bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Characterization of Hemophilia and Implications for Novel Therapies
Authors: Lillicrap David 
Published: October 2019

Abstract
Abstract SCI-9
The genes encoding factor VIII (FVIII) and factor IX (FIX) were cloned 25 years ago. Since then, substantial translational application of this knowledge has been witnessed. First, genetic analysis of hemophilia has enabled a detailed understanding of the types and patterns of mutation responsible for these conditions. This knowledge has provided important biological insights into the mechanisms underlying mutation generation and has also formed the basis for precise molecular diagnosis for hemophilia carriers and potentially affected fetuses. In addition, the hemophilic genotype is the best-characterized and strongest predictor for the development of inhibitor generation in previously untreated patients. The second benefit deriving from the genetic characterization of the hemophilias has been the production of recombinant coagulation factor concentrates. Over the past 20 years, the use of recombinant factor concentrates has increased dramatically and we are currently witnessing a flurry of activity to develop second-generation, enhanced concentrates. The major objective of these projects is to facilitate clotting factor prophylaxis through the production of concentrates whose half-lives are extended, thus reducing the frequency of factor administration. These endeavors are utilizing a variety of approaches, but most are focused on either chemical modification of the factors through polymer conjugates or the generation of fusion proteins with immunoglobulin or albumin to take advantage of the recycling function of the neonatal Fc receptor. The first wave of these new products is now undergoing early phase clinical studies, and while substantial benefits for FIX half-life extension appear achievable, FVIII half-life modification may be more challenging. Finally, molecular genetic knowledge of the hemophilias has resulted in the pursuit of strategies to utilize genetic approaches to effect long-term “cures” of the disease. These initiatives have resulted in several small phase I/II trials of viral vector-based gene transfer but have also formed the basis of mutation-specific therapies such as a translational read-through approach being undertaken in patients with nonsense mutations. Currently, two phase I/II trials are in progress with liver-directed AAV FIX gene transfer in which two different AAV serotypes are being evaluated. These studies follow on from two previous AAV clinical trials in which the major obstacle to clinical benefit appears to have been the host immune response to the vector. There is considerable interest in the outcomes of these new studies.

Disclosures:
No relevant conflicts of interest to declare.
Proton Pump Inhibitor Use and the Ability to Replete Iron Stores
Authors: Huang Alina M , Ho Christine M , Reagan John L. , Winer Eric 
Published: October 2019

Abstract
Abstract 1039

Background:
Oral iron absorption occurs in the duodenum and requires increased gastric acidity to allow iron to remain in the more soluble, ferrous form. Many patients with iron deficiency anemia who require oral iron repletion are on proton pump inhibitors (PPIs) for management of various conditions that require acid suppression for treatment. In vitro studies suggest that inhibition of gastric acid secretion decreases the bioavailability of iron.
In patients with established iron deficiency anemia who are on PPIs, there is a paucity of data regarding the efficacy of iron replacement with concomitant PPI use. Sharma et al. published a case report on 2 patients who were iron deficient and on PPI therapy (Sharma et. al. Southern Medical Journal 2004). Their findings showed that discontinuation of the PPI resulted in improvement of iron deficiency anemia on the same dose of oral iron replacement. Additionally, Hutchinson et al. demonstrated that in individuals with hereditary hemachromatosis, those on PPIs had a significant reduction in phlebotomy requirements needed to keep serum ferritin ∼50μg (Hutchinson et. al. Gut 2007).
We conducted a retrospective study to assess whether a difference exists in terms of iron repletion between patients on a PPI versus those not on a PPI.

Methods:
Patients were selected from the medical primary care clinic from charts dating January 2000 until January 2009. Included patients were adults older than 18 years of age, a diagnosis of iron deficiency based on ferritin < 30 with at least one follow up ferritin value, and recipients of oral iron replacement. Excluded patients were those with chronic active bleeding and anemia of chronic disease. Indices examined include iron (Fe), total iron binding capacity (TIBC), ferritin, transferrin saturation, hemoglobin (Hb), hematocrit (Hct), and mean corpuscular volume (MCV). The primary endpoint of interest is repletion of iron stores defined by ferritin > 30. Those who were able to replete their iron stores are termed “responders” and those who were not are termed “nonresponders”.

