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.

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.

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.

The genetic basis of asymptomatic codon 8 frame-shift (HBB:c25_26delAA) ß(0) -thalassaemia homozygotes.

Two 21-year old dizygotic twin men of Iraqi descent were homozygous for HBB codon 8, deletion of two nucleotides (-AA) frame-shift ß(0) -thalassaemia mutation (FSC8; HBB:c25_26delAA). Both were clinically well, had splenomegaly, and were never transfused. They had mild microcytic anaemia (Hb 120-130 g/l) and 98% of their haemoglobin was fetal haemoglobin (HbF). Both were carriers of Hph α-thalassaemia mutation. On the three major HbF quantitative trait loci (QTL), the twins were homozygous for G>A HBG2 Xmn1 site at single nucleotide polymorphism (SNP) rs7482144, homozygous for 3-bp deletion HBS1L-MYB intergenic polymorphism (HMIP) at rs66650371, and heterozygous for the A>C BCL11A intron 2 polymorphism at rs766432. These findings were compared with those found in 22 other FSC8 homozygote patients: four presented with thalassaemia intermedia phenotype, and 18 were transfusion dependent. The inheritance of homozygosity for HMIP 3-bp deletion at rs66650371 and heterozygosity for Hph α-thalassaemia mutation was found in the twins and not found in any of the other 22 patients. Further studies are needed to uncover likely additional genetic variants that could contribute to the exceptionally high HbF levels and mild phenotype in these twins.

Mutations in Kruppel-like factor 1 cause transfusion-dependent hemolytic anemia and persistence of embryonic globin gene expression.

In this study, we report on 8 compound heterozygotes for mutations in the key erythroid transcription factor Krüppel-like factor 1 in patients who presented with severe, transfusion-dependent hemolytic anemia. In most cases, the red cells were hypochromic and microcytic, consistent with abnormalities in hemoglobin synthesis. In addition, in many cases, the red cells resembled those seen in patients with membrane defects or enzymopathies, known as chronic nonspherocytic hemolytic anemia (CNSHA). Analysis of RNA and protein in primary erythroid cells from these individuals provided evidence of abnormal globin synthesis, with persistent expression of fetal hemoglobin and, most remarkably, expression of large quantities of embryonic globins in postnatal life. The red cell membranes were abnormal, most notably expressing reduced amounts of CD44 and, consequently, manifesting the rare In(Lu) blood group. Finally, all tested patients showed abnormally low levels of the red cell enzyme pyruvate kinase, a known cause of CNSHA. These patients define a new type of severe, transfusion-dependent CNSHA caused by mutations in a trans-acting factor (Krüppel-like factor 1) and reveal an important pathway regulating embryonic globin gene expression in adult humans.

Clinical presentation and molecular identification of four uncommon alpha globin variants in Thailand. Initiation codon mutation of α2-globin Gene (HBA2:c.1delA), donor splice site mutation of α1-globin gene (IVSI-1, HBA1:c.95 + 1G>A), hemoglobin Queens Park/Chao Pra Ya (HBA1:c.98T>A) and hemoglobin Westmead (HBA2:c.369C>G).

Alpha thalassemia is the most common genetic disease in the world with the prevalence of carriers ranging from 5-50% in several populations. Coinheritance of two defective α-globin genes usually gives rise to a symptomatic condition, hemoglobin (Hb) H disease. Previously, it has been suggested from several studies in different populations that nondeletional Hb H disease (--/α(T)α or --/αα(T)) is generally more severe than the deletional type (--/-α). In this report, we describe four rare nondeletional α-thalassemia mutations in Thai individuals, including initiation codon mutation (HBA2:c.1delA), donor splice site mutation (IVSI-1, HBA1:c.95 + 1G>A), Hb Queens Park (HBA1:c.98T>A) [α32(B13)Met>Lys], and Hb Westmead (HBA2:c.369C>G) [α122(H5)His>Gln]. Interactions of the first three mutations with the α(0)-thalassemia resulted in nondeletional Hb H disease; however, their clinical presentations were rather mild and some were detected accidentally. This suggests that a genotype-phenotype correlation of α-thalassemia syndrome might be more heterogeneous and so the type of mutation does not simply imply the prediction of the resulting phenotype. Our data will be of use in future genetic counseling of such conditions that are increasingly identified thanks to the improvement of molecular analysis in routine laboratories.

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.

Interaction between Hb E and Hb Yala (HBB:c.129delT); a novel frameshift beta globin gene mutation, resulting in Hemoglobin E/ß0 thalassemia.

