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THIS ARTICLE WAS WITHDRAWN BY THE PUBLISHER IN October 2021

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor., Nature Communications, 12 (1), ISSN:2041-1723

Embryonic muscle forces are necessary for normal vertebral development and spinal curvature, but their involvement in intervertebral disc (IVD) development remains unclear. The aim of the current study was to determine how muscle contractions affect (1) notochord involution and vertebral segmentation, and (2) IVD development including the mechanical properties and morphology, as well as collagen fibre alignment in the annulus fibrosus. Muscular dysgenesis (mdg) mice were harvested at three prenatal stages: at Theiler Stage (TS)22 when notochord involution starts, at TS24 when involution is complete, and at TS27 when the IVD is formed. Vertebral and IVD development were characterised using histology, immunofluorescence, and indentation testing. The results revealed that notochord involution and vertebral segmentation occurred independently of muscle contractions between TS22 and TS24. However, in the absence of muscle contractions, we found vertebral fusion in the cervical region at TS27, along with (i) a displacement of the nucleus pulposus towards the dorsal side, (ii) a disruption of the structural arrangement of collagen in the annulus fibrosus, and (iii) an increase in viscous behaviour of the annulus fibrosus. These findings emphasise the important role of mechanical forces during IVD development, and demonstrate a critical role of muscle loading during development to enable proper annulus fibrosus formation. They further suggest a need for mechanical loading in the creation of fibre-reinforced tissue engineering replacement IVDs as a therapy for IVD degeneration.

The failure of once promising target-specific therapeutic strategies often arises from redundancies in gene expression pathways. Even with new melanoma treatments, many patients are not responsive or develop resistance, leading to disease progression in terms of growth and metastasis. We previously discovered that the transcription factors ETS1 and PAX3 drive melanoma growth and metastasis by promoting the expression of the MET receptor. Here, we find that there are multiple ETS family members expressed in melanoma and that these factors have redundant functions. The small molecule YK-4-279, initially developed to target the ETS gene–containing translocation product EWS-FLI1, significantly inhibited cellular growth, invasion, and ETS factor function in melanoma cell lines and a clinically relevant transgenic mouse model, BrafCA;Tyr-CreERT2;Ptenf/f. One of the antitumor effects of YK-4-279 in melanoma is achieved via interference of multiple ETS family members with PAX3 and the expression of the PAX3-ETS downstream gene MET. Expression of exogenous MET provided partial rescue of the effects of YK-4-279, further supporting that MET loss is a significant contributor to the antitumor effects of the drug. This is the first study identifying multiple overlapping functions of the ETS family promoting melanoma. In addition, targeting all factors, rather than individual members, demonstrated impactful deleterious consequences in melanoma progression. Given that multiple ETS factors are known to have oncogenic functions in other malignancies, these findings have a high therapeutic impact. Significance: These findings identify YK-4-279 as a promising therapeutic agent against melanoma by targeting multiple ETS family members and blocking their ability to act as transcription factors.

Objectives There is a clinical variability and heterogeneity among Facioscapulohumeral Muscular Dystrophy (FSHD) patients. Escalation after menopause in women, early onset in men suggests that estrogen might be a protective factor on the course of FSHD. In spite of few molecular studies supporting the protective role of estrogen in FSHD in vitro, there is no study revealing the effect of estradiol on the protein levels of DUX4, β-catenin and PAX3/PAX7. In present study, we investigated the effect of estradiol treatment on the expressions of DUX4, β-catenin and PAX3/PAX7 protein levels. Materials and Methods Primary myoblasts of 63 and 71 years old (63yM/71yM) males; 47 years old (47yF) female FSHD patients were used. Cells were processed under these conditions; (i) untreated, (ii) 10 nM-30 min estradiol and (iii) 10 nM-4 h estradiol treated. The expression of DUX4, PAX3/PAX7 and β-catenin were examined by western-blotting. Results Expression of DUX4 significantly downregulated after 4 h treatment of estradiol while PAX3/PAX7 56 kDa variant expression upregulated in 71yM cells. β-catenin and PAX3 expression was variable among the samples. Conclusion Our results suggest that estrogen might be a palliative treatment option via downregulation of DUX4 protein in DUX4 expressing FSHD patients.

Rhabdomyosarcomas (RMS) are group of soft tissue malignant tumours predominantly childhood. Alveolar rhabdomyosarcoma (aRMS) is the second most common variant of RMS. The majority of aRMSs display a translocations of FOXO1 gene. Such tumours are aggressive, metastasize early and are associated with a worse prognosis for the patient. However, some aRMS cases are rhabdomyosarcomas without classic chromosomal rearrangements. These tumors also have alveolar morphology, but neoplastic cells lack the FOXO1 gene translocation. Such fusion-negative round-cell RMSs behave clinically differently and have a better prognosis. The aim of the present study was to assess the prevalence of FOXO1 gene rearrangements in the group of round cell rhabdomyosarcomas with alveolar morphology. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The study group consisted of 250 formalin-fixed paraffin-embedded samples from patients with RMS. The cytogenetic study was performed by fluorescence in situ hybridization with a locus-specific identifier (LSI) for FOXO1 (13q14). The PAX3-FOXO1 (COSF247) and PAX7-FOXO1 (COSF287) fusion transcripts was detected by RT-PCR. In the study group 1 (аRMS), the rearrangement of PAX3/7-FOXO1 was detected in 44% of cases, in 32% of cases was detected a combined rearrangement with amplification of the 3' FOXO1. In one case, the amplification of the 3' end of the FOXO1 gene was detected without rearrangement; this sample was sent for additional PCR study, as a result of which the chimeric PAX3-FOXO1 transcript was detected. In 22% cases cytogenetic abnormalities were not found. has not been identified. In group 2 (embryonal RMS) we did not detect translocation. The group of round-cell PMCs is heterogeneous and is represented by three variants of genetic events that determine the disease prognosis. At the same time, FOXO1 gene abnormalities are not found in the RMS group with non-alveolar morphology.

