Aberrant mRNA Transcripts Research Articles

P1521: A SEVERE MOUSE MODEL OF ALPHA-THALASSEMIA SHOWS ABNORMAL IRON METABOLISM, ERYTHROPOIESIS AND COAGULATION, AND CAN BE RESCUED BY A NOVEL GENE THERAPY APPROACH

Background: Clinical presentation of deletional a-thal varies from an asymptomatic condition (one inactivated a-globin gene) to a complete knockout (Hb Bart’s Hydrops Fetalis). In patients with severe a-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation. Aims: The aims of this study are to develop a novel adult mouse model of a-thal and a gene therapy approach for this disease. Methods: We generated adult animals that do not produce a-globin chains (a-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. To generate a gene therapy tool, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human a-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human a-globin genes. Results: The a-KO animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only b-globin chains (HbH). RBC show aberrant morphology and aggregation of b-globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events, associated with neutrophil infiltration, fibrinogen staining, von Willebrand factor (vWF) released, platelet recruitment and activation. These animals also show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney. We identified ALS20a, a vector where a-globin is under control of the b-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous a-globin at a level comparable to that produced by one endogenous a-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Use of ALS20a resulted in correction of the disease phenotype in a dose-dependent manner in a-KO mice. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels. We tested ALS20a in CD34 cells isolated from four patients with both deletional and non-deletional HbH disease. We measured the change of b/a-globin mRNA ratio (b/aR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial b/aR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a b/aR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced a-thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2). Image:Summary/Conclusion: We generated an adult mouse model of lethal a-thal and, in preliminary experiments, we rescue it with ALS20a. Furthermore, ALS20a successfully improves a-globin levels in patient cells.

A Severe Mouse Model of Alpha-Thalassemia to Study Abnormal Iron Metabolism and Erythropoiesis, Hematopoietic Stem Cell Behavior and Development of a Gene Therapy Approach for Its Treatment

Alpha thalassemia (α-thal) is caused by insufficient production of the α-globin protein because of either deletional or non-deletional inactivation of endogenous α-globin genes. Clinical presentation of deletional α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to a complete knockout (Hb Bart's Hydrops Fetalis). In patients with severe α-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation.The aims of this study are to develop a novel adult mouse model of α-thal and a gene therapy approach for this disease.We generated adult animals that do not produce α-globin chains (α-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. These animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only β-globin chains (HbH). RBC show aberrant morphology and aggregation of α- globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events. These animals show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney.Interestingly, α-KO embryos show lower numbers of FLC compared to WT embryos, lower frequency of engraftable hematopoietic stem cells (HSC; Lin-Sca-1+c-kit+CD48-), decreased clonogenic potential (fewer class 4 CFUs) and elevated erythroferrone. Lethally irradiated mice transplanted with FLC-KO require 5-6x as many cells as those transplanted with FLC-WT for recovery, further suggesting some level of engraftment impairment. Our current hypothesis is that excessive hypoxia in the embryos impairs HSC function and stem cell fitness. Additional assays are in progress to assess the nature of this impairment.To generate a gene therapy tool to rescue these animals and eventually cure severe human α-thal patients, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human α-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human α-globin genes. We identified ALS20α, a vector where α-globin is under control of the β-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous α-globin at a level comparable to that produced by one endogenous α-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Transplantation of ALS20α transduced murine BM-KO results in correction of the disease phenotype in a dose-dependent manner. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels.We tested ALS20α in CD34 cells isolated from four patients with both deletional and non- deletional HbH disease. We measured the change of β/α-globin mRNA ratio (β/αR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial β/αR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a β/αR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced α−thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2).In conclusion, we generated an adult mouse model of lethal α-thal and, in preliminary experiments, we rescue it with ALS20α. Furthermore, ALS20α successfully improves α-globin levels in patient cells. Further experiments are in progress to establish the consistency of our vector's expression in vivo, as well as to demonstrate its ability to transduce bona fide long-term HSCs. DisclosuresKattamis: Agios Pharmaceuticals: Consultancy; IONIS: Consultancy; VIFOR: Consultancy; CRISPR/Vertex: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Chiesi: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy. Rivella: Celgene Corporation: Consultancy; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Incyte: Consultancy; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Drug Repurposing Patent Applications April–June 2021

