PAX5 Research Articles

Frequency and Correlation of Common Genes Copy Number Alterations in Childhood Acute Lymphoblastic Leukemia with Prognosis.

Objective:It was shown by genomic profiling that despite no detectable chromosomal abnormalities a proportion of children with pre-B acute lymphoblastic leukemia harbors copy number alterations (CNA) of genes playing role in B-cell development and function. The aim of the study was to determine the frequency of CNA in pediatric acute lymphoblastic leukemia and correlate these findings with clinical outcome. Methods:DNA extracted from peripheral blood or bone marrow at diagnosis/relapse of fifty newly diagnosed children with precursor B-cell acute lymphoblastic leukemia was analyzed for CNA with multiplex ligation-dependent probe amplification. Results:The analysis revealed 76 CNA in 24 patients most frequently found in PAR1 (17%), CDKN2A/B (15.7%) and PAX5 (14.4%) genes. There were significant CNA co-occurrences between PAX5, CDKN2A/B, BTG1, ETV6, PAR1 or XP22 genes, (p<0.020) and the high-risk group. There was a significant correlation between EBF1, RB1, and IKZF1 alterations and bone marrow relapse. Patients with CNA in screened genes are more likely to succumb to their disease except for those with PAR1 or XP22 genes (p<0.050). Conclusion:The multiplex ligation-dependent probe amplification could be considered as an independent diagnostic tool allowing prompt identification of patients at high risk of treatment failure and, subsequently, a more adequate treatment approach.

Dynamical modeling predicts an inflammation-inducible CXCR7+ B cell precursor with potential implications in lymphoid blockage pathologies.

BackgroundThe blockage at the early B lymphoid cell development pathway within the bone marrow is tightly associated with hematopoietic and immune diseases, where the disruption of basal regulatory networks prevents the continuous replenishment of functional B cells. Dynamic computational models may be instrumental for the comprehensive understanding of mechanisms underlying complex differentiation processes and provide novel prediction/intervention platforms to reinvigorate the system.MethodsBy reconstructing a three-module regulatory network including genetic transcription, intracellular transduction, and microenvironment communication, we have investigated the early B lineage cell fate decisions in normal and pathological settings. The early B cell differentiation network was simulated as a Boolean model and then transformed, using fuzzy logic, to a continuous model. We tested null and overexpression mutants to analyze the emergent behavior of the network. Due to its importance in inflammation, we investigated the effect of NFkB induction at different early B cell differentiation stages.ResultsWhile the exhaustive synchronous and asynchronous simulation of the early B cell regulatory network (eBCRN) reproduced the configurations of the hematopoietic progenitors and early B lymphoid precursors of the pathway, its simulation as a continuous model with fuzzy logics suggested a transient IL-7R+ ProB-to-Pre-B subset expressing pre-BCR and a series of dominant B-cell transcriptional factors. This conspicuous differentiating cell population up-regulated CXCR7 and reduced CXCR4 and FoxO1 expression levels. Strikingly, constant but intermediate NFkB signaling at specific B cell differentiation stages allowed stabilization of an aberrant CXCR7+ pre-B like phenotype with apparent affinity to proliferative signals, while under constitutive overactivation of NFkB, such cell phenotype was aberrantly exacerbated from the earliest stage of common lymphoid progenitors. Our mutant models revealed an abnormal delay in the BCR assembly upon NFkB activation, concomitant to sustained Flt3 signaling, down-regulation of Ebf1, Irf4 and Pax5 genes transcription, and reduced Ig recombination, pointing to a potential lineage commitment blockage.DiscussionFor the first time, an inducible CXCR7hi B cell precursor endowed with the potential capability of shifting central lymphoid niches, is inferred by computational modeling. Its phenotype is compatible with that of leukemia-initiating cells and might be the foundation that bridges inflammation with blockage-related malignancies and a wide range of immunological diseases. Besides the predicted differentiation impairment, inflammation-inducible phenotypes open the possibility of newly formed niches colonized by the reported precursor. Thus, emergent bone marrow ecosystems are predicted following a pro-inflammatory induction, that may lead to hematopoietic instability associated to blockage pathologies.