Pax3 and Pax7 are closely related transcription factors that are widely expressed in the developing nervous system and somites. During the normal development in the central nervous system (CNS), Pax3 and Pax7 are mainly expressed in the dorsal part of the neural tube. Further analysis revealed that Pax3 and Pax7 shared redundant functions in the spinal cord development. However, it is still unknown whether Pax3 and Pax7 play a role in neuronal differentiation. In this study, Pax3 and Pax7 genes were overexpressed in Neuro‐2a, the mouse neuroblastoma cell line. CCK‐8 and EdU assay results showed that overexpression of Pax3 inhibited cell viability and proliferation of Neuro‐2a cells, whereas the overexpression of Pax7 had no significant difference on their cell viability and proliferation. Overexpression of Pax3 not only increased the percentage of cells in the S phase and G0/G1 phase, but also decreased that in the G2 phase. Moreover, the total neurite lengths of Neuro‐2a cells were significantly shorter in Pax3 overexpressed group than those in negative control group and showed no significant difference between Pax7 overexpressed group and negative control group. These results suggested that Pax3 not only inhibited the cell viability and proliferation but also affected the cell cycle and the neurite outgrowth of Neuro‐2a cells. RNA sequencing analysis showed up‐regulated genes in Pax3 overexpressed group were involved in cell cycle machinery, which may reveal the potential mechanism of Neuro‐2a cells proliferation.

Waardenburg syndrome subtypes 1 and 3 are caused by pathogenic variants in PAX3. We investigated 12 individuals from four unrelated families clinically diagnosed with Waardenburg syndrome type 1/3. Novel pathogenic variants identified in PAX3 included single nucleotide variants (c.166C>T, c.829C>T), a 2-base pair deletion (c.366_367delAA) and a multi-exonic deletion. Two novel variants, c.166C>T and c.829C>T and a previously reported variant, c.256A>T in PAX3 were evaluated for their nuclear localization and ability to activate MITF promoter. The coexistence of two subtypes of Waardenburg syndrome with pathogenic variants in PAX3 and EDNRB was seen in one of the affected individuals. Multiple genetic diagnoses of Waardenburg syndrome type 3 and autosomal recessive deafness 1A was identified in an individual. We also review the phenotypic and genomic spectrum of individuals with PAX3-related Waardenburg syndrome reported in the literature.

Waardenburg syndrome type 1 (WS1) is a rare genetic disorder characterized by dystopia canthorum, abnormal iris pigmentation, and congenital hearing loss with variable penetrance.WS1 is caused by mutations in paired box gene 3 (PAX3). The current study aimed to investigate the genetic cause of hearing loss in a four-generation Chinese WS1 family. The phenotype of the study family was characterized using clinical evaluation and pedigree analysis. Target region high-throughput sequencing system was designed to screen the all coding exons and flanking intronic sequences of the six WS-associated genes. Sanger sequencing was used to identify the causative nucleotide changes and perform the co-segregating analysis. The expression, subcellular distribution, and transcriptional activity of the mutant PAX3 protein were analysis to reveal the functional consequences of the mutation. Based on diagnostic criteria, the proband of this pedigree classified as WS1. We identified a novel missense mutation (c.117 C > A, p. Asn39Lys) in exon 2 of the PAX3 gene. In vitro, the Asn39Lys PAX3 retained nuclear distribution ability. However, it failed to activate the melanocyte inducing transcription factor (MITF) promoter and impaired the function of WT PAX3. Our study reports a novel missense PAX3 mutation in a Chinese family and shows haploinsufficiency may be the underlying mechanism for the WS1 phenotype.

Induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells and tissues including skeletal muscle. The approach to convert these stem cells into skeletal muscle cells offers hope for patients afflicted with skeletal muscle diseases such as Duchenne muscular dystrophy (DMD). Several methods have been reported to induce myogenic differentiation with iPSCs derived from myogenic patients. An important point for generating skeletal muscle cells from iPSCs is to understand in vivo myogenic induction in development and regeneration. Current protocols of myogenic induction utilize techniques with overexpression of myogenic transcription factors such as Myod1(MyoD), Pax3, Pax7, and others, using recombinant proteins or small molecules to induce mesodermal cells followed by myogenic progenitors, and adult muscle stem cells. This review summarizes the current approaches used for myogenic induction and highlights recent improvements.

Waardenburg syndrome (WS), also known as auditory-pigmentary syndrome, is the most common cause of syndromic hearing loss. It is responsible for 2–5% of congenital deafness. WS is classified into four types depending on the clinical phenotypes. Currently, pathogenic mutation of PAX3, MITF, EDNRB, EDN3, SNAI2, or SOX10 can cause corresponding types of WS. Among them, SOX10 mutation is responsible for approximately 15% of type II WS or 50% of type IV WS. We report the case of a proband in a Chinese family who was diagnosed with WS type II. Whole exome sequencing (WES) of the proband detected a novel heterozygous spontaneous mutation: SOX10 c.246delC. According to analysis based on nucleic acid and amino acid sequences, this mutation may produce a truncated protein, with loss of the HMG structure domain. Therefore, this truncated protein may fail to activate the expression of the MITF gene, which regulates melanocytic development and plays a key role in WS. Our finding expands the database of SOX10 mutations associated with WS and provides more information regarding the molecular mechanism of WS.

Fusion‐positive rhabdomyosarcoma (FP‐RMS) is an aggressive pediatric cancer. The chromatin factor KDM3A and the downstream transcription factor Ets1 comprise a new epigenetic axis that works together with, as well as independently of, the driver PAX3/FOXO1 oncofusion to upregulate disease‐promoting gene expression and phenotypic properties in FP‐RMS. These findings offer new insights into potential ways to target aggressive properties in this disease., Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and young adults. RMS exists as two major disease subtypes, oncofusion‐negative RMS (FN‐RMS) and oncofusion‐positive RMS (FP‐RMS). FP‐RMS is characterized by recurrent PAX3/7‐FOXO1 driver oncofusions and is a biologically and clinically aggressive disease. Recent studies have revealed FP‐RMS to have a strong epigenetic basis. Epigenetic mechanisms represent potential new therapeutic vulnerabilities in FP‐RMS, but their complex details remain to be defined. We previously identified a new disease‐promoting epigenetic axis in RMS, involving the chromatin factor KDM3A and the Ets1 transcription factor. In the present study, we define the KDM3A and Ets1 FP‐RMS transcriptomes and show that these interface with the recently characterized PAX3/FOXO1‐driven gene expression program. KDM3A and Ets1 positively control numerous known and candidate novel PAX3/FOXO1‐induced RMS‐promoting genes, including subsets under control of PAX3/FOXO1‐associated superenhancers (SE), such as MEST. Interestingly, KDM3A and Ets1 also positively control a number of known and candidate novel FP‐RMS‐promoting, but not PAX3/FOXO1‐dependent, genes. Epistatically, Ets1 is downstream of, and exerts disease‐promoting effects similar to, both KDM3A and PAX3/FOXO1. MEST also manifests disease‐promoting properties in FP‐RMS, and KDM3A and Ets1 each impacts activation of the PAX3/FOXO1‐associated MEST SE. Taken together, our studies show that the KDM3A/Ets1 epigenetic axis plays an important role in disease promotion in FP‐RMS, and provide insight into potential new ways to target aggressive phenotypes in this disease.

