Globin Research Articles

Cross Sectional Analysis indicates that alpha thalassemia deletion mutation is prevalent in a South Indian Population

Thalassemia is a group of genetic disorders characterized by quantitative defects in globin chain synthesis. Deletions of either one (α -thalassemia 2) or both (α -thalassaemia 1) α-globin genes on chromosome 16 account for over 95% of alpha thalassemia cases. The subsequent absence or decrease of hemoglobin production results in microcytosis with varying degrees of anemia. The present study was undertaken to investigate the prevalence of α thalassemia in a South Indian population, Nadars, inhabiting Tamil Nadu and to compare them with Asian Populations. 31 random samples were collected and genotyped using Gap PCR. Statistical analysis was done using Excel, ARLEQUIN, MEGA and GRAPH PAD. The study population Nadars has a frequency 32% of αα/-α3.7. The two-tailed p value for Fisher’s exact equals 0.0033, highly statistically significant. The dendrogram shows that Nadars clubbed with Malay population that showed a double mutation for -4.2. This means that the mutation rate for α thalassemia in both populations is significant and Nadars need to be studied in depth for the -4.2 mutation. The principal component analysis pointed out Nadars is perhaps unique because of αα/-α3.7 genotype and the need of the hour is to screen all south Indian populations for thalassemia mutations in general and alpha thalassemia in particular.

Molecular diagnosis of thalassemia compound heterozygotes in PUMCH from 2012 to 2015

Objective To investigate the genotype distribution of thalassemia intermedia, major and compound thalassemia in Peking Union Medical College Hospital from 2012 to 2015. Methods Retrospectively 1 084 suspected thalassemia cases were analyzed in recent four years. Three common deletions of α globin chain were detected by GAP-PCR. Three common point mutations of α globin chain and seventeen common mutations of β globin chain were identified by PCR reverse dot blot hybridization. Hemoglobin electrophoresis was carried out by Capillary Electrophoresis System. RBC associated parameters and morphology were analyzed by hematology analyzer and blood smear. Results 702 cases were confirmed to be thalassemia, and the positive rate was 64.76% (702/1084). 19 types of gene defects were detected. There were 4 types of gene defects in 23 case with α-thalassemia intermeida, including -α3.7/--SEA, -α4.2/--SEA, αCSα/--SEA and αQSα/--SEA, -α3.7/--SEA to be the most common genotype (18 cases) .3 cases with β-thalassemia intermeida were confirmed and the genotypes were βCD17(A→T)/β-29(A→G), β-28(A→G)/β-28(A→G) and βIVS-Ⅱ-654(C→T)/βCD17(A→T), respectively. There were also 1 βCD41-42(-TTCT)/βCD17(A→T) thalassemia major case. The genotypes of 2 HbE/β-thalassemia cases were βCD41-42(-TTCT)/βE and βCD17(A→T)/βE. 5 αβ-thalassemia including 2 βCD41-42(-TTCT)/βA compounded with αα/-α3.7, 1βIVS-Ⅱ-654(C→T)/βA compounded with --SEA/αCSα, 1βCD17(A→T)/βA compounded with -α4.2/αα and 1βCD41-42(-TTCT)/βA compounded with αCSα/αα. Rare and untypical haematological results were found, such as normal level HbA2 and undetectable HbH, in compound heterozygosity with --SEA/αCSα and βIVS-Ⅱ-654(C→T)/βA. Conclusions The genotypes of thalassemia intermedia, major and compound thalassemia in Peking Union Medical College were highly variable.(Chin J Lab Med, 2016, 39: 491-495) Key words: Thalassemia; alpha-globin; Beta-globin; Genotype; Hemoglobinometry

The molecular characterization of Beta globin gene in thalassemia patients reveals rare and a novel mutations in Pakistani population