REGULATION OF OVIDUCT HOMEOSTASIS AND FERTILITY BY Pax2 AND Pax8 GENES

In mouse and humans, the development regulatory genes Pax2 and Pax8 are expressed in ovarian duct surface epithelia, yet the function of these genes and proteins in adult females remains unclear. By Generating double & single KO mouse modules of Pax2 and Pax8 we investigate their roles in maintaining the integrity of the oviductal cells. Our studies directly address aspects of oviduct epithelial homeostasis and the effects on fertility. To create Pax2, Pax8, and Pax2/8 double mutants specifically in oviduct epithelia and characterize changes in gene expression, epithelial cell integrity, and fertility. We will implement detailed analyses of oviducts isolated at various times post tamoxifen administration from mice with conditional Pax2, Pax8, or both alleles, using the Ovigp1-CreER driver. Mice will also carry reporter allele (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo) to mark all active Cre expressing cells with cell surface EGFP. In addition to defining novel functions for Pax proteins in the oviduct, we will utilize state-of-the-art methods for single cell analyses, transcriptomics, and chromatin remodeling. Mutation of Pax2 and Pax8 in adult female oviducts results in significant changes in gene expression and morphology. Loss of both Pax2 and Pax8 resulted in infertility (Table 1). To date, most of the preliminary data was obtained from Pax2/8 double mutants since we were concerned with redundancy and expected the most significant phenotypes in the double mutants. Analyses of histology, immunohistochemistry and changes in gene expression of total RNA from whole oviducts isolated after tamoxifen administration. For a rapid first pass screen, we compared controls (Pax2f/f;Pax8f/f) to single Pax mutants and Pax2/8 double mutants (Pax2f/f;Pax8f/f; Ovigp1-CreER) by Affymetrix microarrays. These data show hundreds of changes in gene expression levels upon Pax2 or Pax8 deletion, with significant overlap between the Pax2 and Pax8 mutants but also expression changes unique to each single mutant.Tabled 1TABLE 1. Fertility of Oviduct Specific Pax MutantsGenotype # females # plugs Litters Litter sizePax2f/f, Pax8f/f, Ovi-Cre 5 15 0 0Pax2f/f, Pax8f/f, 4 12 12 6-9Pax8f/f, Ovi-Cre 3 6 2 6-7 Open table in a new tab Little is known regarding the mechanisms underlying epithelial homoeostasis, the proteins that determine cell fates, and epithelial integrity in the adult oviduct. Pax8 expressing secretory cells are thought to give rise to ciliated cells, which help move the oocyte down the duct and Pax2 is associated with cilia motility and physiology. Using a conditional KO model of Pax2 & Pax8 will allow us to understand those mechanisms and identify future novel therapeutic targets for diagnostics and treatments

3065 – ETV6-RUNX1 ENHANCES THE SELF-RENEWAL OF HEMATOPOIETIC STEM CELLS AND CORRUPTS EARLY LYMPHOID DIFFERENTIATION

One-third of pediatric cancer patients are diagnosed with acute leukemia. Within this patient cohort, acute lymphoblastic leukemia (ALL) is by far the most predominant, accounting for ∼80% of the cases. Approximately 25% of B-cell ALL cases are caused by the ETV6-RUNX1 (TEL-AML1) translocation, a somatic translocation that emerges in utero. However, the ETV6-RUNX1 fusion gene is insufficient for leukemogenesis, and secondary events/hits are always required for transformation. Previous studies have proposed hematopoietic stem cells as potential targets for ETV6-RUNX1-associated ALL; however, an appropriate experimental model to answer this question definitively has been lacking. Here, we generated a mouse model in which ETV6-RUNX1 can be conditionally activated in any primary cell type of choice upon Tetracycline treatment. In line with previous studies, we observed an expansion of phenotypically defined HSCs and a block in B-cell differentiation upon ETV6-RUNX1 induction. With the benefits offered by our model, in which we can turn off the expression of ETV6-RUNX1, we were able to confirm that the candidate HSCs that expand in response to ETV6-RUNX1 are both phenotypically and functionally similar to normal HSCs. Moreover, when coupled with transheterozygous mutations in the Pax5 and Ebf1 genes, an established murine model system for B-ALL, ETV6-RUNX1 significantly accelerated B-ALL leukemia development. Through our ongoing work, we aim to unravel how ETV6-RUNX1 modulates gene expression to promote HSC expansion and leukemia development. It is our anticipation that these approaches will provide new insights on the biology that underlie the most common ALL translocation found in children.

