ALBERT

Description: Severe forms of α-thalassaemia, haemoglobin H disease and haemoglobin Bart’s hydrops fetalis, are an important public health concern in Southeast Asia. Yet information on the prevalence, genetic diversity and health burden of α-thalassaemia in the region remains limited. We compiled a geodatabase of α-thalassaemia prevalence and genetic diversity surveys and, using geostatistical modelling methods, generated the first continuous maps of α-thalassaemia mutations in Thailand and sub-national estimates of the number of newborns with severe forms in 2020. We also summarised the current evidence-base for α-thalassaemia prevalence and diversity for the region. We estimate that 3595 (95% credible interval 1,717–6,199) newborns will be born with severe α-thalassaemia in Thailand in 2020, which is considerably higher than previous estimates. Accurate, fine-scale epidemiological data are necessary to guide sustainable national and regional health policies for α-thalassaemia management. Our maps and newborn estimates are an important first step towards this aim.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Description: Introduction: Although routine tissue iron monitoring using magnetic resonance imaging (MRI) has become a standard clinical management of both transfusion dependent and non-transfusion dependent thalassemias (TDT & NTDT) in several developed countries since this module can provide a better organ-directed iron measurement and related to clinical outcome including morbidity and mortality secondary to iron overload (IOL). However, accessibility of this monitoring remains limited in several developing countries including Thailand, where thalassemia and hemoglobinopathies are highly prevalent. Earlier in 2016, we have demonstrated that although not so perfectly crafted, a cross-sectional measurement of serum ferritin (SF) can be used for determination of IOL as a predictive marker; in NTDT, MRI for liver iron concentration (LIC) should be performed in those with SF 〉300 ug/L and for TDT, the SF cut off of 〉2,500 and 〉3,500 ug/L are useful to predict patients with severe LIC (〉15 mgFe/g dw) and cardiac siderosis (T2*〈 20 ms) (Ekwattanakit S. et.al., EHA 2016). In this study, we performed a further analysis to evaluate whether a serial measurement of SF and its trend can provide a better prediction. Objectives: To evaluate the clinical utility of serial SF trend compared with a cross-sectional SF cut-off for early detection of IOL in a real-life practice in thalassemic patients in order to select the most vulnerable patients for further MRI evaluation in a resource limited setting. Methods: In this prospective study, total 968 standard MRI for LIC and cardiac T2*were performed at Siriraj hospital during 2009-2014 and paired clinical data including serial SF measurements were collected from 301 thalassemia patients; NTDT (N=76; 109 LIC and 95 cardiac T2* results) and TDT (N=155; 478 LIC and 474 cardiac T2*). In addition, 71 patients were NTDT with regular blood transfusion later on in their life, mainly Hb E/β thalassemia (218 LIC and 210 cardiac T2*). These patients were evaluated for IOL using SF every 4-12 weeks during their follow up. Median follow up time was 96 months. Receiver operating characteristic (ROC) analysis was performed using different SF cut-off levels (1000, 1500 and 2000ug/L) and percentage of serial SF measurements that above each these cut-off levels (50 and 75%) during different durations before MRI (1, 2, or 3 years priori) for predicting liver IOL (LIC 〉5 in NTDT and severe liver siderosis; LIC 〉 15 mgFe/g dw) and cardiac IOL (T2*1000 ug/L over 75% of serial measurements in 1-year period prior to MRI can predict LIC 〉 5 mgFe/g dw with the AUC of 0.59 with PPV 86.7% and NPV 51.1% and serial SF 〉1500 ug/l over 50% of measurement in a year prior predict LIC 〉15 mgFe/g dw (AUC of 0.72, PPV 75% and NPV 89.7%). Only 1 NTDT had cardiac IO. These newly identified criteria do not provide a more sensitive predictor of LIC results than our previous SF cut-off. In TDT population, the majority of patients were HbE/β thal (78%) and cardiac iron overload was detected mainly in patients older than 15 yrs (81/83; 97.6%). The best predictor for LIC〉15 mgFe/g dw was SF 〉2000 ug/L over 75% of serial measurement durations in 3-year period (AUC 0.778, PPV 50.8%, NPV 95.5%). However this cut-off during 1 year priori also provided a similar prediction (AUC 0.769, PPV 51%, NPV 93.6%). This cut-off value also provided the best prediction for cardiac T2* 〈 20 ms in all age group (AUC 0.754, PPV 31.5%, NPV 97.4%) and a higher sensitivity and specificity when it was applied in patients 〉15 yrs of age (AUC 0.764, PPV 41.9%, NPV 96.3%). In NTDT with regular transfusion, all cardiac IOL (N=19) occurred after 15 years of age and again the same criteria was the good predictive cut-off for cardiac IO (AUC 0.788, PPV 19%, NPV 100%) and severe LIC 〉15 mgFe/g dw (AUC 0.728, PPV 52.8%, NPV 87.3%). Conclusions: In a resource limited setting for MRI evaluation, a serial measurement of SF and its values in thalassemic patients who received regular blood transfusion above the cut-off of 2000 ug/L over 75% of measurement in one year priori could be used as a predictive marker for selecting the most vulnerable TDT for MRI evaluation since this criteria is strongly associated with severe liver (LIC 〉 15 mgFe/g dw) and cardiac siderosis (T2*

