibiotics (e.g., progression to fibrosis) is currently lacking.In the USA, with the emergence of the more transmissible Delta variant, a recent rise in s in children has led to overcrowded hospital and intensive care units.39 For hospitalised children, intensive care admission and mortality rates are currently stable at 23% and 0.4%29– 1.8%,30 respectively.

Of note, this has occurred in settings with low treatment coverage in adults and suboptimal preventive measures in place. There are no reports indicating an increase in the severity of buy antibiotics in children since the Delta variant has become dominant.At this time, buy antibiotics treatments only have ‘emergency use authorisation’ in children between 12 and 16 years of age, which is for interventions that address a serious or life-threatening condition. It has been argued that, unless children are at high risk of severe buy antibiotics because of an underlying condition, it is unclear whether the benefits to the individual outweigh the risks in this age group, and approval through the standard regulatory process should be awaited.40There are good reasons to consider offering vaccination to children and adolescents at higher risk of being hospitalised or becoming severely unwell from a antibiotics , as, in their case, the risk of harm from vaccination is estimated to be lower than the risk of harm from buy antibiotics. This includes children with neurodisabilities, Down’s syndrome, immunodeficiencies, malignancies, some cardiac, respiratory and renal diseases, obesity and poorly controlled diabetes.41The low risk of hospitalisation and death from buy antibiotics might not be a good argument against vaccinating against this disease as the risk is similar or even higher than that for other diseases for which treatments are routinely given, such as varicella, rubella, hepatitis A and influenza.42 In addition, if a high proportion of children are infected, even a very low rate of severe illness might translate to a high absolute number of cases.

Moreover, in low/middle-income countries (LMICs), the impact of buy antibiotics in children may be greater due to comorbidities that impact immunity, including diarrhoea, dengue fever, tuberculosis, malnutrition, stunting and anaemia.33 Similary, in high-income countries, children from deprived and ethnic minority groups are more frequently infected with antibiotics, which might be due to a greater likelihood of living with unvaccinated adults or in multigenerational and overcrowded households.43 44 These children have also been reported to have more severe buy antibiotics and to more frequently suffer from PIMS-TS.45–47Protection against PIMS-TSThe risk of PIMS-TS is low, affecting less than 0.1% of antibiotics-infected children. Although up to 70% of children with PIMS-TS are admitted to intensive care units,48 49 almost all patients recover without sequelae.11–20 48 50 51 Between 79% and 100% of abnormal cardiac findings are reported to resolve within 14–30 days after hospital discharge.48 52 53 Six months after discharge, 96% of children have a normal echocardiography, and renal, haematological, otolaryngological and neurological abnormalities have largely resolved.45 However, the long-term consequences of PIMS-TS remain uncertain and the death rate from PIMS-TS is estimated to be 1%–2%.48 49 There is no evidence to date on whether vaccination protects against PIMS-TS. Although by protecting against antibiotics it may well also protect against post-infectious sequelae. Data are needed to confirm this.

Since the pathogenesis of PIMS-TS remains unclear, there is also a theoretical risk that antibodies induced by buy antibiotics vaccination could cause PIMS-TS, though there is no evidence of this to date.Protection against long buy antibioticsWhile vaccination prevents with antibiotics to a degree and thus, presumably, persistent symptoms following the , more data are needed to determine accurately the incidence of long buy antibiotics in children.21 Studies to date report a prevalence ranging from 1.2% to 66%.54–64 However, most of these studies have substantial limitations, including a lack of a clear case definition, the absence of a control group without , inclusion of children without laboratory-confirmed antibiotics , follow-up at arbitrary time points and high non-responder bias.54–63 65–68 Of the five studies to date that have included controls,55 59 61 65 two did not find a difference in the prevalence of persistent symptoms between infected and uninfected children.61 65 This highlights the difficulty of separating buy antibiotics-related symptoms from those attributable to other factors associated with the flagyl, such as lockdowns and school closures. The three that did find a difference had significant limitations, including potential selection bias due to a high non-responder rate, that could lead to an overestimate of the risk of long buy antibiotics.55 59Prevention of community transmissionAnother advantage of vaccinating children is helping decrease transmission and thus reducing severe cases in adults and the risk of new flagyl variants emerging. As well as reducing disease, buy antibiotics treatments also reduce . Initial studies reported that vaccinated individuals who become infected are less likely to transmit the flagyl due to decreased viral load and duration of flagyl shedding69 70 and as a consequence, transmission from vaccinated individual to household contacts is significantly lower71 (by 50% in one study69).