It is hard to supply satellite cells as a cell source for therapy of muscle degenerative disease since the sampling of muscle tissue is very invasive to a patient with muscular disease. Direct conversion allows us to get specific cell types by transduction of defined transcriptional factors. To induce myogenic direct conversion, we transduced five transcriptional factors including Pax3, Sox2, Klf4, c-Myc, and Esrrb into mouse embryonic fibroblasts. We found that the transduction of the five transcriptional factors induced myogenic direct conversion of fibroblast. We revealed that the transduced cells with the five transcriptional factors were converted to myogenic lineage cells through a paraxial mesoderm-like stage. The expression level of myogenic-related genes of the transduced cells gradually increased as the passage increased. The induced myogenic lineage cells differentiated into muscle fibers in virto and in vivo. The current study revealed that the five transcription factors generated myogenic lineage cells from fibroblast via a paraxial mesoderm stage. The induced myogenic lineage cells may have a potential being applied as cell source for degenerative muscle disease.

Aims: To determine the clinical characteristics and genetic cause of Waardenburg syndrome type 1 (WS1) in a Chinese family. Materials and Methods: Evaluations, including history, clinical features, and audiological tests, were performed on the proband and her parents. Genetic analyses were performed targeting 144 known deafness genes using a next-generation sequencing panel. Bioinformatic analyses were used to analyze the candidate mutation. Results: The proband and her parents suffered from congenital bilateral profound hearing loss. Her mother exhibited bilateral blue irides. WS1 was diagnosed in the proband and her mother according to the Waardenburg syndrome consortium criteria: the calculated W index of the proband was 2.39 and that of her mother was 2.31. A novel mutation c.1076_1077del (p.Thr359fs) in exon 7 of the PAX3 gene (paired box 3) was identified in the proband and her mother that was absent in the father and controls. Conclusion: Mutations in exon 7 of the PAX3 gene are rare. We identified a novel frameshift mutation in exon 7 of the PAX3 gene that we determined was responsible for WS1 in this family.

The study is aimed to investigate the expression of PAX3 in bilateral paravertebral muscles in adolescent idiopathic scoliosis (AIS) and controls, and to further clarify its association with the paravertebral muscle volume and curve severity. Ten AIS patients and 10 age-matched controls with lumbar disc herniation were included. Bilateral biopsies of paravertebral muscles were obtained at the apical vertebral level for patients and at L5 level for controls. The concave and convex expression of PAX3 was compared between the two groups. The AIS patients were evaluated with magnetic resonance imaging scan of the spine at the apex. The muscle volume of apical paravertebral muscles were measured and compared between concave and convex side. Correlation between concave/convex PAX3 expression ratio and concave/convex muscle volume ratio was analyzed. AIS patients were found to have significantly lower PAX3 expression than controls (p

Waardenburg syndrome type I (WS1) is a human autosomal dominant genetic disease characterized by sensorineural hearing loss, pigmentary abnormalities, and dystopia canthorum. In this study, we generated an induced pluripotent stem cell (iPSC) line using non-integrative Sendai viral reprogramming technology from the peripheral blood mononuclear cells (PBMCs) of a 29-year-old woman affected with WS1, caused by a heterozygous frameshift mutation in the PAX3 (NM_181459.4:c.123del). This iPSC line carrying the same mutation displays a nomal karyotype, expresses pluripotent markers, and shows differentiation capacity in vitro.

In vertebrates, the skeletal muscles of the body and their associated stem cells originate from muscle progenitor cells, during development. The specification of the muscles of the trunk, head and limbs, relies on the activity of distinct genetic hierarchies. The major regulators of trunk and limb muscle specification are the paired-homeobox transcription factors PAX3 and PAX7. Distinct gene regulatory networks drive the formation of the different muscles of the head. Despite the redeployment of diverse upstream regulators of muscle progenitor differentiation, the commitment towards the myogenic fate requires the expression of the early myogenic regulatory factors MYF5, MRF4, MYOD and the late differentiation marker MYOG. The expression of these genes is activated by muscle progenitors throughout development, in several waves of myogenic differentiation, constituting the embryonic, fetal and postnatal phases of muscle growth. In order to achieve myogenic cell commitment while maintaining an undifferentiated pool of muscle progenitors, several signaling pathways regulate the switch between proliferation and differentiation of myoblasts. The identification of the gene regulatory networks operating during myogenesis is crucial for the development of in vitro protocols to differentiate pluripotent stem cells into myoblasts required for regenerative medicine.

An induced pluripotent stem cell (iPSC) line, in which a H2B-fluorescent protein fusion is temporally expressed, is a valuable tool to track cells and study cell divisions and apoptosis. To this end we introduced a 3rd generation "all-in-one" doxycycline-inducible H2B-mTurquoise2 vector into the AAVS1 locus of PAX3-Venus iPSCs via CRISPR/Cas9. H2B-mTurquoise2 expression is absent but readily induced by doxycycline allowing quantification of cell divisions and imaging of living cells. Besides being a universal reporter in iPSC-based differentiation and toxicity assays, the generated pluripotent and genomically normal LUMCi041-A-2 line is particularly suited to study PAX3-positive stages of development. ispartof: STEM CELL RESEARCH vol:57 ispartof: location:England status: published

Skeletal muscle from meat-producing livestock such as cattle is a major source of food for humans. To improve skeletal muscle growth efficiency or quality in cattle, it is necessary to understand the genetic and physiological mechanisms that govern skeletal muscle composition, development, and growth. Satellite cells are the myogenic progenitor cells in postnatal skeletal muscle. In this study we analyzed the composition of bovine satellite cells with single-cell RNA sequencing (scRNA-seq). We isolated satellite cells from a 2-week-old male calf, cultured them in growth medium for a week, and performed scRNA-seq using the 10x Genomics platform. Deep sequencing of two scRNA-seq libraries constructed from cultured bovine satellite cells yielded 860 million reads. Cell calling analyses revealed that these reads were sequenced from 19,096 individual cells. Clustering analyses indicated that these reads represented 15 cell clusters that differed in gene expression profile. Based on the enriched expression of markers of satellite cells (PAX7 and PAX3), markers of myoblasts (MYOD1, MYF5), and markers of differentiated myoblasts or myocytes (MYOG), three clusters were determined to be satellite cells, two clusters myoblasts, and two clusters myocytes. Gene ontology and trajectory inference analyses indicated that cells in these myogenic clusters differed in proliferation rate and differentiation stage. Two of the remaining clusters were enriched with PDGFRA, a marker of fibro-adipogenic (FAP) cells, the progenitor cells for intramuscular fat, and are therefore considered to be FAP cells. Gene ontology analyses indicated active lipogenesis in one of these two clusters. The identity of the remaining six clusters could not be defined. Overall, the results of this study support the hypothesis that bovine satellite cells are composed of subpopulations that differ in transcriptional and myogenic state. The results of this study also support the hypothesis that intramuscular fat in cattle originates from fibro-adipogenic cells. Published version

