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Interested persons are invited to send comments regarding the burden estimate https://www.msamentoring.com/propecia-pills-for-sale/ or any other aspect of this collection of information, including the necessity and utility of the proposed information collection for the proper performance of the agency's functions, the accuracy of the estimated burden, ways to enhance the quality, utility, and clarity of the information to be collected, and the use buy propecia online no prescription of automated collection techniques or other forms of information technology to minimize the information collection burden. Comments on the collection(s) of information must be received by the OMB desk officer by June 21, 2022. Written comments and recommendations for the proposed information collection should be sent within 30 days of publication of this notice to www.reginfo.gov/​public/​do/​PRAMain. Find this particular information collection by selecting “Currently under 30-day Review—Open for Public Comments” or by using buy propecia online no prescription the search function. To obtain copies of a supporting statement and any related forms for the proposed collection(s) summarized in this notice, you may make your request using one of following.

1. Access CMS' buy propecia online no prescription website address at website address at. Https://www.cms.gov/​Regulations-and-Guidance/​Legislation/​PaperworkReductionActof1995/​PRA-Listing. Start Further Info William Parham at (410) 786-4669. End Further buy propecia online no prescription Info End Preamble Start Supplemental Information Under the Paperwork Reduction Act of 1995 (PRA) (44 U.S.C.

3501-3520), federal agencies must obtain approval from the Office of Management and Budget (OMB) for each collection of information they conduct or sponsor. The term “collection of information” is defined in 44 U.S.C. 3502(3) and 5 CFR 1320.3(c) and includes agency requests or requirements that members of the public submit reports, keep records, or provide information to buy propecia online no prescription a third party. Section 3506(c)(2)(A) of the PRA (44 U.S.C. 3506(c)(2)(A)) requires federal agencies to publish a 30-day notice in the Federal Register concerning each proposed collection of information, including each proposed extension or reinstatement of an existing collection of information, before submitting the collection to OMB for approval.

To comply with this requirement, CMS is buy propecia online no prescription publishing this notice that summarizes the following proposed collection(s) of information for public comment. 1. Type of Information Collection Request. Revision of a currently buy propecia online no prescription approved collection. Title of Information Collection.

Outcome and Assessment Information Set OASIS-E. Use. This request is for OMB approval to modify the Outcome and Assessment Information Set (OASIS) that home health agencies (HHAs) are required to collect in order to participate in the Medicare program. The current version of the OASIS, OASIS-D (0938-1279) data item set was approved by the Office of Management and Budget (OMB) on December 6, 2018 and implemented on January 1, 2019. We are seeking OMB approval for the proposed revised OASIS item set, referred to hereafter as OASIS-E, scheduled for implementation on January 1, 2023.

The OASIS-E includes changes pursuant to the Improving Medicare Post-Acute Care Transformation Act of 2014 (the IMPACT Act). And, to accommodate data element removals to reduce burden. And improve formatting throughout the document. Subsequent to publishing the 60-day Federal Register notice (87 FR 7457), we removed the GG activity items from the Follow-Up time point which resulted in a decrease in the burden hours. Form Number.

CMS-10545 (OMB control number. 0938-1279). Frequency. Occasionally. Affected Public.

Private Sector (Business or other for-profit and Not-for-profit institutions). Number of Respondents. 11,354. Total Annual Responses. 18,030,766.

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First elected propecia pill price in 2017, his re-election by secret ballot, was confirmed during the 75th World Health Assembly in Geneva. He was the sole candidate.The vote was the culmination of an election process that began in April 2021 when Member States were invited to submit proposals for candidates for the post of propecia pill price Director-General. The WHO Executive Board, meeting in January of this year, nominated Dr Tedros to stand for a second term.His re-election was met with wide and loud applause from ministers propecia pill price and others at the Assembly in Geneva.