Drug Repurposing Patent Applications April–June 2021

2018 AHA Late-Breaking Basic Science Abstracts.

2018 AHA Late-Breaking Basic Science Abstracts.

Mis-splicing of the GALNS gene resulting from deep intronic mutations as a cause of Morquio a disease

BackgroundMucopolysaccharidosis-IVA (Morquio A disease) is a lysosomal disorder in which the abnormal accumulation of keratan sulfate and chondroitin-6-sulfate is consequent to mutations in the galactosamine-6-sulfatase (GALNS) gene. Since standard DNA sequencing analysis fails to detect about 16% of GALNS mutant alleles, gross DNA rearrangement screening and uniparental disomy evaluation are required to complete the molecular diagnosis. Despite this, the second pathogenic GALNS allele generally remains unidentified in ~ 5% of Morquio-A disease patients.MethodsIn an attempt to bridge the residual gap between clinical and molecular diagnosis, we performed an mRNA-based evaluation of three Morquio-A disease patients in whom the second mutant GALNS allele had not been identified. We also performed sequence analysis of the entire GALNS gene in two patients.ResultsDifferent aberrant GALNS mRNA transcripts were characterized in each patient. Analysis of these transcripts then allowed the identification, in one patient, of a disease-causing deep intronic GALNS mutation. The aberrant mRNA products identified in the other two individuals resulted in partial exon loss. Despite sequencing the entire GALNS gene region in these patients, the identity of a single underlying pathological lesion could not be unequivocally determined. We postulate that a combination of multiple variants, acting in cis, may synergise in terms of their impact on the splicing machinery.ConclusionsWe have identified GALNS variants located within deep intronic regions that have the potential to impact splicing. These findings have prompted us to incorporate mRNA analysis into our diagnostic flow procedure for the molecular analysis of Morquio A disease.

PTGIS is a Novel Susceptibility Gene for Pulmonary Arterial Hypertension

Background: Genetic variants such as inactivating mutation in the bone morphogenetic protein receptor 2 (BMPR2) gene account for approximately 15-20% of the etiology of idiopathic pulmonary arterial hypertension (IPAH). Increasing evidence indicates the existence of other IPAH susceptibility genes. Methods: The whole-genome sequencing was performed in a discovery cohort including 42 IPAH patients who are lacking BMPR2 mutations. Putative IPAH genes were validated in an independent cohort that included 188 IPAH patients and 968 healthy controls. Functional assessments were conducted to analyze the effects of candidate genetic variants on transcript splicing, enzymatic activity and function. Results: An enrichment of rare variants in the gene encoding prostacyclin synthase (PTGIS) was initially identified by the whole-genome sequencing. The association of PTGIS rare variants with IPAH was confirmed in the replication cohort. In total, fourteen patients (6.1%) were heterozygous for three PTGIS rare variants, including one splice-site variant (c.521 1G>A) and two missense variants (R252Q, A447T). These rare variants conferred a 7.8-fold greater risk of IPAH (95% confidence interval (CI), 3.2 to 18.8; P=5 x 10-6). Inhaled iloprost induced more significantly decrease of pulmonary vascular resistance (P=0.001) and increase of cardiac index (P A variant resulted in aberrant mRNA transcripts. The functional studies showed that the two missense variants caused a decrease in prostacyclin production and increased cell death in pulmonary microvascular endothelial cells. Conclusions: We have identified three loss-of-function rare variants in the PTGIS gene from two independent IPAH cohorts. The genetic variants of PTGIS predispose pulmonary vascular responses to the iloprost stimulation and cause deleterious effects. These results provide a novel finding that PTGIS rare variants are involved in the pathogenesis of IPAH. Funding: The work was supported by the following grants: the Beijing Natural Science Foundation (7181009, 7172180), the National Natural Science Foundation of China (81320108005), the National Science Fund for Distinguished Young Scholars (81425002), CAMS Innovation Fund for Medical Sciences (2016-I2M-1-002, 2016-I2M-4-003), the National Key Research and Development Program of China (2016YFC0901502), Sanming Project of Medicine in Shenzhen-Professor Zhi-Cheng Jing's Group for Precise Medicine Research in Thrombosis and Vascular Disease (SZSM201502001) , and the fund from State Key Laboratory of Cardiovascular Disease (2016-kf04, 2018ZR-02). Declaration of Interest: The authors declare no competing financial interests. Ethical Approval: The study protocol was approved by the ethics committees of Shanghai Pulmonary Hospital and FuWai Hospital. The participants, who were all of Chinese Han ancestry, provided written informed consent.

