bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutations in the Poly(A)-Specific Ribonuclease (PARN) Impair Telomerase RNA 3' End Maturation in Dyskeratosis Congenita Patients
Authors: Moon Diane H. , Segal Matthew , Boyraz Baris , Guinan Eva , Hofmann Inga , Cahan Patrick , Tai Albert K , Agarwal Suneet 
Published: October 2019

Abstract
Mutations in 11 genes have been described in patients with dyskeratosis congenita (DC) and related telomere diseases, and account for ~60% of cases. Amongst these, loss-of-function mutations in the poly(A)-specific ribonuclease (PARN) have most recently been found in patients with DC and idiopathic pulmonary fibrosis. PARN has no known role in telomere biology. Why PARN deficiency should mimic telomere diseases remains unclear. Based on its 7-methylguanylate (m7 G) cap recognition and poly(A) deadenylase functions, PARN's primary role is considered to be in regulating mRNA metabolism. Recent studies implicate PARN in non-coding RNA biogenesis, including maturation of small nucleolar RNAs (snoRNAs) via the deadenylation of oligo-adenylated (oligo(A)) intermediates. In two DC patients with biallelic defects in the PARN gene, we found decreased levels of the telomerase RNA component (TERC). TERC is the essential non-coding RNA template and scaffold of the telomerase holoenzyme. TERC possesses an m7 G cap but unlike mRNAs does not have a long poly(A) tail; rather, TERC contains a box H/ACA motif as is found in some snoRNAs. We hypothesized that TERC is regulated by PARN in a manner similar to snoRNAs, given their shared 3′ box H/ACA architecture. Here, usingsomatic cells and induced pluripotent stem (iPS) cells from DC patients with PARN mutations, we show that PARN is required for the 3′ end maturation and accumulation of TERC RNA. In PARN-mutant iPS cells and PARN-deficient cell lines, we find impaired telomerase activity and telomere maintenance. Deep sequencing of TERC 3' ends reveals that PARN is required for the removal of genomically-encoded extensions and post-transcriptionally acquired oligo(A) tails that target nuclear RNAs for destruction. In keeping with this, TERC transcripts from PARN-mutant patient cells decay at an accelerated rate. The diminished TERC steady-state levels, increased oligo(A) forms of TERC, and defects in telomere maintenance in patient cells are rescued by ectopic expression of PARN. Global transcript analysis by RNA-Seq in patient cells and PARN-deficient cell lines compared to controls reveals no protein coding mRNAs that consistently manifest a fold-change exceeding the change in TERC levels. Our data indicate that PARN functions in TERC biogenesis via deadenylation of oligo-adenylated nascent transcripts, which promotes 3' end maturation and stability. Given these findings and the phenotype of patients with PARN mutations, we speculate that a major, non-redundant function of PARN in human cells is regulating the maturation of TERC and other non-coding RNAs, more so than mRNA metabolism. Our results reveal a novel role for PARN in the biogenesis of TERC, and provide a mechanism linking PARN mutations to defective telomere maintenance in DC and related diseases.

Disclosures
No relevant conflicts of interest to declare.
The Fusion Gene Landscape in Multiple Myeloma, with Clinical Impact
Authors: Cleynen Alice , Szalat Raphaël , Samur Mehmet Kemal , Parmigiani Giovanni , Munshi Nikhil C. , Avet-Loiseau Hervé 
Published: October 2019