Results:
Forty-five patients have been identified who have met the above criteria. Of these patients, 14 were on a PPI and 31 were not on a PPI. Of the patients not on a PPI, 38.7% responded to oral iron. Of the patients on a PPI, 50.0% responded to oral iron. The average initial and final values of indices of interest were compared between the groups (Table 1).

Discussion:
Our data suggests that patients both on and off PPIs show the ability to replete their iron stores. Furthermore, it appears that a slightly greater percentage of patients on a PPI compared with those not on a PPI are able to replete their iron stores, though the numbers remain small. Most importantly, these findings challenge the commonly held belief that PPI use impairs oral iron absorption. A prospective study is warranted to further confirm these observational data.

Disclosures:
No relevant conflicts of interest to declare.
Dosed Deficiency of Iron Restricts Terminal Maturation and Enucleation of Cultured Human Erythroblasts
Authors: Byrnes Colleen , Lee Y.Terry , Meier Emily Riehm , Rabel Antoinette , Miller Jeffery L. 
Published: October 2019

Abstract
Abstract 1041
In addition to reduced hemoglobin production, iron deficiency causes anemia though poorly understood defects in erythropoiesis that reduce the number of erythroid cells released from the marrow space. To study the negative effects of iron deficiency upon erythropoiesis, CD34+ cells from three healthy human donors were cultured in serum-free media supplemented with transferrin-bound iron. Dosed titrations of holo-transferrin (1.2, 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 mg/ml) were balanced by apo-transferrin supplementation to maintain total transferrin levels of 1.2 mg/ml in all cultures. Analyses were performed over the 21-day culture period that included assessment of proliferation, differentiation, enucleation and surface phosphatidylserine expression. Diminution of holo-transferrin in the culture medium caused a decrease in total cell counts and reduced cell divisions detected by CFSE analysis. During the first two weeks of culture, the progenitor cells differentiated into a glycophorin A positive population at all holo-transferrin doses. However, several iron-dependent changes were detected in the final stages of differentiation during culture days 14–21. Hemoglobin accumulation was reduced by 72% at the lowest holo-transferrin dose. Flow cytometry demonstrated a decrease in the loss of surface transferrin receptor in iron-deficient cultures. Thiazole orange analysis also demonstrated significantly lower levels of enucleation from 25.9±8.2% (1.2 mg/ml) to 4.4±4.2% (0.1mg/ml). Microscopic examination of the enucleated cells demonstrated a hypochromatic phenotype at lower holo-transferrin concentrations. Phosphatidylserine on the surface membrane (annexin V staining) increased with reduction of holo-transferrin from 6.3±2.6% (1.2 mg/ml) to 78.3±9.4% (0.1 mg/ml, p=0.003). The effects of iron on surface phosphatidylserine expression were limited to holo-transferrin concentrations of <0.6 mg/ml. Isocitrate supplemented at a concentration of 5.0 mM increased the cell counts in all concentrations of holo-transferrin, and partially rescued the iron deficient phenotype. These data suggest that iron deficiency ex vivo causes decreased proliferation, decreased loss of surface transferrin receptor, and decreased enucleation of normoblasts. There was also a significant increase in phosphatidylserine detected on the surface of the iron deficient normoblasts that provides a mechanism whereby macrophages can identify, phagocytose, and efficiently recycle iron from those cells to other normoblasts in the local marrow environment via erythroblastic islands.