Complete androgen insensitivity syndrome caused by c.1769-1G > C mutation and activation of a cryptic splice acceptor site in the androgen receptor gene

Androgen insensitivity syndrome (AIS) is the most common cause of 46,XY disorders of sex development (46,XY DSD). This syndrome is an X-linked recessive genetic disease characterized by resistance to the actions of androgens in an individual with a male karyotype and it is caused by mutations in the androgen receptor (AR) gene. We evaluated two siblings with primary amenorrhea, normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the AR gene revealed a splice acceptor site mutation in intron 2 (c.1769-1G > C). The analysis of mRNA showed that this mutation resulted in the activation of a cryptic splice acceptor site located in intron 2 and in the synthesis of an aberrant mRNA transcript with 69 nucleotides insertion between exon 2 and exon 3, leading to an insertion of 23 amino acids in the AR protein instead of generating a premature termination codon. The additional 23 amino acids insertion affects AR intracellular trafficking by impairing its translocation from the cytoplasm to the nucleus after hormone stimulation. The c.1769-1G > C mutation provides new insights into the molecular mechanism involved in splicing defects and expands the spectrum of mutations associated with the androgen insensitivity syndrome.

Role of the uridine/cytidine kinase 2 mutation in cellular sensitiveness toward 3'-ethynylcytidine treatment of human cancer cells.

A nucleosidic medicine, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine [3'-ethynylcytidine (ECyd)], is a potent inhibitor of RNA polymerase I and shows anticancer activity to various human solid tumors in vitro and in vivo. ECyd is phosphorylated to 3'-ethyntlcytidine 5'-monophosphate by uridine/cytidine kinase 2 (UCK2) and subsequently further to diphosphate and triphosphate (3'-ethyntlcytidine 5'-diphosphate, 3'-ethyntlcytidine 5'-triphosphate). 3'-Ethyntlcytidine 5'-triphosphate is an active metabolite that can inhibit RNA polymerase I competitively, causing cancer cell death. Here, to identify the UCK2 mutation for detecting responder or nonresponder to ECyd, we investigated the relationship between point mutation of the UCK2 gene and response to ECyd in various human solid tumors. We identified several functional point mutations including the splice-site mutation of the UCK2 gene IVS5+5 G>A. In addition, we found that the IVS5+5 G>A variant generates an aberrant mRNA transcript, namely, truncated mRNA was produced and normal mRNA levels were markedly decreased in the ECyd-resistant cancer cell line HT1080. We concluded that these findings strongly suggest that the IVS5+5 G>A variant would affect the expression level of the UCK2 transcript, resulting in decreased sensitivity to ECyd.