Description: Introduction: Pyruvate kinase-red blood cell (PK-R) enzyme is an isoform of PK-liver (PK-L) expressed by tissue-specific promoter on the PKLR gene. The PK-R functions in the glycolytic pathway to generate pyruvate & adenosine triphosphate (ATP), the energy carrier molecule supporting 120-day red blood cells (RBCs) survival. Patients with PKLR mutations have PK deficiency (PKD) & defective RBCs leading to different degree of chronic hemolysis from non-transfusion to transfusion dependent. A novel synthetic AG-348 compound, an allosteric activator of PK, enhances PK-R activity in patients with PKD (Kung et al., 2017). Previously, our group described a novel form of severe hemolytic anemia due to mutations of the Krϋppel-like factor 1 (KLF1 disease) (Viprakasit et al., 2014). Besides deregulated globin gene switching with persistent embryonic globin expression, all KLF1 patients had low PK-R activity leading to hemolytic anemia & transfusion dependency. Decreased KLF1 expression in trans lead to phenotypes mimic that of PKD since the PKLR genes are regulated by the KLF1. At present, there is no specific treatment for KLF1 disease besides supportive care & RBCs transfusion. Objective: To determine ex vivo effects of AG-348 on PK-R activity, ATP production & potential adverse effects on RBC physiology (hemolysis, inducing reactive oxygen species (ROS) and apoptosis) in RBCs from KLF1 patients. Methods: EDTA-whole blood samples were collected from two non-regularly transfused KLF-1 patients. The genotypes of the PKLR and KLF1 genes were confirmed by Sanger sequencing. RBCs were isolated by cellulose column and prepared into a suspension; 1 x 108 RBCs/mL and subsequently incubated with various concentrations of AG-348 (Agios Co. Ltd.); 0.002 - 10 μM at 37⁰C overnight. The AG-348-treated RBCs were then harvested and determined for PK-R activity by coupled enzyme assay with lactate dehydrogenase and ATP production. Furthermore, we evaluated possible adverse consequences of this compound by evaluating hemolysis, intracellular ROS and apoptosis and numeration by flow cytometry. Results: Two adolescent patients: patient A & B had chronic anemia with baseline hemoglobin (Hb) ≤ 8 g/dL with high reticulocyte count (10 %) and high level of Hb F. The genotypes of KLF1 gene were identified in both subjects as compound heterozygotes of one null and one missense mutation (Table 1). Both patients had low PK-R activity but no PKLR gene mutation identified. Using an ex vivo assay, the PK-R activities of patients' RBCs significantly increased after incubation with AG-348 in a dose-dependent manner for 100% to 150% compared to baseline and 0.1% DMSO as control. Moreover, the ATP production, a direct product from RBC glycolytic pathway driven by PK catalysis also significantly increased in AG-348 incubated RBCs in a similar manner (Figure 1A and 1B). Nevertheless, there were no changes in normal RBC controls for both PK activity and ATP generation. Also, AG-348 did not adversely induce hemolysis in the KLF1-mutated RBCs as determined by Hb released (µg/mL) in the culture medium compared with 0.1% DMSO (Figure 1C). Similarly, intracellular ROS detected by mean fluorescent intensity (MFI) & apoptotic signals did not increase in normal and KLF1-mutated RBCs (data not shown). Conclusion: Our ex vivo study using RBCs derived from KLF-1 patients is the first investigation to suggest a potential clinical role of the AG-348 as a therapeutic intervention in this hereditary condition. Our results were consistent with the previous study that AG-348 enhanced PK-R activity & ATP production in RBCs from patients with classical PKD due to PKLR mutations (Kung et al., 2017) and currently this compound is under investigation in patients with non-transfusion dependent & transfusion dependent PKD (NCT03548220 and NCT03559699, respectively). For the safety profile, the AG-348 does not induce ROS and apoptosis in our ex vivo setting. Interestingly, our data showed that AG-348 might be effective for RBCs with lower PK activity and potentially improves RBCs survival, no matter the causes of PKD. Our study warrants further investigation to determine whether this PK correction could be translated into a clinical benefit in our KLF-1 patients. Finally, our finding suggests that the activity of AG-348 might include those with higher RBCs turnover & exhaust PK activity such as other hemolytic anemias especially thalassemia & hemoglobinopathy. Disclosures No relevant conflicts of interest to declare.

