Genetic regulation of fetal hemoglobin across global populations.

Human genetic variation has enabled the identification of several key regulators of fetal-to-adult hemoglobin switching, including BCL11A, resulting in therapeutic advances. However, despite the progress made, limited further insights have been obtained to provide a fuller accounting of how genetic variation contributes to the global mechanisms of fetal hemoglobin (HbF) gene regulation. Here, we have conducted a multi-ancestry genome-wide association study of 28,279 individuals from several cohorts spanning 5 continents to define the architecture of human genetic variation impacting HbF. We have identified a total of 178 conditionally independent genome-wide significant or suggestive variants across 14 genomic windows. Importantly, these new data enable us to better define the mechanisms by which HbF switching occurs in vivo. We conduct targeted perturbations to define BACH2 as a new genetically-nominated regulator of hemoglobin switching. We define putative causal variants and underlying mechanisms at the well-studied BCL11A and HBS1L-MYB loci, illuminating the complex variant-driven regulation present at these loci. We additionally show how rare large-effect deletions in the HBB locus can interact with polygenic variation to influence HbF levels. Our study paves the way for the next generation of therapies to more effectively induce HbF in sickle cell disease and ß-thalassemia.

Common Single Nucleotide Polymorphism of TMPRSS6, an Iron Regulation Gene, Associated with Variable Red Blood Cell Indices in Deletional α-Globin Genotypes.

Red blood cell (RBC) indices, including mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH), have been widely used for primary screening for thalassaemia (thal) syndromes. Recently, a single nucleotide polymorphism (SNP) rs855791 of TMPRSS6, an iron regulation gene involved in the substitution of a nucleotide between thymine (T) and cytosine (C) in exon 17 resulted in an amino acid change, p.Val736Ala (V736A), has been described to associate with RBC indices. The objective was to study the effects of common SNP V736A on RBC indices in deletional α-thal variations. SNP rs855791 genotypes were identified from 433 Thai volunteers, including 32.6% males and 67.4% females with an average age of 23.0 ± 8.7 years. These populations included individuals (82.4%) who had normal globin genotype (αα/αα, ßß) and α-thal carriers, which were divided into two subgroups, including α+-thal (-α/αα) (14.1%) and αo-thal (--/αα) (3.5%). Among three SNP genotypes, the C allele gradually expressed higher MCV and MCH than those of the T allele in both α+- and αo-thal traits. Importantly, SNP rs855791 of TMPRSS6 responded to α-globin deletions for sustaining RBC sizes and haemoglobinisation in α-thal carriers.

Identification of optimal thalassemia screening strategies for migrant populations in Thailand using a qualitative approach.

BACKGROUND: Thalassemia is a common inherited hemoglobin disorder in Southeast Asia. Severe thalassemia can lead to significant morbidity for patients and economic strain for under-resourced health systems. Thailand's thalassemia prevention and control program has successfully utilized prenatal screening and diagnosis to reduce the incidence of severe thalassemia in Thai populations, but migrant populations are excluded despite having high thalassemia prevalence. We sought to identify key barriers to and facilitators of thalassemia screening and to develop tailored recommendations for providing migrants with access to thalassemia prevention and control. METHODS: We conducted 28 in-depth interviews and 4 focus group discussions (FGDs) in Chonburi, Thailand with Myanmar and Cambodian migrants, Thai healthcare providers, Thai parents of children affected by thalassemia, and migrant agents. RESULTS: Participant narratives revealed that migrants' lack of knowledge about the prevalence, manifestations, severity, and inherited nature of thalassemia led to misconceptions, fear, or indifference toward thalassemia and screening. Negative perceptions of pregnancy termination were based in religious beliefs but compounded by other sociocultural factors, presenting a key obstacle to migrant uptake of prenatal screening. Additionally, structural barriers included legal status, competing work demands, lack of health insurance, and language barriers. Participants recommended delivering public thalassemia education in migrants' native languages, implementing carrier screening, and offering thalassemia screening in convenient settings. CONCLUSIONS: An effective thalassemia prevention and control program should offer migrants targeted thalassemia education and outreach, universal coverage for thalassemia screening and prenatal care, and options for carrier screening, providing a comprehensive strategy for reducing the incidence of severe thalassemia in Thailand and establishing an inclusive model for regional thalassemia prevention and control.

Feasibility of and barriers to thalassemia screening in migrant populations: a cross-sectional study of Myanmar and Cambodian migrants in Thailand.