Disclosures:
No relevant conflicts of interest to declare.
RNAi-Mediated Inhibition of Tmprss6 Elevates Hamp1 Expression and Reduces Serum Iron Levels in Mice
Authors: Toudjarska Ivanka , Cai Zuhua , Racie Tim , Milstein Stuart , Bettencourt Brian R , Hettinger Julia , Sah Dinah WY , Vaishnaw Akshay , Bumcrot David 
Published: October 2019

Abstract
Abstract 1045
The liver hormone Hepcidin (encoded by Hamp1) regulates serum iron levels by controlling the efflux of iron from intestinal enterocytes and macrophages. Maintaining sufficient iron levels to support erythropoiesis while preventing iron overload requires tight control of Hepcidin expression. Transcription of Hamp1 in hepatocytes is stimulated by high serum iron levels, via Transferrin Receptor signaling, as well as by activation of the BMP/SMAD pathway. The membrane serine protease Matriptase-2 (encoded by Tmprss6) inhibits BMP induced Hamp1 induction through the regulation of the BMP co-receptor, Hemojuvelin. In humans, loss of function mutations in TMPRSS6 lead to elevated Hepcidin levels resulting in iron-resistant iron-deficiency anemia (IRIDA). In diseases associated with iron overload, such as Thalassemia intermedia (TI) and Familial Hemochromatosis (FH), Hepcidin levels are low despite elevated serum iron concentrations. Studies in murine models of TI and FH have shown that elevating Hepcidin levels by genetic inactivation of Tmprss6 can prevent iron overload and correct aspects of the disease phenotype. Therefore, therapeutic strategies aimed at specifically inhibiting Tmprss6 expression could prove efficacious in these, and other, iron overloading diseases. Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to durable inhibition of Tmprss6 mRNA in the mouse liver, with concomitant elevation of Hamp1 expression. This leads to significant decreases in serum iron concentration and Transferrin saturation, along with changes in hematologic parameters consistent with iron restriction. Further testing in mouse genetic models of TI and FH will support the rationale for developing LNP formulated Tmprss6 siRNA as a novel therapeutic modality.

Disclosures:
Toudjarska: Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment.
Increased Incidence of Hematological Malignancies Among Californians with Sickle Cell Disease
Authors: Mahajan Anjlee , Brown Monica , Brunson Ann , Wun Ted 
Published: October 2019

Abstract
Abstract 1073

Background:
High cellular turnover and inflammation are associated with the development of malignancy. Both conditions exist in sickle cell disease (SCD). We used a population-based epidemiological approach to determine the incidence of hematological malignancy in patients with SCD.

Methods:
The California (CA) Office of Statewide Healthcare Planning and Development (OSHPD) operates a Patient Discharge Dataset (PDD) and Emergency Department Utilization (EDU) dataset that records the discharge diagnoses and procedures for all non-federal hospitalizations or ED visits in CA. Cases coded as SCD (282.60, 282.61, 282.62, 282.63, 282.68, 282.69, 282.41, 282.42) from 1991–2007 (PDD) or 2005–2007 (EDU) were found. Cases were excluded if the SCD code did not appear in the principal position and appeared in the secondary diagnosis position only once; or age < 2 years (lacked social security number for linkage). These cases were then linked to the California Cancer Registry (CCR) to determine cases with leukemia, myeloma, or lymphoma (including Hodgkin). Standardized incidence ratios (SIR) were calculated. Sensitivity analysis was also performed to determine the effect of doubling the CA SCD population on SIR.

Results:
There were 46 total cases of hematological malignancies identified: 25 females and 21 males. Fifty nine percent of the SCD patients with hematological malignancies were ages 30–59 years. The cohort was 78% Black, 11% White, 7% Hispanic, and 4% Asian.
The remaining cases had chronic myelogenous leukemia (n=4) and other leukemia not otherwise specified (n=5). Limiting analysis to only Non-Hispanic Blacks did not appreciably change the SIR estimates. Even if the SCD population was underestimated by half, sensitivity analysis showed the observed cases would yield an SIR for all hematopoietic malignancies of 19 (95% CI 14–25). The individual sub-type SIR would also still range from 13–24 and all would be statistically significant.