IntroductionA multicentre study (including four cities in Pakistan) aimed to investigate the frequency and spectrum of alpha and beta thalassemia genetic mutations and XmnI polymorphism of the Gamma Globin gene. MethodsOne hundred and sixty one beta thalassemia patients, identified on the ground of haematological parameters, were screened for mutations of the Alpha (HBA2 and HBA1) and Beta (HBB) Globin genes as well as Gamma (HBG2) Globin gene, -158 Gγ XmnI polymorphism, using a combination of multiplex GAP polymerase chain reaction (PCR), Sanger sequencing and restriction fragment length polymerase (RFLP) based PCR. ResultsMutations of at least one HBB gene was identified in 157 of 161 patients screened. Among 16 identified mutations in the beta gene, HBB:c.27_28insG (p. Ser10Valfs*14) was the most prevalent. α−3.7 and α−4.2 deletions were co-inherited with beta thalassemia mutations. Rare mutations such as HBB:c.–138C > T and HBB:c.315 + 1G > A were also identified. One novel variant (HBB:c.-148T > A), two rare mutations [HBB:c.332T > C (p.Leu111Pro); HBB:c.92G > C (p.Arg31Thr] and a novel association, HBB:c.[92G > C (p.Arg31Thr)] and [-92C > G], were reported for the first time in our study. HBG2:c.-211C > T base-pair substitution (historically described as -158 GγXmnI polymorphism) was present in 36% of the patients. ConclusionHeterogeneity in clinical and haematological parameters in TM, show that monogenic disorders can present with a wide spectrum of disease severity. Our studies identified rare and novel mutations that will be useful in the prevention of highly prevalent disease of thalassemia in Pakistan following nationwide awareness campaign.

Association between white blood cell count and non-alcoholic fatty liver disease in urban Han Chinese: a prospective cohort study

ObjectivesThe white blood cell (WBC) count is a simple and convenient marker of inflammation for use in medical practice; however, its association with non-alcoholic fatty liver disease (NAFLD) has not...

562. Fetal Hemoglobin Expression Can Be Increased by Targeted Disruption of the Gamma Hemoglobin Promoter in Human Peripheral Blood Stem Cells

Hemoglobinopathies such as sickle cell disease and β-thalassemia are both common and morbid inherited genetic disorders. Mutations in the β -globin (HBB) gene result in the expression of pathologic globin molecules or the reduction in expression of the wildtype allele. Despite the high prevalence of these disorders, current treatment options are limited. Patients with a hereditary persistence of fetal hemoglobin (HPFH) have disease modifying mutations that result in the compensatory expression of gamma or delta hemoglobin and these patients tend to have a much less severe phenotype. A previously reported unique 13bp deletion identified in the gamma hemoglobin promoter of two sickle cell patients with HPFH (HbF >30%) offers a novel target for gene editing using targeted endonucleases. This region contains known binding sites for multiple regulatory factors including the DRED repressor complex and the CAAT-box binding factor (CBF). TALENs designed to target this 13bp site have been generated and the mRNA successfully transfected into human peripheral blood CD34 cells. Genomic analysis of TALEN transfected cells reveals the introduction of INDELs by NHEJ in both the γ1 (HBG1) and γ2 (HBG2) genes (33% and 8% respectively). TALEN edited human peripheral blood stem cells were then differentiated into erythroid progeny that express increased levels of fetal hemoglobin detected by both flow cytometry (>50% increase over control) and HPLC (>5-fold increase over control). CRISPR guides (traditional and chemically modified RNA guides) targeting the same locus are currently being compared to TALENs. These data suggest that the introduction of INDELs in this locus is capable of de-repressing the gamma globin gene in human CD34 cells. In vivo mouse xenografts are underway to further assess this approach as a means for a functional cure of a wide array of hemoglobinopathies.

The application of multicolor probe melting curve analysis for the prenatal diagnosis of β thalassemia

Objective To establish a method of multicolor melting curve analysis for the prenatal diagnosis of β thalassemia. Methods Methodology establishment. A total of 95 cases, including 9 fetal villi samples(10-13 weeks)and 86 amniotic fluid samples(18-24 weeks)were collected by Center for Prenatal Diagnosis of Shenzhen Maternity and Child Healthcare Hospital between January 2014 and December 2014. A double-blind test was done to detect the mutations of beta globin gene by means of reverse dot (RDB) blot and multicolor melting curve analysis(MMCA). The consistency of the two methods is compared. Results The results of 93 cases detected by MMCA and RDB are completely consistent. The results of the 2 cases detected by MMCA after correction are the same as the results detected by RDB. Finally, the coincidence rate of the result was 100%. Conclusion MMCA can be applied to the prenatal diagnosis of β thalassemia as an effective supplement to RDB.(Chin J Lab Med, 2016, 39: 192-196) Key words: beta-Thalassemia; Prenatal diagnosis; Reverse transcriptase polymerase chain reaction; Nucleic Acid Hybridization

Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ9 desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ9-desaturase inhibited cells 48h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer

Novel Epigenetic Modulators That Promote Fetal Hemoglobin Production for the Prevention of Sickle Cell Disease Related Complications