BACKGROUND: Thalassemia, an inherited hemoglobin disorder, has become a global public health problem due to population migration. Evidence-based strategies for thalassemia prevention in migrants are lacking. We characterized barriers to thalassemia screening and the burden of thalassemia in migrant workers in Thailand. METHODS: Multilingual demographic and KAP surveys were completed by 197 Thai, 119 Myanmar, and 176 Cambodian adults residing in Thailand. Thalassemia awareness, socio-demographic predictors, and knowledge and attitude scores were compared between migrant and Thai subjects. Comprehensive thalassemia testing was performed for migrants. RESULTS: Migrants had extremely poor thalassemia awareness (4.1%) compared to Thai subjects (79.6%) and had lower thalassemia knowledge scores but similar attitude scores. Surveys identified differing sociodemographic factors predicting awareness in Thai and migrant subjects, as well as key misconceptions likely to hinder thalassemia screening uptake. Nearly all migrants consented to thalassemia testing. We identified abnormal hemoglobin profiles in 52.7% of migrants and a higher projected rate of severe thalassemia births in migrants. CONCLUSIONS: The high burden of thalassemia and tremendous knowledge gap in migrants needs urgent attention. Thalassemia screening was feasible and acceptable in our migrant population. Sociocultural and structural barriers merit further attention when designing thalassemia screening and prevention policies for migrants in Thailand and globally.

Paper-based microchip electrophoresis for point-of-care hemoglobin testing.

Nearly 7% of the world's population live with a hemoglobin variant. Hemoglobins S, C, and E are the most common and significant hemoglobin variants worldwide. Sickle cell disease, caused by hemoglobin S, is highly prevalent in sub-Saharan Africa and in tribal populations of Central India. Hemoglobin C is common in West Africa, and hemoglobin E is common in Southeast Asia. Screening for significant hemoglobin disorders is not currently feasible in many low-income countries with the high disease burden. Lack of early diagnosis leads to preventable high morbidity and mortality in children born with hemoglobin variants in low-resource settings. Here, we describe HemeChip, the first miniaturized, paper-based, microchip electrophoresis platform for identifying the most common hemoglobin variants easily and affordably at the point-of-care in low-resource settings. HemeChip test works with a drop of blood. HemeChip system guides the user step-by-step through the test procedure with animated on-screen instructions. Hemoglobin identification and quantification is automatically performed, and hemoglobin types and percentages are displayed in an easily understandable, objective way. We show the feasibility and high accuracy of HemeChip via testing 768 subjects by clinical sites in the United States, Central India, sub-Saharan Africa, and Southeast Asia. Validation studies include hemoglobin E testing in Bangkok, Thailand, and hemoglobin S testing in Chhattisgarh, India, and in Kano, Nigeria, where the sickle cell disease burden is the highest in the world. Tests were performed by local users, including healthcare workers and clinical laboratory personnel. Study design, methods, and results are presented according to the Standards for Reporting Diagnostic Accuracy (STARD). HemeChip correctly identified all subjects with hemoglobin S, C, and E variants with 100% sensitivity, and displayed an overall diagnostic accuracy of 98.4% in comparison to reference standard methods. HemeChip is a versatile, mass-producible microchip electrophoresis platform that addresses a major unmet need of decentralized hemoglobin analysis in resource-limited settings.

Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target.

BACKGROUND: Based on resistance of currently used anti-malarials, a new anti-malarial drug target against Plasmodium falciparum is urgently needed. Damaged DNA cannot be transcribed without prior DNA repair; therefore, uracil-DNA glycosylase, playing an important role in base excision repair, may act as a candidate for a new anti-malarial drug target. METHODS: Initially, the native PfUDG from parasite crude extract was partially purified using two columns, and the glycosylase activity was monitored. The existence of malarial UDG activity prompted the recombinant expression of PfUDG for further characterization. The PfUDG from chloroquine and pyrimethamine resistant P. falciparum strain K1 was amplified, cloned into the expression vector, and expressed in Escherichia coli. The recombinant PfUDG was analysed by SDS-PAGE and identified by LC-MS/MS. The three dimensional structure was modelled. Biochemical properties were characterized. Inhibitory effects of 12 uracil-derivatives on PfUDG activity were investigated. Inhibition of parasite growth was determined in vitro using SYBR Green I and compared with results from human cytotoxicity tests. RESULTS: The native PfUDG was partially purified with a specific activity of 1,811.7 units/mg (113.2 fold purification). After cloning of 966-bp PCR product, the 40-kDa hexa-histidine tagged PfUDG was expressed and identified. The amino acid sequence of PfUDG showed only 24.8% similarity compared with the human enzyme. The biochemical characteristics of PfUDGs were quite similar. They were inhibited by uracil glycosylase inhibitor protein as found in other organisms. Interestingly, recombinant PfUDG was inhibited by two uracil-derived compounds; 1-methoxyethyl-6-(p-n-octylanilino)uracil (IC50 of 16.75 µM) and 6-(phenylhydrazino)uracil (IC50 of 77.5 µM). Both compounds also inhibited parasite growth with IC50s of 15.6 and 12.8 µM, respectively. Moreover, 1-methoxyethyl-6-(p-n-octylanilino)uracil was not toxic to HepG2 cells, with IC50 of > 160 µM while 6-(phenylhydrazino)uracil exhibited cytoxicity, with IC50 of 27.5 µM. CONCLUSIONS: The recombinant PfUDG was expressed, characterized and compared to partially purified native PfUDG. Their characteristics were not significantly different. PfUDG differs from human enzyme in its size and predicted amino acid sequence. Two uracil derivatives inhibited PfUDG and parasite growth; however, only one non-cytotoxic compound was found. Therefore, this selective compound can act as a lead compound for anti-malarial development in the future.