Clear cell renal cell carcinoma (ccRCC) carrying wild-type Von Hippel–Lindau (VHL) tumor suppressor are more invasive and of high morbidity. Concurrently, competing endogenous RNA (ceRNA) network has been suggested to play an important role in ccRCC malignancy. In order to understand why the patients carrying wild-type VHL gene have high degrees of invasion and morbidity, we applied bioinformatics approaches to identify 861 differentially expressed RNAs (DE-RNAs) between patients carrying wild-type and patients carrying mutant VHL from The Cancer Genome Atlas (TCGA) database, established a ceRNA network including 122 RNAs, and elected six survival-related DE-RNAs including Linc00942, Linc00858, RP13_392I16.1, hsa-miR-182-5p, hsa-miR-183-5p, and PAX3. Examining clinical samples from our hospital revealed that patients carrying wild-type VHL had significantly higher levels of all six RNAs than those carrying mutant VHL. Patients carrying wild-type VHL had significantly higher risk scores, which were calculated based on expression levels of all six RNAs, than those carrying mutant VHL. Patients with higher risk scores had significantly shorter survival times than those with lower risk scores. Therefore, the risk scores serve well to predict malignancy and prognosis.

In this study, we identified active enhancers in the mouse cerebellum at embryonic and postnatal stages establishing the first catalog of enhancers active during embryonic cerebellum development. The majority of cerebellar enhancers have dynamic activity between embryonic and postnatal development. Cerebellar enhancers were enriched for neural transcription factor binding sites with temporally specific expression. Putative gene targets displayed spatially restricted expression patterns, indicating cell-type specific expression regulation. Functional analysis of target genes indicated that enhancers regulate processes spanning several developmental epochs such as specification, differentiation and maturation. We use these analyses to discover one novel regulator and one novel marker of cerebellar development: Bhlhe22 and Pax3, respectively. We identified an enrichment of de novo mutations and variants associated with autism spectrum disorder in cerebellar enhancers. Our study provides insight into the dynamics of gene expression regulation by enhancers in the developing brain and delivers a rich resource of novel gene-enhancer associations providing a basis for future in-depth studies in the cerebellum.

Mouse models provide opportunities to investigate genetic interactions that cause or modify the frequency of neural tube defects (NTDs). Mutation of the PAX3 transcription factor prevents neural tube closure, leading to cranial and spinal NTDs whose frequency is responsive to folate status. Canonical Wnt signalling is implicated both in regulation of Pax3 expression and as a target of PAX3. This study investigated potential interactions of Pax3 mutation and canonical Wnt signalling using conditional gain- and loss-of-function models of β-catenin. We found an additive effect of β-catenin gain of function and Pax3 loss of function on NTDs and neural crest defects. β-catenin gain of function in the Pax3 expression domain led to significantly increased frequency of cranial but not spinal NTDs in embryos that are heterozygous for Pax3 mutation, while both cranial and spinal neural tube closure were exacerbated in Pax3 homozygotes. Similarly, deficits of migrating neural crest cells were exacerbated by β-catenin gain of function, with almost complete ablation of spinal neural crest cells and derivatives in Pax3 homozygous mutants. Pax3 expression was not affected by β-catenin gain of function, while we confirmed that loss of function led to reduced Pax3 transcription. In contrast to gain of function, β-catenin knockout in the Pax3 expression domain lowered the frequency of cranial NTDs in Pax3 null embryos. However, loss of function of β-catenin and Pax3 resulted in spinal NTDs, suggesting differential regulation of cranial and spinal neural tube closure. In summary, β-catenin function modulates the frequency of PAX3-related NTDs in the mouse.

Waardenburg syndrome (WS) is a group of autosomal-dominant hereditary conditions with a global incidence of 1/42,000. WS can be categorized into at least four types: WS1–4, and these are characterized by heterochromia iridis, white forelock, prominent nasal root, dystopia canthorum, hypertrichosis of the medial part of the eyebrows, and deaf-mutism. WS3 is extremely rare, with a unique phenotype (upper limb abnormality). Heterozygous mutations of PAX3 are commonly associated with WS1, whereas partial or total deletions of PAX3 are often observed in WS3 cases. Deletions, together with insertions, translocations, inversions, mobile elements, tandem duplications, and complexes, constitute structural variants (SVs), which can be fully and accurately detected by third-generation sequencing (TGS), a new generation of high-throughput DNA sequencing technology. In this study, after failing to identify the causative gene by Sanger sequencing, SNP-array, and whole-exome sequencing (WES), we finally detected a heterozygous gross deletion of PAX3 (10.26kb, chr2: 223153899-223164405) in a WS family by TGS. Our description would enrich the genetic map of WS and help us to further understand this disease. Our findings also demonstrated the value of TGS in clinical genetics researches.

Purpose: Primary central nervous system (CNS) rhabdomyosarcomais a rare mesenchymal tumor predominantly seen in children and associated with a poor outcome. We report a case of primary CNS rhabdomyosarcoma with PAX3-NCOA2 fusion and presenta systematic meta-review of primary CNS rhabdomyosarcoma to characterize this rare tumor.Methods: We present the case of a 6-year-old boy with primary CNS rhabdomyosarcoma in the posterior fossa. In a systematic meta-review, we compare the demographic data of primary CNS rhabdomyosarcoma with data of rhabdomyosarcoma at all sites from the SEER database and analyze clinical factors associated with survival outcome.Results: Our patient underwent gross total resectionand receivedvincristine, actinomycin-D, cyclophosphamide with early introduction of concurrent focal radiation and remained alive with no evidence of disease for 2 years after the end of therapy. Histopathological review revealed embryonal-type rhabdomyosarcoma, and whole-transcriptome analysis revealed PAX3 (EX6)-NCOA2 (EX12) fusion. In all, 77 cases of primary CNS rhabdomyosarcoma were identified through the meta-review. The demographic data of primary CNS rhabdomyosarcoma were similar to dataof rhabdomyosarcoma at all sites. Overall and event-free survival outcomes were available for 64 and 56 patients, respectively, with a 3-year OS of 29.5%and a 3-year EFS of 26.2%. The group that received trimodal treatment exhibited better survival outcomes, with a 3-year OS of 57.4%and a 3-year EFS of 46.3%.Conclusions: Primary CNS rhabdomyosarcoma shares common histological, molecular, and demographic features with non-CNS rhabdomyosarcoma. A trimodal treatment approach with early introduction of radiation therapy may result in favorable survival outcomes.