However, more recent studies done since the Delta variant became dominant show similar viral loads in vaccinated and unvaccinated individuals.72–75Children, including young children, can transmit antibiotics.76 Nonetheless, even though transmission in schools can contribute to the circulation of antibiotics,77 the rate of transmission in educational settings is low and index cases are often adults.78–81 The risk of in schools correlates strongly with local community rates, which can be reduced by vaccinating older age groups. Nevertheless, the risk of transmission in different age groups and settings might change with the emergence of new flagyl variants of concern. For the Delta variant, it has been suggested that infected fully vaccinated individuals are as likely to transmit antibiotics as infected unvaccinated individuals, although for shorter duration.82 83 However, recent data from Australia reported a low risk of transmission in educational settings with protection measurements in place, even with the Delta variant (the transmission rate from adults to children was 8%, from children to adults 1.3% and from children to other children 1.8%).84Earlier in the flagyl, it was reported that index cases in households were more likely to be a parent or adolescent than a young child.6 85–87 However, one study suggests that children and adolescents are more likely to infect others.88 Another study reported that household transmission was more common from children aged 0–3 years than from children aged 14–17 years.89 However, this might change with the Delta or other new variants. In a population with low numbers of vaccinated adults, infected children transmitted the Delta variant to 70% of households (in 57% of households all members became infected).84 Nevertheless, once a large proportion of the adult population is vaccinated, preventing transmission to them from unvaccinated children becomes less important.

There is a stronger argument for vaccinating children and adolescents who live with immunosuppressed or other high-risk household members, not only for the protection of the latter but also to benefit the mental health of the former. Also, in LMICs children under 12 years of age form a larger proportion of the population and might therefore have a larger role in tranmission.Another consideration is that, once antibiotics becomes endemic, primary antibiotics in early childhood, when buy antibiotics is mild, with subsequent boosting from ongoing exposure at older ages, may bring about population immunity, as seen with common circulating antibioticses, more effectively than mass immunisation.90Avoidance of indirect (population-level) harmsVaccinating children and adolescents might help reduce the indirect harms caused by quarantine, lockdowns, repeat testing, school exclusion and closures, and other policies aimed at reducing community transmission, although the extent to which mass vaccination is necessary to achieve this remains unclear. Also, if the purpose of lockdowns and school closures is to protect adults, the incremental benefit of vaccinating children will be minimal once most adults are protected through vaccination. The possibility that vaccination might become a requirement for children for international travel is another consideration.Potential risks of vaccinating childrenRisk of adverse effectsAs with any treatment, there are potential rare adverse effects of buy antibiotics treatments.

The development of myocarditis or pericarditis after mRNA treatments has been a recent concern,91 92 particularly in male adolescents (studies reporting 6.3–6.7 cases per 100 000 second treatment doses in males aged 12–17 years,91 93 and 15.1 cases per 100 000 second treatment doses in males aged 16–19 years94). Another study reported an incidence of 10.7 cases per 100 000 persons in males aged 16–29 years.95 Of these patients, approximately 6% required intensive care admission.96 However, most recovered without sequelae (86% had resolution of symptoms after mean duration of 35 days).97 98 Importantly, even in this age group, recent reports suggest the risk of myocarditis associated with buy antibiotics is higher (see above).The risk of thrombosis after viral vector treatments observed rarely in adults also needs to be considered. The thrombotic risk in children or adolescents is less99 and no cases have been reported to date in this age group. However, since the pathogenesis underlying thrombosis associated with buy antibiotics treatments is thought to differ from that for clots from other causes, such as stasis and the contraceptive pill, further data from children are necessary.

As thrombotic events have either not been observed or appear to be very rare in Asia, Africa and Latin America, some countries are considering these treatments as an option. The theoretical risk of buy antibiotics treatments triggering PIMS-TS has been raised but there are no reports of this to date.100Long-term safetyThe lack of long-term safety data is another consideration. Longer term follow-up of myocarditis cases is needed to exclude any possibility of myocardial fibrosis and associated dysfunction or arrhythmia risk. Two studies showed a high prevalence of late gadolinium enhancement in MRIs in patients suffering from post-treatment myocarditis.97 101 Further studies are needed to establish whether this resolves or evolves into fibrosis.