According to news reports he received 155 out of 160 votes cast, although he did not win the support of his native Ethiopia, due to opposing views over the Tigray conflict.The WHO chief’s new mandate officially commences on 16 August. A Director-General can be re-appointed once, in accordance with World Health Assembly rules and procedures.‘Humbled and honoured’In a tweet following the vote, Tedros said that he was “humbled and honoured” by the vote of confidence, adding that he was “deeply grateful for the trust and confidence of Member States.”“I thank all health workers and my WHO colleagues around the world”, he continued saying propecia pill price he was looking forward to “continuing our journey together.”In remarks after the vote, he said his re-election was a vote of confidence in the whole WHO adding. €œthis is for the whole team.”He acknowledged the pressure and attacks from “many quarters” during the propecia, saying that despite the insults and attacks, he and the organization always kept an open mind and did not take it personally.“We have to focus on promoting health…number two, we have to propecia pill price focus on primary healthcare” and thirdly, he cited the importance of emergency preparedness and response, being dependent on the first two priorities.TransformationDuring his first term, Tedros instituted a wide-ranging transformation of the WHO, the agency said in a press release, “aimed at increasing the Organization’s efficiency driving impact at country level to promote healthier lives, protect more people in emergencies and increase equitable access to health.”Tedros guided WHO’s response to the unprecedented hair loss treatment propecia, where he sometimes faced criticism, most notably, from former United States President, Donald Trump, who took the decision to withdraw the US from the WHO – a move since reversed.The WHO chief also steered the response to outbreaks of Ebola in the Democratic Republic of the Congo (DRC) and led the agency dealing with the health impacts of multiple other humanitarian crises, most recently the war in Ukraine.Ministerial careerBefore first being appointed WHO Director-General, Dr Tedros served as Minister of Foreign Affairs for Ethiopia between 2012 and 2016 and as Minister of Health prior to that, from 2005.He had also served as chair of the Board of the Global Fund to Fight AIDS, Tuberculosis and Malaria.

As chair propecia pill price of the Roll Back Malaria (RBM) Partnership Board. And as co-chair of the Board of the Partnership for Maternal, Newborn and Child Health.“Not only are the majority of rich countries failing to provide healthy environments for children within their borders, they are also contributing to the destruction of children’s environments in other parts of the world,” said Gunilla Olsson, Director of the UNICEF Office of Research – Innocenti.Unhealthy. Dangerous.

Toxic.This is the world the majority of wealthy countries are creating for children.Our latest report. #PlacesAndSpaces— UNICEF (@UNICEF) May 24, 2022 Urgent policy shiftThe latest Innocenti Report Card 17. Places and Spaces compares how 39 countries in the Organization for Economic Co-operation and Development (OECD) and European Union (EU) impact children’s environments.Indicators include exposure to harmful pollutants, such as toxic air, pesticides, damp and lead.

Access to light, green spaces and safe roads. And countries’ contributions to the climate crisis, resource consumption, and e-waste dumping.The report states that if the entire world consumed resources at the rate of OECD and EU countries, the equivalent of 3.3 earths would be needed to keep up with consumption levels.If it were at the rate at which people in Canada, Luxembourg and the United States do, at least five earths would be needed, according to the report.Not in your own backyardWhile Spain, Ireland and Portugal feature at the overall top of the list, all OECD and EU countries are failing to provide healthy environments for all children across all indicators.Based on CO2 emissions, e-waste and overall resource consumption per capita, Australia, Belgium, Canada and the United States are among other wealthy countries that rank low on creating a healthy environment for children within and beyond their borders. Meanwhile, Finland, Iceland and Norway are among those that provide healthier environments for their country’s children but disproportionately contribute to destroying the global environment.“In some cases we are seeing countries providing relatively healthy environments for children at home while being among the top contributors to pollutants that are destroying children’s environments abroad,” attested Gunilla Olsson, Director of UNICEF Office of ResearchIn contrast, the least wealthy OECD and EU countries in Latin America and Europe, have a much lower impact on the wider world.Harmful exposuresOver 20 million children in this group, have elevated levels of lead – one of the most dangerous environmental toxic substances – in their blood.In Iceland, Latvia, Portugal and the United Kingdom, one in five children is exposed to damp and mould at home.