IDENTIFICACIÓN DE ALTERACIONES MOLECULARES EN PACIENTES VENEZOLANOS CON DIAGNÓSTICO DE LEUCEMIA LINFOBLÁSTICA AGUDA

Acute Lymphoblastic Leukemia (ALL) is the most common neoplasm in pediatric age. In recent years, between 15 and 20% of patients failed in their treatments. Knowledge on cytogenetics and molecular biology has an important impact on the determination of the prognosis and the appropriate treatment scheme. In Venezuela there is limited knowledge regarding the molecular genetics of this onco-hematological alteration. The aim of this work was to evaluate the most frequent genetic alterations in Venezuelan patients with a clinical diagnosis of acute lymphoblastic leukemia. A cross-sectional, descriptive and prospective study was carried out from 2006 to 2014, in which the translocations ETV6/RUNX1, MLL/AF4, TCF3/PBX1, BCR/ABL1, as well as mutations in the PAX5 and FLT3 genes were evaluated through the use of different types of PCR. One hundred and thirty patients with a clinical diagnosis of acute lymphocytic leukemia were included in the study. Molecular alterations were identified in 56 patients (43.1%), in which we observed the presence of one or several alterations in conjunction in the same patient. The alterations identified were t(12; 21) (11.5%), t(4; 11) (8.5%), t(1; 19) (10%), t(9; 22) (20.8%), ITD-FLT3 (14.8%), P80S mutation (4.2%) and S77del (4.2%) in the PAX5 gene. The prevalence of BCR/ABL is one of the highest described so far in cases of ALL where most of the population is made up of pediatric patients. These results represent the first molecular study of ALL in Venezuela, laying the foundations for the diagnosis and monitoring of the disease in its population. Key words: Acute Lymphoblastic Leukemia; Translocations; ETV6/RUNX1; MLL/AF4; TCF3/PBX1; BCR/ABL1; PAX5; FLT3.

Variable Expression of Notch1 and Pax5 in Classical Hodgkin Lymphoma and Infection with Epstein-Barr in Pediatric Patients.

NOTCH1 and PAX5 participate in the proliferation and differentiation of B and T lymphocytes. Their expression can be modified by activation of NOTCH1, induced by the Epstein–Barr (EBV) viral proteins identified as LMP1 and LMP2. To identify whether PAX5, NOTCH1, and EBV latency genes participate in the oncogenic process of pediatric patients with classical Hodgkin lymphoma (cHL), the present study aimed to identify the variable expression of NOTCH1 among disease subtypes and to assess its effect on PAX5 expression. A total of 41 paraffin-embedded tissues from Mexican pediatric patients with cHL were analyzed. The expression of CD30, CD20, NOTCH1, PAX5, and LMP1 was evaluated by immunohistochemistry and immunofluorescence. EBV detection was performed by in situ hybridization. Out of all cases, 78% (32/41) of the cHL cases were EBV positive. NOTCH1 expression was detected in 78.1% (25/32) of EBV-positive cases, nodular sclerosis being the most frequent subtype (11/25, 44%). In cases where the expression of both genes was identified, double immunofluorescence assays were conducted, finding no colocalization. We found that Reed–Sternberg cells had aberrant expression compared to their cells of origin (B lymphocytes) due to the molecular mechanisms involved in the loss of expression of PAX5 and that the identification of NOTCH1 could be considered as a candidate diagnostic/prognostic marker and a therapeutic target in pediatric cHL.

Quercetin treatment reduces the severity of renal dysplasia in a beta-catenin dependent manner.

Renal dysplasia, the major cause of childhood renal failure, is characterized by defective branching morphogenesis and nephrogenesis. Beta-catenin, a transcription factor and cell adhesion molecule, is markedly increased in the nucleus of kidney cells in human renal dysplasia and contributes to its pathogenesis by altering target genes that are essential for kidney development. Quercetin, a naturally occurring flavonoid, reduces nuclear beta-catenin levels and reduces beta-catenin transcriptional activity. In this study, we utilized wild type and dysplastic mouse kidney organ explants to determine if quercetin reduces beta-catenin activity during kidney development and whether it improves the severity of renal dysplasia. In wild type kidney explants, quercetin treatment resulted in abnormal branching morphogenesis and nephrogenesis in a dose dependent manner. In wild type embryonic kidneys, quercetin reduced nuclear beta-catenin expression and decreased expression of beta-catenin target genes Pax2, Six2, and Gdnf, which are essential for kidney development. Our RDB mouse model of renal dysplasia recapitulates the overexpression of beta-catenin and histopathological changes observed in human renal dysplasia. RDB kidneys treated with quercetin resulted in improvements in the overall histopathology, tissue organization, ureteric branching morphogenesis, and nephrogenesis. Quercetin treatment also resulted in reduced nuclear beta-catenin and reduced Pax2 expression. These improvements were associated with the proper organization of vimentin, NCAM, and E-cadherin, and a 45% increase in the number of developing and maturing nephrons. Further, our results show that in human renal dysplasia, beta-catenin, vimentin, and e-cadherin also have abnormal expression patterns. Taken together, these data demonstrate that quercetin treatment reduces nuclear beta-catenin and this is associated with improved epithelial organization of developing nephrons, resulting in increased developing nephrons and a partial rescue of renal dysplasia.