Background: Sickle cell disease (SCD) is the most common inherited blood disorder in the United States. Affected patients are at risk of multi-organ complications, significant morbidity and early mortality with an average age at death of 42 and 48 years for men and women, respectively. Complications include acute and chronic pain due to vascular occlusion, end organ damage, acute chest syndrome and increased risk of sepsis. SCD is caused by a point mutation on chromosome 11, which codes for the beta chain of adult hemoglobin. Fetal hemoglobin is not affected by the mutation. Persons with SCD who maintain persistent fetal hemoglobin demonstrate a decreased number of pain crises and acute chest episodes along with a decreased risk of mortality compared to other patients with SCD. The only FDA approved fetal hemoglobin inducer, Hydroxyurea, remains highly underutilized due to the risk of complications and unwanted side effects. The discovery of a new agent that promotes re-expression of fetal hemoglobin could revolutionize the care of affected patients by reducing morbidity and mortality. Epigenetic modulation is one possible mechanistic approach to accomplish this goal. Recent studies with lysine specific demethylase-1 (LSD1) inhibitors, including tranylcypromine (TCP) have shown promising results. Unfortunately, TCP also affects monoamine oxidase and thus has significant side effects. Developing a molecule with high specificity, low cross reactivity and negligible toxicity would increase desired effects while reducing unwanted side effects.Objectives: To discover small-molecule inhibitors of LSD1 that exhibit high selectivity for LSD1 and low in-vivo toxicity that can be used to promote re-expression of fetal hemoglobin in SCD patients.Design/Methods: Our laboratory at the Medical University of South Carolina has recently described a library of potent, non-toxic LSD1 inhibitors. Our preliminary results suggest that these inhibitors show promise as fetal hemoglobin inducers. Using K562 cells, an erythroleukemic cell line known to model in-vivo hemoglobin production, we are screening this library to identify the most effective compounds using Western blotting for the gamma globin chain that is unique to fetal hemoglobin. Following identification of the most effective compounds in initial screens, we will confirm results with RT-qPCR. Subsequent studies will involve culturing and treating erythroid progenitor cells from healthy subject samples and from patients with SCD obtained through bone marrow sampling. HPLC will be used for identification and quantification of hemoglobin chains, including fetal hemoglobin, after ex-vivo treatment with selected compounds.Results: Initial results from Western blots for fetal hemoglobin (gamma globin protein) demonstrate that TCP, as well as the experimental compounds C1, 107-3 and 107-15, promote the re-expression of fetal hemoglobin in K562 cells. Experimental compounds were compared to DMSO as negative control, and hemin (10 mM) and hydroxyurea (200 mM) as positive controls. All experimental compounds evaluated are significantly less toxic than hydroxyurea, and do not impact cell viability in trypan blue exclusion studies.Conclusion: Initial studies show promising results for fetal hemoglobin production using these novel LSD-1 inhibitors. Viability data is also encouraging. Continued investigation is warranted to further investigate the efficacy of these compounds. DisclosuresNo relevant conflicts of interest to declare.

Temporally Distinct Developmental Waves of Erythropoiesis from Human Pluripotent Stem Cells

Mammalian erythropoiesis during embryogenesis occurs in several distinct stages or "waves" that vary according to timing, site of production, gene expression and physiology. The ontogeny of mammalian erythropoiesis is most thoroughly studied in mice where the earliest circulating erythroblasts released from the yolk sac are termed primitive. Later, the first definitive erythroid lineage is established by erythro-myeloid progenitors (EMPs) that originate in the yolk sac and migrate to the fetal liver for terminal differentiation. A second wave of definitive erythropoiesis is established from hematopoietic stem/progenitor cells that originate in the dorsal aorta and migrate to later stage fetal liver for terminal differentiation. Finally around birth, definitive erythropoiesis shifts to the bone marrow. The ontogeny of erythropoiesis overlaps in mice and humans, although less is known about the latter, as hematopoietic tissues from precisely staged early human embryos are difficult to obtain. We hypothesized that the initial steps of human erythroid ontogeny could be recapitulated by induced pluripotent stem cells (iPSCs) induced to undergo hematopoietic differentiation. We used a serum- and feeder-free protocol to differentiate iPSCs into embryoid bodies (EBs) that produced two sequential waves of distinctly different erythroid precursors. At day 8 of differentiation, EBs began to release hematopoietic precursors. Thereafter, erythroid precursors were released from the EBs in the presence of stem cell factor (SCF), erythropoietin (EPO) and insulin-like growth factor 1 (IGF-1). Erythroid precursors produced during wave 1 (days 12-23 of differentiation) were relatively large and expressed embryonic-type globins (zeta and epsilon), resembling those produced during primitive erythropoiesis. In contrast, wave 2 erythroblasts (day 27 and later) were smaller and expressed mainly gamma and alpha globins with some beta globin, suggestive of fetal-type definitive erythropoiesis. To investigate further the similarity of wave 1 and wave 2 erythroblasts to cells at the primitive and definitive stages of ontogeny, respectively, we used Affymetrix Genechips to analyze the global transcriptomes of stage-matched (CD235+ CD71high) cells. As primary human primitive erythroblasts were not available for comparison, we compared the transcriptomes from the iPSC-derived erythroblasts with those of primary murine definitive and primitive erythroblasts that were flow cytometry-purified from embryonic day 15.5 (E15.5) fetal liver and E10.5 bloodstream, respectively. The comparisons showed that wave 1 erythroblasts from human pluripotent cells resembled more closely the erythroid primitive lineage from mice, while wave 2 erythroblasts from the human cells resembled the erythroid definitive lineage of mice (P-value < 0.05 by a modified Kolmogorov-Smirnov test). For example, SOX6 and BCL11A, preferentially expressed during definitive erythropoiesis, were expressed at relatively high levels in wave 2 erythroblasts. In addition, gene set enrichment analysis (GSEA) demonstrated that wave 2 human iPSC-derived erythroblasts and primary murine definitive erythroblasts expressed numerous genes related to immune/inflammatory pathways that were shown previously to be important for the formation of definitive hematopoietic stem and progenitor cells in zebrafish and mouse embryos. Our findings demonstrate that human iPSC-derived embryoid bodies recapitulate early stages of erythroid ontogeny with respect to the timing of emerging lineages and their gene expression. Additionally, gene expression studies of human iPSC-derived primitive and definitive erythroblasts indicate inflammatory signaling as a potential regulator of the later stage of erythroid development. DisclosuresNo relevant conflicts of interest to declare.