While in Cyprus, Hungary and Turkey, that number rises to more than one in four.Many children are breathing toxic air both in and outside of their homes.More than one in 12 children in Belgium, Czech Republic, Israel and Poland and are exposed to high pesticide pollution, which has been linked with cancer – including childhood leukaemia – and can harm vital body systems. Source. WHORoutes of children’s exposure to toxic substances..

First elected in 2017, his go to website re-election by secret ballot, was confirmed buy propecia online no prescription during the 75th World Health Assembly in Geneva. He was buy propecia online no prescription the sole candidate.The vote was the culmination of an election process that began in April 2021 when Member States were invited to submit proposals for candidates for the post of Director-General. The WHO Executive Board, meeting in January buy propecia online no prescription of this year, nominated Dr Tedros to stand for a second term.His re-election was met with wide and loud applause from ministers and others at the Assembly in Geneva. According to news reports he received 155 out of 160 votes cast, although he did not win the support of his native Ethiopia, due to opposing views over the Tigray conflict.The WHO chief’s new mandate officially commences on 16 August. A Director-General can be re-appointed once, in accordance with World Health Assembly rules and procedures.‘Humbled and honoured’In a tweet following the vote, Tedros said that he was “humbled and honoured” by the vote of confidence, adding that he was “deeply grateful for the trust and confidence of Member States.”“I thank all health buy propecia online no prescription workers and my WHO colleagues around the world”, he continued saying he was looking forward to “continuing our journey together.”In remarks after the vote, he said his re-election was a vote of confidence in the whole WHO adding.

€œthis is for the whole team.”He acknowledged the pressure and attacks from “many quarters” during the propecia, saying that despite the insults and attacks, he and the organization always kept an open mind and did not take it personally.“We have to focus on promoting health…number two, we have to focus on primary healthcare” and thirdly, he cited the importance of emergency preparedness and response, being dependent on the first two priorities.TransformationDuring his first term, Tedros instituted a wide-ranging transformation of the WHO, the agency said in a press release, “aimed at increasing the Organization’s efficiency driving impact at country level to promote healthier lives, protect more people in emergencies and increase equitable access to buy propecia online no prescription health.”Tedros guided WHO’s response to the unprecedented hair loss treatment propecia, where he sometimes faced criticism, most notably, from former United States President, Donald Trump, who took the decision to withdraw the US from the WHO – a move since reversed.The WHO chief also steered the response to outbreaks of Ebola in the Democratic Republic of the Congo (DRC) and led the agency dealing with the health impacts of multiple other humanitarian crises, most recently the war in Ukraine.Ministerial careerBefore first being appointed WHO Director-General, Dr Tedros served as Minister of Foreign Affairs for Ethiopia between 2012 and 2016 and as Minister of Health prior to that, from 2005.He had also served as chair of the Board of the Global Fund to Fight AIDS, Tuberculosis and Malaria. As chair buy propecia online no prescription of the Roll Back Malaria (RBM) Partnership Board. And as co-chair of the Board of the Partnership for Maternal, Newborn and Child Health.“Not only are the majority of rich countries failing to provide healthy environments for children within their borders, they are also contributing to the destruction of children’s environments in other parts of the world,” said Gunilla Olsson, Director of the UNICEF Office of Research – Innocenti.Unhealthy. Dangerous. Toxic.This is the world the majority of wealthy countries are creating for children.Our latest report.