RNA sequencing of synaptic and cytoplasmic Upf1-bound transcripts supports contribution of nonsense-mediated decay to epileptogenesis

The nonsense mediated decay (NMD) pathway is a critical surveillance mechanism for identifying aberrant mRNA transcripts. It is unknown, however, whether the NMD system is affected by seizures in vivo and whether changes confer beneficial or maladaptive responses that influence long-term outcomes such the network alterations that produce spontaneous recurrent seizures. Here we explored the responses of the NMD pathway to prolonged seizures (status epilepticus) and investigated the effects of NMD inhibition on epilepsy in mice. Status epilepticus led to increased protein levels of Up-frameshift suppressor 1 homolog (Upf1) within the mouse hippocampus. Upf1 protein levels were also higher in resected hippocampus from patients with intractable temporal lobe epilepsy. Immunoprecipitation of Upf1-bound RNA from the cytoplasmic and synaptosomal compartments followed by RNA sequencing identified unique populations of NMD-associated transcripts and altered levels after status epilepticus, including known substrates such as Arc as well as novel targets including Inhba and Npas4. Finally, long-term video-EEG recordings determined that pharmacologic interference in the NMD pathway after status epilepticus reduced the later occurrence of spontaneous seizures in mice. These findings suggest compartment-specific recruitment and differential loading of transcripts by NMD pathway components may contribute to the process of epileptogenesis.

P2.03b-042 MET exon 14 Mutations Encode a Hyperactive Kinase and Therapeutic Target in Lung Adenocarcinoma: Topic: Biomarkers

Activation of the MET tyrosine kinase receptor can drive oncogenesis and metastasis in certain cancer types. Somatic mutations at or around the splice junctions for MET exon 14 (METΔ14) are a recurrent mechanism of MET activation. METΔ14 mutations lead to aberrant messenger RNA (mRNA) splicing resulting in a METΔ14 protein lacking the juxtamembrane domain. We analyzed RNAseq data across 12 cancer types to define the prevalence of METΔ14 in solid tumors. We explored the driver role of METΔ14 both in vitro and in vivo. Finally, we explored acquired resistance mechanisms in a patient treated with crizotinib. The METΔ14 mutation and aberrant mRNA transcript are most common in lung adenocarcinoma and also identified in other cancers. Endogenous levels of METΔ14 transcript transform human epithelial lung cells in an HGF-dependent manner. This allele also induces lung cancer in mice that is responsive to a clinically available MET inhibitor. Finally, we document clinical response to crizotinib in a patient with a METΔ14-driven NSCLC lung cancer. Upon clinical progression on crizotinib, indicating acquired resistance, we detected acquired MET mutations in cell-free DNA from the patient that converge on critical drug-binding residues in the MET activation loop. These findings qualify METΔ14 mutations as drivers of lung adenocarcinoma, demonstrate the utility of a new animal model of MET-driven disease and identify a subpopulation of patients who may benefit from further development of targeted MET/HGF therapies.

Abstract 2124: Analysis of the functional relevance of novel alternative splicing events in non-small cell lung cancer