Abstract
Background: Gene fusions play an important role in aberrant cellular biology as well as development and progression of cancer. Expression of fusion genes such as PML-RAR drives the transformation in APL and provides important targets for therapy. However, in multiple Myeloma (MM), a heterogeneous disease characterized by genomic instability, frequent gains and losses of DNA, and a diverse mutational landscape, only the well characterized MMSET-IGH fusion product has been reported. Here we investigate the fusion gene landscape in multiple myeloma, and its possible impact on survival.
Method: Deep RNA-Seq was performed on purified MM cells from 430 newly-diagnosed MM patients, 20 normal individuals and 71 cell-lines; data were analyzed for gene expression profiles, long-non coding RNA signatures, and both novel and known fusion genes using two common algorithms: TopHat and MapSplice. MM characteristics, cytogenetic and FISH as well as clinical survival outcomes were also analyzed and correlated with genomic data.
Results: After filtering candidate fusions linking genes belonging to the same family, we identified 416 different candidates in myeloma patients, 40 % of which identified either IGH or Kappa as a partner. IGH fusion partners included the previously described and validated WHSC1 and B2M genes, as well as over 50 new candidates, while more than 70 different partners were found to be fused with Kappa. These genes exhibit functional enrichment of positive regulators of the cytokine-mediated signaling pathway, negative regulation of myeloid cell differentiation, negative regulation of interleukin-6 production, as well as others. 31% of patients presented no fusions, and another 32% presented a single fusion event. The other 37% presented at least 2 fusion candidates, with up to 27 different candidates. Similar patterns were observed in cell-lines, with 196 unique candidates identified, only 16% of which involving IGH or kappa. However, all partners were found in at least one patient as well. Only 12% of cell-lines exhibited no fusion, and another 14% presented only one fusion. On average, 3 fusions were identified per cell-line, with a maximum of 10. Validation of some these fusion genes is required to understand their functional role. Importantly, although having IgH-or kappa-related fusions did not affect patient outcome by themselves, patients with high numbers of fusion candidates had worse event-free survival.
Conclusion: Our data describes a diverse and rich fusion gene landscape in Multiple Myeloma. Similar to mutational profiles, there is no predominant fusion gene driving the disease process. Association of poor prognosis with a higher number of fusions may indicate that genomic instability plays an important role in the biology of Multiple Myeloma.

Disclosures
No relevant conflicts of interest to declare.
Mesenchymal Stem Cell As a Salvage Treatment for Patients with Refractory Bronchiolitis Obliterans Syndrome after Allogenetic Hematopoietic Stem Cell Transplantation
Authors: Zhao Ke , Fan Zhiping , Huang Fen , Xiang Peng , Liu Qifa 
Published: October 2019

Abstract
Background Bronchiolitis obliterans syndrome (BOS) is the most detrimental late pulmonary complications after allogeneic hematopoietic stem cell transplantation, as it is characterized by non-responsiveness to treatment, leading to high morbidity and mortality. Mesenchymal stem cell (MSC) has been considered as an effective treatment for refractory aGVHD, but the response to treat cGVHD, especially refractory BOS, is rarely reported. This study evaluated the efficacy of MSC from bone marrow (BM) of a third-party donor to BOS patients.
Methods Fifty-three patients with refractory BOS were enrolled in our prospective study, including 29 patients in MSC group and 24 patients in non-MSC group. All patients had previously failed to at least 2 lines of immunosuppressive therapy. MSCs were given at a median dose of 1×106 cells/kg once weekly for 4 weeks as one cycle treatment. The responsiveness of MSC was evaluated after one cycle, non-responsive patients discontinued MSC treatments, others continued to accept another cycle of MSC treatments.
Results Fifty-three patients developed BOS at a median time of 172 days (range, 94 to 398). After a total of 169 doses of MSC were administered, with a median of 6 (range:3-11) doses per patient, the overall response (OR) rate in MSC group was 75.9%, including CR in 13.8% and partial response (PR) in 62.1%. Compared with MSC group, OR rate in non-MSC group was 16.7%, including CR in 4.2% and PR in 12.5% (P=0.000; P=0.233; P=0.000, respectively). Furthermore, the efficacy of MSC to refractory BOS was significantly related with the severity of pulmonary function (mild VS severity, P=0.039). During the median follow-up time of 814 (range:228-2499) days post-transplantation, 7 patients were dead in MSC group, while 18 patients died in non-MSC group. The 5-year OS post-transplantation in MSC group were 68.6%±5.7%, compared with 21.2%±7.1% in non-MSC group (P=0.033). No patients experienced any toxic side effects and other secondary tumors after MSC treatment.
Conclusion MSC derived from BM of third-party donors is a promising treatment for patients with refractory BOS.

Disclosures
No relevant conflicts of interest to declare.
Hematopoietic Peripheral Circulating Blood Stem Cells As an Independent Marker of Good Transfusion Management in Patients with Beta-Thalassemia
Authors: Napolitano Mariasanta , Gerardi Calogera , Di Lucia Anna , Accardo Pietro Andrea , Rizzuto Luigi , Maria Ferraro , Tarantino Giuseppe , Siragusa Sergio , Buscemi Filippo 
Published: October 2019