#PlacesAndSpaces— UNICEF (@UNICEF) May 24, 2022 Urgent policy shiftThe latest Innocenti Report Card 17. Places and Spaces compares how 39 countries in the Organization for Economic Co-operation and Development (OECD) and European Union (EU) impact children’s environments.Indicators include exposure to harmful pollutants, such as toxic air, pesticides, damp and lead. Access to light, green spaces and safe roads. And countries’ contributions to the climate crisis, resource consumption, and e-waste dumping.The report states that if the entire world consumed resources at the rate of OECD and EU countries, the equivalent of 3.3 earths would be needed to keep up with consumption levels.If it were at the rate at which people in Canada, Luxembourg and the United States do, at least five earths would be needed, according to the report.Not in your own backyardWhile Spain, Ireland and Portugal feature at the overall top of the list, all OECD and EU countries are failing to provide healthy environments for all children across all indicators.Based on CO2 emissions, e-waste and overall resource consumption per capita, Australia, Belgium, Canada and the United States are among other wealthy countries that rank low on creating a healthy environment for children within and beyond their borders. Meanwhile, Finland, Iceland and Norway are among those that provide healthier environments for their country’s children but disproportionately contribute to destroying the global environment.“In some cases we are seeing countries providing relatively healthy environments for children at home while being among the top contributors to pollutants that are destroying children’s environments abroad,” attested Gunilla Olsson, Director of UNICEF Office of ResearchIn contrast, the least wealthy OECD and EU countries in Latin America and Europe, have a much lower impact on the wider world.Harmful exposuresOver 20 million children in this group, have elevated levels of lead – one of the most dangerous environmental toxic substances – in their blood.In Iceland, Latvia, Portugal and the United Kingdom, one in five children is exposed to damp and mould at home.

While in Cyprus, Hungary and Turkey, that number rises to more than one in four.Many children are breathing toxic air both in and outside of their homes.More than one in 12 children in Belgium, Czech Republic, Israel and Poland and are exposed to high pesticide pollution, which has been linked with cancer – including childhood leukaemia – and can harm vital body systems. Source. WHORoutes of children’s exposure to toxic substances..

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IntroductionSOX10 belongs to the SOX family of transcription factors, of which the members are defined propecia prostate problems based on the presence of a 79 amino acid DNA-binding domain with homology 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 propecia prostate problems 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 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) propecia prostate problems 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 a complete description of the human gene 5’ non-coding exon(s) has given rise to two propecia prostate problems coexisting exon numbering systems. Historically, exons 1 and 2 are non-coding, the initiation codon is found in exon 3, and the stop codon in exon 5.8 The second system is based on the reference transcript NM_006941, with only one non-coding exon in the 5’UTR (untranslated transcribed region) and a total of four exons.

A major transcript of ~3 kb is detected in most tissues tested, consistent with the predicted SOX10 mRNA sequence.9 10The protein’s structure is propecia prostate problems 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 propecia prostate problems 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-143585296" 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)..

IntroductionSOX10 belongs to the SOX family of transcription factors, of which the members are defined based on the presence of a buy propecia online no prescription 79 amino acid DNA-binding domain with this hyperlink homology 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 buy propecia online no prescription 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) buy propecia online no prescription 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 a complete description of the human gene 5’ non-coding exon(s) buy propecia online no prescription has given rise to two coexisting exon numbering systems. Historically, exons 1 and 2 are non-coding, the initiation codon is found in exon 3, and the stop codon in exon 5.8 The second system is based on the reference transcript NM_006941, with only one non-coding exon in the 5’UTR (untranslated transcribed region) and a total of four exons. A major transcript of ~3 kb is detected in most tissues tested, consistent with the predicted SOX10 mRNA sequence.9 10The protein’s structure is buy propecia online no prescription 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 buy propecia online no prescription import (nuclear localisation signal) and one export (nuclear export signal) signals.12 1310 samples in various tissues (including the brain, gut, nerves (tibial), breast and salivary glands)/total number of A-to-I modifications reported is. AluY. 67/224. AluSx. 25/87.

