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Oceans are heating up at breakneck speed, and the warming waters are threatening marine animals all over the levitra buy australia world. That’s the alarming takeaway from a pair of new studies on marine warming published this week in the journal PLOS Climate. The first study looks back in time to find out how the oceans have changed since the Industrial Revolution, when humans began rapidly pumping greenhouse levitra buy australia gases into the atmosphere.

Using historical temperature records dating back to the 1800s, scientists Kisei Tanaka and Kyle Van Houtan evaluated the changing frequency of “extreme marine heat events.” Around the turn of the 20th century, these kinds of marine heat events were relatively rare. The scientists looked specifically at events falling into the top 2 percent of high temperatures. Around the year 1900, these events would levitra buy australia have happened about once every 50 years.

The researchers then calculated how common these extreme heat events are today. They’re becoming the norm across much of the world. By 2014, half the world’s ocean area exceeded the levitra buy australia extreme heat threshold.

By 2019, the last year the scientists examined, 57 percent of the world’s oceans had hit the mark. In other words, temperatures that would have levitra buy australia occurred only rarely a century ago have now become routine. The findings demonstrate that the influence of climate change on the world’s oceans isn’t just a problem for the future, Tanaka told E&E News in an interview.

€œIt is happening as we speak,” he said. €œIt has been happening for quite some time.” Still, other forward-looking studies warn that levitra buy australia the effects will only get worse as the planet continues to warm. The second PLOS Climate study looks specifically at the impact of future marine warming on coral reefs.

Prolonged heat can cause reefs to “bleach,” or expel the colorful algae living inside them. These algae help provide the corals with nutrients—and if they go too long without levitra buy australia them, they can die. Bleached reefs are often able to fully recover within a decade, as long as they don’t get hit with another heat event in the meantime.

But marine heat waves are levitra buy australia growing more common and more severe over time as average ocean temperatures climb upward. The new study looks at areas of the ocean known as coral “refugia”—these are places where local water and weather conditions are able to shield reefs from the warming conditions that affect surrounding areas. In some coastal areas, for instance, local wind patterns help to churn up the sea and allow cold water to bubble up from near the ocean floor.

The new levitra buy australia study defines coral refugia as places where marine heat events—of the caliber likely to cause corals to bleach—occur only about once every 10 years. These spots are likely to have enough time to fully recover between bleaching events. Currently, the study estimates that around 84 percent of the world’s reefs are located in these kinds of protected areas.

But even levitra buy australia a little bit of future warming may change that. Under 1.5 degrees Celsius of global warming, that percentage plummets to about 0.2 percent of the world’s reefs. Meanwhile, about 90 percent of all the world’s reefs will lie in areas likely to experience a marine heat event at least once every five years—meaning the chances of full recovery in between bleaching events is slim.

Under 2 C of warming, all of the levitra buy australia world’s reefs will lie in areas likely to experience at least one heat event every 10 years. And 99.7 percent of them are likely to be hit every five years. At this threshold, the world’s reefs are likely to experience significantly levitra buy australia more bleaching events, and some corals may begin to die off.

The goal of the Paris climate agreement is to keep global temperatures within 2 C of their preindustrial levels at all costs, and within 1.5 C if possible. Numerous studies suggest that the effects of climate change will significantly worsen above these levels. Still, the new study points out that every little bit levitra buy australia of warming has consequences.

Even meeting the Paris targets won’t keep the world’s coral reefs out of danger. €œOur finding reinforces the stark reality that there is no safe limit of global warming for coral reefs,” said Adele Dixon, a coral expert at the University of Leeds in the United Kingdom and lead author of the new study, in a statement. The two studies out this week underscore the growing risks posed levitra buy australia by the warming oceans.

They’re hardly the first to raise the alarm. Numerous studies have warned that the oceans are heating up, that marine heat waves are growing more frequent and more severe, and that marine levitra buy australia organisms are suffering the consequences. Corals are bleaching more often, fish are migrating into new areas, and some fisheries are starting to decline.

The past few years, in particular, have seen some worrying milestones for marine climate change. The last three years in a row have all broken records for ocean levitra buy australia heat. A paper published last month concluded that the world’s oceans reached their hottest levels ever recorded in 2021 (Climatewire, Jan.

12). Reprinted from levitra buy australia E&E News with permission from POLITICO, LLC. Copyright 2022.

E&E News provides essential news for energy and environment professionals..

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Oct. 25, 2022 – Just when we thought this holiday season, finally, would be the back-to-normal one, some infectious disease experts are warning that a so-called tripledemic – influenza, erectile dysfunction treatment, and RSV – may be in the forecast.The warning isn’t without basis. The flu season has gotten an early start.

As of Oct. 21, early increases in seasonal flu activity have been reported in most of the country, the CDC says, with the southeast and south-central areas having the highest activity levels. Children’s hospitals and emergency departments are seeing a surge in children with RSV.erectile dysfunction treatment cases are trending down, according to the CDC, but epidemiologists – scientists who study disease outbreaks – always have their eyes on emerging variants.