Abstract
Aim Aim of the current study was to prospectively evaluate the potential role of peripheral circulating CD34+ stem cells as new independent marker of appropriate hemopoietic balance in patients with thalassemia major and intermedia.
Materials and methods Peripheral blood samples from patients with thalassemia major (TM) and intermedia (TI) were drawn. Peripheral circulating CD34+ stem cells, CF-GEMM, CFU-GM and BFU-GM were assayed with monoclonal antibodies for CD34 and clonogenic tests, according to standard procedures and ISHAGE method (BD stem cell enumeration kit, Becton Dickinson; H4434, Stem Cell Technology). Demographic and clinical data were recorded from each enrolled subject. Results were compared with healthy controls.
Results Overall, 56 patients with thalassemia major (median age:35 years, range:13-52 years) and 13 with thalassemia intermedia (median age:44 years, range:27-67) were evaluated. Annual red blood cells transfusion requirements ranged from 10 to 65 in all the patients except three with thalassemia intermedia, that were transfusion independent. One patient with TM did not accept transfusion for religion (Table 1). A statistically significant increase in peripheral circulating stem cells was observed in all the patients, in comparison with healthy controls. Mean peripheral circulating CD34+cells were 6.9±4.5/mmc in patients with TM and 11.8±14.8/mmc in patients with TI, significantly higher than mean CD34+ cells of healthy volunteer blood donors values (3.5±2.9/mmc, p=0.014 and p= 0.051 respectively for TM and TI). Only in patients with TI an increase in CFU-GEMM (3.0±4.8 vs 0.75±2.05, p=0.0001) was observed. Patients not treated with transfusions showed the mean highest levels of circulating stem cells (CD34: 32.5±14.8/mmc, BFU-E: 41.3±22.8, CFU-GM: 19.6±5.6, CFU-GEMM 9.0±6.1). At multivariate analysis, peripheral circulating CD34+ stem cells did not correlate with age, sex, number of red blood cells units transfused, hemoglobin levels, history of splenectomy and hypothyroidism.
Conclusion Circulating peripheral CD34+stem cells are increased in patients with the highest hemopoietic stress (TI, transfusion independent), thus their determination could represent a useful independent marker of clinical response to transfusion in patients with beta thalassemia.
Table 1. Baseline data of patients with thalassemia Thalassemia major (n=56) Thalassemia intermedia (n=13) Males 28 7 Females 28 6 Mean Age (range) 35 (13-52) 42(27-67) BMI (range) 21.5 (15.7-27.3) 21.4 (14.7-33.5) Splenectomy 31 13 Hypothyroidism 9 1 Smokers 32 31

Disclosures
No relevant conflicts of interest to declare.
First Case of Leukemia in a Child Suffering from Cyclic Neutropenia with ELANE Mutation
Authors: Zeidler Cornelia , Mellor-Heineke Sabine , Klimiankou Maksim , Skokowa Julia , Welte Karl 
Published: October 2019

Congenital neutropenia (CN) and Cyclic neutropenia (CyN) are rare hematological conditions in which ELANE mutations have been found. The discrimination of Cyn from CN is based on the cycling neutrophil counts which decrease to <0.2 x 109/L, usually at 21 day intervals. Infectious episodes are typically less severe in CyN compared to infections in CN patients. While in patients suffering from ELANE-CN an increased risk of leukemic transformation of more than 10 percent is well documented, no AML has been reported in the European SCNIR cohort of 38 patients with ELANE -CYN so far.
Here we report the first case of AML secondary to ELANE -CyN in a 17 year old girl. Severe neutropenia was first diagnosed at the age of four weeks during a septicemia with pseudomonas, but no serial blood counts were collected. G-CSF treatment was initiated at an age of 2 years after recurrent otitis media, pneumonia and skin abscesses. Infectious events occurred infrequently under G-CSF treatment. At an age of 14 years she was referred to us after a severe infectious event (liver abscess) and an unexplained high range of ANCs (0 to more than 10x109/µl) under G-CSF treatment with varying doses. Sequential blood counts confirmed the diagnosis of CyN. Molecular genetic testing revealed a heterozygous ELANE mutation, both parents are ELANE negative. Previous bone marrows varied between a maturation arrest on the promyelocyte stage with absence of neutrophils and up to 16 percent segmented neutrophils. No cytogenetic aberrations were detected prior to AML. AML was diagnosed recently when she presented with decreasing platelets and hemoglobin. Bone marrow at this time showed 14% blasts and presence of monosomy 7 plus trisomy 21 in the cytogenetic evaluation.
Molecular genetic evaluation of the bone marrow revealed presence of a CSFR3 and RUNX1 mutations in the leukemic cells.
Conclusion
Our patient presented with a secondary AML after ELANE positive cyclic neutropenia. Cytogenetics, CSFR3 and RUNX1 mutations in the bone marrow at the time of leukemia diagnosis have been described in the pathway of leukemogenesis in Congenital neutropenia with ELANE, HAX1 or other underlying mutation.
Our patient is the first case of leukemia in ELANE -CyN and proofs that ELANE -CyN is another pre-leukemic condition. However, the risk of myeloid transformation is very low.