As discussed above, information on this risk is also needed for myocarditis resulting from antibiotics .Although the majority of adverse treatment effects occur early after vaccination, any unforeseen adverse effects could undermine treatment confidence and reduce vaccination rates against other diseases.102treatment supplyThe currently limited global buy antibiotics treatment supply is another factor to consider. To date, many LMICs have only been able to vaccinate less than 5% of their population despite the COVAX programme. At this time, available supplies might be better prioritised for vaccinating adults with a higher risk of severe buy antibiotics and death, including healthcare workers.103 Another consideration is the higher immunogenicity of mRNA treatments in children, meaning that one dose or a reduced dose might be sufficient to protect this age group.25 On the other hand, the infrastructure to upscale the production of buy antibiotics treatments already exists and strategies for boosting global supply have been outlined.104CostSince the risks of intensive care admission or death in children are so low, the cost–benefit ratio of buy antibiotics vaccination in children is higher. However, the emergence of new variants might change this if these variants cause more frequent or more severe disease in children.105 The cost of vaccination also needs to be balanced against the reduction in community transmission that might be achieved through vaccinating children, which would enable a faster return to pre-flagyl economic stability with associated benefits to children.Other immunisation programmesRoutine immunisation programmes for children and adolescents have been disrupted by the flagyl.106 107 Implementing a universal buy antibiotics treatment programme for these age groups runs the risk of causing further delays by using up existing delivery resources and personnel.

This in turn may harm children by resulting in more cases of treatment-preventable s and diseases such as cervical cancer, meningitis, measles and pertussis. However, if buy antibiotics vaccination is combined with the administration of other routine treatments, this problem might be reduced..

Control https://swifamilies.org/how-to-get-zithromax-prescription/ flagyl online canada. A brief revisitLet me start by clarifying that I’m not claiming novelty for a concept (the importance of flagyl online canada control on life quality) in circulation at least since Aristotle’s halcyon period between 300 and 400 BC. I think, though, emphasising the notion does no harm when it occasionally gets lost in the maelstrom of ‘activity’ inherent to clinical processes. So, in quasi- bullet point terms my argument is along the lines flagyl online canada of.

Whichever route individually chosen, we share the need for our lives to ‘have flagyl online canada meaning’. One spoke of meaningfulness is the ability to influence one’s own trajectory, in other words, to exert a degree of control and, by extrapolation, autonomy in our/a child’s/a family’s particular circumstances. In broad terms this involves the means to alter the environment and the individual-environmental relationship flagyl online canada. So, what’s flagyl online canada the environment?.

So broad as to be unanswerable, but arguably, encompasses all exposures from mitochondrial DNA to societal laws to ozone layer protection.Discussion space here is limited but I hope you get the idea. The papers I’ve chosen are very different in terms of content, but are strongly connected by the flavour of sense of control and quality of life.Ethnicity and healthThe complex composite exposures inherent to racism are put under the microscope of Heather Burris (I’m delighted to say, our new advocacy editor) in a blistering, appropriately unsettling, complacency-busting and (excuse the school masterly phraseology) obligatory reading piece.Without divulging too much, the comparable birth outcomes in black women born outside the US and white US flagyl online canada born women with a change within a generation, strongly implicate environment. Again, the flagyl online canada ‘what is environment’ question rears its head. Yet to be fully decodified, it includes segregation, stress, pollution, and the corrosive psychological effect of chronic discrimination.

See page 212Global child healthZika in the normocephalic flagyl online canada childIt was clear from the start of the Zika epidemic that microcephaly after fetal exposure is a harbinger of poor outcome. What is unclear is whether exposure without flagyl online canada a measurable abnormality in brain growth (the head circumference) has similar implications or none. Karen Blackmon and colleagues tested this by enrolling seropositive pregnant women and comparing normocephalic Zika-positive children with an unexposed group in Grenada, West Indies. Gratifyingly, there flagyl online canada were no differences in neuro-cognitive outcomes, but some subtle ophthalmological discrepancies – grounds then for positivity, but not nonchalance.

