CRISPR-Cas9-Mediated Bioluminescent Tagging of Endogenous Proteins by Fluorescent Protein-Assisted Cell Sorting.

Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central "driver" roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC). HiBiT is an 11-amino acid peptide derived from the NanoLuc luciferase that produces a luminescence signal which is ~100-fold brighter than firefly or Renilla luciferases through high-affinity binding to a complementary NanoLuc peptide fragment called LgBiT. To facilitate single-cell clonal isolation of knock-ins, the homology-directed repair template encoding HiBiT was followed by a P2A self-cleaving peptide for coexpression of an mCherry fluorescent protein as a fluorescence-activated cell sorter (FACS)-selectable marker. HiBiT tagging thus allows highly sensitive luminescence detection of endogenous PAX3-FOXO1 levels permitting quantitative high-throughput screening of large compound libraries for the discovery of PAX3-FOXO1 inhibitors and degraders.

Transforming Growth Factor Beta and Alveolar Rhabdomyosarcoma: A Challenge of Tumor Differentiation and Chemotherapy Response.

Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-ß). This overexpression of TGF-ß1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-ß also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-ß in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-ß1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.

KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

Reduction of lens size in PAX6-related aniridia.

Heterozygous mutation of PAX6 in humans leads to congenital aniridia (OMIM 106210) which is typified by congenital iris and foveal defects, and later onset glaucoma, aniridic keratopathy, and cataract. Mice heterozygous for Pax6 mutations phenocopy many aspects of aniridia including the iris defects, keratopathy and cataract, although Pax6 mutant mice have small lenses, a phenotype which is not typically reported in human aniridia, perhaps due to difficulties in measuring lens diameter during typical ophthalmic examinations as the lens periphery is shielded by the iris. In order to overcome this, records of patients diagnosed with congenital aniridia between April 2015 and May 2021 at the Necker-Enfants Malades Hospital, and genetically confirmed with a disease-causing PAX6 variant, were retrospectively reviewed for those with normal axial length whose iris defects allowed visualization of the lens margins and corneal diameter to allow calculation of a lens/corneal diameter ratio. This value was compared with values obtained from a cohort of patients with Sjödell grade IV oculocutaneous albinism type 1 (OCA1; OMIM 203100) which allowed visualization of the lens periphery via iris transillumination. This analysis revealed that patients with congenital aniridia had a significantly lower lens/corneal ratio when compared to those with albinism, suggesting that humans haploinsufficient for PAX6, like mice, rats, frogs, and zebrafish, exhibit reductions in lens size.

Characterization of neural damage and neuroinflammation in Pax6 small-eye mice.

Aniridia is a panocular condition characterized by a partial or complete loss of the iris. It manifests various developmental deficits in both the anterior and posterior segments of the eye, leading to a progressive vision loss. The homeobox gene PAX6 plays an important role in ocular development and mutations of PAX6 have been the main causative factors for aniridia. In this study, we assessed how Pax6-haploinsufficiency affects retinal morphology and vision of Pax6Sey mice using in vivo and ex vivo metrics. We used mice of C57BL/6 and 129S1/Svlmj genetic backgrounds to examine the variable severity of symptoms as reflected in human aniridia patients. Elevated intraocular pressure (IOP) was observed in Pax6Sey mice starting from post-natal day 20 (P20). Correspondingly, visual acuity showed a steady age-dependent decline in Pax6Sey mice, though these phenotypes were less severe in the 129S1/Svlmj mice. Local retinal damage with layer disorganization was assessed at P30 and P80 in the Pax6Sey mice. Interestingly, we also observed a greater number of activated Iba1+ microglia and GFAP + astrocytes in the Pax6Sey mice than in littermate controls, suggesting a possible neuroinflammatory response to Pax6 deficiencies.

PAX3-FOXO1 dictates myogenic reprogramming and rhabdomyosarcoma identity in endothelial progenitors.

Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.