The Histone Deacetylase 1 and 2 (HDAC1/2) Inhibitor ACY-957 Increases Epsilon (HbE) and Gamma (HbG) Globin mRNA in the Peripheral Blood of Non-Anemic Rats and Monkeys

The Histone Deacetylase 1 and 2 (HDAC1/2) Inhibitor ACY-957 Increases Epsilon (HbE) and Gamma (HbG) Globin mRNA in the Peripheral Blood of Non-Anemic Rats and Monkeys

Clonal Analysis of Human Bone Marrow CD34+ Cells Edited By BCL11A-Targeting Zinc Finger Nucleases Reveals Clinically Relevant Levels of Fetal Globin Expression in Edited Erythroid Progeny

Sickle cell disease (SCD) is one of the most common inherited blood disorders and is caused by a mutation at the adult beta globin gene resulting in substitution of valine for glutamic acid at position 6 in the encoded protein. While SCD can be cured by hematopoietic stem cell transplant (HSCT), complete donor chimerism is not required to achieve clinical benefits. Stable mixed chimerism of 10-15% in bone marrow or peripheral blood nucleated cells with >70% donor-derived RBCs has been reported to achieve transfusion independence and a symptom-free state in a SCD patient. It has also been proposed that SCD can be treated by reactivating developmentally silenced fetal gamma globin to form fetal hemoglobin (alpha2gamma2, HbF), which inhibits polymerization of HbS. The effect of HbF is predicted to be maximal when HbF content per cell exceeds 10 pg (~30% of total Hb). Furthermore, pathology is prevented when protective F cells (>30% HbF per cell) constitute >70% of total RBCs. We hypothesize that in a gene therapy setting, if >15% of SCD patients' autologous HSCs are programmed to produce protective F cells during erythropoiesis, it will translate into >70% protective F cells in circulation and provide significant alleviation of clinical symptoms.Genome wide association studies have identified BCL11A as a major modifier of HbF levels. Subsequent studies have shown that BCL11A plays a critical role in the fetal to adult globin developmental switch and in repressing fetal globin expression in adult erythroid cells. Conditional inactivation of BCL11A in adult erythroid cells leads to high levels of pan-cellular fetal globin expression and correction of hematologic and pathologic defects in a humanized SCD mouse model. Previously, we have reported that zinc finger nucleases (ZFNs) targeting BCL11A either in the coding region or the GATAA motif in the erythroid-specific enhancer efficiently disrupt the BCL11A locus in human primary CD34+ cells following electroporation of ZFN-encoding mRNA. Elevated fetal globin expression in bulk erythroid cultures was observed following disruption. To determine what percentage of HSPCs have been modified and whether the HbF/F cell content has reached the hypothesized therapeutic level, we analyzed erythroid cells clonally derived from ZFN-transfected CD34+ cells. Genotype of each clonal culture was determined by deep sequencing and globin production was analyzed by a highly sensitive UPLC method. We found that up to 80% of the BFU-Es had both BCL11A alleles edited, half of which had KO/KO alleles (either out of frame mutations for coding region or elimination of the GATAA motif in the enhancer). BCL11A coding KO/KO cells expressed on average 79.1% ± 12.2% fetal globin (Mean ± SD) whereas GATAA motif enhancer region KO/KO cells expressed approximately 48.4% ± 14.1% fetal globin, in comparison with 14.5% ± 9.6% in WT/WT cells . These levels of fetal globin should be sufficiently high to confer protection against HbS polymerization in sickle cells. WT/KO cells in both coding and enhancer editing experiments showed an intermediate phenotype with fetal globin averaging 26.9%± 9.9% and 25.79% ± 12.6%, respectively. Interestingly, when background (WT/WT) fetal globin level was subtracted, the fetal globin levels in WT/KO cells are comparable to those observed in patients with BCL11A haploinsufficiency, which average 14.6%± 10.3%. Together, our data demonstrate that genome editing of BCL11A using highly efficient ZFNs can lead to clinically relevant levels of fetal globin expression in KO/KO erythroid cells. If the frequency of KO/KO BFU-Es we observed in vitro reflects the frequency of KO/KO HSCs in bone marrow after autologous transplantation, genome editing of BCL11A has the potential to provide significant clinical benefit for patients with SCD. DisclosuresChang:Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Liu:Biogen: Employment, Equity Ownership. Yang:Biogen: Employment, Equity Ownership. Sun:Biogen: Employment, Equity Ownership. Vieira:Biogen: Employment, Equity Ownership. Zhang:Biogen: Employment. Hong:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Tan:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.