Genetic predisposition to childhood cancer: A retrospective cohort analysis in a multidisciplinary genetic cancer counseling Unit.

e13643 Background: The incidence of childhood cancer in Spain is 18, 2 cases per 100.000 inhabitants per year. The presence of predisposing genetic factors has been revealed in approximately 10% of patients, in them the detection of genetic mutations associated is essential in order to establish the correct clinical management of patients and their relatives. However, genetic counseling in this population is currently underestimated since, not all patients with pediatric malignancies have a family history of cancer or other clinical signs to suspect and inheritance syndrome. Methods: The Unit for Predisposition to Child and Adolescent Cancer of the University Hospital La Paz in Madrid, Spain, is composed by the Units of Clinical Oncology, Pediatric Hemathology-Oncology, and Genetics. Inclusion criteria are based on the characteristics of the individual's neoplasia, family characteristics and other phenotypic characteristics.The molecular study is carried out through Next Generation Sequencing, Direct Sequencing and / or MLPA. In the present study, a retrospective and descriptive analysis of the cases studied from September 2018 to December 2019 was carried out. Results: The Pediatric Cancer Predisposition Functional Unit of the La Paz University Hospital attended during the years 2018 and 2019 a 58 patients with suspected predisposition syndromes to childhood cancer belonging to 41 families. The following inheritance syndromes have been identified: hereditary retinoblastoma in 14 families with mutation in the RB1 gene, Li Fraumeni syndrome in 1 family with mutation in the TP53 gene, multiple endocrine neoplasia type 2A by RET gene mutation in 1 family, 1 hereditary melanoma syndrome with a CDKN2A mutation, and inheritance family leukemia in 1 family with a PAX5 gene mutation. Other cases studied have been negative-non informative, with the following neoplasms of the index cases: unilateral retinoblastoma, hepatoblastoma, hamartomatous colon polyposis, lymphoma, leukemia and soft tissue sarcoma. In addition to the molecular diagnosis of the index case, both cases and family members received genetic counseling and adequate clinical management, adapted to each syndrome. Conclusions: The multidisciplinary and specialized approach improves the diagnosis and monitoring of patients with suspected syndromes of predisposition to childhood cancer and allows the establishment of prevention strategies and early diagnosis individualized to each family.

Characterization of new circular RNA products from the Pax-5 gene in B-cells

Abstract Circular RNAs (circRNAs) represent a new class of RNAs which are highly expressed and conserved in mammalian cells. CircRNAs regulate many biological processes and their aberrant expression is often associated with disease progression. We have identified and characterized circRNAs generated from the Pax-5 gene, an essential transcription factor for B-cell lineage commitment and also, a potent oncogene. Given the pivotal role of Pax-5 gene products in B-cell development and function, we set out to profile Pax-5 circular RNA (circPax5) expression and elucidate their role in B-cell biology. We identify and map five major circPax5 transcript isoforms which are found in B-cell lines and primary B-cells isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. These circPax5 variants consists of circular single-stranded RNA sequences corresponding to: i) exons 2–3; ii) exons 2–4; iii) exons 2–5; iv) exons 2–6; and, v) exons 2–8 of the human Pax-5 gene. Using TaqMan assays designed to target each unique circPax5 junction region, we found that circPax5 expression profiles vary with B-cell developmental stages. Furthermore, circPax5 exons 2–5 and 2–6 are overexpressed in patients suffering from chronic lymphocytic leukemia (CLL). Functionally, we observe that circPax5 products modulate B-cell processes and interact by sponging of specific micro-RNAs. Altogether, we identify new genetic products (i.e. circRNAs) generated from a prominent immune-regulating gene (Pax-5). The functional elucidation of circPax5 products will potentially bring insight into the function and signaling of B-cell processes and disease.