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

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

TAM, transactivation domain in the middle of the protein." class="highwire-fragment fragment-images colorbox-load" rel="gallery-fragment-images-143585296" data-figure-caption="Schematic of the SOX10 protein and post-translational modifications. Domains of human SOX10. The numbers refer to amino acid residues. The pink lines above the HMG domain represent the NLS sequences, one at each end of the HMG domain, and the light pink line the NES sequence. Note that although nucleocytoplasmic shuttling of the protein has been well documented for several SOX factors,12 13 in vivo regulation of SOX10 through nuclear translocation is yet to be clarified.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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NIH study finds Black women may be more affected due to higher use Women who propecia for sale canada 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 new study generic of propecia 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 generic of propecia of NIH, that seeks 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 generic of propecia users, that risk goes 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 to put this information generic of propecia 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 generic of propecia 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 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 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 generic of propecia 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 generic of propecia. Che-Jung Chang, Katie M. O’Brien, Alexander generic of propecia P.

Keil, Symielle A http://bookcollaborative.com/artists/jo-frederiks/. Gaston, Chandra generic of propecia L. Jackson, Dale P.

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

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

Gaston, Chandra generic of propecia L. Jackson, Dale P. Sandler, Alexandra generic of propecia J.

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

NIH study finds Black women may propecia usa buy be more affected due to higher use Women who used chemical hair buy propecia online no prescription straightening products were at higher risk for uterine cancer compared to women who did not report using these products, according to a 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 buy propecia online no prescription (NIEHS), part of NIH, that seeks 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 buy propecia online no prescription Environment and Cancer Epidemiology 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 the female buy propecia online no prescription 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 Branch.The findings are consistent with prior studies showing straighteners can increase buy propecia online no prescription 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 straighteners, could be more concerning than other personal care products due to increased absorption through the scalp buy propecia online no prescription 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 buy propecia online no prescription. Che-Jung Chang, Katie M. O’Brien, Alexander buy propecia online no prescription P.

Keil, Symielle A. Gaston, Chandra buy propecia online no prescription L. Jackson, Dale P. Sandler, Alexandra buy propecia online no prescription J. White.

Use of Straighteners and Other Hair Products and Incident Uterine Cancer buy propecia online no prescription. Journal of the National Cancer Institute [Full Text Che-Jung Chang, Katie M. O’Brien, Alexander buy propecia online no prescription P. Keil, Symielle A. Gaston, Chandra buy propecia online no prescription L.

Jackson, Dale P. Sandler, Alexandra J buy propecia online no prescription. White. Use of Straighteners and Other Hair Products and Incident Uterine Cancer. Journal of the National Cancer Institute].

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How to propecia results 1 year find out cite this article:Singh OP. Comprehensive Mental Health Action Plan 2013–2030. We must rise to the challenge propecia results 1 year.

Indian J Psychiatry 2021;63:415-7In May 2013, WHO's Mental Health Action Plan 2013-2020 was adopted at the 66th World Health Assembly which was extended until 2030 by the 72nd World Health Assembly in May 2019 with modifications of some of the objectives and goal targets to ensure its alignment with the 2030 Agenda for Sustainable Development. Further, in September 2021, the 74th World Health Assembly accepted the updates to the action plan, including updates to the target options for indicators and implementation. This is an opportunity for the psychiatric propecia results 1 year community to rise to the challenge and work towards the realization of these objectives and in turn to integrate psychiatry with the mainstream of medicine.The change in objectives and targets is summarized in [Table 1].Table 1.

Comparison between Mental Health Action Plans 2013-20 and 2013-30Click here to viewAs it is obvious that there is an enormous opportunity for the psychiatric community to implement things that we always have been talking about like:Global target 2.2 – Target's doubling of community-based mental health facilities by 2030 in 80% of countries. It would be a substantial achievement for the psychiatric community for its implementation will lead to significant service to psychiatric patientsGlobal target 2.3 – Integration of mental health care into primary healthcareGlobal target 3.2 – Reduction in suicide rate by one-third by 2030Global target 3.3 – Psychological care for disasterGlobal target 4.2 – Mental health research to be doubled by 2030.What has brought about profound change is target 3.4 of Sustainable Development Goal, which is to reduce premature death by NCD by one-third by promoting mental health and wellbeing. It is propecia results 1 year an opportunity for us to expand psychiatry by being involved in general medical care and reduce stigma.