Alveolar rhabdomyosarcoma (ARMS) is associated with PAX3/PAX7-FOXO1 fusion, which confers specific clinic and biologic characteristics with inferior outcomes. A minority of tumors still histologically classified as "true" ARMS lack the canonical PAX-FOXO1 fusion but have new molecular alterations. We present the first case of PAX3-NCOA1 ARMS with clinical data and follow-up in a two-year-old girl with ARMS of the tongue and nodal extension, treated with chemotherapy, hemi glossectomy, lymph node dissection, and brachytherapy to conserve oral function and limit long-term sequelae. Given the rarity of such variant fusion in ARMS, international collaboration is required to evaluate its prognostic value.

ObjectiveThis study aimed to explore the genetic causes of probands who were diagnosed with Waardenburg syndrome (WS) or congenital sensorineural hearing loss.MethodsA detailed physical and audiological examinations were carried out to make an accurate diagnosis of 14 patients from seven unrelated families. We performed whole-exome sequencing in probands to detect the potential genetic causes and further validated them by Sanger sequencing in the probands and their family members.ResultsThe genetic causes for all 14 patients with WS or congenital sensorineural hearing loss were identified. A total of seven heterozygous variants including c.1459C > T, c.123del, and c.959-409_1173+3402del of PAX3 gene (NM_181459.4), c.198_262del and c.529_556del of SOX10 gene (NM_006941.4), and c.731G > A and c.970dup of MITF gene (NM_000248.3) were found for the first time. Of these mutations, we had confirmed two (c.1459C > T and c.970dup) are de novo by Sanger sequencing of variants in the probands and their parents.ConclusionWe revealed a total of seven novel mutations in PAX3, SOX10, and MITF, which underlie the pathogenesis of WS. The clinical and genetic characterization of these families with WS elucidated high heterogeneity in Chinese patients with WS. This study expands the database of PAX3, SOX10, and MITF mutations and improves our understanding of the causes of WS.

Context Kallmann syndrome (KS) is a rare, genetically heterogeneous Mendelian disorder. Structural defects in KS patients have helped define the genetic architecture of gonadotropin-releasing hormone (GnRH) neuronal development in this condition. Objective Examine the functional role a novel structural defect affecting a long noncoding RNA (lncRNA), RMST, found in a KS patient. Design Whole genome sequencing, induced pluripotent stem cells and derived neural crest cells (NCC) from the KS patient were contrasted with controls. Setting The Harvard Reproductive Sciences Center, Massachusetts General Hospital Center for Genomic Medicine, and Singapore Genome Institute. Patient A KS patient with a unique translocation, t(7;12)(q22;q24). Interventions/Main Outcome Measure/Results A novel translocation was detected affecting the lncRNA, RMST, on chromosome 12 in the absence of any other KS mutations. Compared with controls, the patient’s induced pluripotent stem cells and NCC provided functional information regarding RMST. Whereas RMST expression increased during NCC differentiation in controls, it was substantially reduced in the KS patient’s NCC coincident with abrogated NCC morphological development and abnormal expression of several “downstream” genes essential for GnRH ontogeny (SOX2, PAX3, CHD7, TUBB3, and MKRN3). Additionally, an intronic single nucleotide polymorphism in RMST was significantly implicated in a genome-wide association study associated with age of menarche. Conclusions A novel deletion in RMST implicates the loss of function of a lncRNA as a unique cause of KS and suggests it plays a critical role in the ontogeny of GnRH neurons and puberty.

Summary Bone marrow stromal cells (BMSCs) that express high levels of stem cell factor (SCF) and CXC chemokine ligand 12 (CXCL12) are one crucial component of the hematopoietic stem cell (HSC) niche. While the secreted factors produced by BMSCs to support HSCs have been well described, little is known regarding the transcriptional regulators controlling the cell fate of BMSCs and thus indirectly maintaining HSCs. BMI1 is a polycomb group protein that regulates HSCs both cell intrinsically and extrinsically, but it is unknown in which cell type and how BMI1 functions to maintain HSCs extrinsically. Here we show that Bmi1 maintains HSCs by preventing adipogenic differentiation of BMSCs. Bmi1 is highly expressed in BMSCs but becomes downregulated upon adipogenic differentiation and during aging. Deleting Bmi1 from BMSCs increased marrow adipocytes, induced HSC quiescence and depletion, and impaired hematopoiesis. We found that BMI1 repressed multiple developmental programs in BMSCs by safeguarding the repressive epigenetic marks histone H2A ubiquitylation and H3 lysine 27 trimethylation. We identified a novel adipogenic program governed by Pax3, which BMI1 repressed in BMSCs. Our results establish Bmi1 as a critical regulator of BMSC cell fate that suppresses marrow adipogenesis to create a supportive niche for HSCs., Graphical Abstract, Highlights • Bmi1 prevents adipogenesis and promotes osteogenesis by bone marrow stromal cells • Adipocyte expansion after Bmi1 loss in stroma depletes hematopoietic stem cells • Loss of Bmi1 reduces repressive histone marks on target genes Ink4a/Arf and Pax3 • Derepressed Pax3 after Bmi1 ablation promotes adipocytic differentiation, Nakada and colleagues show that Bmi1 is highly expressed in bone marrow stromal cells, loss of which caused marrow adipocyte expansion and hematopoietic stem cell depletion. Bmi1 deletion diminished repressive histone marks on Pax3, leading to its expression and adipogenesis. Reduced Bmi1 expression with age may contribute to age-associated increase of marrow adipocytes.