Long-Term Increase in Fetal Hemoglobin Expression in Nonhuman Primates Following Transplantation of Autologous Bcl11a Nuclease-Edited HSCs

Elevated levels of fetal hemoglobin (HbF) ameliorate the clinical symptoms of beta-thalassemia and sickle cell anemia. The transcription factor B-cell lymphoma/leukemia 11A (BCL11A) is required for silencing of gamma-globin expression in adult erythroid cells and functions as a switch from fetal to adult hemoglobin production in humans. BCL11A therefore constitutes a therapeutic target for the treatment of hemoglobinopathies. We inactivated BCL11A function by double-strand DNA break-induced mutagenesis using Transcription Activator-Like Effector Nucleases (TALENs). 20 to 30% gene editing could be achieved in vitro in human and nonhuman primate CD34+ cells by TALEN mRNAs electroporation targeting exon 2 of Bcl11a. Colony-forming efficiency was slightly lower in Bcl11a-edited CD34+ cells but lineage differentiation potential was unchanged. Erythroid differentiation of CD34+ cells in culture showed increased Fetal to Beta hemoglobin ratio in both human and primate Bcl11a-modified cells as compared to control cells, thus validating our editing approach to increase HbF production.To determine if Bcl11a-edited hematopoietic stem cells (HSCs) could be engrafted and give rise to HbF-producing erythrocytes, we transplanted a pigtail macaque with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. We detected about 1 % gene disruption in vivo early post-transplant and disruption frequency gradually declined to reach a set point of about 0.3% starting at day 28 post-transplantation. In this analysis, which we have so far taken out to 42 days, single clones could be tracked based on their mutation signature, and we found that several clones persisted over time, confirming engraftment of Bcl11a-modified cells. Since the transplantation procedure and chemo-radiotherapy conditioning can raise HbF production, three control animals that were transplanted using similar conditions as with the Bcl11a-edited HSCs and one untransplanted animal were also included in our analysis. Flow cytometry measurement of HbF in peripheral blood showed a rapid increase in F-cell production in all animals, reaching levels that ranged from 10% to 40% by 30 days, while the untransplanted control showed basal HbF expression of about 0.5% (Fig. 1A). The peak for HbF expression lasted for about 140 days and eventually returned to basal levels that averaged 0.5% for all control animals. In comparison, the animal transplanted with Bcl11a-edited cells showed significantly higher HbF levels starting at day 140 post-treatment (1-1.5%), and HbF production has remained constant for at least 150 days. This result was confirmed by hemoglobin mRNA analysis in peripheral blood using real-time PCR. We found a rapid increase in gamma globin expression following transplantation, before returning to near basal levels. As compared to controls, the animal transplanted with Bcl11a-edited cells showed a 5 to 10-fold increase in gamma to beta globin ratio at day 140 and this ratio has remained constant ever since (Fig. 1B). We are currently working on ways to enhance Bcl11a-editing and to select for Bcl11a-modified HSCs using targeted integration of the chemoselection cassette P140K MGMT to ultimately achieve curative HbF production. Potential TALEN off-target sites will also be examined as well as any side effect associated with the inactivation of BCL11A. Overall, our data demonstrate that transplantation of Bcl11a-edited HSCs results in elevated HbF production in nonhuman primates. Furthermore, we show that nonhuman primates can serve as a useful model for novel gene editing strategies toward the treatment of hemoglobinopathies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pathophysiological role of cytoglobin, the fourth globin in mammals, in liver diseases.

