Development of molecular diagnostic platform for α0 -thalassemia 44.6 kb (Chiang Rai, --CR ) deletion in individuals with microcytic red blood cells across Thailand.

INTRODUCTION: The α0 -thalassemia 44.6 kb or Chiang Rai (--CR ) deletion has been reported in northern Thailand and is capable of causing hemoglobin (Hb) H disease and a lethal α-thalassemia genotype, Hb Bart's hydrops fetalis, in this region. However, there are no current data regarding the frequency of --CR nationwide due to a lack of effective diagnostic assay. Therefore, this study aimed to develop a reliable platform for simultaneous genotyping of --CR and two common α0 -thalassemias in Thailand (--SEA and --THAI ) and investigate the frequency of --CR across Thailand. METHODS: Multiplex gap-PCR assay and five renewable plasmid DNA controls for --CR , --SEA , --THAI , α2-globin (HBA2), and ß-actin (ACTB) were newly developed and validated with reference methods. The developed assay was further tested on 1046 unrelated individuals with a reduced mean corpuscular volume (MCV) of less than 75 fl for investigating genotypic and allelic spectrum of --CR . RESULTS: Our developed assay showed 100% concordance with reference methods. The results were valid and reproducible throughout hundreds of reactions. Comparison of the genotypic and allelic spectra revealed that heterozygous --SEA (--SEA /αα) and --SEA alleles were dominant with the frequency of 22.85% (239/1046) and 13.34% (279/2092), respectively. Of these, --THAI and --CR were relatively rare in this population and comparable to each other with the allelic frequency of 0.14% (3/2092). CONCLUSION: This study successfully established a reliable molecular diagnostic platform for genotyping of --CR , --SEA , and --THAI in a single reaction. Additionally, we demonstrated the frequency of --CR in Thailand for the first time and provided knowledge basis for the planning of severe α-thalassemia prevention and control programs in Thailand, where thalassemia is endemic.

Visual genotyping of thalassemia by using pyrrolidinyl peptide nucleic acid probes immobilized on carboxymethylcellulose-modified paper and enzyme-induced pigmentation.

A simple probe pair was designed for the detection of hemoglobin E (HbE) genotype, a single-point mutation that leads to abnormal red blood cells commonly found in South East Asia. The key to differentiation is the use of a conformationally constrained peptide nucleic acid (PNA) that was immobilized on carboxymethylcellulose-modified paper. This was then used for target DNA binding and visualization by an enzyme-catalyzed pigmentation. The biotinylated target DNA bound to the immobilized probe was visually detected via alkaline phosphatase-linked streptavidin. This enzyme conjugate catalyzed the dephosphorylation of the substrate 5-bromo-4-chloro-3-indolyl phosphate, leading to a series of reactions that generate an intense, dark blue pigment. The test was validated with 100 DNA samples, which shows good discrimination among different genotypes (normal, HbE, and heterozygous) with 100% accuracy when optimal conditions of analysis were applied. The method does not require temperature control and can be performed at ambient temperature. This is an attractive feature for diagnosis in primary care, which accounts for a large part of affected population. Graphical abstract Schematic representation of a paper-based sensor for the detection of the gene Hemoglobin E. The interaction between an immobilized peptide nucleic acid and a DNA target leads to enzymatic pigmentation, allowing simple visual readout with up to 100% accuracy.

Immunostick Test for Detecting ζ-Globin Chains and Screening of the Southeast Asian α-Thalassemia 1 Deletion.

BACKGROUND: Couples who carry α-thalassemia-1 deletion are at 25% risk of having a fetus with hemoglobin Bart's hydrops fetalis. Southeast Asian deletion (--(SEA)) is the most common type of α-thalassemia 1 among Southeast Asian populations. Thus, identification of the (--(SEA)) α-thalassemia 1 carrier is necessary for controlling severe α-thalassemia in Southeast Asian countries. RESULTS: Using our generated anti ζ-globin chain monoclonal antibodies (mAbs) clones PL2 and PL3, a simple immunostick test for detecting ζ-globin chain presence in whole blood lysates was developed. The procedure of the developed immunostick test was as follows. The immunostick paddles were coated with 50 µg/mL of mAb PL2 as capture mAb, or other control antibodies. The coated immunostick was dipped into cocktail containing tested hemolysate at dilution of 1:500, 0.25 µg/mL biotin-labeled mAb PL3 and horseradish peroxidase-conjugated streptavidin at dilution of 1:1000. The immunostick was then dipped in precipitating substrate and the presence of ζ-globin chain in the tested sample was observed by the naked eye. Upon validation of the developed immunostick test with various types of thalassemia and normal subjects, 100% sensitivity and 82% specificity for detection of the (--(SEA)) α-thalassemia-1 carriers were achieved. The mAb pre-coated immunostick can be stored at room temperature for at least 20 weeks. CONCLUSION: In this study, a novel simple immunostick test for the screening of (--(SEA)) α-thalassemia 1 carriers was presented. The developed immunostick test, within a single test, contains both positive and negative internal procedural controls.

Molecular Epidemiology of Hemoglobinopathies in Cambodia.

