bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular characterization of the First Italian Variant of Phosphoglycerate Kinase Deficiency
Authors: Bianchi Paola , Fermo Elisa , Mandarà Luana , Vercellati Cristina , Marcello Anna Paola Maria Luisa , Cortelezzi Agostino , Barcellini Wilma , Zanella Alberto 
Published: October 2019

Abstract
Abstract 5270
Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate (1,3-BPG) to ADP to form 3-phosphoglycerate (3-PG) and ATP. It is a small monomeric molecule characterized by two hinge-bent domains, with a highly conserved structure. The N-terminal domain binds 1,3-BPG or 3-PG, whereas the C-terminal domain binds Mg-ADP or Mg-ATP. Two isozymes, PGK1 and PGK2, are present in humans, encoded by two distinct genes. Whereas PGK2 is a testis-specific enzyme, PGK1 is expressed in all the somatic cells.
The PGK1 gene is located on the Xq-13.1 chromosome, and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in enzyme deficiency characterized at clinical level by mild to severe hemolytic anemia, neurological dysfunctions and myopathy. Patients rarely exhibit all three clinical features. Since the first description by Kraus et al, nearly 40 patients with PGK deficiency have been reported, 27 of them characterized at the DNA or protein level. To date, 20 different mutations with worldwide distribution have been described.
The aim of the study was to characterize the molecular defect in an Italian patient affected by phosphoglycerate kinase deficiency.
The patient, born from unrelated parents with negative family history of neurological defects, showed at birth neonatal jaundice. At the age of four years, in concomitance of an infective episode, he displayed hemolytic anemia (Hb 8.6 g/dL, reticulocytes 19%, unconjugated bilirubin 0.91 mg/dL, LDH 445 u/l, aptoglobin absent) and increased CPK values (2483U/L). The patient showed respiratory distress. The study of red cell glycolytic enzymes displayed a drastic reduction of PGK activity (41.8 UI/gHb ref. values 287–392). We examined again the patient at the age of 25 yrs in occasion of his sister's first pregnancy. The patient displayed compensated hemolytic anemia (Hb 14.1 g/dL, reticulocytes 6.6%) and severe myopathy.
Sequencing analysis of the entire coding region and flanking intronic sequences of PGK1 gene showed the presence of a novel missense mutation c.1112 (ATA>AAA) responsible for amino acid substitution Ile371Lys. Although the mutation falls in the third last nucleotide of exon 9, it doesn't alter the splicing as confirmed by patient cDNA analysis that showed a normal transcript. The new mutation is located in a highly conserved region among species close to the ATP binding site and it was not found among the 100 normal alleles examined thus excluding the possibility of a polymorphism. Family study performed in the parents, the two healthy sisters and maternal uncle confirmed the X-linked transmission of the disease.

Disclosures:
No relevant conflicts of interest to declare.
An ANK1 Mutation Study in Chinese Patients with Hereditary Spherocytosis
Authors: So Jason CC , Wong Patricia , Chan Purdy , Chiang Alan KS , Chan Li Chong 
Published: October 2019