Allelic Variants of PAX5 and MEF2C-AS2 Genes are Associated with Depression in Temporal Lobe Epilepsy (4185)

Allelic Variants of PAX5 and MEF2C-AS2 Genes are Associated with Depression in Temporal Lobe Epilepsy (4185)

Comprehensive chromosomal aberrations in a case of a patient with TCF3-HLF-positive BCP-ALL

BackgroundThe use of high-throughput analytical techniques has enabled the description of acute lymphoblastic leukaemia (ALL) subtypes. The TCF3-HLF translocation is a very rare rearrangement in ALL that is associated with an extremely poor prognosis. The TCF3-HLF fusion gene in the described case resulted in the fusion of the homeobox-related gene of TCF3 to the leucine zipper domain of HLF. The TCF3-HLF fusion gene product acts as a transcriptional factor leading to the dedifferentiation of mature B lymphocytes into an immature state (lymphoid stem cells). This process initiates the formation of pre-leukaemic cells. Due to the rarity of this chromosomal aberration, only a few cases have been described in the literature. The advantage of this work is the presentation of an interesting case of clonal evolution of cancer cells and the cumulative implications (diagnostic and prognostic) of the patient’s genetic alterations.Case presentationThis work presents a patient with diagnosed with TCF3-HLF-positive ALL. Moreover, the additional genetic alterations, which play a key role in the pathogenesis of ALL, were detected in this patient: deletion of a fragment from the long arm of chromosome 13 (13q12.2-q21.1) containing the RB1 gene, intragenic deletions within the PAX5 gene and NOTCH1 intragenic duplication.ConclusionsA patient with coexistence of chromosomal alterations and the TCF3-HLF fusion has not yet been described. Identifying all these chromosomal aberrations at the time of diagnosis could be sufficient to determine the cumulative effects of the described deletions on the activity of other oncogenes or tumour suppressors, as well as on the clinical course of the disease. On the other hand, complex changes in the patient’s karyotype and clonal evolution of cancer cells call into question the effectiveness of experimental therapy.

Analysis of multi-lineage gene expression dynamics during primordial germ cell induction from human induced pluripotent stem cells

BackgroundIn mammals, specification of primordial germ cells (PGCs) is established in the early post­implantation embryo. The bone morphogenetic protein (BMP)-SMAD and WNT3-β-catenin signaling initiate the gene regulatory network for PGC specification. The activation of SOX17-BLIMP1 axis is critical for human PGC program. Moreover, EpCAM and INTEGRINα6 were identified as surface markers of human PGC-like cells (PGCLCs) recently. However, the signaling mechanism for PGC specification in non­rodent mammals remains to be clarified.MethodsWe differentiated human induced pluripotent stem cells (hiPSCs) into PGCLCs in vitro in response to Activin A and BMP4. The percentage of EpCAM/INTEGRINα6 double-positive cells (PGCLCs) was analyzed by flow cytometry. The expression of PGC genes was evaluated by qRT-PCR and immunofluorescence. The expression dynamic of multi-lineage genes during the differentiation process was evaluated by qRT-PCR.ResultsUnder the stimulation for PGCLC induction, the embryoids derived from hiPSCs initiated significant upregulation of the early PGC genes (BLIMP1, TFAP2C, and NANOS3), but maintained low or no levels of DPPA3 and late PGC genes (DAZL and DDX4). The percentage of EpCAM/INTEGRINα6 double-positive PGCLCs reached the highest at day 6 of induction. After pre-induction, the incipient mesoderm-like cells (iMeLCs) upregulated most of the mesoderm genes (EOMES, T, MSXI, RUNX2, and MIXL1). The differentiating embryoids showed high levels of key pluripotency genes, OCT4 and NANOG, but became negative for SOX2. In contrast to iMeLCs, the differentiating embryoids downregulated mesoderm genes RUNX2 and EOMES, and ectoderm gene PAX6, but increased the expression of endoderm gene SOX17.ConclusionsDuring PGCLC induction process in vitro, the differentiating embryoids not only activated the PGC-related genes, but also displayed complex regulation of pluripotency genes and multi-lineage genes. These results would be meaningful for future research investigating the regulation of human early germ line development.

Behavioural response of different epithelial tissues of sea cucumber (Apostichopus japonicus) to light and differential expression of the light-related gene Pax6

ABSTRACT In this study, we evaluated the behavioural characteristics of the epithelial tissues of sea cucumber (Apostichopus japonicus) subjected to illumination. The behavioural results indicated that the tentacles, tube feet, and spines were sensitive to light with structural changes. Among the three epithelial tissues, the tentacles were the most sensitive with the shortest reaction and recovery times, whereas the tube feet and spines had longer reaction times. Quantitative real-time PCR (q-PCR) was used to analyse the changes of Pax6 expressions before and after illumination in the tentacles, tube feet and spines. Q-PCR results indicated that Pax6 expressions in the three above tissues after illumination were higher than that before illumination. We hypothesised that sea cucumber may not have independent photoreceptors, but the epithelial tissues were capable of exhibiting a behavioural response to light. This will allow us to re-examine the functions of all epithelial tissues of sea cucumbers.