See page 244Lead exposureWithout willfully trying to delude myself, I think I’m in a majority in that chronic lead toxicity should feel like the domain of old (1960s) black and white textbooks, complete with grainy bone changes and basophilic stippling on the blood film and the attendant negative cognitive effects, because ‘the environment has improved- flagyl online canada right?. €™Not quite. We know, objectively, that isn’t the whole story and Tharwat El Zharman and colleagues in Beirut, Lebanon emphasise this message by flagyl online canada revisiting the prevalence and predictors of high serum lead some 18 years after non-leaded petrol legislation became statute in the country. Levels in hospitalised flagyl online canada children are now markedly lower than they were during the fuel transition period, but that doesn’t equate with equity.

Smaller houses, time elapsed since last painted and maternal education not including college predicted higher blood levels. See page 251Real lifeTwo oncology manuscripts keep up flagyl online canada recent momentum in the area. In the first, Hadeel Hassan and colleagues describe in intricate detail the obstacles encountered in a feasibility study addressing flagyl online canada probiotic prophylaxis and mucositis at the start of chemotherapy. In short, recruitment proved very difficult indeed, reasons (from diary records) including unpalatability, to WiFi issues with the study app, to a sense among some, of trial overload.I can’t remember seeing a feasibility study that demonstrates better why feasibility studies are so important.

They reflect real life flagyl online canada much more closely than the (often quite blunt) tool that is the actual RCT. We get insights into recruitment, into tolerability and into retention, issues that were they to appear after launching a trial could land a fatal blow. Should feasibility studies simply be part of the CONSORT checklist as much for the children, as researchers as donors? flagyl online canada. Looking at flagyl online canada post treatment life, Amanda Friend and colleagues assessed provision at UK tertiary cancer centres for fertility preservation, an area one would naturally assume to be equitable.

This isn’t, however, the case. Though sperm and ovarian storage are standard, oocyte preservation is patchy, and many centres are (eye-openingly) reliant on charity rather than flagyl online canada central funding. See pages 259 and 265Ambulating or flagyl online canada residentEven without the buy antibiotics contribution to the already complex equation, trends in admissions have proved surprisingly resistant to interventions. Smita Dick’s systematic review of RCTs and before and after studies, shows that other than asthma pathways, none of the broad ranging interventions resulted in a reduction in admissions.

My interpretation is that to flagyl online canada some extent this mirrors society. Changes in primary, secondary, ED and out of hours care healthcare, the perception of flagyl online canada greater vulnerability to criticism. Whatever the underpinning reasons, this isn’t a direction we expected to veer towards when the ambulatory care philosophy garnered momentum and we shouldn’t lose sight of this goal being laudable.I’d be interested in Trousseau’s angle on this, but this will need to wait for another day—or at least the Atoms podcast. See page 234Ethics statementsPatient consent for publicationNot applicable.Ethics approvalThis study does not involve human participants.Whether all children should be offered vaccination against antibiotics has been controversial in children aged 12–15 years old, and remains so for those under 12 years of age, partly because the balance of risk and benefit in this age group is more complex (see figure 1).The risk of severe acute buy antibiotics in healthy children infected with antibiotics is much lower than in adults.1–10 Two longer term consequences of flagyl online canada antibiotics might therefore be more of a concern in this age group.