Abstract
Abstract 5277
Hereditary spherocytosis (HS) is the commonest hereditary hemolytic anemia due to a red cell membrane defect. Multiple proteins of the vertical component of the cytoskeleton are implicated. They include ankyrin-1, protein 4.2, band 3 protein, alpha and beta spectrin. Previous studies in Caucasians (Eber et al Nat Genet 1996) and Japanese (Nakanishi et al Int J Hematol 2001) showed that mutations in ANK1, the gene coding for ankyrin-1, was the most prevalent defect in HS. There is no published data on mutations in Chinese patients with HS.
Thirteen Chinese HS patients from 9 unrelated families and 28 normal Chinese subjects were studied. Fourteen of the 42 exons of ANK1 (exon 1, 2, 4, 5, 6, 16, 17, 20, 21, 22, 26, 33, 38, 39) were screened for mutation using direct nucleotide sequencing. Selection of exons for study was based on the frequencies of mutation detected in a large study in an Asian population (Nakanishi et al Int J Hematol 2001). Two novel nonsense mutations were detected in 2 unrelated patients in heterozygous state – codon 968 GAG>TAG and codon 1762 TGG>TAG. Family studies showed that 1 was a de novo mutation and the other was autosomal dominant. The overall prevalence of ANK1 mutations in this cohort was 22%. The actual prevalence may be higher as pathogenic mutations may also lie in the unscreened promoter and exons. Ten benign polymorphisms were detected in 8 of the 14 exons screened. Nine were synonymous mutations and 1 was a reported missense mutation. All but 1 were seen in our normal controls, including the missense mutation.
Our study shows that ANK1 mutation is a genetic cause of HS in Chinese but other candidate genes are definitely implicated. In consistence with published data in Caucasians pathogenic mutations are usually private. Many benign polymorphisms exist and they are commonly found in the general population. These features present significant challenges for screening of mutations and their interpretation in HS patients.

Disclosures:
No relevant conflicts of interest to declare.
In Vitro GDF15 Expression During Thalassemic Erythroid Differentiation and Maturation
Authors: Colancecco Alessandra , Ronzoni Luisa , Duca Lorena , Sonzogni Laura , Nava Isabella , Graziadei Giovanna , Cappellini Maria Domenica 
Published: October 2019

Abstract
Abstract 5285

INTRODUCTION:
The growth differentiation factor-15 (GDF-15), a member of the transforming growth factor-β superfamily, is thought to be related to ineffective or apoptotic erythropoiesis. GDF-15 levels were found to be significantly elevated in sera of patients with β thalassemia major (TM), however little is known about the GDF15 expression during thalassemic erythropoiesis. Non-transfusion dependent thalassemia intermedia (NTDT) represents the model of thalassemic erythropoiesis not affected by transfusions.

AIM:
To determine the GDF15 gene expression profile during normal and thalassemic erythroid differentiation in standard cultures and under different iron conditions, from CD34+ of normal and thalassemia intermedia subjects.

METHODS:
After informed consent, the CD34+ cells were obtained from peripheral blood of healthy volunteers and from patients with NTDT by positive selection using anti-CD34-tagged magnetic beads and cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. Each culture was split in 3 flasks: standard condition, with addition of deferoxamine (DFO 4 mM) as iron chelating agent and ferric ammonium citrate (FAC 100 mM) at day 0 of culture. The expression profiling of GDF15 gene was evaluated at baseline, day 7 and day 14 by real-time PCR (2̂-dCt). GDF15 concentrations in culture supernatants were also evaluated by enzyme-linked immunosorbent assay using DuoSet Sandwich ELISA Kit (R&D Systems, Minneapolis, MN).

RESULTS:
GDF15 expression and secretion increased significantly during erythroid differentiation either in normal and in NTDT cultures. At day 14 in thalassemia intermedia cultures GDF15 expression as well as the concentrations in supernatant were higher (althoug not statistically significant) compared to control (Table 1). At day 14 in control cultures GDF15 expression is up-regulated by DFO and down-regulated by FAC addition. In NTDT GDF15 expression was influenced by iron addition in cultures, but was not increased by iron depletion (Table 2).

CONCLUSIONS:
GDF15 levels in erythroid cultures are related to the erythropoietic stage of differentiation. In NTDT cultures the GDF15 gene profile and protein levels in supernatants are higher than in normal cultures. GDF15 levels seem to be modulated by iron in normal cultures whereas in NTDT cultures they seem to be independent from iron availability. This in vitro study supports that signals different than iron, such as erythropoietic stress, could be the major factor regulating GDF15 expression.