PAX4 loss of function increases diabetes risk by altering human pancreatic endocrine cell development.

The coding variant (p.Arg192His) in the transcription factor PAX4 is associated with an altered risk for type 2 diabetes (T2D) in East Asian populations. In mice, Pax4 is essential for beta cell formation but its role on human beta cell development and/or function is unknown. Participants carrying the PAX4 p.His192 allele exhibited decreased pancreatic beta cell function compared to homozygotes for the p.192Arg allele in a cross-sectional study in which we carried out an intravenous glucose tolerance test and an oral glucose tolerance test. In a pedigree of a patient with young onset diabetes, several members carry a newly identified p.Tyr186X allele. In the human beta cell model, EndoC-ßH1, PAX4 knockdown led to impaired insulin secretion, reduced total insulin content, and altered hormone gene expression. Deletion of PAX4 in human induced pluripotent stem cell (hiPSC)-derived islet-like cells resulted in derepression of alpha cell gene expression. In vitro differentiation of hiPSCs carrying PAX4 p.His192 and p.X186 risk alleles exhibited increased polyhormonal endocrine cell formation and reduced insulin content that can be reversed with gene correction. Together, we demonstrate the role of PAX4 in human endocrine cell development, beta cell function, and its contribution to T2D-risk.

Expanding the clinical and immunological phenotypes of PAX1-deficient SCID and CID patients.

Paired box 1 (PAX1) deficiency has been reported in a small number of patients diagnosed with otofaciocervical syndrome type 2 (OFCS2). We described six new patients who demonstrated variable clinical penetrance. Reduced transcriptional activity of pathogenic variants confirmed partial or complete PAX1 deficiency. Thymic aplasia and hypoplasia were associated with impaired T cell immunity. Corrective treatment was required in 4/6 patients. Hematopoietic stem cell transplantation resulted in poor immune reconstitution with absent naïve T cells, contrasting with the superior recovery of T cell immunity after thymus transplantation. Normal ex vivo differentiation of PAX1-deficient CD34+ cells into mature T cells demonstrated the absence of a hematopoietic cell-intrinsic defect. New overlapping features with DiGeorge syndrome included primary hypoparathyroidism (n = 5) and congenital heart defects (n = 2), in line with PAX1 expression during early embryogenesis. Our results highlight new features of PAX1 deficiency, which are relevant to improving early diagnosis and identifying patients requiring corrective treatment.

Piperacetazine Directly Binds to the PAX3::FOXO1 Fusion Protein and Inhibits Its Transcriptional Activity.

The tumor-specific chromosomal translocation product, PAX3::FOXO1, is an aberrant fusion protein that plays a key role for oncogenesis in the alveolar subtype of rhabdomyosarcoma (RMS). PAX3::FOXO1 represents a validated molecular target for alveolar RMS and successful inhibition of its oncogenic activity is likely to have significant clinical applications. Even though several PAX3::FOXO1 function-based screening studies have been successfully completed, a directly binding small-molecule inhibitor of PAX3::FOXO1 has not been reported. Therefore, we screened small-molecule libraries to identify compounds that were capable of directly binding to PAX3::FOXO1 protein using surface plasmon resonance technology. Compounds that directly bound to PAX3::FOXO1 were further evaluated in secondary transcriptional activation assays. We discovered that piperacetazine can directly bind to PAX3::FOXO1 protein and inhibit fusion protein-derived transcription in multiple alveolar RMS cell lines. Piperacetazine inhibited anchorage-independent growth of fusion-positive alveolar RMS cells but not embryonal RMS cells. On the basis of our findings, piperacetazine is a molecular scaffold upon which derivatives could be developed as specific inhibitors of PAX3::FOXO1. These novel inhibitors could potentially be evaluated in future clinical trials for recurrent or metastatic alveolar RMS as novel targeted therapy options. SIGNIFICANCE: RMS is a malignant soft-tissue tumor mainly affecting the pediatric population. A subgroup of RMS with worse prognosis harbors a unique chromosomal translocation creating an oncogenic fusion protein, PAX3::FOXO1. We identified piperacetazine as a direct inhibitor of PAX3::FOXO1, which may provide a scaffold for designing RMS-specific targeted therapy.