The first is ‘paediatric inflammatory multisystem syndrome-temporally associated with antibiotics (PIMS-TS)’, also known as ‘multisystem inflammatory syndrome in children’, an immune-mediated disease that occurs in a small proportion of children 2–6 weeks after being infected with antibiotics.11–20 The second is long buy antibiotics, flagyl online canada the persistence of symptoms following antibiotics , a heterogeneous group of conditions.21Aside from potential long-term consequences, other considerations in deciding on buy antibiotics treatment policy for children include safety (both common reactions and rare serious side effects), population-level factors, such as reducing community transmission, treatment supply, cost of vaccination, the avoidance of quarantine, school closures and other lockdown measures, and the potential impact on routine immunisation programmes.In this review, we do not argue for or against vaccinating children against buy antibiotics but rather outline the points to consider to highlight the complexity of policy decisions on buy antibiotics vaccination in this age group.Benefits and risks of vaccinating children against buy antibioticsThe main question for implementing any treatment is ‘do the benefits of the treatment in preventing the harms of the disease outweigh any known or potential risks associated with vaccination?. €™ To date, two buy antibiotics treatments have been shown to be effective in children aged 12–17 years, and have been authorised for emergency use and subsequently recommended for this age group in many countries.22–26 Both treatments are currently being evaluated in children aged 6 months–12 years and it is likely that emergency authorisation will be sought in this age group soon. Nevertheless, buy antibiotics flagyl online canada treatment trials in adolescents so far include less than 4000 participants and appropriately focus on efficacy, immunogenicity and rates of common reactions.25 26 A phase 2/3 trial in children 5–12 years of age recently reported that a messenger RNA (mRNA) treatment was safe, well tolerated and induced robust neutralising antibodies.27 Results from the same trial in children under 5 years of age are expected by the end of 2021. Rare adverse effects are difficult to detect with such sample sizes, and are often seen only flagyl online canada after large-scale use.

Outside clinical trials, millions of adolescents between 12 and 18 years of age have been vaccinated, including 13 million in the USA.28 Arguments for and against vaccinating children against buy antibiotics are summarised in table 1.View this table:Table 1 Arguments for and against vaccinating children against buy antibioticsPotential benefits of vaccinating childrenProtection against buy antibioticsbuy antibiotics is generally a mild disease in children with less than 2% of symptomatic children requiring hospital admission.1–10 The rate of intensive care admission of hospitalised children ranges between 2% and 13%.1 7 8 29 30 Higher rates (10%–25%,31 32 up to 33% in some studies33 34) are reported from the USA. However, these numbers often include children who are hospitalised with buy antibiotics and not because of flagyl online canada buy antibiotics, and therefore overestimate the severity. In children and adolescents, the risk of death from antibiotics is 0.005%,35 and in those who are hospitalised with buy antibiotics it is 0%–0.7%.1 7 8 29 30 33 34 However, again, these numbers often include children flagyl online canada who died with a antibiotics and not because of it (a recent population-based study showed that only 41% of child deaths reported from antibiotics s were from buy antibiotics).35 Therefore, the prevention of antibiotics is not as strong an argument for vaccinating all healthy children as it is for adults. Nevertheless, this might change if new variants emerge which cause more severe disease in otherwise healthy children.There are insufficient data to estimate the risk of myocarditis in children and adolescents with buy antibiotics, although one report from the USA suggested a risk of 876 cases per million.36 Another study reported an adjusted risk ratio for myocarditis from patients with buy antibiotics compared with patients without buy antibiotics of 36.8 in children less than 16 years of age and 7.4 in adolescents 16–24 years of age.37 A third study reported an 8.2-fold increase in myocarditis admissions during the flagyl, but no cases among the 1371 children and adolescents less than 18 years of age.38 Information on the long-term outcome of myocarditis resulting from antibiotics (e.g., progression to fibrosis) is currently lacking.In the USA, with the emergence of the more transmissible Delta variant, a recent rise in s in children has led to overcrowded hospital and intensive care units.39 For hospitalised children, intensive care admission and mortality rates are currently stable at 23% and 0.4%29– 1.8%,30 respectively.

Of note, flagyl online canada this has occurred in settings with low treatment coverage in adults and suboptimal preventive measures in place. There are no reports indicating an increase in the severity of buy antibiotics in children since the Delta variant has become dominant.At this time, buy antibiotics treatments only have ‘emergency use authorisation’ in children between 12 and 16 years of age, which is for flagyl online canada interventions that address a serious or life-threatening condition. It has been argued that, unless children are at high risk of severe buy antibiotics because of an underlying condition, it is unclear whether the benefits to the individual outweigh the risks in this age group, and approval through the standard regulatory process should be awaited.40There are good reasons to consider offering vaccination to children and adolescents at higher risk of being hospitalised or becoming severely unwell from a antibiotics , as, in their case, the risk of harm from vaccination is estimated to be lower than the risk of harm from buy antibiotics. This includes flagyl online canada children with neurodisabilities, Down’s syndrome, immunodeficiencies, malignancies, some cardiac, respiratory and renal diseases, obesity and poorly controlled diabetes.41The low risk of hospitalisation and death from buy antibiotics might not be a good argument against vaccinating against this disease as the risk is similar or even higher than that for other diseases for which treatments are routinely given, such as varicella, rubella, hepatitis A and influenza.42 In addition, if a high proportion of children are infected, even a very low rate of severe illness might translate to a high absolute number of cases.