Cytoglobin (Cygb), a stellate cell-specific globin, has recently drawn attention due to its association with liver fibrosis and cancer. In human and rodent livers, Cygb is expressed only in stellate cells and can be utilized as a marker to distinguish stellate cells from hepatic fibroblast-derived myofibroblasts. Loss of Cygb accelerates liver fibrosis and cancer development despite its etiology in mouse models of chronic liver injury. This review discusses the current perception of the distribution, regulation and function of Cygb with regard to liver diseases, with an emphasis on its role in tumorigenesis. Further investigation of Cygb may shed new light on the biology of organ carcinogenesis.

Drosophila glob1 expresses dynamically and is required for development and oxidative stress response.

Biological significance of the globin protein family could be ascertained by their conservation through archaea to human. Globin(s) have been "classically" studied as oxygen binding protein(s), with recent implications in a host of other physiological functions. Drosophila melanogaster possesses three globin genes (glob1, glob2, glob3) located at different cytogenetic positions. We have performed a comprehensive investigation on the cellular expression profile and functional relevance of glob1 in Drosophila development. A profound level of maternally contributed glob1 gene products was found during early embryogenesis. Subsequently, commencement of zygotic transcription leads to its strong expression in somatic muscles, gut primordia, fat bodies, tracheal cells, etc. Similarly, dynamic expression of glob1 was evident in most of the larval tissues, interestingly with high expression in dividing cells. Reduced expression of glob1 leads to various impairments and lethality during embryogenesis and larval development. A substantial increase in level of cellular ROS was also evident due to reduced expression of glob1 which consequently leads to locomotor impairment and early aging in surviving adult flies. To best of our knowledge, this is the first report which demonstrates that in addition to oxygen management, globin gene(s) are also involved in regulating various aspects of development in Drosophila.

Application effect of short-form mini-nutritional assessment on malnutrition evaluation of elderly patients with cardiovascular

Objective To evaluate the nutritional status of elderly patients with cardiovascular by short-form mini-nutritional assessment (MNA-SF), implement nursing interventions for malnourished patients, in order to reduce their risk of malnutrition and to improve their nutritional status. Methods The MNS-SF was used to evaluate the nutritional level of hospitalized patients, and mini-nutritional assessment (MNA) was utilized for malnutrition patients. The nursing interventions were implemented among malnutrition or malnutrition potential patients. Results Comparing before and after nursing interventions, the scores of MNA-SF and MNA had high uniformity (r=0.773, 0.805; P<0.01). After interventions, the scores of MNA-SF and MNA were (12.48±0.65) and (25.34±1.29) higher than the scores when admitted (t=12.377, 15.120; P<0.01). After nursing interventions, the leukomonocyte was (1.44±0.66)×109/L, red cell (3.90±0.52)×1012/L, hemoglobin (120.26±15.93)g/L, hematocrit (0.360±0.055), albumin (36.27±3.78)g/L, globin (29.92±7.44)g/L, which were all higher than those before interventions (t=2.529, 4.937, 5.726, 3.947, 5.547, 2.125; P<0.05); the patients′ blood lipid level was (3.93±0.90) mmol/L, TG (1.02±0.53) mmol/L, LDL-C (1.09±0.29) mmol/L and HDL-C (2.08±0.58) mmol/L better than those before interventions (t=2.875, 3.633, 3.557, 5.727; P<0.01). Conclusions The utilization of MNA-SF and MNA can simple, effective screen out malnutrition risk patients, and then the nursing interventions can reduce the risk of malnutrition in elderly patients with cardiovascular and improve the nutritional status of the patients. Key words: Elderly; Cardiovascular; Malnutrition; Nursing intervention; Short-form mini-nutritional assessment

AB044. Update in the management of thalassemia.