We must also utilize this opportunity to press for the greater representation of psychiatry in MBBS curriculum throughout the country and stop not till it gets a separate subject status in undergraduate medical studies.Now is the time for us to strive to achieve all the objectives which provide an opportunity to expand mental health care, reduce stigma, and translate all the talk of furthering the growth of mental health into action.[2] References 1.World Health Organization. Mental Health propecia results 1 year Action Plan 2013-2020. Geneva.

World Health Organization. 2013. 2.World Health Organization.

Comprehensive Mental Health Action Plan 2013-2030. Geneva. World Health Organization.

2021. Correspondence Address:Om Prakash SinghDepartment of Psychiatry, WBMES, Kolkata, West Bengal. AMRI Hospitals, Kolkata, West Bengal IndiaSource of Support.

None, Conflict of Interest. NoneDOI. 10.4103/indianjpsychiatry.indianjpsychiatry_811_21 Tables [Table 1].

How to buy propecia online no prescription this page cite this article:Singh OP. Comprehensive Mental Health Action Plan 2013–2030. We must buy propecia online no prescription rise to the challenge. Indian J Psychiatry 2021;63:415-7In May 2013, WHO's Mental Health Action Plan 2013-2020 was adopted at the 66th World Health Assembly which was extended until 2030 by the 72nd World Health Assembly in May 2019 with modifications of some of the objectives and goal targets to ensure its alignment with the 2030 Agenda for Sustainable Development. Further, in September 2021, the 74th World Health Assembly accepted the updates to the action plan, including updates to the target options for indicators and implementation.

This is an opportunity for the psychiatric buy propecia online no prescription community to rise to the challenge and work towards the realization of these objectives and in turn to integrate psychiatry with the mainstream of medicine.The change in objectives and targets is summarized in [Table 1].Table 1. Comparison between Mental Health Action Plans 2013-20 and 2013-30Click here to viewAs it is obvious that there is an enormous opportunity for the psychiatric community to implement things that we always have been talking about like:Global target 2.2 – Target's doubling of community-based mental health facilities by 2030 in 80% of countries. It would be a substantial achievement for the psychiatric community for its implementation will lead to significant service to psychiatric patientsGlobal target 2.3 – Integration of mental health care into primary healthcareGlobal target 3.2 – Reduction in suicide rate by one-third by 2030Global target 3.3 – Psychological care for disasterGlobal target 4.2 – Mental health research to be doubled by 2030.What has brought about profound change is target 3.4 of Sustainable Development Goal, which is to reduce premature death by NCD by one-third by promoting mental health and wellbeing. It is buy propecia online no prescription an opportunity for us to expand psychiatry by being involved in general medical care and reduce stigma. We must also utilize this opportunity to press for the greater representation of psychiatry in MBBS curriculum throughout the country and stop not till it gets a separate subject status in undergraduate medical studies.Now is the time for us to strive to achieve all the objectives which provide an opportunity to expand mental health care, reduce stigma, and translate all the talk of furthering the growth of mental health into action.[2] References 1.World Health Organization.

Mental Health Action Plan buy propecia online no prescription 2013-2020. Geneva. World Health Organization. 2013. 2.World Health Organization.

Comprehensive Mental Health Action Plan 2013-2030. Geneva. World Health Organization. 2021. Correspondence Address:Om Prakash SinghDepartment of Psychiatry, WBMES, Kolkata, West Bengal.

AMRI Hospitals, Kolkata, West Bengal IndiaSource of Support. None, Conflict of Interest. NoneDOI. 10.4103/indianjpsychiatry.indianjpsychiatry_811_21 Tables [Table 1].