Moreover, in low/middle-income countries (LMICs), the impact of buy antibiotics in children may be greater due to comorbidities that impact immunity, including diarrhoea, dengue fever, tuberculosis, malnutrition, stunting and anaemia.33 Similary, in high-income countries, children from deprived and ethnic minority groups are more frequently infected with antibiotics, which might be due to a greater likelihood of living with unvaccinated adults or in multigenerational and overcrowded households.43 44 These children have also been reported to have more severe buy antibiotics and to more frequently suffer from PIMS-TS.45–47Protection against PIMS-TSThe risk of PIMS-TS is low, affecting flagyl online canada less than 0.1% of antibiotics-infected children. Although up to 70% of children with PIMS-TS are admitted to intensive care units,48 49 almost all patients recover without sequelae.11–20 48 50 51 Between 79% and 100% of abnormal cardiac findings are reported to resolve within 14–30 days after hospital discharge.48 52 53 Six months after discharge, 96% of children have a normal echocardiography, and renal, haematological, otolaryngological and neurological abnormalities have largely resolved.45 However, the long-term consequences of PIMS-TS remain uncertain and the death rate from PIMS-TS is estimated to be 1%–2%.48 49 There is no evidence to date on whether vaccination protects against PIMS-TS. Although by protecting against antibiotics it may well also flagyl online canada protect against post-infectious sequelae. Data are needed to confirm this.

Since the pathogenesis of PIMS-TS remains unclear, there is also a theoretical risk that antibodies induced by buy antibiotics vaccination could cause PIMS-TS, though there is no evidence of this to date.Protection against long buy antibioticsWhile vaccination prevents with antibiotics to a degree and thus, presumably, persistent symptoms following the , more data are needed to determine accurately the incidence of long buy antibiotics in children.21 Studies to date report a prevalence ranging from 1.2% to 66%.54–64 However, most of these studies have substantial limitations, including a lack of a clear case definition, the absence of a control group without , inclusion of children without laboratory-confirmed antibiotics , follow-up at arbitrary time points and high non-responder bias.54–63 65–68 flagyl online canada Of the five studies to date that have included controls,55 59 61 65 two did not find a difference in the prevalence of persistent symptoms between infected and uninfected children.61 65 This highlights the difficulty of separating buy antibiotics-related symptoms from those attributable to other factors associated with the flagyl, such as lockdowns and school closures. The three that did find a difference had significant limitations, including potential selection bias due to a high non-responder rate, that could lead to an overestimate of the risk of long buy antibiotics.55 59Prevention of community transmissionAnother advantage of vaccinating children is helping decrease transmission and thus reducing severe cases in adults and the flagyl online canada risk of new flagyl variants emerging. As well as reducing disease, buy antibiotics treatments also reduce . Initial studies reported that vaccinated individuals who become infected are less likely to transmit the flagyl due to decreased viral load and duration of flagyl flagyl online canada shedding69 70 and as a consequence, transmission from vaccinated individual to household contacts is significantly lower71 (by 50% in one study69).

However, more recent studies done since the Delta variant became dominant show similar viral loads in vaccinated and unvaccinated individuals.72–75Children, including young children, can transmit antibiotics.76 Nonetheless, even though transmission in schools can contribute to the circulation of antibiotics,77 the rate of transmission in educational settings is low and index cases are often adults.78–81 The risk of in schools correlates strongly with local community flagyl online canada rates, which can be reduced by vaccinating older age groups. Nevertheless, the risk of transmission in different age groups and settings might change with the emergence of new flagyl variants of concern. For the Delta variant, it has been suggested that infected fully vaccinated individuals are as likely to transmit antibiotics as infected unvaccinated individuals, although for shorter duration.82 83 However, recent data from Australia reported a low risk of transmission in educational settings with protection measurements in place, even with the Delta variant (the transmission rate from adults to children was 8%, from children to adults 1.3% and from children to other children 1.8%).84Earlier in the flagyl, it was reported that index cases in households were more likely to be a parent or adolescent than a young child.6 85–87 flagyl online canada However, one study suggests that children and adolescents are more likely to infect others.88 Another study reported that household transmission was more common from children aged 0–3 years than from children aged 14–17 years.89 However, this might change with the Delta or other new variants. In a population with low numbers of vaccinated adults, infected children transmitted the Delta variant to 70% of households (in 57% of households all members became infected).84 Nevertheless, once a large proportion of the adult population is vaccinated, preventing transmission flagyl online canada to them from unvaccinated children becomes less important.