Conclusions:
The CA SCD cohort had elevated SIR for hematological malignancies suggesting higher incidence in this population. Limitations include those inherent in query of any large administrative dataset. However, our SCD population estimate is consistent with that of others and the CCR has validated robust tumor data. Current efforts to develop a US registry for SCD patients should include tracking for hematological, and perhaps all malignancies. Further studies using independent datasets are important to confirm these provocative findings.

Disclosures:
Wun: Glycomimetics, Inc.: Research Funding; Eli Lilly, Inc.: Research Funding.
Does Quantitative Heterogeneity of Human Fetal Hemoglobin (Hb F) Reveal Friends or Foes of KLF1 in Globin Gene Switching ?
Authors: Felice Alexander E. , Galdies Ruth , Borg Joseph , Grech Godfrey , Cassar Wilma , Scerri Christian A , Patrinos George P , Philipsen Sjaak 
Published: October 2019

Abstract
Abstract 1092
The chemical heterogeneity of fetal hemoglobin (Hb F) due to variable ratios of the Gγ and Aγ globin subunits reflects genetic complexity because of common dimorphisms such as Hb F Sardegna (or Aγ75(E19) Ile>Thr; also known as AγT) in Caucasians, and common variants such the Gγ globin variant, Hb F Malta I (or Gγ117(G19) His>Arg) that is in tight linkage disequilibrium with the β globin variant Hb Valletta (or β87(F3) Thr>Pro) and is found in 1.8% of neonates from Malta. Comprehensive and integrated maternal and neonatal testing has led to the finding of triple compound heterozygotes with Hb F Malta I in association with Hb F Sardegna and Hb Valletta in whom all globin genes that are functional in the transition from fetal to adult Hemoglobin profiles are genetically tagged, and quantifiable with High Performance Liquid Chromatography in the neonate or mRNA in the adult in the context of diverse XMNI −158 C>T Gγ globin and (AT)XTY −540 β globin haplotypes. The interaction between XMNI and (AT)XTY revealed “conditional” cis-trans interplay that appeared to be under developmental control. A family with members carrying Hb F Malta I in association with a rare form of the Hereditary Persistence of Fetal Hemoglobin has also been found. The genetic cause has been traced to haplo-insufficiency of the putative erythroid master regulator KLF1, and, as confirmed by functional assays in vitro. However, levels of Hb F expression varied considerably (3.3% – 19.5%) while a second family from Malta with the same KLF1 mutation (p.K288X) had normal Hb F indicating interplay of KLF1 with modifying genes. These data, together with comparative expression profiling of human erythroid progenitors, indicated a small set of additional gene products that may interact positively (friends) or negatively (foes) at the level of commitment or expression in globin gene switching with significant effects on the Mean Corpuscular - Hb F. Whole genome sequencing on critically informative family members currently in progress may further uncover the complex genetic interactions in developmental globin gene control.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of Sialic Acid Loss Greatly Improves Survival of Refrigerated Platelets
Authors: Jansen Gerard , Josefsson Emma C , Liu Qiyong Peter , Rumjantseva Viktoria , Falet Herve , Grozovsky Renata , Bergmeier Wolfgang , Wagner Denisa D. , Hartwig John H. , Hoffmeister Karin M. 
Published: October 2019