Abstract Lung cancer is the most common cause of death by cancer worldwide. Deregulation of alternative splicing (AS) is becoming increasingly relevant for the understanding of the molecular mechanisms of lung tumor progression. Genome-wide microarrays containing probes in exons and junctions can be used to identify differential AS events. We developed an algorithm, called ExonPointer, which was optimized to detect AS events using this kind of platforms. Using this technique, we successfully detected splicing events regulated by the oncogene serine/arginine-rich splicing factor 1 (SRSF1) in lung cancer cell lines. Moreover, four of these genes, ATP11C, IQCB1, TUBD1 and PRRC2C, showed a significantly different pattern of AS in primary non-small cell lung cancer (NSCLC) compared with normal lung tissue. In the case of PRRC2C, we also demonstrated that the deregulation of the splicing event was important for tumor progression. We next applied the same technique to target differential AS events in NSCLC primary tissues (13 lung squamous cell carcinomas and 9 lung adenocarcinomas). Their paired normal lung tissues were used as reference. The validation rate for the top 20 events identified by ExonPointer was 70%. Gene cluster analyses showed an enrichment of clusters implicated in cancer development such as Cellular Growth and Proliferation, or Cell Death and Survival. Among others, we observed accumulation of a tumor-specific splice variant in Mitogen-Activated Protein Kinase 9 (MAPK9 or JNK2). The tumor-specific variant for this gene contains an alternative exon 2 which causes a frame shift introducing a premature stop codon. Functional analyses using isoform-specific siRNAs showed that this variant did not affect tumor growth or progression. However, its expression significantly correlated with the expression of Regulator of Nonsense Transcripts 3A and B (UPF3A and UPF3B) in 42 lung cancer cell lines. These proteins belong to the Nonsense Mediated Decay (NMD) pathway, which eliminates aberrant mRNA transcripts, suggesting that the presence of some aberrant AS variants in lung cancer are caused by a malfunction of NMD. In conclusion, we have developed a reliable tool for the high throughput analysis of AS, and have identified new splicing events in lung cancer that can be studied as potential diagnostic markers and therapeutic targets. Citation Format: Fernando J. de Miguel, Ravi D. Sharma, Pablo Reclusa, María J. Pajares, Angel Rubio, Ruben Pio, Luis M. Montuenga. Analysis of the functional relevance of novel alternative splicing events in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2124. doi:10.1158/1538-7445.AM2015-2124

Genetic Testing Confirmed the Early Diagnosis of X-Linked Hypophosphatemic Rickets in a 7-Month-Old Infant.

Loss-of-function mutations in the phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) have been causally associated with X-linked hypophosphatemic rickets (XLHR). The early diagnosis of XLHR in infants is challenging when it is based solely on clinical features and biochemical findings. We report a 7-month-old boy with a family history of hypophosphatemic rickets., who demonstrated early clinical evidence of rickets, although serial biochemical findings could not definitively confirm rickets. A sequencing assay targeting the PHEX gene was first performed on the mother’s DNA to screen for mutations in the 5′UTR, 22 coding exons, and the exon-intron junctions. Targeted mutation analysis and mRNA studies were subsequently performed on the boys’ DNA to investigate the pathogenicity of the identified mutation. Genetic screening of the PHEX gene revealed a novel mutation, c.1080-2A>C, at the splice acceptor site in intron 9. The detection of an aberrant mRNA transcript with skipped (loss of) exon 10 establishes its pathogenicity and confirms the diagnosis of XLHR in this infant. Genetic testing of the PHEX gene resulted in early diagnosis of XLHR, thus enabling initiation of therapy and prevention of progressive rachitic changes in the infant.

RNA and protein expression of HLA-A∗23:19Q

The assignment of null alleles is clinically relevant in stem cell transplantation, in particular for donor selection. It is unclear how questionable (Q) alleles, having an unknown expression profile, should be considered in matching criteria. In this study we analyzed the RNA and protein expression profile of a questionable allele encountered in a sample of the Guadeloupe population: GD23Q, HLA-A∗23:19Q, 29:02:01. Full-length DNA sequencing of HLA-A∗23:19Q revealed a single polymorphism at position 619 (G>A) compared to HLA-A∗23:01:01. Serological typing showed only the presence of HLA-A29; HLA-A∗23:19Q was not detected on the cell surface. The absence of HLA-A∗23:19Q surface expression was shown by flow cytometry using a directly labeled monoclonal antibody and a panel of five indirectly labeled polyclonal antibodies all directed against HLA-A23 (HLA-A9) molecules. Allele specific amplification revealed the absence of intact full-length mRNA, but the presence of two major alternatively spliced mRNAs: sequencing identified that in one variant exon 3 is missing and in the other variant introns 2 and 3 are retained. Based upon the lack of HLA-A∗23:19Q surface expression and the presence of aberrant mRNA transcripts only, this study shows that HLA-A∗23:19Q is non-expressed.