Alveolar rhabdomyosarcoma (RMS) is associated with an underlying pathogenic translocation involving either PAX3 or PAX7 and FOXO1. The presence or absence of this fusion defines the biology and clinical behavior of this subtype of RMS and its identification in tumors is relevant to prognostication and treatment planning. To further explore the unique characteristics of fusion-driven RMS, we leveraged a published gene expression data set to perform an unbiased comparison of 33 fusion-positive and 25 fusion-negative RMS cases. Our analyses revealed 1790 expressed loci with more than two-fold differential expression at a threshold of P .05. Genes with increased expression in fusion-positive relative to fusion-negative RMS were significantly enriched for those involved in "nervous system development," "neuron differentiation," and "neurogenesis," highlighting a neurodevelopmental gene expression signature driven by the alveolar RMS-associated fusion protein. We show that neurodevelopmental genes are enriched near PAX3-FOXO1 fusion protein binding sites, suggesting a genome-wide fusion protein-mediated activation of cis regulatory elements. Among the genes with differential expression in fusion-positive versus fusion-negative RMS, we identified expression of the transcriptional regulator of motor neuron and oligodendrocyte development, OLIG2, as a marker of the fusion protein-dependent neurodevelopmental signature. Immunohistochemical analysis of a cohort of 73 RMS specimens revealed OLIG2 expression in 96.4% of fusion-positive RMS (N = 27/28), but only in 6.7% of fusion-negative RMS (N = 3/45; P .001). The proportion of OLIG2-expressing cells in fusion-negative cases did not exceed 5%, while 92.9% of fusion-positive cases showed expression in at least 5% of cells. Our findings identify OLIG2 expression as a unique manifestation of a neurodevelopmental gene expression signature driven by the oncogenic fusion protein characteristic of alveolar RMS, which may aid in the diagnostic and prognostic distinction of fusion-positive cases.

Summary The use of adult skeletal muscle stem cells (MuSCs) for cell therapy has been attempted for decades, but still encounters considerable difficulties. MuSCs derived from human induced pluripotent stem cells (hiPSCs) are promising candidates for stem cell therapy to treat Duchenne muscular dystrophy (DMD). Here we report that four transcription factors, HEYL, KLF4, MYOD, and PAX3, selected by comprehensive screening of different MuSC populations, enhance the derivation of PAX3-positive myogenic progenitors from fibroblasts and hiPSCs, using medium that promotes the formation of presomitic mesoderm. These induced PAX3-positive cells contribute efficiently to the repair of DMD-damaged myofibers and also reconstitute the MuSC population. These studies demonstrate how a combination of core transcription factors can fine-tune the derivation of MuSCs capable of contributing to the repair of adult skeletal muscle., Highlights • Persistent single MyoD can induce myogenic cells, not muscle stem cells • The combination of Heyl, Klf4, Pax3, and transient MyoD can induce muscle stem cells • Induced PAX3+ cells revealed incorporation into regenerating myofibers of DMD mice, In this article, Sato and colleagues show that four transcription factors, Heyl, Klf4, MyoD, and Pax3, selected by comprehensive screening of mouse muscle stem cells, enhance the derivation of PAX3-positive myogenic progenitors from fibroblasts and hiPSCs. These induced PAX3-positive cells contribute efficiently to the repair of DMD-damaged myofibers and also reconstitute stem cell population.

The purpose of this study is to profile the clinical and genetic features of Japanese Waardenburg syndrome (WS) patients and validate the W index. Sixteen Japanese WS families with congenital sensorineural hearing loss were included in the study. The inner canthal, interpupillary, and outer canthal distances (ICD, IPD, and OCD) were measured for all patients, and patients were screened for presence of PAX3, MITF, SOX10, and EDNRB mutations. The WS patients were clinically classified under the current W index as follows: 13 families with WS1, 2 families with WS2, and 1 family with WS4. In the 13 WS1 families, genetic tests found PAX3 mutations in 5 families, MITF mutations in 4 families, SOX10 mutations in 3 families, and EDNRB mutations in 1 family. 61% of clinically classified WS1 patients under the current W index conflicted with the genetic classification, which implies W index is not appropriate for Japanese population. Resetting the threshold of W index or novel index formulated with ethnicity matched samples is necessary for clinical classification which is consistent with genetic classification for WS patients with distinct ethnicity.

Our studies conducted on reptilian limb muscle development revealed, for the first time, early forelimb muscle differentiation at the morphological and molecular level. Sand lizard skeletal muscle differentiation in the early forelimb bud was investigated by light, confocal, and transmission electron microscopy as well as western blot. The early forelimb bud, filled with mesenchymal cells, is surrounded by monolayer epithelium cells. The immunocytochemical analysis revealed the presence of Pax3- and Lbx-positive cells in the vicinity of the ventro-lateral lip (VLL) of the dermomyotome, suggesting that VLL is the source of limb muscle progenitor cells. Furthermore, Pax3- and Lbx-positive cells were observed in the dorsal and ventral myogenic pools of the forelimb bud. Skeletal muscle development in the early limb bud is asynchronous, which is manifested by the presence of myogenic cells in different stages of differentiation: multinucleated myotubes with well-developed contractile apparatus, myoblasts, and mitotically active premyoblasts. The western blot analysis revealed the presence of MyoD and Myf5 proteins in all investigated developmental stages. The MyoD western blot analysis showed two bands corresponding to monomeric (mMyoD) and dimeric (dMyoD) fractions. Two separate bands were also detected in the case of Myf5. The observed bands were related to non-phosphorylated (Myf5) and phosphorylated (pMyf5) fractions of Myf5. Our investigations on sand lizard forelimb myogenesis showed that the pattern of muscle differentiation in the early forelimb bud shares many features with rodents and chicks. Electronic supplementary material The online version of this article (10.1007/s00427-019-00635-7) contains supplementary material, which is available to authorized users.

The PAX3 (paired box 3) gene plays an important role in embryonic development, diseases, and cancer formation. Our preliminary studies have shown that PAX3 gene is upregulated in glioma cells, which is associated with a worse prognosis. Moreover, PAX3, by facilitating cell proliferation and invasion and inhibiting cell apoptosis, plays an oncogenic role in glioma. However, the specific molecular mechanism of PAX3 acting as an oncogene in glioma remains unclarified. In the present study, we have found that PAX3 overexpression was observed in high grade glioma and predicted a worse prognosis. PAX3 overexpression did not correlate significantly to IDH1 mutation and MGMT methylation. Moreover, the expression of PAX3 was positively correlated with that of β-catenin. In U87 glioma cells, PAX3 interacted with β-catenin, as was confirmed by CO-IP. Besides, PAX3 overexpression promoted cell proliferation and cell cycle progression, while it inhibited cell apoptosis by altering the expressions of important molecules associated with the Wnt signaling pathway, including β-catenin, Myc, VEGF, cyclinD1, MMP7, and Wnt1. In the meantime, it was also proved that PAX3 correlated to β-catenin through a negative regulatory mechanism with respect to the promotion of U87 glioma cell proliferation and cell cycle progression and inhibition of the cell apoptosis. Our experiment demonstrated the role of PAX3 in promoting glioma growth and development, possibly by interacting directly with β-catenin and regulating the Wnt signaling pathway.