Predicting exactly when cases will peak is difficult, says Justin Lessler, PhD, a professor of epidemiology at the University of North Carolina at Chapel Hill. Lessler is on the coordinating team for the erectile dysfunction treatment Scenario Modeling Hub, which aims to predict the course erectile dysfunction treatment, and the Flu Scenario Modeling Hub, which does the same for influenza. For erectile dysfunction treatment, some models are predicting some spikes before Christmas, he says, and others see a new wave in 2023.

For the flu, the model is predicting an earlier-than-usual start, as the CDC has reported. While flu activity is relatively low, the CDC says, the season is off to an early start. For the week ending Oct.

21, 1,674 patients were hospitalized for flu, higher than in the summer months but fewer than the 2,675 hospitalizations for the week of May 15, 2022. As of Oct. 20, erectile dysfunction treatment cases have declined 12% over the last 2 weeks, nationwide.

But hospitalizations are up 10% in much of the Northeast, The New York Times reports, and the improvement in cases and deaths has been slowing down. As of Oct. 15, 15% of RSV tests reported nationwide were positive, compared with about 11% at that time in 2021, the CDC says.

The surveillance collects information from 75 counties in 12 states. Experts point out that the levitraes -- all three are respiratory levitraes -- are simply playing catchup. “They spread the same way and along with lots of other levitraes, and you tend to see an increase in them during the cold months," says Timothy Brewer, MD, professor of medicine and epidemiology at UCLA.The increase in all three levitraes “is almost predictable at this point in the levitra,” says Dean Blumberg, MD, a professor and chief of pediatric infectious diseases at the University of California Davis Health.

€œAll the respiratory levitraes are out of whack.” Last year, RSV cases were up, too, and began to appear very early, he says, in the summer instead of in the cooler months. Flu also appeared early in 2021, as it has this year. That contrasts with the flu season of 2020-2021, when erectile dysfunction treatment precautions were nearly universal, and cases were down.

At UC Davis, “we didn’t have one pediatric admission due to influenza in the 2020-2021 [flu] season,” Blumberg says. The number of pediatric flu deaths usually range from 37 to 199 per year, according to CDC records. But in the 2020-2021 season, the CDC recorded one pediatric flu death in the U.S.

Both children and adults have had less contact with others the past 2 seasons, Blumberg says, “and they don’t get the immunity they got with those s [previously]. That’s why we are seeing out-of-season, early season [levitraes].” Eventually, he says, the cases of flu and RSV will return to previous levels. €œIt could be as soon as next year,” Blumberg says.

And erectile dysfunction treatment, hopefully, will become like influenza, he says.“RSV has always come around in the fall and winter,” says Elizabeth Murray, DO, a pediatric emergency medicine doctor at the University of Rochester Medical Center and a spokesperson for the American Academy of Pediatrics. This year, children are back in school and for the most part not masking, she says. €œIt’s a perfect storm for all the germs to spread now.

They’ve just been waiting for their opportunity to come back.” Self-Care vs. NotRSV can pose a risk for anyone, but most at risk are children under age 5, especially infants under age 1, and adults over age 65. There is no treatment for it.

Symptoms include a runny nose, decreased appetite, coughing, sneezing, fever, and wheezing. But in young infants, there may only be decreased activity, crankiness, and breathing issues, the CDC says. Keep an eye on the breathing if RSV is suspected, Murray tells parents.

If your child can’t breathe easily, is unable to lie down comfortably, can’t speak clearly, or is sucking in the chest muscles to breathe, get medical help. Most kids with RSV can stay home and recover, she says, but often will need to be checked by a medical professional.She advises against getting an oximeter to measure oxygen levels for home use. €œThey are often not accurate,” she says.

If in doubt about how serious your child’s symptoms are, “don’t wait it out,” she says, and don’t hesitate to call 911.Symptoms of flu, erectile dysfunction treatment, and RSV can overlap. But each can involve breathing problems, which can be an emergency. €œIt’s important to seek medical attention for any concerning symptoms, but especially severe shortness of breath or difficulty breathing, as these could signal the need for supplemental oxygen or other emergency interventions,” says Mandy De Vries, a respiratory therapist and director of education at the American Association for Respiratory Care.

Inhalation treatment or mechanical ventilation may be needed for severe respiratory issues. PrecautionsTo avoid the tripledemic – or any single – Timothy Brewer, MD, a professor of medicine and epidemiology at UCLA, suggests some familiar measures. €œStay home if you’re feeling sick.

Make sure you are up to date on your vaccinations. Wear a mask indoors.”.

Oct. 25, 2022 – Just when we thought this holiday season, finally, would be the back-to-normal one, some infectious disease experts are warning that a so-called tripledemic – influenza, erectile dysfunction treatment, and RSV – may be in the forecast.The warning isn’t without basis. The flu season has gotten an early start. As of Oct. 21, early increases in seasonal flu activity have been reported in most of the country, the CDC says, with the southeast and south-central areas having the highest activity levels.