Multifactorial Likelihood Assessment of BRCA1 and BRCA2 Missense Variants Confirms That BRCA1:c.122A&gt;G(p.His41Arg) Is a Pathogenic Mutation

Rare exonic, non-truncating variants in known cancer susceptibility genes such as BRCA1 and BRCA2 are problematic for genetic counseling and clinical management of relevant families. This study used multifactorial likelihood analysis and/or bioinformatically-directed mRNA assays to assess pathogenicity of 19 BRCA1 or BRCA2 variants identified following patient referral to clinical genetic services. Two variants were considered to be pathogenic (Class 5). BRCA1:c.4484G> C(p.Arg1495Thr) was shown to result in aberrant mRNA transcripts predicted to encode truncated proteins. The BRCA1:c.122A>G(p.His41Arg) RING-domain variant was found from multifactorial likelihood analysis to have a posterior probability of pathogenicity of 0.995, a result consistent with existing protein functional assay data indicating lost BARD1 binding and ubiquitin ligase activity. Of the remaining variants, seven were determined to be not clinically significant (Class 1), nine were likely not pathogenic (Class 2), and one was uncertain (Class 3).These results have implications for genetic counseling and medical management of families carrying these specific variants. They also provide additional multifactorial likelihood variant classifications as reference to evaluate the sensitivity and specificity of bioinformatic prediction tools and/or functional assay data in future studies.

Identification and expression of a novel MDM4 splice variant in human glioma

The product of the MDMX (or MDM4) gene is structurally related to the MDM2 oncoprotein and is also capable of interacting with the tumor suppressor protein p53. The MDM4 gene is overexpressed in several human tumors, while its product can be detected as various isoforms. This study was aimed to find the presence of aberrant mRNA transcripts of MDM4 in human glioma and their association with the clinicopathological characteristics of glioma patients. 42 glioma tissues were examined for MDM4 mRNA splicing variants by RT-PCR. A total of four distinct transcript sizes (full length-MDM4 851bp, MDM4-S 783bp, MDM4-A 701bp, MDM4-B 540bp) were detected. In the present study, we first report the novel alternative splicing form of MDM4, MDM4-B (GenBank accession no.KC479043.1). Expression of MDM4-B was present in various stages of human gliomas, but no significant correlation between presence of MDM4-B and malignancy of glioma was observed. The expression level of MDM4-B mRNA detected by real-time PCR was not only significantly associated with tumor stages, but also with p53 mutation and Ki-67 status which are important clinical molecular markers of glioma. Our data indicate that the novel variant MDM4-B may play a role in glioma tumorigenesis or cancer progression.

Mutations in the interleukin receptor IL11RA cause autosomal recessive Crouzon‐like craniosynostosis

We have characterized a novel autosomal recessive Crouzon-like craniosynostosis syndrome in a 12-affected member family from Antakya, Turkey, the presenting features of which include: multiple suture synostosis, midface hypoplasia, variable degree of exophthalmos, relative prognathism, a beaked nose, and conductive hearing loss. Homozygosity mapping followed by targeted next-generation sequencing identified a c.479+6T>G mutation in the interleukin 11 receptor alpha gene (IL11RA) on chromosome 9p21. This donor splice-site mutation leads to a high percentage of aberrant IL11RA mRNA transcripts in an affected individual and altered mRNA splicing determined by in vitro exon trapping. An extended IL11RA mutation screen was performed in a cohort of 79 patients with an initial clinical diagnosis of Crouzon syndrome, pansynostosis, or unclassified syndromic craniosynostosis. We identified mutations segregating with the disease in five families: a German patient of Turkish origin and a Turkish family with three affected sibs all of whom were homozygous for the previously identified IL11RA c.479+6T>G mutation; a family with pansynostosis with compound heterozygous missense mutations, p.Pro200Thr and p.Arg237Pro; and two further Turkish families with Crouzon-like syndrome carrying the homozygous nonsense mutations p.Tyr232* and p.Arg292*. Using transient coexpression in HEK293T and COS7 cells, we demonstrated dramatically reduced IL11-mediated STAT3 phosphorylation for all mutations. Immunofluorescence analysis of mouse Il11ra demonstrated specific protein expression in cranial mesenchyme which was localized around the coronal suture tips and in the lambdoidal suture. In situ hybridization analysis of adult zebrafish also detected zfil11ra expression in the coronal suture between the overlapping frontal and parietal plates. This study demonstrates that mutations in the IL11RA gene cause an autosomal recessive Crouzon-like craniosynostosis.