Disclosures:
No relevant conflicts of interest to declare.
In Vitro Ferroportin Expression in Thalassemia Intermedia During Erythroid Differentiation
Authors: Ronzoni Luisa , Colancecco Alessandra , Sonzogni Laura , Graziadei Giovanna , Cappellini Maria Domenica 
Published: October 2019

Abstract
Abstract 5287

INTRODUCTION:
Ferroportin (FPN) is the sole mammalian iron exporter protein known and it plays a critical role in iron metabolism. It is expressed in various types of cells including duodenal enterocytes, hepatocytes, erythroblasts cells, syncytiotrophoblasts and reticuloendothelial macrophages. Ferroportin is expressed in multiple alternative transcripts: with (FPN1A) or without (FPN1B) an iron-responsive element (IRE). The expression of one form rather than the other depends on cell type and iron availability. The expression of ferroportin in thalassemia intermedia (TI), characterized by iron overload, is not yet fully elucidated.

AIM:
To investigate the different expression profile of ferroportin isoforms during erythroid differentiation in control and TI cell cultures.

METHODS:
After informed consent, the CD34+ cells were obtained from peripheral blood of healthy volunteers and from patients with thalassemia intermedia by positive selection using anti-CD34-tagged magnetic beads and cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. The expression profiling of FPN1A and FPN1B was evaluated at baseline, day 7 and day 14 of culture by real-time PCR (2̂-dCt).

RESULTS:
In control cultures, FPN1A isoform was highly expressed at erythroid progenitors stage (day 0 of culture), decreased at early erythroblasts stage (day 7) and increased again at late erythroblasts stage (day 14). In TI cultures, the FPN1A isoform expression remained high even in early erythroblasts (Table 1). In control cultures the FPN1B isoform expression was very low at any stage of erythroid differentiation, whereas in TI cultures it was highly expressed at baseline and, althoug decreased during differentiation, remained always higher than control (Table 2).

CONCLUSIONS:
In thalassemic conditions the FPN1B is the major expressed ferroportin isoform, possibly contributing to iron overload. In control cultures, FPN1A was mainly expressed in undifferentiated erythroid progenitors and in mature erythroblasts, suggesting a functional role at these stages of erythroid differentiation. In TI cultures, the persistent expression of FPN1A at early erythroblasts stage was probably due to thalassemic erythropoiesis. These data suggest that in TI condition other signals, such as the erythropoiesis status, can override iron overload in regulating ferroportin expression.

Disclosures:
No relevant conflicts of interest to declare.
Right Ventricular Volumes and Function Normalized to Body Surface Area, Age and Sex in a Large Cohort of Well-Treated Thalassemia Major without Myocardial Iron Overload
Authors: Meloni Antonella , Aquaro Giovanni , Ait-Ali Lamia , Campisi Saveria , D'Ascola Domenico Giuseppe , Quarta Antonella , Bitti Pier Paolo , Roccamo Gaetano , Bisconte Maria Grazia , Piraino Basilia , Lai Maria Eliana , Peluso Angelo , Renne Stefania , De Franceschi Lucia , Positano Vincenzo , Lombardi Massimo , Pepe Alessia 
Published: October 2019

Abstract
Abstract 5293

Introduction.
Cardiovascular Magnetic Resonance (CMR) has provided the opportunity to quantify right ventricular (RV) parameters with excellent reproducibility and accuracy. The role of the RV is gaining ground in thalassemia major (TM) patients and this population could experience different “normal” RV values due to chronic anemia and eventually pre-existing iron burdens. The aim of this study was to establish the ranges for normal RV volumes, mass and ejection fraction (EF) normalized to the influence of body surface area (BSA), age and sex from CMR in a large cohort of well-treated TM patients without myocardial iron overload.

Methods.
Among the 923 TM patients enrolled in the Myocardial Iron Overload (MIOT) network who underwent CMR for the assessment of cardiac iron overload, function and fibrosis, we selected 142 patients with no known risk factors or history of cardiac disease, normal electrocardiogram, no myocardial iron overload (all the cardiac segments with a normal T2* value) and no myocardial fibrosis. All patients had been regularly transfused and chelated since early childhood. Moreover, we studied 71 healthy subjects matched for age and sex. RV function parameters were quantitatively evaluated in a standard way by SSFP cine images using MASS® software. RV end-diastolic volume (EDV), end-systolic volume (ESV) and stroke volume (SV) were normalized by body surface area (EDVI, ESVI, SVI).