Multi-channel collection of G-quadruplex transducers for amplified signaling of Pax-5 based on target-triggered split-to-intact remodeling of dual-G-rich duplex probe

Nucleic acids-sensing methods provide the potential to enable and hasten early disease detection and to rationalize treatments, especially for cancers. In this work, a colorimetric label-free sensing system based on target-triggered split-to-intact remodeling of dual-G-rich duplex probe (Dual-GDP) has been developed for amplified signaling of Pax-5 gene. In this sensing system, the Dual-GDP is splited into one hairpin-structured G-rich probe (HGP) and one duplex-shaped G-rich probe (DGP), which can be re-built to an intact form through target gene triggered accurate base-pairing. Significantly, this unique split-to-intact remodeling behavior allows the occurrence of a well-designed active isothermal amplification that can amplify target response with seven stages to multi-channel accumulate numbers of G-quadruplex transducers. When specifically combined with hemin, the formed G-quadruplex-hemin complexes, also known as DNAzymes, can strongly catalyze the oxidization of TMB to a colored TMB+ and robustly reflect the target gene even in complex biological environments. We demonstrate that this method offers simplified operations and improved assay abilities for Pax5 gene analysis. We envision these findings might provide new insights to exploit versatile oligonucleotide probes and nucleic acid-based amplification strategies for diverse bioanalytical applications.

Modeling ocular lens disease in Xenopus.

Ocular lens clouding is termed as cataract, which depending on the onset, is classified as congenital or age-related. Developing new cataract treatments requires new models. Thus far, Xenopus embryos have not been evaluated as a system for studying cataract. We characterized the developmental process of lens formation in Xenopus laevis tailbuds and tadpoles, and we disrupted the orthologues of three mammalian cataract-linked genes in F0 by CRISPR/Cas9. We assessed the consequences of gene inactivation by combining external examination with histochemical analyses and functional vision assays. Inactivating the key metazoan eye development transcription factor gene pax6 produces a strong eye phenotype including an absence of eye tissue. Inactivating the genes for gap-junction protein and a nuclease, gja8 and dnase2b, produces lens defects that share several features of human cataracts, including impaired vision acuity, nuclei retention in lens fiber cells, and actin fibers disorganization. We tested the potential improvement of the visual acuity of gja8 crispant tadpoles upon treatment with the molecular chaperone 4-phenylbutyrate. Xenopus is a valuable model organism to understand the molecular pathology of congenital eye defects, including cataracts, and to screen molecules with a potential to prevent or reverse cataracts.

The Role of Genetic Mutations on Genes PAX2 &amp; PAX6 in Cyclopia Syndrome

Cyclopia syndrome typically coexists with holoprosencephaly. This ocular lesion may present as complete ocular fusion in a single orbit or as two eyes in a single orbit. Eye defects linked with cyclopia syndrome include colobomas (gaps) in the iris, retina, and optic nerve; inconstant optic nerve numbers (either one or two is possible), and an absent or abnormal optic chiasm. Cyclopia syndrome is caused by mutations in the PAX2 and PAX6 genes. Cyclopia syndrome occurs when the rostral (anterior) portion of the notochord and adjacent mesoderm are deficient in mass. This shortage leads to the aberrant induction of the forebrain tissues followed by severe derangement of midline facial development.

The role of genetic mutations on genes PAX2 &amp; PAX6 in cyclopia syndrome

The role of genetic mutations on genes PAX2 &amp; PAX6 in cyclopia syndrome

Liquid Biopsy Is Useful to Identify the Genetic Profile of NHL-B at Diagnosis in Different Histological Subtypes