There is a stronger argument for vaccinating children and adolescents who live with immunosuppressed or other high-risk household members, not only for the protection of the latter but also to benefit the mental health of the former. Also, in LMICs children under 12 years of age form a larger proportion of the population and might therefore have a larger role in tranmission.Another consideration is that, once antibiotics becomes endemic, primary antibiotics in early childhood, when buy antibiotics is mild, with subsequent boosting from ongoing exposure at older ages, may bring about population immunity, as seen with common circulating antibioticses, more effectively than mass immunisation.90Avoidance of indirect (population-level) harmsVaccinating children and adolescents might help reduce the indirect harms caused by quarantine, lockdowns, repeat testing, school exclusion and closures, flagyl online canada and other policies aimed at reducing community transmission, although the extent to which mass vaccination is necessary to achieve this remains unclear. Also, if the purpose of lockdowns and school closures is to flagyl online canada protect adults, the incremental benefit of vaccinating children will be minimal once most adults are protected through vaccination. The possibility that vaccination might become a requirement for children for international travel is another consideration.Potential risks of vaccinating childrenRisk of adverse effectsAs with any treatment, there are potential rare adverse effects of buy antibiotics treatments.

The development of myocarditis or pericarditis flagyl online canada after mRNA treatments has been a recent concern,91 92 particularly in male adolescents (studies reporting 6.3–6.7 cases per 100 000 second treatment doses in males aged 12–17 years,91 93 and 15.1 cases per 100 000 second treatment doses in males aged 16–19 years94). Another study reported an incidence of 10.7 cases per 100 000 persons in males aged 16–29 years.95 Of these patients, approximately 6% required intensive care admission.96 However, most recovered without sequelae (86% had resolution of symptoms after mean duration of 35 days).97 98 Importantly, even in this age group, recent reports suggest the risk of myocarditis associated with buy antibiotics is higher (see above).The risk of thrombosis after flagyl online canada viral vector treatments observed rarely in adults also needs to be considered. The thrombotic risk in children or adolescents is less99 and no cases have been reported to date in this age group. However, since the pathogenesis underlying thrombosis associated with buy antibiotics treatments is thought to differ from flagyl online canada that for clots from other causes, such as stasis and the contraceptive pill, further data from children are necessary.

As thrombotic events have either not been observed or appear to be very rare in Asia, Africa flagyl online canada and Latin America, some countries are considering these treatments as an option. The theoretical risk of buy antibiotics treatments triggering PIMS-TS has been raised but there are no reports of this to date.100Long-term safetyThe lack of long-term safety data is another consideration. Longer term follow-up of myocarditis cases is needed to exclude any possibility of myocardial fibrosis and flagyl online canada associated dysfunction or arrhythmia risk. Two studies showed a high prevalence of late gadolinium enhancement in MRIs in patients suffering from post-treatment myocarditis.97 101 Further studies flagyl online canada are needed to establish whether this resolves or evolves into fibrosis.

As discussed above, information on this risk is also needed for myocarditis resulting from antibiotics .Although the majority of adverse treatment effects occur early after vaccination, any unforeseen adverse effects could undermine treatment confidence and reduce vaccination rates against other diseases.102treatment supplyThe currently limited global buy antibiotics treatment supply is another factor to consider. To date, many LMICs flagyl online canada have only been able to vaccinate less than 5% of their population despite the COVAX programme. At this time, available supplies might be better prioritised for vaccinating adults with a higher risk of severe buy antibiotics and death, including healthcare workers.103 Another consideration is the higher immunogenicity of mRNA treatments in children, meaning that one dose or a reduced dose might be sufficient to protect this age group.25 On the other hand, the infrastructure to upscale the production of buy antibiotics treatments already exists and strategies for flagyl online canada boosting global supply have been outlined.104CostSince the risks of intensive care admission or death in children are so low, the cost–benefit ratio of buy antibiotics vaccination in children is higher. However, the emergence of new variants might change this if these variants cause more frequent or more severe disease in children.105 The cost of vaccination also needs to be balanced against the reduction in community transmission that might be achieved through vaccinating children, which would enable a faster return to pre-flagyl economic stability with associated benefits to children.Other immunisation programmesRoutine immunisation programmes for children and adolescents have been disrupted by the flagyl.106 107 Implementing a universal buy antibiotics treatment programme for these age groups runs the risk of causing further delays by using up existing delivery resources and personnel.