Results.
The table shows the comparison of the CMR parameters with differentiation for sex and age in TM patients and healthy subjects and the cut-off of normality defined as mean – 2 standard deviation (SD). TM patients showed significantly lower BSA than the controls (P<0.0001). TM males (except age group 14–20 yrs) showed significantly higher RV EF compared to controls. In TM patients all LV volumes indexes were significantly larger in males than in females (P<0.0001 in all age groups). The EF was not different between the sexes. In males as well as in females the RV volumes were no significant different among the age groups, while in males the EF was significant different (P=0.004).

Conclusion.
In a large cohort of well-treated TM patients males showed significantly higher RV EF compared to controls. Due to the influence of BSA, sex and age, appropriate “normal” reference ranges normalized to these variables should be used to avoid misdiagnosis of cardiomyopathy in the clinical arena in TM patients.

Disclosures:
No relevant conflicts of interest to declare.
Relationship of Being a Beta Globin Gene Carrier with Insulin Resistance
Authors: Bahar Adeleh , Kashi Zahra , Sohrab Mehrnoush , Kowsarian Mehrnoush , Janbabaei Ghasem , Ghavamzadeh Ardeshir 
Published: October 2019

Abstract
Abstract 5305

Objective:
There was a paper in favor of beta-globin gene carrier and insulin resistance. Considering the high prevalence of these individuals in north of Iran, the present study was designed to assess the relationship between a beta-globin gene carrier and developing insulin resistance.

Methods:
This historical cohort study was conducted on 164 people, including 82 healthy controls and 82 individuals with thalassemia minor. The two study groups were matched for age, body mass index(BMI) and family history of diabetes mellitus. Blood samples were taken for; CBC, fasting blood sugar(FBS), liver enzymes (AST, ALT), high sensitive C- reactive protein(CRP), serum insulin and a standard OGTT were performed in all. Insulin resistance was diagnosed based on homeostasis model assessment method(HOMA). TM was diagnosed if microcytic(MCV<80 fl) hypochromia(MCH<25 pg) was detected on CBC and HbA2 ≥3.5% using HPLC method. Controls had negative past medical history and normal CBC. Student T- Test and and Chi-Square test were used to compare demographic data. Relative Risk was measured to test the hypothesis. P<0.05 was considered as significant.

Results:
Age, gender, BMI, were similar. CRP, and AST were significantly higher in case group. (p value <0.05). The Relative Risk for diabetes mellitus and insulin resistance in the cases with minor thalassemia was 2 (CI % 95:1.8–2.5) and 2.02 (CI % 95:1.7–2.4), respectively.

Conclusion:
The risk of developing diabetes and insulin resistance in patients with thalassemia minor is two times greater than the general population. Considering the high serum levels of CRP in these cases, the inflammation noted in liver cells could be considered as the underlying cause of insulin resistance, impaired glucose tolerance and diabetes in these patients.

Disclosures:
No relevant conflicts of interest to declare.
Nitrite Oxidase Activities of Cytochrome P450 and Mitochondria
Authors: Curtis Erin , Hsu Lewis L , Hon Yuen Yi , Geary Lisa , Noguchi Audrey C , Shiva Sruti 
Published: October 2019