Background. Waardenburg syndrome (WS) is one of the most common forms of syndromic deafness with heterogeneity of loci and alleles and variable expressivity of clinical features. Methods. The technology of single-nucleotide variants (SNV) and copy number variation (CNV) detection was developed to investigate the genotype spectrum of WS in a Chinese population. Results. Ninety WS patients and 24 additional family members were recruited for the study. Fourteen mutations had not been previously reported, including c.808C>G, c.117C>A, c.152T>G, c.803G>T, c.793-3T >G, and c.801delT on PAX3; c.642_650delAAG on MITF; c.122G>T and c.127C>T on SOX10; c.230C>G and c.365C>T on SNAI2; and c.481A>G, c.1018C>G, and c.1015C>T on EDNRB. Three CNVs were de novo and first reported in our study. Five EDNRB variants were associated with WS type 1 in the heterozygous state for the first time, with a detection rate of 22.2%. Freckles occur only in WS type 2. Yellow hair, amblyopia, congenital ptosis, narrow palpebral fissures, and pigmentation spots are rare and unique symptoms in WS patients from China. Conclusions. EDNRB should be considered as another prevalent pathogenic gene in WS type 1. Our study expanded the genotype and phenotype spectrum of WS, and diagnostic next-generation sequencing is promising for WS.

Background Glioma is identified as a broad category of brain and spinal cord tumors. MiR-362-3p is important in regulating the genesis of different cancers; however, the mechanism of miR-362-3p in the progression of glioma remains largely unknown. Objectives This study aimed to elucidate pathobiological functions of miR-362-3p by targeting PAX3 in glioma. Method qRT-PCR and western blotting were used to examine miR-362-3p and PAX3 expression in glioma tissues and cells. CCK-8 assay and transwell assays were used to examine the functions of miR-362-3p on human glioma. Two bioinformatics analysis software and luciferase reporter assay were performed to analyze the relationship between miR-362-3p and PAX3. Results MiR-362-3p was downregulated, and PAX3 was upregulated in glioma tissues and cells. Functional assays revealed that ectopic expression of miR-362-3p inhibited glioma cell proliferation and migration. Further, PAX3 was confirmed as direct target gene of miR-362-3p, and downregulation of PAX3 reversed the suppressive effects of miR-362-3p in glioma. In addition, miR-362-3p also exhibited suppressive effect on epithelial-mesenchymal transition and Wnt/β-catenin pathway. Conclusions MiR-362-3p downregulation or PAX3 overexpression predicted poor prognosis in glioma. MiR-362-3p played a role in the suppressive effect on glioma by targeting PAX3 through suppressing Wnt/β-catenin pathway.

Background: Waardenburg Syndrome Type 1 (WS1) is a rare hereditary disease, which is usually caused by the mutations of PAX3 (paired box 3). Here, we reported a pedigree with WS1, which was caused by a novel mutation in PAX3.Case Report: In this present report, a 10-year-old boy and his twin sister from a Han Chinese family presented with iris pigmentary abnormality, synophrys, and broad and high nasal root. Their father presented premature whitening of the hair, but no iris pigmentary abnormality. Their aunts presented the same clinical characteristics with the twins and premature graying of hair. However, none of the patients reported hearing loss. The clinical diagnosis of the four patients from this pedigree was WS1. The whole exome sequencing (WES) revealed a novel mutation (c.959-5T>G) in the PAX3 gene, which could be responsible for the observed pathogenic of WS1 in this pedigree. The genetic test confirmed the diagnosis of WS1 in the four patients from the studied pedigree.Conclusion: This present study demonstrated that genetic test based on WES, an effective alternative to regular clinical examinations, helps diagnose WS1. The newly identified PAX3 gene mutation can expand the understanding of WS1.

Waardenburg syndrome (WS) is a genetic disorder characterized by sensorineural deafness. It has a variable presentation of pigmentation defects. Here, we generated an induced pluripotent stem cell (iPSC) line using episomal plasmid vectors from the fibroblasts of a 4-year-old boy affected with WS type II, caused by a novel mutation in microphthalmia-associated transcription factor (MITF) (NM_000248.3: exon6:c.626A>T). The patient-specific iPSC line (CSUXHi003-A) carrying the same MITF mutation showed normal karyotype, expressed pluripotent markers, and presented differentiation capacity in vitro. It may be a useful tool for in vitro modeling of WS.

Background Waardenburg syndrome (WS) is a rare genetic disorder. The purpose of this study was to investigate clinical and molecular characteristics of WS in four probands from four different Iranian families. Case presentation The first patient was a 1-year-old symptomatic boy with congenital hearing loss and heterochromia iridis with a blue segment in his left iris. The second case was a 1.5-year-old symptomatic girl who manifested congenital profound hearing loss, brilliant blue eyes, and skin hypopigmentation on the abdominal region at birth time. The third patient was an 8-month-old symptomatic boy with developmental delay, mild atrophy, hypotonia, brilliant blue eyes, skin hypopigmentation on her hand and foot, Hirschsprung disease, and congenital profound hearing loss; the fourth patient was a 4-year-old symptomatic boy who showed dystopia canthorum, broad nasal root, synophrys, skin hypopigmentation on her hand and abdomen, brilliant blue eyes, and congenital profound hearing loss. Whole exome sequencing (WES) was used for each proband to identify the underlying genetic factor. Sanger sequencing was performed for validation of the identified mutations in probands and the available family members. A novel heterozygous frameshift mutation, c.996delT (p.K334Sfs*15), on exon 8 of the MITF gene was identified in the patient of the first family diagnosed with WS2A. Two novel de novo heterozygous mutations including a missense mutation, c.950G > A (p.R317K), on exon 8 of the MITF gene, and a frameshift mutation, c.684delC (p.E229Sfs*57), on the exon 3 of the SOX10 gene were detected in patients of the second and third families with WS2A and PCWH (Peripheral demyelinating neuropathy, Central dysmyelinating leukodystrophy, Waardenburg syndrome, Hirschsprung disease), respectively. A previously reported heterozygous frameshift mutation, c.1024_1040del AGCACGATTCCTTCCAA, (p.S342Pfs*62), on exon 7 of the PAX3 gene was identified in the patient of the fourth family with WS1. Conclusions An exact description of the mutations responsible for WS provides useful information to explain the molecular cause of clinical features of WS and contributes to better genetic counseling of WS patients and their families.