Treatment of thalassemia depends on the clinical diagnosis. Almost all severe alpha thalassemia, Hb Bart’s hydrops fetalis, die intra utero or a few minute after birth. However, pregnant woman with Hb Bart’s hydrops fetus may develop toxemia of pregnancy and antepartum and postpartum hemorrhage. Those homozygous beta thalassemias (transfusion dependent thalassemia, TDT) are severely anemic after 6 months. At birth the baby is asymptomatic because Hb F level is high. As Hb F production waning off, replaced by inefficient beta globin chain production, at the age of 6 to 12 months, the baby begins to be anemic with hepatosplenomegaly. A group of non transfusion dependent thalassemia (NTDT) includes the majority of beta thalassemia/Hb E, Hb H disease and a few cases of homozygous beta thalassemias. The thalassemia minor including both alpha and beta thalassemia carriers and some homozygous state like homozygous alpha+ thalassemia, homozygous Hb E are asymptomatic and does not need any regular treatment. All thalassemia major, TDT, patients need regular blood transfusion, every 3-4 weeks, to maintain the hemoglobin around 10-12 g/dL. For those NTDT blood is given only when necessary. Usually patients with hemoglobin level higher than 6 g/dL require no blood transfusion. In infants and children if the hemoglobin levels can be maintained at 7 g/dL or above, defective physical development and bone changes can be prevented until they reached third or fourth decades of life that osteoporosis is almost always presented in those with low hemoglobin level. Iron overload occurs in moderate and severe case without exception. This will lead to dark skin, liver cirrhosis, cardiac arrhythmia and congestive heart failure. Diabetes mellitus secondary to iron deposition in the pancreas and other endocrine dysfunction does develop if the patients live long enough. Iron chelation with 1-2 gm/day of desferrioxamine intravenously or intramuscularly every day, at least 5 days a week, is recommended for those patients who have hemochromatosis. Oral iron chelator such as deferiprone and deferasirox is recently available. This helps patients to have better compliance with the iron chelator. Stem cell transplantation has been tried with a very good result in class I cases. Because some difficulty in finding appropriated HLA matched donor lately people try to perform haploidentical stem cell transplantation with some good results. Lupstaercept (ACE-536) and Sotatarcept (ACE-011), a recombinant fusion protein containing modified activin receptor type IIB and IgG Fc, is being developed for the treatment of anemia due to ineffective erythropoiesis. Preliminary data showed that the compound could increase hemoglobin levels 1.5 g/dL after two weeks of treatment in NTDT case and decrease blood transfusion in 60% of cases with TDT. Research is in progress to find better agents to enhance Hb F production.

AB027. Developing capacity for variant data sharing in low and middle income countries: HVP’s Global Globin 2020 Challenge

The hemoglobinopathies, collectively, are cause for significant morbidity and mortality. Children are the most severely affected. Despite much of the genetics and biology of hemoglobinopathies being known for a long time, and being used successfully in some countries to systematically reduce burden of disease, many low and medium income countries remain practically untouched by recent developments in human genomics involving the systematic collection and sharing of variation data to fighting hemoglobinopathies (notably thalassaemias and sickle cell disease, but also G6PD). Commitment to systematic variant data collection is increasing, but this is occurring mostly in high-income countries where much of the diagnosis and testing takes place. There is a risk that countries with the highest burden of these diseases are being left behind in a form of “genomic divide”. Capacity to generate quality data on variants, to store this information so that it can be shared internationally, needs to be built in these countries. Tackling hemoglobinopathies is an ideal entry point for these countries to develop the necessary infrastructure and expertise that can expand into other areas of health. This genomic capacity will enable building: (I) the genetic evidence base for better management of delivery of local treatment, care and eventually even cure; (II) a foundation for genomic medicine by working with national, regional and local health care professionals to raise public awareness of the genetic basis of hemoglobinopathies. Global Globin 2020 Challenge has been initiated with two goals: (I) to see growth in the quality and quantity of curated inputs into internationally recognized genetic databases from low- and middle-income countries participating in the project, and to harmonize the sharing of all relevant variant data between countries in accordance with international best practice that integrates all the relevant ethical and regulatory frameworks and policies required to protect patients at the same time that the biotechnical procedures are developed; (II) to ensure that the storage, curation and sharing of the relevant DNA variation information is sustainable in the medium and longer term by expanding and strengthening the international network of professionals, including curators, researchers, clinicians, bioinformaticians, counsellors, patient groups and policymakers. Pursuit of these goals will raise the profile of genomic medicine in low and middle income countries in national, regional and international research organizations. It will also develop the capability of professionals required for diagnosing, treating and counseling carriers in low and middle income countries thus giving them a greater voice and profile among genomic researchers globally so they can actively participate in regional and international partnerships related to genomic research. Initially the GG2020 Challenge will focus on a group of countries that have already formed groups of the relevant professionals including: Belgium, China, Cyprus, Egypt, France, Malaysia, Mexico, Mozambique, Nigeria, South Africa, Venezuela, Vietnam, Portugal, and The Netherlands. Other countries are ready to be included as the project expands. HVP will utilize its relationship with both UNESCO and WHO to ensure that the necessary international standards and procedures are developed in a consultative and harmonized manner.