Abstract
Abstract 5310
Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO2−) is now accepted as an endocrine reserve of NO that elicits a number of fundamental biological responses in all major organ systems. While it is known that tissue nitrite is derived from both oxidation of NO and from dietary nitrite and nitrate, much less is known about how nitrite is metabolized by tissue or about the factors that influence this metabolism. Here we investigate the rates and mechanisms by which nitrite is metabolized by tissue over a range of oxygen tensions in rats and mice. We show that the rate of nitrite metabolism differs in heart, liver, lung and brain tissue. Further, oxygen regulates the rate and products of nitrite metabolism in each of these tissues. In hypoxic tissue, nitrite is predominantly reduced to NO, with significant formation of iron-nitrosyl heme proteins and S-nitrosothiols. Interestingly, this hypoxic nitrite metabolism is mediated by different sets of nitrite reductase enzymes in each tissue. In contrast, tissue consumption of nitrite is more rapid in normoxia and the major end product is nitrate. While cytochrome P450s and myoglobin contributed in the liver and heart respectively, mitochondrial cytochrome c oxidase played a significant role in this normoxic nitrite oxidation, which could be completely inhibited by cyanide in all tissues. We used cyanide-based nitrite preservation solution to measure the pharmacokinetics of oral and intraperitoneally administered nitrite in vivo. Using this methodology, we measured basal levels of nitrite in the major tissues and confirm that the heart contains the greatest concentration of nitrite, followed by the liver and finally the lung. We demonstrate that intraperitoneal administration of nitrite to mice increases nitrite levels most significantly in the liver and heart, where nitrite uptake is rapid (5–10 min) and steadily decreases thereafter, such that levels are back to baseline by 30 min. Little to no increase was observed in the lung. In these studies, changes in nitrate were difficult to detect due to the high levels of basal nitrate present in vivo and low concentration of nitrite administered. However, the rapid metabolism of nitrite in the tissue suggests that oxidation is at least partially responsible. In contrast to intraperitoneal nitrite, oral nitrite increased nitrite levels in all organs of mice, an effect which peaked in all organs except liver at 3 days. Collectively, these data provide insight into the fate of nitrite in tissue, the enzymes involved in hypoxic and normoxic nitrite metabolism and the role of oxygen in regulating these processes.

Disclosures:
No relevant conflicts of interest to declare.
Deficiency of Superoxide Dismutase Impairs Generation of Activated Protein C and Enhances Susceptibility to Experimental Thrombosis in Mice
Authors: Dayal Sanjana , Wilson Katina M , Hutchins Ryan , Lentz Steven R. 
Published: October 2019

Abstract
Abstract 535
In vitro studies have suggested that reactive oxygen species such as superoxide can produce several potentially prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin that impairs its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis in vivo. Using a murine model that is genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), we tested the hypothesis that lack of superoxide dismutase enhances susceptibility to thrombosis. Additionally, we investigated the mechanisms of superoxide-enhanced thrombosis. First, we examined the susceptibility to carotid artery thrombosis in a photochemical injury model. We found that Sod1−/− mice formed stable occlusions significantly faster than Sod1+/+ or Sod1+/− mice (P<0.05). Further, using an inferior vena cava (IVC) stasis method we observed that Sod1−/− mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 compared with Sod1+/+ or Sod1+/− mice). These findings suggest that deficiency of SOD1 leads to increased susceptibility to both arterial and venous thrombosis in mice. To address the mechanism of accelerated thrombosis, we first examined activation of platelets in response to multiple agonists using flow cytometry. After activation by thrombin (0.5 U/ml) and convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen to activated platelets were observed between Sod1−/−, Sod1+/+, or Sod1+/− mice, suggesting that increased susceptibility to thrombosis in Sod1−/− mice is not platelet mediated. Next, we measured expression of TF mRNA in lung by real time qPCR. TF mRNA levels in Sod1−/− mice were similar to those in Sod1+/+ mice, suggesting that deficiency of SOD1 does not influence TF expression in mice. Finally, we measured the activation of protein C in vivo in response to infusion of thrombin (40 U/Kg). Generation of activated protein C was significantly lower in Sod1−/− mice compared with Sod1+/+ mice (P<0.05). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in Sod1−/− mice compared with Sod1+/+ mice (P=0.4 and 0.6 respectively), suggesting that altered generation of activated protein C in Sod1−/− mice may be related to a direct oxidative effect on thrombomodulin rather than to decreased expression of thrombomodulin or EPCR. We conclude that lack of SOD1 in mice accelerates thrombosis and impairs the protein C anticoagulant response to thrombin.

Disclosures:
No relevant conflicts of interest to declare.