Description: Background Transfusion-dependent patients with severe cardiac siderosis often require intensive iron chelation therapy for a limited time to facilitate rapid removal of iron from the heart, allowing patients to move from a high-risk (cardiac T2*

Description: Hereditary hemochromatosis is an iron overload disorder that can lead to the impairment of multiple organs and is caused by mutations in one or more different genes. Type 1 hemochromatosis is the most common form of the disease and results from mutations in the HFE gene. Juvenile hemochromatosis (JH) is the most severe form, usually caused by mutations in hemojuvelin (HJV) or hepcidin (HAMP). The autosomal dominant form of the disease, type 4, is due to mutations in the SLC40A1 gene, which encodes for ferroportin (FPN). Hereditary hemochromatosis is commonly found in populations of European origin. By contrast, hemochromatosis in Asia is rare and less well understood and can be masked by the presence of iron deficiency and secondary iron overload from thalassemia. Here, we provide a comprehensive report of hemochromatosis in a group of patients of Asian origin. We have identified novel mutations in HJV, HAMP, and SLC40A1 in countries not normally associated with hereditary hemochromatosis (Pakistan, Bangladesh, Sri Lanka, and Thailand). Our family studies show a high degree of consanguinity, highlighting the increased risk of iron overload in many countries of the developing world and in countries in which there are large immigrant populations from these regions.

Description: Abstract 4272 Background: Globally, a number of management guidelines provide recommendations for transfusion and iron chelation therapy across various transfusion-dependent anemias. Most treatment guidelines aim to control body iron burden by maintaining serum ferritin 2500 ng/mL across all anemias. Serum ferritin levels were ≥2500 ng/mL in 61.3% of patients across regions (50.6% [Europe], 59.3% [Middle East/Africa] and 74.3% [Asia-Pacific]). For patients with TM, TI, AA and SCD, serum ferritin levels were substantially higher in the Asia-Pacific region compared with other regions (Figure). In the Asia-Pacific region, the proportion of patients with serum ferritin levels ≥4000 ng/mL varied between 31.1% and 53.6% across anemias, compared with 14.3–37.5% in Europe. Conclusions: There are many differences in transfusion and iron chelation practices across regions, with most prominent differences in the Asia-Pacific region. Factors contributing to these differences might include regional variations in specific disease characteristics (severity, transfusion requirement), treatment practices (eg, hemoglobin level at which transfusion is initiated), the availability and accessibility of transfusion and iron chelation therapy including patients' compliance and physician attitude and adherence to treatment guidelines. The high proportion of patients with baseline serum ferritin 〉2500 ng/mL suggests that previous iron chelation regimens with DFO and/or deferiprone prior to the EPIC study were suboptimal with limitations for adequate control of iron burden across geographical regions. A greater improvement in iron chelation practices is warranted across the globe with an immediate focus on the Asia-Pacific region. Disclosures: Viprakasit: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gattermann:Novartis: Honoraria, Research Funding. Porter:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Taher:Novartis: Honoraria, Research Funding. Habr:Novartis: Employment. Roubert:Novartis: Employment. Domokos:Novartis: Employment. Cappellini:Novartis: Speakers Bureau.