Children’s hospitals and emergency departments are seeing a surge in children with RSV.erectile dysfunction treatment cases are trending down, according to the CDC, but epidemiologists – scientists who study disease outbreaks – always have their eyes on emerging variants. Predicting exactly when cases will peak is difficult, says Justin Lessler, PhD, a professor of epidemiology at the University of North Carolina at Chapel Hill. Lessler is on the coordinating team for the erectile dysfunction treatment Scenario Modeling Hub, which aims to predict the course erectile dysfunction treatment, and the Flu Scenario Modeling Hub, which does the same for influenza. For erectile dysfunction treatment, some models are predicting some spikes before Christmas, he says, and others see a new wave in 2023. For the flu, the model is predicting an earlier-than-usual start, as the CDC has reported.

While flu activity is relatively low, the CDC says, the season is off to an early start. For the week ending Oct. 21, 1,674 patients were hospitalized for flu, higher than in the summer months but fewer than the 2,675 hospitalizations for the week of May 15, 2022. As of Oct. 20, erectile dysfunction treatment cases have declined 12% over the last 2 weeks, nationwide.

But hospitalizations are up 10% in much of the Northeast, The New York Times reports, and the improvement in cases and deaths has been slowing down. As of Oct. 15, 15% of RSV tests reported nationwide were positive, compared with about 11% at that time in 2021, the CDC says. The surveillance collects information from 75 counties in 12 states. Experts point out that the levitraes -- all three are respiratory levitraes -- are simply playing catchup.

“They spread the same way and along with lots of other levitraes, and you tend to see an increase in them during the cold months," says Timothy Brewer, MD, professor of medicine and epidemiology at UCLA.The increase in all three levitraes “is almost predictable at this point in the levitra,” says Dean Blumberg, MD, a professor and chief of pediatric infectious diseases at the University of California Davis Health. €œAll the respiratory levitraes are out of whack.” Last year, RSV cases were up, too, and began to appear very early, he says, in the summer instead of in the cooler months. Flu also appeared early in 2021, as it has this year. That contrasts with the flu season of 2020-2021, when erectile dysfunction treatment precautions were nearly universal, and cases were down. At UC Davis, “we didn’t have one pediatric admission due to influenza in the 2020-2021 [flu] season,” Blumberg says.

The number of pediatric flu deaths usually range from 37 to 199 per year, according to CDC records. But in the 2020-2021 season, the CDC recorded one pediatric flu death in the U.S. Both children and adults have had less contact with others the past 2 seasons, Blumberg says, “and they don’t get the immunity they got with those s [previously]. That’s why we are seeing out-of-season, early season [levitraes].” Eventually, he says, the cases of flu and RSV will return to previous levels. €œIt could be as soon as next year,” Blumberg says.

And erectile dysfunction treatment, hopefully, will become like influenza, he says.“RSV has always come around in the fall and winter,” says Elizabeth Murray, DO, a pediatric emergency medicine doctor at the University of Rochester Medical Center and a spokesperson for the American Academy of Pediatrics. This year, children are back in school and for the most part not masking, she says. €œIt’s a perfect storm for all the germs to spread now. They’ve just been waiting for their opportunity to come back.” Self-Care vs. NotRSV can pose a risk for anyone, but most at risk are children under age 5, especially infants under age 1, and adults over age 65.

There is no treatment for it. Symptoms include a runny nose, decreased appetite, coughing, sneezing, fever, and wheezing. But in young infants, there may only be decreased activity, crankiness, and breathing issues, the CDC says. Keep an eye on the breathing if RSV is suspected, Murray tells parents. If your child can’t breathe easily, is unable to lie down comfortably, can’t speak clearly, or is sucking in the chest muscles to breathe, get medical help.

Most kids with RSV can stay home and recover, she says, but often will need to be checked by a medical professional.She advises against getting an oximeter to measure oxygen levels for home use. €œThey are often not accurate,” she says. If in doubt about how serious your child’s symptoms are, “don’t wait it out,” she says, and don’t hesitate to call 911.Symptoms of flu, erectile dysfunction treatment, and RSV can overlap. But each can involve breathing problems, which can be an emergency. €œIt’s important to seek medical attention for any concerning symptoms, but especially severe shortness of breath or difficulty breathing, as these could signal the need for supplemental oxygen or other emergency interventions,” says Mandy De Vries, a respiratory therapist and director of education at the American Association for Respiratory Care.

Inhalation treatment or mechanical ventilation may be needed for severe respiratory issues. PrecautionsTo avoid the tripledemic – or any single – Timothy Brewer, MD, a professor of medicine and epidemiology at UCLA, suggests some familiar measures. €œStay home if you’re feeling sick. Make sure you are up to date on your vaccinations. Wear a mask indoors.”.

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Keep out of the reach of children. Store at room temperature between 15 and 30 degrees C (59 and 86 degrees F). Throw away any unused medicine after the expiration date.

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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 Buy kamagra pills the high mobility group (HMG) box of SRY (sex-determining pastillas levitra 20mg 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 neural tube, and migrate through several routes to reach target tissues and give rise to neurons and glia of pastillas levitra 20mg 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) pastillas levitra 20mg initial 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 pastillas levitra 20mg mouse Sox10 genes encode 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 pastillas levitra 20mg sequence.9 10The protein’s structure 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 pastillas levitra 20mg signal) signals.12 1310 samples in various tissues (including the brain, gut, nerves (tibial), 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-1933801000" 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)..