MicroRNA 125 represses nonsense-mediated mRNA decay by regulating SMG1 expression

Nonsense-mediated mRNA decay (NMD) is a cellular response mechanism that eliminates aberrant mRNA transcripts and thereby prevents the production of potentially deleterious C-terminally truncated proteins. The phosphatidylinositol 3-kinase-related protein kinase SMG1 is considered to be an essential factor in the NMD pathway. We demonstrate that the brain-enriched microRNA, miRNA-125 (miRNA-125a and miRNA-125b) is a bona fide negative regulator of SMG1 in humans. Down-regulation of SMG1 expression is mediated by miRNA-125 binding to a microRNA response element in the 3′ untranslated region of SMG1 mRNA, which leads to degradation of the SMG1 mRNA. In human cells, overexpression of miR-125 represses the endogenous levels of SMG1 protein and suppresses the NMD pathway; however, knockdown of miR-125 up-regulates the NMD pathway. These results suggest the existence of an RNA circuit linking the microRNA and NMD pathways.

Branchio-Oto-Renal Syndrome (BOR) associated with focal glomerulosclerosis in a patient with a novel EYA1 splice site mutation

BackgroundBranchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial, ear, and renal anomalies. The most common gene mutated in BOR patients is EYA1, the human homolog of the Drosophila eyes absent gene, while mutations in SIX1 gene, the human homolog of sine oculis, encoding a DNA binding protein interacting with EYA1, have been reported less frequently. Recently, mutations in another SIX family member, SIX5, have been described in BOR patients, however, this association has not been confirmed by other groups.Case presentationIn this study, we have clinically and genetically characterized a proband that displayed hearing loss, pre-auricular pits, branchial fistulae, hypoplasia of the left kidney, bilateral mild hydronephrosis, progressive proteinuria and focal glomerulosclerosis. Mutational analysis of EYA1 gene revealed a novel splice site mutation, c.1475 + 1G > C, that affects EYA1 splicing and produces an aberrant mRNA transcript, lacking exon 15, which is predicted to encode a truncated protein of 456 aa.ConclusionThis report provided the functional description of a novel EYA1 splice site mutation and described for the first time a case of BOR syndrome associated with the atypical renal finding of focal glomerulosclerosis, highlighting the importance of molecular testing and detailed clinical evaluation to provide accurate diagnosis and appropriate genetic counselling.

Next-Generation Sequencing Reveals Deep Intronic Cryptic ABCC8 and HADH Splicing Founder Mutations Causing Hyperinsulinism by Pseudoexon Activation

Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ~10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease.

The role of mammalian poly(A)-binding proteins in co-ordinating mRNA turnover

The function of cytoplasmic PABPs [poly(A)-binding proteins] in promoting mRNA translation has been intensively studied. However, PABPs also have less clearly defined functions in mRNA turnover including roles in default deadenylation, a major rate-limiting step in mRNA decay, as well as roles in the regulation of mRNA turnover by cis-acting control elements and in the detection of aberrant mRNA transcripts. In the present paper, we review our current understanding of the complex roles of PABP1 in mRNA turnover, focusing on recent progress in mammals and highlighting some of the major questions that remain to be addressed.