Disclosures
No relevant conflicts of interest to declare.
DNA Editing: A Primer
Authors: Mali Prashant 
Published: October 2019

Abstract
RNA-guided Cas9 nucleases based on the prokaryotic CRISPR-Cas systems provide an unprecedented ease in being able to edit the genomes of diverse organisms. As a single unifying factor capable of co-localizing RNA, DNA, and protein, I believe tools and techniques based on Cas9 will grant exquisite control over cellular organization, regulation, and behavior. Here I will describe work on development of the CRISPR-Cas9 targeting methodology, and detail current and prospective genome-engineering methodologies.

Disclosures
No relevant conflicts of interest to declare.
Modified IPS Cells for Hemoglobinopathies
Authors: Townes Tim M. , Ding Lei , Chang Chia-Wei , Lai Yi-Shin , Sun Joe , Pawlik Kevin 
Published: October 2019

Abstract
We have recently used CRISPR-Cas to correct the HBB gene in induced Pluripotent Stem Cells (iPSC) derived from patients with Sickle Cell Disease (SCD) and to correct the JAK3 gene in iPSC derived from patients with Severe Combine Immune Deficiency (SCID). Off-target mutations were minimized, if not eliminated, by use of paired guide RNAs and the Cas9 nickase. When erythroid progenitors (EP) produced from corrected SCD iPSC are transplanted into NSG mice, a complete switch from gamma- to beta-globin gene expression occurs within 24 hours and high levels of beta mRNA are synthesized; the betaA:betaS ratio is 60:40. Hematopoietic progenitors produced from corrected SCID iPSC can be differentiated into T cell populations that express a full repertoire of T Cell Receptors (TCRs). These results suggest that CRISPR-Cas enhanced gene replacement may provide safe and effective therapies for many erythroid and lymphoid disorders. In an alternative approach, we electroporated a preformed, biochemical complex composed of a guide RNA (gRNA), a modified recombinant Cas9 (mrCas9) and a single stranded oligodeoxnucleotide (ssODN) into sickle iPSC or primary sickle CD34+ hematopoietic progenitors. Sixty-five percent of sickle iPSC colonies contained at least one corrected allele. When homozygous corrected colonies were differentiated into erythroid progenitors and transplanted into NSG mice, human erythroid cells expressed 100% HbA. When primary sickle CD34+ hematopoietic progenitors were electroporated with the gRNA/mrCas9/ssODN complex and differentiated into erythroid cells in vitro, HbA was expressed at 30-40%. These results suggest that primary bone marrow CD34+ cells that are isolated from patients with sickle cell disease, beta-thalassemia, SCID, DBA and other hematopoietic disorders may be electroporated with specific gRNA/mrCas9/ssODN complexes and transplanted into patients to rapidly, safely and effectively treat these debilitating disorders.

Disclosures
No relevant conflicts of interest to declare.
Engineering the Cancer Genome
Authors: Jacks Tyler 
Published: October 2019

Abstract
Over the past several years, my laboratory has employed gene targeting technology to create a series of mouse models of cancer that share genetic and pathological features of the cognate diseases in humans. We have focused particularly on models of lung cancer, both adenocarcinoma and small cell lung cancer. These models have been subjected to various types of molecular characterization, including mRNA profiling and exome/whole-genome sequencing. These efforts have defined genes and pathways that promote tumor progression and metastasis, including in the case of the adenocarcinoma model down-regulation of the lineage-restricted transcription factor Nkx2.1 and other transcriptional regulators that are involved in inducing and enforcing various differentiation states that might limit tumor progression. We have also studied the involvement of subpopulations of tumor cells in this model in which the Wnt signaling pathway is active. Data from lineage tracing experiments are consistent with this population having increased tumor propagating potential. In order to functionally characterize these and other cancer genes, we have recently developed methods to mutate genes in developing tumors using the CRISPR/Cas9 system. Using tri-functional lentiviral, we have developed tumors that have undergone Cas9-mediated mutation of a series of genes of interest. Several examples of the application of this method will be reviewed. As another example of cancer genome manipulation, we have engineered tumors of the lung and other sites to express specific T cell antigens and/or NK cell ligands. These models have allowed a detailed investigation of adaptive and innate immune responses to a developing tumor. We have used the models to explore mechanisms of immune suppression in cancer and develop methods to elicit improved anti-tumor immune responses.