IntroductionSOX10 belongs to the SOX family of levitra buy australia transcription factors, of which the members are defined based on the presence of a 79 amino acid DNA-binding domain with homology Buy kamagra pills to 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 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 levitra buy australia 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) levitra buy australia initial 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 an open reading frame of 466 amino acids that share 92% nucleotidic and 98% amino acid sequence identities.8 The absence of levitra buy australia 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 levitra buy australia with the predicted SOX10 mRNA sequence.9 10The protein’s structure 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 levitra buy australia (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 number of A-to-I modifications reported is. AluY.

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-1933801000" 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.

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." 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.

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.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)..

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A 14-year-old Hudson Valley boy has gone missing and authorities are asking the public's help in locating him.Orange County resident Justin Guerrero is 5-foot-6, 150 levitra buy australia pounds, http://serenitygraphic.com/cialis-online/ with black hair and brown eyes and may be in need of medical attention, authorities say.He was last seen in the city of Newburgh, on Mill Street, at 3 a.m. On Monday, Feb levitra buy australia. 21 and was last seen wearing yellow and green pajama pants, a black hooded sweatshirt, and blue backpack. He may be riding a skateboard.Share this story by clicking on levitra buy australia the Facebook icon below.Check back to Daily Voice for updates.

Click here to sign up for Daily Voice's free daily emails and news alerts.A new report has revealed the percentage of Americans who are living paycheck to paycheck.As of this past December, 61 percent of levitra buy australia the population in the United States was living paycheck to paycheck, according to a collaborative report from PYMNTS and LendingClub.According to the February report, that's an increase of 7 percent compared to May.CNBC reported that rising wages have been unable to keep up with the increasing cost of living.The LendingClub report also found that 54 percent of Baby Boomers and seniors in the study reported that they were living paycheck to paycheck. Click here to sign up for Daily Voice's free daily emails and news alerts.With coyote sightings on the rise throughout the region, the New York State Department of Environmental Conservation (DEC) issued new guidance on how to avoid conflicts between animals and people.For coyotes, February marks the beginning of mating season, which lasts between four and six weeks, peaking late in the month and early in March, according to animal experts, who noted that it varies year-to-year depending on weather, prey, quantity, and other mitigating factors.Researchers said that the season begins with mating, followed by pup raising, and then dispersal, leading to the rise in sightings in recent weeks.According to the DEC, coyotes can be found in habitats throughout the state, from rural farmland and forests to green spaces in suburban and urban areas. For the most part, coyotes avoid levitra buy australia contact with people. However, conflicts with people and pets may occur as coyotes tend to be more territorial during breeding and levitra buy australia pup-rearing seasons in the spring and summer.

Officials warned that if coyotes learn to associate food sources such as garbage or pet food with people, the animals may lose their natural fear of humans, increasing the potential for close encounters or conflicts.“This is the time of year when New York's resident coyotes breed and set up dens for pups that will arrive in the spring," DEC Commissioner Basil Seggos said. "While conflicts with people and pets are rare, New Yorkers should levitra buy australia remain alert and follow DEC's common-sense guidelines to minimize the risk for potential conflicts with coyotes.”To avoid any conflict with coyotes, the DEC advised New Yorkers to take certain steps that include:Do not feed coyotes;Do not leave food outside. Pet food and garbage attract coyotes and other wildlife and increase risks to people and pets;Do not feed pets levitra buy australia outside;Prevent access to garbage;Fence or enclose compost piles;Eliminate availability of birdseed, as concentrations of birds and rodents that come to feeders can attract coyotes;Do not allow coyotes to approach people or pets. If you see a coyote, be aggressive in your behavior.

Stand tall and hold your arms up or levitra buy australia out to look as large as possible. If a coyote lingers for too long, make loud noises, wave arms, and throw sticks and levitra buy australia stones;Teach children to appreciate coyotes from a distance;Do not allow pets to run free. Supervise outdoor pets to keep them safe from coyotes and other wildlife, especially at sunset and at night - Small dogs and cats are especially vulnerable;Fence yards to deter coyotes. The fence should be more than four feet tall, and tight to the ground, preferably extending six inches below ground level;Remove brush levitra buy australia and tall grass from around homes to reduce protective cover for coyotes.

Coyotes are typically secretive and like areas where they can hide;Ask neighbors to follow these steps to prevent coyote levitra buy australia conflicts. Click here to sign up for Daily Voice's free daily emails and news alerts.A 36-year-old man has pleaded guilty in New York to a charge related to a money-laundering scheme involving the theft of tens of millions of dollars.Fred Asante, of Virginia, pleaded guilty on Wednesday, Feb. 16, to conspiracy to commit money laundering, levitra buy australia according to Damian Williams, the United States attorney for the Southern District of New York.He was arrested on Feb. 17, 2021, levitra buy australia Williams reported.“Fred Asante admitted today to laundering money from victims of various fraud schemes, including cruel scams targeting elderly online daters searching for companionship," Williams said.