Description: Hemoglobin (Hb) switching is described as temporal, tissue- and stage-specific patterns of globin gene expression; from embryonic to fetal and adult Hb in parallel to developmental stages of erythropoiesis. DNA methylation, one of the epigenetic mechanisms, was associated with inactivated chromatin domain and repressive transcription. To study the role of the DNA methylation on the beta (β)-globin genes, we analyzed CpG dinucleotides in 87 kb regions around β-globin gene cluster, including 5’upstream locus control regions (LCR; DNAse I Hypersensitive site (HS) 1–5), 3’HS1, the promoter regions of the G-and A-gamma (Gγ and Aγ), and β-globin genes, in several representative cells. These cells were primary adult erythroid cells culture (three different stages: early, intermediate, and late), fetal cord blood DNA, and neutrophil cell line (non-erythroid). Using bisulphite modification, followed by nested PCR and in vitro translation, the cleavage products were analysed by MALDI-TOF Mass Spectrometry to quantify the DNA methylation level. The results were consistent with bisulphite sequencing. We found that the promoters of Gγ and Aγ-globin genes were significantly hypomethylated in fetal cells (44% and 47% global methylation), when γ-globin genes were fully expressed, while they were heavily methylated in non-erythroid (86% and 95%). There was also a decreasing trend of the DNA methylation level at Gγ and Aγ-globin genes during adult erythroid differentiation from 80% and 82%, in early stage, to 67% and 66% in late stage (p=0.12 and 0.04). At β-globin promoter, the global methylation level changed from 90% in non-erythroid to 81%, 42%, and 26% in fetal, early and late adult erythroid cells, respectively. Moreover, we found the significant changes at 5’HS4, 3, and 1 as all erythroid cells were hypomethylated compare to non-erythroid. While at the insulators, 5’HS5 and 3’HS1, all tested CpG dinucleotides were heavily methylated in all cells. This is the first report that demonstrates the differences in DNA methylation at β-globin LCR between erythroid and non-erythroid cells. These epigenetic marks were associated with globin genes expression and might be useful to predict clinical severity in patients with β-thalassemia intermedia.

Description: The analysis of globin gene regulation has elucidated many of the principles underlying mammalian gene expression. To understand how the epigenetic program that unfolds during hematopoiesis impacts on alpha (α)-globin gene expression, we analyzed DNA methylation at over 200 CpG dinucleotides in 130 kb around the human α-globin gene cluster focussing particularly on a region (12 kb) covering the α2 and α1-globin genes (HBA2 and HBA1), and the upstream regulatory sites (DNAse I hypersensitive sites (HS) -48, HS-40, HS-33, and HS-10); so-called multi-species conserved sequencesregulatory element1-4 (MCS-R1-4). Cultures of primary adult erythroid cells (at three stages: early, intermediate, and late) were analyzed together with non-erythroid human cells (neutrophils, peripheral blood mononuclear cells, and a human embryonic stem (ES) cell line). Using a recently published method involving bisulphite modification and MALDI-TOF Mass Spectrometry we quantified the level of DNA methylation across the alpha globin cluster. Most sequences outside of the CpG islands were methylated. However, the DNA methylation levels between erythroid and non-erythroid cells differed at regulatory elements. MCS-R1 contained only 1 CpG site and the average percentage of DNA methylation for erythroid and non-erythroid was 8% and 40%, respectively. While they were 8% vs. 42% (p=0.428), 11% vs. 99% (p=0.054), and 14% vs. 97% (p=0.001) in MCS-R2 to 4, respectively. To study changes in DNA methylation in a comprehensive, developmental and tissue-specific manner, we used a humanized-mouse model, in which the conserved α-globin syntenic region of mouse genomic sequence is replaced by that of human. This contains all cis-acting sequences required for fully regulated expression of the α-globin genes. Although the alpha globin cluster is expressed efficiently in this model, DNA methylation patterns differed in the 20 kb region encompassing the α-globin genes when comparing primary human cells and primary humanized cells. DNA in humanized cells were relatively hypomethylated both in erythroid and non-erythroid tissues. The methylation levels were higher in testis of the humanized mouse than in other tissues but still not as high as those in normal human non-erythroid cells. The cause of these differences is being investigated. Despite these differences, the humanized-mouse mimicked the patterns of methylation found in regulatory elements in erythroid and nonerythroid cells. This study shows that despite appropriate regulation of gene expression, epigenetic templating may differ between species (human and mouse). DNA methylation at the upstream regulatory elements might be involved in the regulation of α-globin gene expression during erythropoiesis, although it is equally possible that these changes in methylation are generated by passive demethylation secondary to transcription factor binding in erythroid cells. The mechanism(s) underlying demethylation of regulatory elements during differentiation remains to be clarified.