Functional Characteristics of Diverse PAX6 Mutations Associated with Isolated Foveal Hypoplasia.

The human fovea is a specialized pit structure in the central retina. Foveal hypoplasia is a condition where the foveal pit does not fully develop, and it is associated with poor vision. Autosomal dominant isolated foveal hypoplasia (FVH1) is a rare condition of foveal hypoplasia (FH) that lacks any other ocular manifestations. FVH1 is associated with hypomorphic mutations in the PAX6 gene that encodes a sequence-specific DNA-binding transcription factor for morphogenesis and evolution of the eye. We report our findings in 17 patients with PAX6 mutations associated with FVH1 or FH with aniridia and corneal opacities. Patients with three mutations, p.V78E, p.V83F and p.R128H, in the C-terminal subdomain of the paired domain (CTS) consistently have severe FH. Luciferase assays for a single reporter containing a representative PAX6 binding site indicated that the transcriptional activities of these mutations were significantly reduced, comparable to that of the truncation mutation of p.G65Rfs*5. Patients with p.P20S in the N-terminal subdomain of the paired domain, and a patient with p.N365K in the proline-serine-threonine-rich domain (PSTD) had mild FH. A patient with p.Q255L in the homeodomain had severe FH. The P20S and Q255L mutants did not affect the transcriptional activity. Mutant N365K has a retained DNA-binding activity but a reduced transcriptional activity, due to a low PSTD transactivation. These findings demonstrated that mutations associated with FVH1 underlie a functional divergence between DNA-binding ability and transcriptional activity. We conclude that a wide range of mutations in the PAX6 gene is not limited to the CST region and are responsible for FVH1.

Long-read genome sequencing identifies cryptic structural variants in congenital aniridia cases.

BACKGROUND: Haploinsufficiency of the transcription factor PAX6 is the main cause of congenital aniridia, a genetic disorder characterized by iris and foveal hypoplasia. 11p13 microdeletions altering PAX6 or its downstream regulatory region (DRR) are present in about 25% of patients; however, only a few complex rearrangements have been described to date. Here, we performed nanopore-based whole-genome sequencing to assess the presence of cryptic structural variants (SVs) on the only two unsolved "PAX6-negative" cases from a cohort of 110 patients with congenital aniridia after unsuccessfully short-read sequencing approaches. RESULTS: Long-read sequencing (LRS) unveiled balanced chromosomal rearrangements affecting the PAX6 locus at 11p13 in these two patients and allowed nucleotide-level breakpoint analysis. First, we identified a cryptic 4.9 Mb de novo inversion disrupting intron 7 of PAX6, further verified by targeted polymerase chain reaction amplification and sequencing and FISH-based cytogenetic analysis. Furthermore, LRS was decisive in correctly mapping a t(6;11) balanced translocation cytogenetically detected in a second proband with congenital aniridia and considered non-causal 15 years ago. LRS resolved that the breakpoint on chromosome 11 was indeed located at 11p13, disrupting the DNase I hypersensitive site 2 enhancer within the DRR of PAX6, 161 Kb from the causal gene. Patient-derived RNA expression analysis demonstrated PAX6 haploinsufficiency, thus supporting that the 11p13 breakpoint led to a positional effect by cleaving crucial enhancers for PAX6 transactivation. LRS analysis was also critical for mapping the exact breakpoint on chromosome 6 to the highly repetitive centromeric region at 6p11.1. CONCLUSIONS: In both cases, the LRS-based identified SVs have been deemed the hidden pathogenic cause of congenital aniridia. Our study underscores the limitations of traditional short-read sequencing in uncovering pathogenic SVs affecting low-complexity regions of the genome and the value of LRS in providing insight into hidden sources of variation in rare genetic diseases.