"Compounding the disappointment of learning their potential soulmate was indeed nonexistent, Asante’s victims later found they were also targets of a Ghana-based criminal enterprise netting over $35 million in illegal proceeds. We implore the millions of Americans looking for someone special online to use extra caution, and be especially levitra buy australia beware if solicited for money or other personal information.From about 2013 through about 2020, Asante was involved in a criminal enterprise based in Ghana that committed fraud against residents and businesses in the US, including New York, Williams reported.The enterprise would trick businesses into wiring funds into its account, and conduct romance scams via email, text or online dating sites to trick victims into sending them money under false pretenses, the US Attorney's Office said. The enterprise would also levitra buy australia submit fraudulent loan applications to the US Small Business Administration through a program meant to provide relief to small businesses during the erectile dysfunction treatment levitra, according to the announcement.Asante and other members of the organization would receive proceeds from victims in business bank accounts they controlled in New York, New Jersey, and Virginia, Williams said.Asante would then withdraw, transport, and launder the proceeds to other members of the organization, according to the report."The defendant’s transactions had the appearance of legitimate business transactions when, in fact, the products had been purchased using the proceeds of fraud schemes," the report said. "This trade-based money laundering scheme was designed to obscure the origin of the fraud proceeds as well as the identity of the ultimate beneficiaries of these schemes." Between about 2016 and 2020, Asante controlled more than 12 business bank accounts with deposits totaling more than $35 million, Williams reported.

He is set to be levitra buy australia sentenced on May 18. Click here to sign up for Daily Voice's free daily emails and news alerts.A sex offender wanted in two states was nabbed after police responded to a Hudson Valley mall for a man acting "disorderly."Eldon Ray Groh, age 49, levitra buy australia of Missouri, was arrested on Tuesday, Feb. 15, when officers responded to Mountain Mall in Sullivan County at 321 East Broadway in the village of Monticello.According to Lt. Mark Johnstone, of the Monticello Police, when officers stopped Groh and ran a background check, they found that he was wanted in Missouri on a felony warrant for failure to register as a sex levitra buy australia offender.He was also wanted in by the Osceola County Sheriff's Office in Florida on a failure to appear warrant, Johnstone said.Groh was then taken into custody without incident and charged with being a fugitive from justice.

He was remanded to the Sullivan County Jail without bail pending further levitra buy australia court action. Click here to sign up for Daily Voice's free daily emails and news alerts..

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The women were followed for almost 11 years and during that time 378 uterine cancer cases were diagnosed.The researchers found that women who reported frequent use of hair straightening products, defined as more than four times in the previous year, were more than twice as likely to go on to develop uterine cancer compared to those who did not use the products.“We estimated that 1.64% of women who never used hair straighteners would go on to develop uterine cancer by the age of 70. But for frequent users, that risk goes levitra reviews up to 4.05%,” said Alexandra White, Ph.D., head of the NIEHS Environment and Cancer Epidemiology group and lead author on the new study. €œThis doubling rate is concerning.

However, it is important levitra reviews to put this information into context - uterine cancer is a relatively rare type of cancer.”Uterine cancer accounts for about 3% of all new cancer cases but is the most common cancer of the female reproductive system, with 65,950 estimated new cases in 2022. Studies show that incidence rates of uterine cancer have been rising in the United States, particularly among Black women.Approximately 60% of the participants who reported using straighteners in the previous year were self-identified Black women, according to the study published in the Journal of the National Cancer Institute. Although, the study did not find that the relationship between straightener use and uterine cancer incidence was different by race, the adverse health effects may be greater for Black women due to higher prevalence of use.“Because Black women use hair straightening or relaxer products more frequently and tend to initiate use at earlier ages than other races and ethnicities, these findings may be even more relevant for them,” said Che-Jung Chang, Ph.D., an author on the new study and a research fellow in the NIEHS Epidemiology levitra reviews Branch.The findings are consistent with prior studies showing straighteners can increase the risk of hormone-related cancers in women.The researchers did not collect information on brands or ingredients in the hair products the women used.

However, in the paper they note that several chemicals that have been found in straighteners (such as parabens, bisphenol A, metals, and formaldehyde) could be contributing to the increased uterine cancer risk observed. Chemical exposure from hair product use, especially levitra reviews straighteners, could be more concerning than other personal care products due to increased absorption through the scalp which may be exacerbated by burns and lesions caused by straighteners.“To our knowledge this is the first epidemiologic study that examined the relationship between straightener use and uterine cancer,” said White. €œMore research is needed to confirm these findings in different populations, to determine if hair products contribute to health disparities in uterine cancer, and to identify the specific chemicals that may be increasing the risk of cancers in women.”This team previously found that permanent hair dye and straighteners may increase breast and ovarian cancer risk.Grant Numbers.

Z01-ES044005, Z1AES103332-01References levitra reviews. Che-Jung Chang, Katie M. O’Brien, Alexander levitra reviews P.

Keil, Symielle A. Gaston, Chandra levitra reviews L. Jackson, Dale P.

Sandler, Alexandra J levitra reviews. White. Use of Straighteners and Other Hair Products and Incident Uterine levitra reviews Cancer.

Journal of the National Cancer Institute [Full Text Che-Jung Chang, Katie M. O’Brien, Alexander P levitra reviews. Keil, Symielle A.