Abstract
Abstract 1133
Platelets have the shortest shelf life of all major blood components and are the most difficult to store, a fact that complicates platelet transfusion practices. Platelet refrigeration could slow bacterial growth and possibly retard the loss of platelet function following storage. However, in contrast to other blood components, platelets do not tolerate refrigeration and are rapidly cleared from the circulation. We demonstrated that two distinct pathways recognizing GPIba remove refrigerated platelets in recipient's livers: 1) αMβ2 integrins (Mac-1) on hepatic resident macrophages (Kupffer cells) selectively recognize irreversibly clustered b-N-acetylglucosamine (β-GlcNAc)–terminated glycans on GPIbα, and 2) hepatic Asialoglycoprotein (Asg) receptors (Ashwell Morell receptors) recognize desialylated GPIba. We here investigated the mechanism of sialic acid loss during refrigeration. We show, that when refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1 that remove sialic acid from platelet receptors, specifically from GPIbα. Platelets also express Neu3 on their surfaces, however Neu3 expression appears to be unaffected by platelet refrigeration. Importantly, the recovery and circulation of refrigerated platelets is greatly improved by storage in the presence of the competitive sialidase inhibitor N-Acetylneuraminic Acid, 2,3-Dehydro-2-deoxy-Sodium Salt (DANA). Desialylated von Willebrand receptor (vWfR) complex is also a target for metalloproteinases (MMPs), as GPIbα and GPV are cleaved from the surface of refrigerated platelets. Receptor shedding is inhibited by the metalloproteinase inhibitor GM6001 and does not occur in ADAM17ΔZn/ΔZn platelets expressing inactive ADAM17. Critically, desialylation in the absence of metalloproteinase-mediated receptor shedding is sufficient to induce the rapid clearance of platelets from circulation. Desialylation of platelet vWfR therefore triggers platelet clearance, and primes GPIbα and GPV for metalloproteinase-dependent cleavage. We conclude that desialylation of platelets is caused by increased surface sialidase activity following refrigeration and desialylation of glycoproteins, specifically of GPIbα, promotes receptor cleavage by MMPs.

Disclosures:
Liu: Velicomedical, Inc: Employment.
Noval Mutation in Four Saudi Families with Glanzmann Thrombasthenia
Authors: Owaidah Tarek , Abalkhail Hala , Musa Abdulrahman Al , Mosmali Hasan , Abdulmajeed Albanyan , Jefri Abdullah Al , alNounou Randa , Zahrani Hazzaa Al , Saleh Mahasen 
Published: October 2019

Abstract
Abstract 1136

Introduction:
Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation and variable bleeding tendency. Inherited genetic mutations in integrin alpha IIb and beta3 (ITGA2B, ITGB3) result in a heterogeneity of the thrombasthenia phenotypes. It is phenotypically expressed in homozygotes or compound heterozygotes, given that 50% of normal aIIbb3 is sufficient to guarantee unimpaired platelet function that result in asymptomatic carriers. Defects in ITGB3 result in failure of binding of B3 and alpha IIb. These defects had been reported in Arabs (Iraqi Jews). We are reporting some results of Saudi GT genotype project.

Materials & Methods:
In this study, we analyzed the entire coding region ITGB3 gene using polymerase chain reaction (PCR) and direct sequencing with primers specifically designed to amplify the coding region of exon 1–15 and exon /Intron boundaries in a cohort of 51 GT patients diagnosed and treated in our institute.

Results:
Out of 51 cases from 20 families had mutational screening of the ITGB3 gene with the aim to detect the causative pathogenic mutations to enable the pre-symptomatic diagnosis in at risk family members. In this study we detect 1 novel germline mutation c.2190delC (p.Ser703fs) in exon 13. The mutation is predicted to result in premature stop codon and protein truncation. The mutation was detected in 6 patients in homozygous stat (3 males and 3 females). Three tested samples from the patients family members detected the mutation in heterozygous state and all of them were asymptomatic with normal PFA and Intact expression of Platelet Glycoprotiens CD41(Gpllb), CD42a(GPIX), CD42b(GPlb), and CD61(Gpllla). All the GT patients with this mutation were type I GT with Prolonged PFA and complete absence of CD41(Gpllb) and CD61(Gpllla) glycoprotein.

Conclusion:
The result of this study represents the first Molecular analysis of ITGB3 gene in Saudi Arabia and displays the existence of novel pathogenic and possibly a founder effect in Saudi families.

Disclosures:
No relevant conflicts of interest to declare.