Pax4 in Health and Diabetes.

Paired box 4 (Pax4) is a key transcription factor involved in the embryonic development of the pancreatic islets of Langerhans. Consisting of a conserved paired box domain and a homeodomain, this transcription factor plays an essential role in early endocrine progenitor cells, where it is necessary for cell-fate commitment towards the insulin-secreting ß cell lineage. Knockout of Pax4 in animal models leads to the absence of ß cells, which is accompanied by a significant increase in glucagon-producing α cells, and typically results in lethality within days after birth. Mutations in Pax4 that cause an impaired Pax4 function are associated with diabetes pathogenesis in humans. In adulthood, Pax4 expression is limited to a distinct subset of ß cells that possess the ability to proliferate in response to heightened metabolic needs. Upregulation of Pax4 expression is known to promote ß cell survival and proliferation. Additionally, ectopic expression of Pax4 in pancreatic islet α cells or δ cells has been found to generate functional ß-like cells that can improve blood glucose regulation in experimental diabetes models. Therefore, Pax4 represents a promising therapeutic target for the protection and regeneration of ß cells in the treatment of diabetes. The purpose of this review is to provide a thorough and up-to-date overview of the role of Pax4 in pancreatic ß cells and its potential as a therapeutic target for diabetes.

Novel variants in the PAX6 gene related to isolated aniridia.

Aniridia, which is a rare congenital defect of the eye, consists of iris hypoplasia or aplasia, and additional ocular abnormalities. It is most commonly caused by autosomal dominant PAX6 gene mutations. However, in about 30% of cases, it is associated with chromosomal rearrangements in the 11p13 region. The aim of this study was to identify the potential PAX6 gene variants, which could cause the isolated aniridia. Eight patients with isolated aniridia were included in this study. MLPA analysis allowed in the past to exclude large structural rearrangements of the PAX6 and adjacent genes like WT1. Blood samples were collected from the patients (and their families in familial cases) and genomic DNA was extracted from peripheral blood leukocytes and buccal cells. The amplification of the 11 exons of the PAX6 gene was performed. Bidirectional Sanger Sequencing was conducted for the identification of the potentially pathogenic variants, and for the segregation analysis of the identified variant in the family. The results were analyzed with the use of CodonCode Aligner software. In three patients, aniridia was sporadic, whereas in another five cases, the eye defect was familial. The potentially pathogenic variants in the PAX6 gene were found in 6 out of 8 patients with aniridia. We identified four known (c.781C > T, c.607C > T, and c.949C > T twice), and two novel variants (c.258_265del and c.495_496insG). Point mutations in the PAX6 gene are the most frequent cause of aniridia. The investigation of the genetic background of the disease is essential for patients to evaluate recurrence risk in the offspring.

[Alveolar rhabdomyosarcoma: novel surrogate markers associated with oncogenic translocation]. / Al'veolyarnaya rabdomiosarkoma: novye vspomogatel'nye surrogatnye markery onkogennykh translokatsii.

BACKGROUND: Anomalies of the FOXO1 gene in alveolar rhabdomyosarcoma are associated with a worse clinical prognosis, which determines the high value of studying the status of this gene when choosing a therapy strategy. The «gold standard¼ for determining FOXO1 gene rearrangements is currently the fluorescent in situ hybridization (FISH) technique. OBJECTIVE: Study of the relationship between canonical FOXO1 translocation and immunohistochemical expression of new surrogate markers in alveolar rhabdomyosarcoma to determine their predictive value. MATERIAL AND METHODS: 139 cases of rhabdomyosarcoma were retrospectively studied. The study used tissue matrix technology (TMA). On sections obtained from TMA blocks, the FISH technique was implemented using the locus-specific probe MetaSystems XL FOXO1 Break Apart (Metasystems, Germany). Immunohistochemical studies were performed on similar sections from TMA blocks with OLIG2 (Cell Marque Antibodies, clone 211F1.1) and MUC4 (Cell Marque Antibodies, clone 8G7) antibodies. RESULTS: The final expression analysis and statistical processing using a 2x2 contingency table and Fisher's exact test passed 111 cases (76 without FOXO1 rearrangement and 35 with rearrangement). The specificity of OLIG2 and MUC4 expression for FOXO1-rearranged alveolar rhabdomyosarcoma was 85.53% and 80.26%, respectively (p<0.01). CONCLUSION: The present study confirms the high predictive value of the expression of surrogate markers OLIG2 and MUC4 in determining the genetic status of alveolar rhabdomyosarcoma, which makes it possible to predict with high specificity the detection of the FOXO1 gene rearrangement.