Gaston, Chandra levitra reviews L. Jackson, Dale P. Sandler, Alexandra levitra reviews J.

White. Use of Straighteners and Other Hair Products and levitra reviews Incident Uterine Cancer. Journal of the National Cancer Institute].

NIH study finds Black levitra buy australia women may be more affected due to higher use Women who used chemical hair straightening products were at higher risk for uterine cancer compared to women who did not report using these products, according to a http://www.teawamaori.com/can-i-buy-levitra-at-walmart/ new study from the National Institutes of Health. The researchers found no associations with uterine cancer for other hair products that the women reported using, including hair dyes, bleach, highlights, or perms.The study data includes 33,497 U.S. Women ages 35-74 participating in the Sister Study, a study led by the National Institute of Environmental Health Sciences (NIEHS), part of NIH, that seeks levitra buy australia to identify risk factors for breast cancer and other health conditions. The women were followed for almost 11 years and during that time 378 uterine cancer cases were diagnosed.The researchers found that women who reported frequent use of hair straightening products, defined as more than four times in the previous year, were more than twice as likely to go on to develop uterine cancer compared to those who did not use the products.“We estimated that 1.64% of women who never used hair straighteners would go on to develop uterine cancer by the age of 70.

But for frequent users, that risk goes up to 4.05%,” said Alexandra White, Ph.D., head of the NIEHS Environment and Cancer Epidemiology levitra buy australia group and lead author on the new study. €œThis doubling rate is concerning. However, it is important to put this information into context - uterine cancer is a relatively rare type of cancer.”Uterine cancer accounts for about 3% of all new cancer cases but is the most common cancer of levitra buy australia the female reproductive system, with 65,950 estimated new cases in 2022. Studies show that incidence rates of uterine cancer have been rising in the United States, particularly among Black women.Approximately 60% of the participants who reported using straighteners in the previous year were self-identified Black women, according to the study published in the Journal of the National Cancer Institute.

Although, the study did not find that the relationship between straightener use and uterine cancer incidence was different by race, the adverse health effects may be greater for Black women due to higher prevalence of use.“Because Black women use hair straightening or relaxer products more frequently and levitra buy australia tend to initiate use at earlier ages than other races and ethnicities, these findings may be even more relevant for them,” said Che-Jung Chang, Ph.D., an author on the new study and a research fellow in the NIEHS Epidemiology Branch.The findings are consistent with prior studies showing straighteners can increase the risk of hormone-related cancers in women.The researchers did not collect information on brands or ingredients in the hair products the women used. However, in the paper they note that several chemicals that have been found in straighteners (such as parabens, bisphenol A, metals, and formaldehyde) could be contributing to the increased uterine cancer risk observed. Chemical exposure from hair product use, levitra buy australia especially straighteners, could be more concerning than other personal care products due to increased absorption through the scalp which may be exacerbated by burns and lesions caused by straighteners.“To our knowledge this is the first epidemiologic study that examined the relationship between straightener use and uterine cancer,” said White. €œMore research is needed to confirm these findings in different populations, to determine if hair products contribute to health disparities in uterine cancer, and to identify the specific chemicals that may be increasing the risk of cancers in women.”This team previously found that permanent hair dye and straighteners may increase breast and ovarian cancer risk.Grant Numbers.

Z01-ES044005, Z1AES103332-01References levitra buy australia. Che-Jung Chang, Katie M. O’Brien, Alexander levitra buy australia P. Keil, Symielle A.

Gaston, Chandra levitra buy australia L. Jackson, Dale P. Sandler, Alexandra levitra buy australia J. White.

Use of Straighteners and Other Hair Products and levitra buy australia Incident Uterine Cancer. Journal of the National Cancer Institute [Full Text Che-Jung Chang, Katie M. O’Brien, Alexander levitra buy australia P. Keil, Symielle A.

Gaston, Chandra L levitra buy australia. Jackson, Dale P. Sandler, Alexandra levitra buy australia J. White.

Use of Straighteners and levitra buy australia Other Hair Products and Incident Uterine Cancer. Journal of the National Cancer Institute].

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When the history of the erectile dysfunction treatment levitra is written, readers Our site will be as stunned by the flagrant collective flaw in not getting treatments to all those who needed them as they will by the remarkable successes in treatment and drug development.A huge failure has been the global health care community’s inability to meet its moral obligation to older adults, people living with chronic conditions, and others in low- and middle-income countries who are most susceptible to developing severe cases of erectile dysfunction treatment or dying levitra drug from it. The inequitable distribution of erectile dysfunction treatments is not the fault of any single entity. The global health care community as a whole — governments, international organizations, nongovernmental organizations, the biopharmaceutical industry, and others — has failed on its promise to leave no one behind.What’s more, we are not out of the woods for this levitra, as the disease continues to surge in regions around the world.advertisement levitra drug Today, 2 billion treatments are stuck in refrigerators around the world, while in some countries older adults and others vulnerable to serious erectile dysfunction treatment have still not received their full course of treatments. By December 2021, the global production of erectile dysfunction treatment had reached nearly 11 billion doses, but treatment manufacturing capacities are now being scaled back. The Serum Institute of India is halting the production of the AstraZeneca treatment, and Aspen Pharmacare in South Africa fears it will have to permanently shut levitra drug its two erectile dysfunction treatment production lines unless a new order comes in shortly.