OBJECTIVES: There are more than 200 known mutations found in patients with ß-thalassemia, a possibility to identify an unknown or novel mutation becomes less possible. Here, we report a novel mutation in a patient from Thailand who presented with chronic hemolytic anemia. METHODS: A comprehensive hematology and DNA analysis was applied in the index patient and her mother. RESULTS: Hematological and hemoglobin analyses were consistent with the clinical diagnosis of Hb E/ß0-thalassemia. However, we could find only Hb E heterozygous mutation using our common polymerase chain reaction-based mutation detection of the ß-globin genes. Furthermore, the molecular analysis demonstrated a novel T-deletion at codon 42 of the second exon of the ß-globin gene which we named 'Hb Yala' according to the origin of this index family. DISCUSSION: This mutation was assumed to generate a truncated ß-globin chain terminating at codon 60 with possible unstable variant leading to a 'null' or ß0-thalassemia. However, the clinical phenotype was surprisingly mild and no other ameliorating genetic factors, including co-inheritance of α-thalassemia and high propensity of Hb F by Xmn I polymorphism, were found. CONCLUSION: This report has provided evidence that genotype-phenotype correlation in thalassemia syndromes is highly complex and a correct clinical severity classification of thalassemia should be mainly based on clinical evaluation.

Human primary erythroid cells as a more sensitive alternative in vitro hematological model for nanotoxicity studies: Toxicological effects of silver nanoparticles.

Although immortalized cells established from cancerous cells have been widely used for studies in nanotoxicology studies, the reliability of the results derived from immortalized cells has been questioned because of their different characteristics from normal cells. In the present study, human primary erythroid cells in liquid culture were used as an in vitro hematological cell model for investigation of the nanotoxicity of silver nanoparticles (AgNPs) and comparing the results to the immortalized hematological cell lines HL60 and K562. The AgNPs caused significant cytotoxic effects in the primary erythroid cells, as shown by the decreased cell viability and induction of intracellular ROS generation and apoptosis, whereas they showed much lower cytotoxic and apoptotic effects in HL60 and K562 cells and did not induced ROS generation in these cell lines. Scanning electron microcopy revealed an interaction of AgNPs to the cell membrane in both primary erythroid and immortalized cells. In addition, AgNPs induced hemolysis in the primary erythroid cells in a dose-dependent manner, and transmission electron microcopy analysis revealed that AgNPs damaged the erythroid cell membrane. Taken together, these results suggest that human primary erythroid cells in liquid culture are a more sensitive alternative in vitro hematological model for nanotoxicology studies.

Diagnostic applications of newborn screening for α-thalassaemias, haemoglobins E and H disorders using isoelectric focusing on dry blood spots.

BACKGROUND: Neonatal screening for haemoglobin (Hb) disorders is a standard of care in several developed countries with the main objective to detect Hb S. Such practice has not been established in Thailand where α-thalassaemia and haemoglobin E (Hb E) are highly prevalent. Early identification of thalassaemias could be helpful and strengthen the programme for prevention and control for severe thalassaemias. METHODS: Data from isoelectric focusing (IEF) and Isoscan® for detecting types and amount (%) of each haemoglobin in 350 newborn's dried blood spots were analysed and compared with the comprehensive genotype analysis by DNA studies as a gold standard. RESULTS: Based on genetic profiles, there were 10 different categories: (1) normal (n = 227), (2) α(+)-thalassaemia trait (n = 14), (3) α(0)-thalassaemia trait (n = 13), (4) ß(0)-thalassaemia trait (n = 7), (5) Hb E trait (n = 72), (6) Hb E trait with α(0)-thalassaemia or homozygous α(+)-thalassaemia (n = 5), (7) Hb E trait with α(+)-thalassaemia trait (n = 5), (8) homozygous Hb E (n = 3), (9) homozygous Hb E with α(0)-thalassaemia trait (n = 1) and (10) Hb H disease (n = 3). The presence of Hb Bart's and Hb E were used to identify cases with α-thalassaemia and Hb E, respectively. We set 0.25% of Hb Bart's and 1.5% of Hb E as a cut-off level to detect α(+)-thalassaemia trait (sensitivity 92.86% and specificity 74.0%) and Hb E trait with 100% of both sensitivity and specificity for IEF diagnosis. CONCLUSION: Although molecular diagnosis seems to be better for definitive diagnosis of thalassaemia syndromes at birth, however, using our reference range described herein, IEF can be applied in a resource-limiting setting with acceptable reliability.

Association of xmn I polymorphism and hemoglobin e haplotypes on postnatal gamma globin gene expression in homozygous hemoglobin e.

Background and Objectives. To explore the role of cis-regulatory sequences within the ß globin gene cluster at chromosome 11 on human γ globin gene expression related to Hb E allele, we analyze baseline hematological data and Hb F values together with ß globin haplotypes in homozygous Hb E. Patients and Methods. 80 individuals with molecularly confirmed homozygous Hb E were analyzed for the ß globin haplotypes and Xmn I polymorphism using PCR-RFLPs. 74 individuals with complete laboratory data were further studied for association analyses. Results. Eight different ß globin haplotypes were found linked to Hb E alleles; three major haplotypes were (a) (III), (b) (V), and (c) (IV) accounting for 94% of Hb E chromosomes. A new haplotype (Th-1) was identified and most likely converted from the major ones. The majority of individuals had Hb F < 5%; only 10.8% of homozygous Hb E had high Hb F (average 10.5%, range 5.8-14.3%). No association was found on a specific haplotype or Xmn I in these individuals with high Hb F, measured by alkaline denaturation. Conclusion. The cis-regulation of γ globin gene expression might not be apparent under a milder condition with lesser globin imbalance such as homozygous Hb E.