Disclosures
No relevant conflicts of interest to declare.
New Approaches for Mapping Epigenome Dynamics
Authors: Henikoff Steven 
Published: October 2019

Abstract
The protein complexes that package our genomes must be mobilized for active processes to occur, including replication and transcription, but until recently we have only had a static, low resolution view of the "epigenome". Genomes are packaged into nucleosomes, octamers of four core histones wrapped by 147 base pairs of DNA. Nucleosomes present obstacles to transcription, which over genes is the RNA Polymerase II (RNAPII) complex, and one current challenge is to understand what happens to a nucleosome when RNAPII transcribes through the DNA that it occupies. We study this process by developing methods for following nucleosomes as they are evicted and replaced. Among the factors that we have implicated in the process is torsional stress, which we can now measure genome-wide. RNAPII movement can unwrap nucleosomes and thus destabilize them, causing them to be occasionally evicted and replaced. Interestingly, we find that destabilization of nucleosomes during transcription is enhanced by anthracycline compounds, widely used chemotherapeutic drugs that intercalate between DNA base pairs, thus suggesting a new mechanism for cell killing during chemotherapy. We are also interested in what happens to RNAPII during its encounter with a nucleosomes. In vitro, RNAPII cannot transcribe completely through a nucleosome, but rather stalls as it tries to unwrap the DNA from around the core. We have been studying this process in vivo, and have developed a simple method for precisely mapping RNAPII genome-wide. We have used this method to show exactly where RNAPII stalls as it unwraps a nucleosome in vivo, surprisingly in a different place in vivo from where it stalls in vitro. We also have discovered that a variant histone, H2A.Z, which is found in essentially all eukaryotes, helps to reduce the nucleosome barrier to transcription, and in this way may modulate transcription. Other protein components of the epigenome involved in dynamic processes are nucleosome remodelers, which use the energy of ATP to slide or even evict nucleosomes from DNA. Some remodelers help RNAPII get started and others help it overcome the nucleosome barrier to transcription, and by mapping them at base-pair resolution, we can gain insight into how they act. We have also applied our high-resolution mapping tools to transcription factors, which bind DNA at specific sites to regulate transcription and other processes. Our ability to achieve high spatial and temporal resolution mapping of the binding and action of nucleosomes, transcription factors, remodelers and RNAPII provides us with a detailed picture of epigenome dynamics. By using these tools we are beginning to understand how DNA sequence and conformation are recognized for regulation of transcription and other epigenomic processes.

Disclosures
No relevant conflicts of interest to declare.
Noncellular Niche Influences: Nervous System Mediators, Metabolic Mediators, and Hypoxia/Oxidative Stress
Authors: Méndez-Ferrer Simón 
Published: October 2019

Hematopoietic stem cells (HSCs) traffic between bone marrow and circulation, what allows for life-saving clinical transplantation. Our previous work has shown that HSC numbers in blood follow circadian oscillations that are regulated by the central pacemaker in the brain, which reaches bone marrow nestin+ mesenchymal stem cells through peripheral sympathetic nerves. In the perinatal bone marrow, HSC-niche forming mesenchymal stem cells might be different from those that form the skeleton and some of them might be neural crest-derived, like peripheral neurons and supporting glial cells. Thus, tight regulation of the bone marrow stem-cell niche in vertebrates might build upon developmental relationships of its cellular components. We have found recently that cholinergic nerves regulate HSC maintenance, proliferation and migration in divergent niches. We will present unpublished evidence of how both branches of the autonomic nervous system cooperate to regulate HSC maintenance and function in spatially and temporally distinct niches. Moreover, we have shown recently that damage to this regulatory network is essential for the manifestation of myeloproliferative neoplasms. In these diseases, previously thought to be driven solely by mutated HSCs, protecting the HSC niche might represent a novel therapeutic strategy. Patients with myeloproliferative neoplasms have a higher risk of developing acute leukemia. However, at this stage, leukemic cells might be less sensitive to the normal control by the microenvironment and, instead, acute myelogenous leukemic cells might transform the bone marrow niches to support their own survival. We will discuss potential contributions of HSC niches to myeloproliferative neoplasms and MLL-AF9-driven acute myeloid leukemia.

Disclosures
Off Label Use: Potential use of selective estrogen receptor modulators and beta3-adrenergic agonists in myeloproliferative neoplasms.