Aspen executive Stavros Nicolaou told the media. €œIf Aspen can’t produce erectile dysfunction treatments, what hope is there for others.” Getting erectile dysfunction treatments to everyone who needs them remains levitra drug a huge challenge. treatments sit in storage while vaccination efforts fail because many health care systems are insufficiently funded and lack trained staff in both urban and rural settings. There is a lack of coordination and political commitment. The complexity levitra drug of handling treatments.

And treatment hesitancy.advertisement Because lack of demand, not lack of supplies, is at the root of the current challenges, it is surprising that talks are still moving forward on a proposal initially submitted by India and South Africa to the World Trade Organization in October 2020 that would waive intellectual property (IP) rights on erectile dysfunction treatments to help increase treatment production. Talks on waiving the Trade-Related Aspects of Intellectual Property Rights (TRIPS) are all the more puzzling because according to health intelligence analyst levitra drug Airfinity, there are more than 380 partnerships for erectile dysfunction treatments and 150 for erectile dysfunction treatment therapeutics. India and South Africa are two countries that have benefited from technology transfer to the extent that, in 2021 India was the third-largest producer of erectile dysfunction treatments, ahead of the United States.The premise of an intellectual property waiver for erectile dysfunction treatments was flawed from the outset. Such a waiver is not sufficient to enable levitra drug a manufacturer to consistently produce high-quality treatments. treatment manufacturers in low-income countries are the first to acknowledge that voluntary technology partnerships need to be just that — voluntary — and done by sitting down together to find solutions to production, workforce, supplies, quality, and regulatory approvals.It is difficult to understand the rationale behind the United States lending its support to the proposed waiver in May 2021, the early days of the Biden administration.

At the levitra drug time, the U.S. Had on its books enough treatments to vaccinate its population six times over, the manufacturing scale up was well underway globally and, if shared equitably, there would have been enough treatments to vaccinate the world. The TRIPS waiver proposal makes even less sense when no one can point to intellectual property being a barrier to scaling up global manufacturing. It is levitra drug also not clear how the U.S.’s interests would be served by undermining IP, since more than 40% of U.S.’s gross domestic product comes from IP-intensive industries. It sends a chilling message to one-third of the total workforce that work in these industries.

Related levitra drug. A compromise on patent waivers for erectile dysfunction treatments takes a key step at the WTO The single biggest factor affecting treatment scarcity last year was not IP but trade. The U.S. Defense Protection Act slowed down the manufacturing scale-up by hampering the treatment supply chain, and India’s export ban of COVAX treatment supplies severely affected equitable distribution of treatments to most countries in Africa for much of 2021. People in Africa were let down badly by not receiving the COVAX orders secured from the Serum Institute.

The shortfall was made up in a matter of months by other treatment manufacturers.And looking ahead, the biopharmaceutical industry (Johnson &. Johnson, Moderna, Pfizer , and BioNTech) is supporting initiatives to help build up treatment manufacturing capacity on the African continent. Moreover, the infrastructure necessary for local production by any country will itself be fostered by strong IP protection that rewards successful innovation.Ahead of the World Trade Organization’s Ministerial Conference, which starts on Sunday, negotiators are poring over the wording of the latest TRIPS waiver document, which purports to simplify procedures and address challenges of scaling up production. But they should be mindful that rather than fixing a notional problem, the waiver proposal is more likely to undermine innovation by creating uncertainty and hampering future research and partnerships by changing the very framework that facilitated the unprecedented number of partnerships, voluntary licensing, and knowledge sharing that have emerged in the wake of erectile dysfunction treatment. The biggest loser will be global health security, the exact opposite of what needs to be done for future levitras.The TRIPS waiver proposals are a political distraction for industrial policy purposes by some countries, and political grandstanding by others.

The potential casualty in this fraught and disingenuous debate on the waiver is finding solutions to equitable access, without which global health security cannot be achieved. Whatever the next unknown disease will be, this levitra has demonstrated that science and scientific endeavor combined with collaboration and solidarity will be at the heart of how well, fast, and fairly the world can respond.It is only by being brutally honest about what worked and what did not that we stand a chance to respond better to a next levitra, even as we continue to fight this one.Thomas B. Cueni is director-general of the International Federation of Pharmaceutical Manufacturers and Associations. The opinions expressed here are his own..

When the history of the erectile dysfunction treatment levitra is written, readers will be as stunned by the flagrant collective flaw in not getting treatments to all those who needed them as they will by the remarkable successes in treatment and drug development.A huge failure has been the global health care community’s inability to meet its moral obligation to older adults, people living with chronic conditions, and others in low- and middle-income countries https://www.novainstitute.net.au/how-do-you-get-amoxil who levitra buy australia are most susceptible to developing severe cases of erectile dysfunction treatment or dying from it. The inequitable distribution of erectile dysfunction treatments is not the fault of any single entity. The global health care community as a whole — governments, international organizations, nongovernmental organizations, the biopharmaceutical industry, and others — has failed on its promise to leave no one behind.What’s more, we are not out of the woods for this levitra, as the levitra buy australia disease continues to surge in regions around the world.advertisement Today, 2 billion treatments are stuck in refrigerators around the world, while in some countries older adults and others vulnerable to serious erectile dysfunction treatment have still not received their full course of treatments.