This in turn may harm children by resulting in more cases of treatment-preventable s and diseases such as cervical cancer, meningitis, measles and pertussis. However, if buy antibiotics vaccination is combined with the administration of other routine treatments, this problem might be reduced..

How long after taking flagyl can you drink

BIRMINGHAM, AL – Two Birmingham delivery companies, employed https://mind-3.com/accessibility/ by FedEx how long after taking flagyl can you drink Corp. As subcontractors, shortchanged 235 workers after wrongly claiming the workers were not entitled to overtime pay under motor carrier regulations, a U.S. Department of Labor investigation has how long after taking flagyl can you drink found.

The department’s Wage and Hour Division determined Steel City Couriers Inc. And Cahaba how long after taking flagyl can you drink Valley Couriers Inc. Paid workers straight time for all hours or a flat weekly rate without overtime compensation.

They also failed to maintain accurate records of hours worked. These practices how long after taking flagyl can you drink violated the Fair Labor Standards Act. Division investigators found employers misapplied an FLSA exemption from overtime requirements when workers duties involve driving on interstate highways, operating delivery trucks weighing at least 10,000 pounds, or when working for an employer who falls under the regulations governed by the U.S.

Department of how long after taking flagyl can you drink Transportation. The investigation led to the recovery of $181,379 in back wages for 235 workers. “Misapplying overtime how long after taking flagyl can you drink rules deprives workers of their legal right to be paid time-and-a-half for hours over 40 in a workweek,” said Wage and Hour Division District Director Kenneth Stripling in Birmingham, Alabama.

€œEmployers who ignore their obligations and violate workers’ rights can find their mistakes costly and their ability to recruit and retain the people they need to run a successful business difficult, if not impossible.” “Violations like this could have been avoided by contacting the Wage and Hour Division, whose staff can help employers understand their responsibilities under the law,” Stripling added. The division offers multiple compliance assistance resources, including a fact sheet on the FLSA’s motor carrier exemption to provide employers the information they need to comply with the law..

BIRMINGHAM, AL – Two Birmingham delivery companies, http://bobmackin.ca/?p=190 employed flagyl online canada by FedEx Corp. As subcontractors, shortchanged 235 workers after wrongly claiming the workers were not entitled to overtime pay under motor carrier regulations, a U.S. Department of Labor investigation has found flagyl online canada. The department’s Wage and Hour Division determined Steel City Couriers Inc.

And Cahaba flagyl online canada Valley Couriers Inc. Paid workers straight time for all hours or a flat weekly rate without overtime compensation. They also failed to maintain accurate records of hours worked. These practices violated the Fair flagyl online canada Labor Standards Act.

Division investigators found employers misapplied an FLSA exemption from overtime requirements when workers duties involve driving on interstate highways, operating delivery trucks weighing at least 10,000 pounds, or when working for an employer who falls under the regulations governed by the U.S. Department of flagyl online canada Transportation. The investigation led to the recovery of $181,379 in back wages for 235 workers. “Misapplying overtime rules deprives workers of their legal right to be paid time-and-a-half for hours over 40 flagyl online canada in a workweek,” said Wage and Hour Division District Director Kenneth Stripling in Birmingham, Alabama.

€œEmployers who ignore their obligations and violate workers’ rights can find their mistakes costly and their ability to recruit and retain the people they need to run a successful business difficult, if not impossible.” “Violations like this could have been avoided by contacting the Wage and Hour Division, whose staff can help employers understand their responsibilities under the law,” Stripling added. The division offers multiple compliance assistance resources, including a fact sheet on the FLSA’s motor carrier exemption to provide employers the information they need to comply with the law..