Introduction: Non-Hodgkin B lymphomas (NHL-B) are a large group of heterogeneous diseases, with well-defined, histological and clinical features. Currently, the lymph node biopsy is essential for diagnosis, often obtained from hard to reach areas. Our aims with this study was to analyze genetics variants at diagnosis trying to discriminate between different NHL-B histologic subtypes using formalin fixed paraffin embedded (FFPE) tissue sections and circulating tumor free DNA (ctDNA) samples. Material and Methods: This is a retrospective, cross-sectional, single-center study. Sixty patients were selected with a diagnosis of different NHL-B subtypes: Diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal lymphoma (ML), mantle cell lymphoma (MCL) and plasmablastic lymphoma (PL). Sixty FFPE tissue samples and 23 ctDNA specimens obtained at diagnosis were selected. We performed an enrichment panel of 54 genes recurrently mutated in lymphomas (Lymphoma Solution; Sophia Genetics) by next generation sequencing (NGS; NextSeq; Illumina). The depth of 90% of the readings was greater than 2600x. Quantitative variables were expressed as median and range. Categorical variables were expressed as frequency and percentage. The Fisher exact test was used to compare the distribution of categorical variables. The Mann-Whitney test was used to compare differences between quantitative variables. Statistical significance was set at p &lt; 0.05. Results: Our study shows that 97% (58/60) of patients presented any variant. The most frequently mutated genes were KMT2D, EP300 and SOCS1. Exclusive variants were detected in FL in the genes CCND1, PAX5, CREBBP, BCL2 and REL genes. In the same way, in DLCBL the variants detected in the BRAF, TCF3, XPO1, PIM1, CHD2, ID3 and BCL6 genes were exclusive of this subtype. Furthermore, the genes BCL2 (p=0.0021), CREBBP (p=0.0004), NOTCH2 (p=0.03), PAX5 (p=0.01) and TNFRSF14 (p=0.04) are more frequently altered in patients with FL, ATM (p=0.02) gene in MCL; NFKBIE (p=0.03), CIITA (p=0.02), MYC (p=0.009) and PIM1 (p=0.04) in DLCBL, among which it was found that high-grade lymphomas are more frequently associated with mutations in CHD2 (p=0.02), MYC (p=0.03) and MYD88 (p=0.02; data not shown). In the subgroup of 23 patients with paired samples (FFPE/ctDNA), 95% (22/23) of patients had mutations in any of the genes on the panel in FFPE tissue samples and 82% (19/23) in ctDNA specimens. The number of variants detected was different depending on the type of sample analysed, 52% of the variants were detected in both specimens, 39 only in FFPE tissue and 9% in ctDNA; Figure 1). In variants detected in both samples, the average value of the VAF was higher that when is detected only in one of the samples (FFPE: 28.1 vs 5.8 (p&lt;0.00001); ctDNA: 8.7 vs. 2.3 ((p&lt;0.0009); Figure 2). Patients with MCL presented lower number of variants and primary mediastinal lymphoma presented higher number of variants compared with the others subtypes (Table 1). We did not find significant differences in the number of variants depending on the stage, classification, tumour burden or bulky mass (Table 1). Conclusion: In our study, we were able to identify genetic variables that could characterize different histological groups of NHL-B in FFPE tissue and ctDNA samples, by using a commercial gene panel of NGS. The mutational profile obtained from ctDNA and FFPE tissue is comparable in all histological subtypes, regardless tumour burden, aggressiveness or stage, throwing each one of them complemental information. These results support the hypothesis that could be possible to distinguish different histologies through non-invasive strategies, specially oriented to lesions where obtaining a tissue sample is difficult. We are working on the development of clinical-genetic algorithms that allow us to achieve our goal. Disclosures No relevant conflicts of interest to declare.

Pre-Clinical Efficacy of the Novel Kinase Inhibitor Nintedanib on PAX5 Fusion Genes in Pediatric Ph-like B-Cell Precursor Acute Lymphoblastic Leukemia