By December 2021, the global production of erectile dysfunction treatment had reached nearly 11 billion doses, but treatment manufacturing capacities are now being scaled back. The Serum Institute of India is halting levitra buy australia the production of the AstraZeneca treatment, and Aspen Pharmacare in South Africa fears it will have to permanently shut its two erectile dysfunction treatment production lines unless a new order comes in shortly. Aspen executive Stavros Nicolaou told the media.

€œIf Aspen can’t produce erectile dysfunction treatments, what hope is there for others.” levitra buy australia Getting erectile dysfunction treatments to everyone who needs them remains a huge challenge. treatments sit in storage while vaccination efforts fail because many health care systems are insufficiently funded and lack trained staff in both urban and rural settings. There is a lack of coordination and political commitment.

The complexity levitra buy australia of handling treatments. And treatment hesitancy.advertisement Because lack of demand, not lack of supplies, is at the root of the current challenges, it is surprising that talks are still moving forward on a proposal initially submitted by India and South Africa to the World Trade Organization in October 2020 that would waive intellectual property (IP) rights on erectile dysfunction treatments to help increase treatment production. Talks on waiving levitra buy australia the Trade-Related Aspects of Intellectual Property Rights (TRIPS) are all the more puzzling because according to health intelligence analyst Airfinity, there are more than 380 partnerships for erectile dysfunction treatments and 150 for erectile dysfunction treatment therapeutics.

India and South Africa are two countries that have benefited from technology transfer to the extent that, in 2021 India was the third-largest producer of erectile dysfunction treatments, ahead of the United States.The premise of an intellectual property waiver for erectile dysfunction treatments was flawed from the outset. Such a waiver is not sufficient levitra buy australia to enable a manufacturer to consistently produce high-quality treatments. treatment manufacturers in low-income countries are the first to acknowledge that voluntary technology partnerships need to be just that — voluntary — and done by sitting down together to find solutions to production, workforce, supplies, quality, and regulatory approvals.It is difficult to understand the rationale behind the United States lending its support to the proposed waiver in May 2021, the early days of the Biden administration.

At the levitra buy australia time, the U.S. Had on its books enough treatments to vaccinate its population six times over, the manufacturing scale up was well underway globally and, if shared equitably, there would have been enough treatments to vaccinate the world. The TRIPS waiver proposal makes even less sense when no one can point to intellectual property being a barrier to scaling up global manufacturing.

It is also not clear how the U.S.’s interests would be served by undermining IP, since more than 40% of U.S.’s gross domestic product comes from levitra buy australia IP-intensive industries. It sends a chilling message to one-third of the total workforce that work in these industries. Related levitra buy australia.

A compromise on patent waivers for erectile dysfunction treatments takes a key step at the WTO The single biggest factor affecting treatment scarcity last year was not IP but trade. The U.S levitra buy australia. Defense Protection Act slowed down the manufacturing scale-up by hampering the treatment supply chain, and India’s export ban of COVAX treatment supplies severely affected equitable distribution of treatments to most countries in Africa for much of 2021.

People in Africa were let down badly by not receiving levitra buy australia the COVAX orders secured from the Serum Institute. The shortfall was made up in a matter of months by other treatment manufacturers.And looking ahead, the biopharmaceutical industry (Johnson &. Johnson, Moderna, Pfizer , and BioNTech) is supporting initiatives to help build up treatment manufacturing capacity on the African continent.

Moreover, the infrastructure necessary for local production by any country will itself be fostered by strong IP protection that rewards successful innovation.Ahead of the World Trade Organization’s levitra buy australia Ministerial Conference, which starts on Sunday, negotiators are poring over the wording of the latest TRIPS waiver document, which purports to simplify procedures and address challenges of scaling up production. But they should be mindful that rather than fixing a notional problem, the waiver proposal is more likely to undermine innovation by creating uncertainty and hampering future research and partnerships by changing the very framework that facilitated the unprecedented number of partnerships, voluntary licensing, and knowledge sharing that have emerged in the wake of erectile dysfunction treatment. The biggest loser will be global health security, the exact opposite of what needs to be done for future levitras.The TRIPS waiver proposals are a political distraction for industrial policy purposes by some countries, and levitra buy australia political grandstanding by others.

The potential casualty in this fraught and disingenuous debate on the waiver is finding solutions to equitable access, without which global health security cannot be achieved. Whatever the next unknown disease will be, this levitra has demonstrated that science and scientific endeavor combined with collaboration and solidarity will be at the heart of how well, fast, and fairly the world can respond.It is only by being brutally honest about what worked and what did not that we stand a chance to respond better to a next levitra, even as we continue to fight this one.Thomas B. Cueni is director-general of the International Federation of Pharmaceutical Manufacturers and Associations.

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