ΔNp63 targets cytoglobin to inhibit oxidative stress-induced apoptosis in keratinocytes and lung cancer.

During physiological aerobic metabolism, the epidermis undergoes significant oxidative stress as a result of the production of reactive oxygen species (ROS). To maintain a balanced oxidative state, cells have developed protective antioxidant systems, and preliminary studies suggest that the transcriptional factor p63 is involved in cellular oxidative defence. Supporting this hypothesis, the ΔNp63α isoform of p63 is expressed at high levels in the proliferative basal layer of the epidermis. Here we identify the CYGB gene as a novel transcriptional target of ΔNp63 that is involved in maintaining epidermal oxidative defence. The CYGB gene encodes cytoglobin, a member of the globin protein family, which facilitates the diffusion of oxygen through tissues and acts as a scavenger for nitric oxide or other ROS. By performing promoter activity assays and chromatin immunoprecipitation, reverse transcriptase quantitative PCR and western blotting analyses, we confirm the direct regulation of CYGB by ΔNp63α. We also demonstrate that CYGB has a protective role in proliferating keratinocytes grown under normal conditions, as well as in cells treated with exogenous hydrogen peroxide. These results indicate that ΔNp63, through its target CYGB has an important role in the cellular antioxidant system and protects keratinocytes from oxidative stress-induced apoptosis. The ΔNp63-CYGB axis is also present in lung and breast cancer cell lines, indicating that CYGB-mediated ROS-scavenging activity may also have a role in epithelial tumours. In human lung cancer data sets, the p63-CYGB interaction significantly predicts reduction of patient survival.

Inactivation of HDAC1 or HDAC2 induces gamma globin expression without altering cell cycle or proliferation.

Other than hydroxyurea, no pharmacologic agents are clinically available for fetal hemoglobin (HbF) induction in sickle cell disease (SCD). An optimal candidate would induce HbF without causing cell cycle inhibition and would act independently of hydroxyurea in order to yield additional HbF induction when combined. We explored whether inhibition of histone deacetylase (HDAC) 1 or HDAC2 could achieve these goals. In human erythroid progenitor cells, shRNA knockdown of the HDAC1 or HDAC2 genes induced gamma globin, without altering cellular proliferation in vitro, and without altering cell cycle phase. Treatment with hydroxyurea in combination with HDAC2 knockdown yielded a further increase in gamma globin expression. Additionally, when CD34+ cells were treated with both hydroxyurea and MS-275 (an inhibitor of HDAC 1, 2, and 3), an additive induction of relative gamma globin expression was achieved. Our findings support further clinical investigation of HDAC inhibitors in combination with hydroxyurea in SCD patients.

Intermediaries of branched chain amino acid metabolism induce fetal hemoglobin, and repress SOX6 and BCL11A, in definitive erythroid cells.

High levels of fetal hemoglobin (HbF) can ameliorate human β-globin gene disorders. The short chain fatty acid butyrate is the paradigmatic metabolic intermediary that induces HbF. Inherited disorders of branched-chain amino acid (BCAA) metabolism have been associated with supranormal HbF levels beyond infancy, e.g., propionic acidemia (PA) and methylmalonic acidemia (MMA). We tested intermediaries of BCAA metabolism for their effects on definitive erythropoiesis. Like butyrate, the elevated BCAA intermediaries isovalerate, isobutyrate, and propionate, induce fetal globin gene expression in murine EryD in vitro, are associated with bulk histone H3 hyperacylation, and repress the transcription of key gamma globin regulatory factors, notably BCL11A and SOX6. Metabolic intermediaries that are elevated in Maple Syrup Urine Disease (MSUD) affect none of these processes. Percent HbF and gamma (γ) chain isoforms were also measured in non-anemic, therapeutically optimized subjects with MSUD (Group I, n=6) or with Isovaleric Acidemia (IVA), MMA, or PA (Group II, n=5). Mean HbF was 0.24 ± 0.15% in Group I and 0.87 ± 0.13% in Group II (p=.01); only the Gγ isoform was detected. We conclude that a family of biochemically related intermediaries of branched chain amino acid metabolism induces fetal hemoglobin during definitive erythropoiesis, with mechanisms that mirror those so far identified for butyrate.