Despite the current risk-based stratification protocol, 15% of pediatric patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) still experience relapse. In the large subset of 'B-other' patients (negative for common fusion transcripts, non-high hyperdiploid and non-Down Syndrome), 'Ph-like' is a high risk subgroup with high incidence of relapses, which represents 30% of B-others or 10-15% of BCP-ALL patients. The PAX5 gene, encoding for a B-cell related transcription factor, is frequently involved in several translocations in Ph-like patients, determining the formation of fusion genes encoding for aberrant proteins. Our previous studies showed that PAX5 fusions sustain survival of leukemic cells by Lymphocyte kinase (LCK) hyperactivation, which can be targeted by the kinase inhibitor Nintedanib/BIBF1120. This study aims (i) to unravel the functional features of PAX5 fusion genes, elucidating the involved signaling pathways; (ii) to develop new pre-clinical strategies to target PAX5 fusion genes, testing the efficacy of the LCK inhibitor BIBF1120. We selected a cohort of 138 B-other cases among 400 childhood BCP-ALL cases enrolled in Italy in AIEOP-BFM ALL2000/R2006 protocols; by gene expression analysis (HG-U133Plus2.0 Affymetrix array), 59/138 presented as Ph-like, and by NGS, a driver fusion gene was identified in 34/59 (58%). Beside known genetic lesions, such as P2RY8/CLRF2 (N=9), EBF1 fusions (N=3), TCF3/HLF (N=1), IKZF1-fusion (N=1) and BCL9/MEF2D (N=1), and single cases with novel fusions, seven cases were carrying a PAX5 fusion gene, representing the most frequent rearrangement. Ex-vivo inhibition with BIBF1120 was setup on primary cells from 5 PAX5-fusion BCP-ALL samples, demonstrating significant efficacy in inducing leukemic cell apoptosis, both as single agent and in combination with standard chemotherapeutic agents (Annexin V viability assay of leukemic cells in co-culture on human bone marrow stroma). Strikingly, in 3/5 cases dexamethasone and BIBF1120 had a synergistic effect and were further tested in in vivo assays. A daily treatment was performed at bulk disease detection (BM aspiration, mean engraftment 20% CD10/CD19+) in patient-derived xenotransplanted NSG mice from 2 different PAX5 fusion cases. At the endpoint (after two weeks), in the PAX5/AUTS2 PDX mice we detected a mild effect in the BM by BIBF1120 alone (disease reduction 24%, p=0.057), further enhanced in combination with dexamethasone (-49%, p=0.005, with a mean engraftment in vehicle mice of 82.6%). In the spleen, the efficacy was highly significant both for BIBF1120 (-52%, p=0.025) and the combination (-91%, p=0.015, mean engraftment vehicle 69.5%). A similar statistical significant effect was observed also in peripheral blood, whilst, BIBF1120 alone showed a specific significant efficacy in CNS meninges. Analogous results have been observed in PDX from the PAX5/DACH2 PDX mice; BM leukemia decreased 47% using BIBF1120 alone (p=0.004), further diminished by the combination (-65%, p=0.0004, with a mean engraftment in vehicle mice of 65%). In the spleen, the efficacy was highly significant both with BIBF1120 (-45.6%, p=0.04) and for the combination (-96.3%, p=0.0008, mean engraftment vehicle 72.4%). Strikingly, BIBF1120 treatment alone showed analogous efficacy in PB and CNS, with leukemia decrease as low as -45% (p=0.04) and -76% (p=0.007), respectively, and the combination nearly achieved remission in PB (-94%, p=0.0001) and it was significant in CNS (-81.2%, p=0.03). Dexamethasone alone was not effective in the BM and spleen, whereas it decreased the leukemia bulk both in PB (-65%, p=0.0004) and CNS (-52.8%, p=0.03). Overall, BIBF1120 treatment was more effective than dexamethasone. Interestingly, phosphoflow analysis showed a marked inhibition by BIBF1120 of pAkt-Thr308 and its downstream effectors, such as pS6 and 4pEBP1, in ex vivo BM and spleen cells. In conclusions, PAX5 fusion genes are highly recurrent among Ph-like patients and can potentially be targeted by Nintendanib/BIBF1120, that showed a significant effect ex vivo and in vivo, even as a single agent. Disclosures No relevant conflicts of interest to declare.

Folate deficiency induced H2A ubiquitination to\xa0lead to downregulated expression of genes involved in neural tube defects

BackgroundNeural tube defects (NTDs) are common congenital malformations resulting in failure of the neural tube closure during early embryonic development. Although it is known that maternal folate deficiency increases the risk of NTDs, the mechanism remains elusive.ResultsHerein, we report that histone H2A monoubiquitination (H2AK119ub1) plays a role in neural tube closure. We found that the folate antagonist methotrexate induced H2AK119ub1 in mouse embryonic stem cells. We demonstrated that an increase in H2AK119ub1 downregulated expression of the neural tube closure-related genes Cdx2, Nes, Pax6, and Gata4 in mouse embryonic stem cells under folate deficiency conditions. We also determined that the E3 ligase Mdm2 was responsible for the methotrexate-induced increase in H2AK119ub1 and downregulation of neural tube closure-related genes. Surprisingly, we found that Mdm2 is required for MTX-induced H2A ubiquitination and is recruited to the sites of DSB, which is dependent on DNA damage signaling kinase ATM. Furthermore, folic acid supplementation restored H2AK119ub1 binding to neural tube closure-related genes. Downregulation of these genes was also observed in both brain tissue of mouse and human NTD cases, and high levels of H2AK119ub1 were found in the corresponding NTDs samples with their maternal serum folate under low levels. Pearson correlation analysis showed a significant negative correlation between expression of the neural precursor genes and H2AK119ub1.ConclusionOur results indicate that folate deficiency contributes to the onset of NTDs by altering H2AK119ub1 and subsequently affecting expression of neural tube closure-related genes. This may be a potential risk factor for NTDs in response to folate deficiency.