Novel Missense Variants in PAX8 and NKX2-1 Cause Congenital Hypothyroidism.

Primary congenital hypothyroidism (CH) is a common neonatal endocrine disorder characterized by elevated concentrations of thyroid stimulating hormone (TSH) and low concentrations of free thyroxine (FT4). PAX8 and NKX2-1 are important transcription factors involved in thyroid development. In this study, we detected three novel variants in PAX8 (c.149A > C and c.329G > A) and NKX2-1 (c.706A > G) by whole exome sequencing (WES) in three unrelated CH patients with variable phenotypes. The results of Western blot and immunofluorescence analysis showed that the three variants had no effect on protein expression and subcellular localization. However, the results of the electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay suggested that the three variants in PAX8 and NKX2-1 both affected their DNA-binding ability and reduced their transactivation capacity. Moreover, a dominant-negative effect in K236E−NKX2-1 was identified by dual-luciferase reporter assay. To sum up, our findings extend our knowledge of the current mutation spectrum of PAX8 and NKX2-1 and provide important information for diagnosing, treating, and preventing CH in these families.

Rhabdomyosarcoma With Epithelioid Features And NSD3::FOXO1 Fusion: Evidence For Reconsideration Of Previously Reported FOXO1::FGFR1 Fusion.

Epithelioid rhabdomyosarcoma is a rare rhabdomyosarcoma variant for which no diagnostic recurrent driver genetic events have been identified. Here we report a rapidly progressive and widely metastatic rhabdomyosarcoma with epithelioid features that arose in the thigh of a male infant. Conventional cytogenetics revealed a t(8;13)(p11.2;q14) translocation. Fluorescence in situ hybridization studies showed rearrangement of FOXO1 and amplification of its 3" end, and rearrangement of NSD3 and amplification of its 5` end. Next generation sequencing identified a NSD3::FOXO1 fusion, which is a previously unreported gene fusion. We also review the historic report of a FOXO1::FGFR1 fusion in a solid variant of alveolar rhabdomyosarcoma and propose that NSD3::FOXO1 fusion may have been the more appropriate interpretation of the data presented in that report.

ZNF582 hypermethylation as a prognostic biomarker for malignant transformation of oral lesions.

OBJECTIVES: This hospital-based cohort study evaluated whether ZNF582 and PAX1 methylation levels at baseline can be used as biomarkers to identify lesions with a high potential for malignant transformation in patients with normal mucosa and oral potentially malignant disorders. PATIENTS AND METHODS: We recruited 171 adult patients with normal mucosa and oral potentially malignant disorders in 2012-2014. They were followed until 2017. Outcomes, including advanced histopathological findings and oral cancer occurrence, were obtained from medical charts, the Taiwan Cancer Registry, and cause-of-death data. Kaplan-Meier analysis and Cox proportional hazards regression models were used to examine the association of ZNF582 and PAX1 methylation levels at baseline with subsequent outcome occurrences. RESULTS: After 260,192 days of follow-up, 11 cases of oral cancer and 4 cases of advanced histopathological progression occurred. Patients with higher ZNF582 and PAX1 methylation levels at baseline had a higher incidence of disease progression. After adjustment for all studied factors using Cox proportional hazards regression models, ZNF582m level (adjusted hazard ratio, 11.41; 95% CI, 2.05-63.36; p = 0.005) was the only significant and independent predictor of disease progression. CONCLUSIONS: ZNF582 hypermethylation can be an effective and noninvasive biomarker for identifying oral lesions with a high potential for malignant transformation.