Background/aim Fusions of the paired box 3 gene (PAX3 in 2q36) with different partners have been reported in rhabdomyosarcomas and biphenotypic sinonasal sarcomas. We herein report the myocardin (MYOCD on 17p12) gene as a novel PAX3-fusion partner in a pediatric tumor with adverse clinical outcome. Materials and methods A rhabdomyo-sarcoma found in a 10-year-old girl was studied using a range of genetic methodologies. Results The karyotype of the tumor cells was 48,XX,add(2)(q11),+del(2)(q35),add(3)(q?25),-7, del(8)(p 21),-15, add(17)(p 11), + 20, +der(?) t(?; 15) (?;q15),+mar[8]/46,XX[2]. Fluorescence in situ hybridization detected PAX3 rearrangement whereas array comparative genomic hybridization revealed genomic imbalances affecting hundreds of genes, including MYCN, MYC, FOXO3, and the tumor suppressor gene TP53. A PAX3-MYOCD fusion transcript was found by RNA sequencing and confirmed by Sanger sequencing. Conclusion The investigated rhabdomyosarcoma carried a novel PAX3-MYOCD fusion gene and extensive additional aberrations affecting the allelic balance of many genes, among them TP53 and members of MYC and FOXO families of transcription factors.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood characterized by the inability to exit the proliferative myoblast-like stage. The alveolar fusion positive subtype (FP-ARMS) is the most aggressive and is mainly caused by the expression of PAX3/7-FOXO1 oncoproteins, which are challenging pharmacological targets. Here, we show that the DEAD box RNA helicase 5 (DDX5) is overexpressed in alveolar RMS cells and that its depletion and pharmacological inhibition decrease FP-ARMS viability and slow tumor growth in xenograft models. Mechanistically, we provide evidence that DDX5 functions upstream the EHMT2/AKT survival signalling pathway, by directly interacting with EHMT2mRNA and modulating its protein expression. We found that EHMT2 in turns regulates PAX3-FOXO1 activity in a methylation-dependent manner, thus sustaining FP-ARMS myoblastic state. Together, our findings identify a novel survival-promoting loop in FP-ARMS and highlight DDX5 as potential therapeutic target to arrest rhabdomyosarcoma growth.

Background Migrating muscle progenitors delaminate from the somite and subsequently form muscle tissue in distant anatomical regions such as the paired appendages, or limbs. In amniotes, this process requires a signaling cascade including the transcription factor paired box 3 (Pax3). Results In this study we found that, unlike in mammals, pax3a/3b double mutant zebrafish develop near to normal appendicular muscle. By analyzing numerous mutant combinations of pax3a, pax3b and pax7a and pax7b, we determined that there is a feed-back system and a compensatory mechanism between Pax3 and Pax7 in this developmental process, even though Pax7 alone is not required for appendicular myogenesis. pax3a/3b/7a/7b quadruple mutant developed muscle-less pectoral fins. Conclusions We found that Pax3 and Pax7 are redundantly required during appendicular myogenesis in zebrafish, where Pax7 is able to activate the same developmental programs as Pax3 in the pre-migratory progenitor cells. This article is protected by copyright. All rights reserved.

The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS., Author summary The fusion-positive subtype of rhabdomyosarcoma (FP-RMS) is a rare malignant paediatric cancer, whose induction and evolution still remain to be deciphered. Out of the gross genetic aberrations found in these cancers, t(2:13) and t(1,13) chromosome translocations are the first to appear and lead to the expression of fusion proteins made of the DNA binding domains of either PAX3 or PAX7 and the transactivation domain of FOXO1. Both PAX3-FOXO1 and PAX7-FOXO1 have a strong impact on gene transcription, yet they only inefficiently promote the transformation of healthy cells into tumorigenic cells. To address this issue, we have used chick embryos to monitor in vivo the early response of cells to PAX-FOXO1 chimeric proteins. We showed that both proteins, but not the normal PAX3 and PAX7, transform neural cells into cells with FP-RMS molecular features. The PAX-FOXO1s also force polarized epithelial neural cells to adopt a mesenchymal phenotype with tissue invasive properties. However, the PAX-FOXO1s inhibit cell division and hence tumour growth. Genetically re-activating core cell cycle regulators rescues PAX-FOXO1 mediated cell cycle inhibition. Together, our findings bring further support to the idea that the PAX-FOXO1s are stricto sensu oncoproteins, whose oncogenicity is limited by negative effects on cell cycle.

Waardenburg syndrome (WS), also known as auditory-pigmentary syndrome, is the most common cause of syndromic hearing loss (HL), which accounts for approximately 2-5% of all patients with congenital hearing loss. WS is classified into four subtypes depending on the clinical phenotypes. Currently, pathogenic mutations of PAX3, MITF, SOX10, EDN3, EDNRB or SNAI2 are associated with different subtypes of WS. Although supportive techniques like hearing aids, cochlear implants, or other assistive listening devices can alleviate the HL symptom, there is no cure for WS to date. Recently major progress has been achieved in preclinical studies of genetic HL in animal models, including gene delivery and stem cell replacement therapies. This review focuses on the current understandings of pathogenic mechanisms and potential biological therapeutic approaches for HL in WS, providing strategies and directions for implementing WS biological therapies, as well as possible problems to be faced, in the future.

Background Waardenburg syndrome (WS) is a rare disorder characterized by varying combinations of sensorineural hearing loss and abnormal pigmentation of the hair and skin. WS is classified into four subtypes (WS1-WS4) based on additional symptoms. Dystopia canthorum is a hallmark of WS type 1. There are two genes linked to WS type 1, including PAX3 and EDNRB. Objective This study aimed to investigate the genetic etiology of WS type 1 in a pair of twins from China with profound hearing loss, blond hair and eyebrows, dystopia canthorum, and brown irides. Methods The target capture sequencing and Whole-exome sequencing were performed to detect mutations in WS-related genes. Results A novel de novo frameshift mutation, p.L341Rfs*18 in MITF was identified in the twins. Hearing thresholds showed substantial improvements following cochlear implantation with a pure-tone average of 30 dB in free-field conditions. Conclusions The study showed the new genotype-phenotype correlations of MITF to WS type 1. Further molecular analysis is necessary to reappraise the current classification on WS.