Retinoic acid control of pax8 during renal specification of Xenopus pronephros involves hox and meis3.

Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 â€‹kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 â€‹kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.

Isolated aniridia caused by a novel PAX6 heterozygous deletion mediated by multi-exon complex rearrangement.

PURPOSE: Mutations in PAX6 gene (chromosome 11p13) encoding a transcriptional regulator involved in oculogenesis mostly present with aniridia. Aniridia is not uncommon in the Philippines but only limited information is available as yet. The purpose of this study was to present a novel, deletion mediated by complex rearrangement in PAX6 gene causing an isolated aniridia in a Filipino girl. PATIENTS AND METHODS: The patient is an 8-year-old girl who came in due to leukocoria with associated nystagmus and esotropia. She presented with subnormal vision, nystagmus, aniridia, and cataractous lenses in both eyes. The family history reveals presence of the aniridia and cataract with the mother and a sibling. The patient underwent lens extraction without intraocular lens implantation bilaterally, where patient subsequently underwent intraocular lens implantation on her left eye. Systemic workup was performed including whole abdomen, renal ultrasound, blood chemistry, and urinalysis. Targeted cataract panel with WT1 and PAX6 genes revealed a novel, heterozygous PAX6-inherited mutation from the mother. This variant is a complex rearrangement in PAX6 involving partial deletions of exons 3-5, including the initiator codon. Deletions of PAX6 are part of a contiguous gene deletion syndrome - Wilms tumor, aniridia, genitourinary anomalies, and intellectual disability syndrome - and therefore evaluation of the WT1 gene was necessary to rule out this life-threatening syndrome. CONCLUSION: This rare, complex rearrangement of multiple exons and deletions in PAX6 causing an isolated aniridia phenotype is probably the first reported case. The patient was managed by a multidisciplinary team and the guardians were counseled regarding the prognosis and complications.

Predicting Molecular Subtype and Survival of Rhabdomyosarcoma Patients Using Deep Learning of H&E Images: A Report from the Children's Oncology Group.

PURPOSE: Rhabdomyosarcoma (RMS) is an aggressive soft-tissue sarcoma, which primarily occurs in children and young adults. We previously reported specific genomic alterations in RMS, which strongly correlated with survival; however, predicting these mutations or high-risk disease at diagnosis remains a significant challenge. In this study, we utilized convolutional neural networks (CNN) to learn histologic features associated with driver mutations and outcome using hematoxylin and eosin (H&E) images of RMS. EXPERIMENTAL DESIGN: Digital whole slide H&E images were collected from clinically annotated diagnostic tumor samples from 321 patients with RMS enrolled in Children's Oncology Group (COG) trials (1998-2017). Patches were extracted and fed into deep learning CNNs to learn features associated with mutations and relative event-free survival risk. The performance of the trained models was evaluated against independent test sample data (n = 136) or holdout test data. RESULTS: The trained CNN could accurately classify alveolar RMS, a high-risk subtype associated with PAX3/7-FOXO1 fusion genes, with an ROC of 0.85 on an independent test dataset. CNN models trained on mutationally-annotated samples identified tumors with RAS pathway with a ROC of 0.67, and high-risk mutations in MYOD1 or TP53 with a ROC of 0.97 and 0.63, respectively. Remarkably, CNN models were superior in predicting event-free and overall survival compared with current molecular-clinical risk stratification. CONCLUSIONS: This study demonstrates that high-risk features, including those associated with certain mutations, can be readily identified at diagnosis using deep learning. CNNs are a powerful tool for diagnostic and prognostic prediction of rhabdomyosarcoma, which will be tested in prospective COG clinical trials.