Determining the magnitude of the thalassemia problem in a country is important for implementing a national prevention and control program. In order to acquire accurate thalassemia prevalence data, the gene frequency of α- and ß-thalassemia (α- and ß-thal) in different regions of a country should be determined. The molecular basis of thalassemia in Cambodia was performed by polymerase chain reaction (PCR)-based techniques in a community-based cross-sectional survey of 1631 unrelated individuals from three regions, Battambang, Preah Vihear and Phnom Penh. Thalassemia mutations were detected in 62.7% of the three studied population of Cambodia. Hb E (HBB: c.79G > A) was the most common ß-globin gene mutation with a frequency ranging from 0.139 to 0.331, while the most frequent α-globin gene mutation was the -α(3.7) (rightward) deletion (0.098-0.255). The other frequencies were 0.001-0.003 for ß-thal, 0.008-0.011 for α-thal-1 (- -(SEA)), 0.003-0.008 for α-thal-2 [-α(4.2) (leftward deletion)], 0.021-0.044 for Hb Constant Spring (Hb CS, HBA2: c.427T > C) and 0.009-0.036 for Hb Paksé (HBA2: c.429A > T). A regional specific thalassemia gene frequency was observed. Preah Vihear had the highest prevalence of Hb E (55.9%), α-thal-2 (24.0%) and nondeletional α-thal (15.1%), whereas Phnom Penh had the lowest frequency of thalassemia genes. Interestingly, in Preah Vihear, the frequency of Hb Paksé was extremely high (0.036), almost equivalent to that of Hb CS (0.044). Our results indicate the importance of micromapping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in Cambodia.

Diagnosis of α-thalassaemia by colorimetric gap loop mediated isothermal amplification.

α-Thalassaemia is an inherited haemoglobin disorder that results from the defective synthesis of α-globin protein. Couples whom both carry the α-thalassaemia 1 gene are at risk of having a foetus with the most severe thalassaemia, Hb Bart's hydrops fetalis, with a risk of maternal mortality. However, haematological parameters alone cannot distinguish between a α-thalassaemia 1 carrier and a homozygous α-thalassaemia 2, in which one α-globin gene has been deleted on each chromosome. A rapid and accurate molecular detection assay is essential for prevention of the disease in populations where α-thalassaemia 1 is common. Multiplex Gap-PCR analysis is widely used for diagnosis of α-thalassaemia. However, the technique requires a thermocycler and post-amplification processing, which limits its application in primary care or in rural areas in developing countries. Loop mediated isothermal amplification (LAMP) amplifies target DNA at a constant temperature and does not require a thermocycler. This study developed a colorimetric Gap-LAMP using malachite green to allow naked eye visualization of two deletional α-thalassaemia 1 commonly found in Asian populations, the Southeast Asian type (--SEA) and the Thai type (--THAI) deletions. The Gap-LAMP was performed on DNA samples from 410 individuals carrying various α-thalassaemia gene defects with 100% concordance with conventional Gap-PCR analysis. This method eliminates post-amplification processing or the use of expensive sophisticated equipment and allows screening large populations for the prevention and control of α-thalassaemia.

Generation of human induced pluripotent stem cell line (MUi033-A) from a male with homozygous for Hemoglobin E.

Hemoglobin E (HbE), a common variant in Southeast Asian populations, results from a G to A substitution at codon 26 of the HBB gene, causing abnormal Hb and mild ß-thalassemia-like symptoms. Here, we derived an induced pluripotent stem cell (iPSC) line, named MUi033-A, from a male homozygous for HbE. The iPSC line demonstrates a normal karyotype and embryonic stem cell-like properties including pluripotency gene expression, and tri-lineage differentiation potential. This iPSC resource holds the potential for investigating gene therapy targeting HbE mutation.

Generation of human induced pluripotent stem cell line (MUi034-A) from an unusual case of hydrops fetalis associated with homozygous hemoglobin Constant Spring.

Hemoglobin Constant Spring (HbCS) is unstable hemoglobin resulting from a nucleotide substitution at the termination codon of the HBA2 gene (c.427 T > C). The homozygous state for HbCS is non-transfusion dependent in adults. Nevertheless, severe anemia is often observed in fetuses. Here, human induced pluripotent stem cell line MUi034-A was generated from peripheral blood CD34+ hematopoietic stem/progenitor cells (HSPCs) derived from a 14-year-old female with homozygous HbCS who had a history of severe anemia and hydrops during fetal period. The MUi034-A cell line represented embryonic-like characteristics as they expressed specific pluripotency markers, differentiated into the three germ layers, and retained normal karyotyping.

Molecular analysis of globin gene expression in different thalassaemia disorders: individual variation of ß(E) pre-mRNA splicing determine disease severity.

Thalassaemia is characterized by the reduced or absent production of globins in the haemoglobin molecule leading to imbalanced α-globin/non α-globin chains. HbE, the result of a G to A mutation in codon 26 of the HBB (ß-globin) gene, activates a cryptic 5' splice site in codon 25 leading to a reduction of correctly spliced ß(E) -globin (HBB:c.79G>A) mRNA and consequently ß(+) -thalassaemia. A wide range of clinical severities in bothα- and ß-thalassaemia syndromes, from nearly asymptomatic to transfusion-dependent, has been observed. The correlation between clinical heterogeneity in various genotypes of thalassaemia and the levels of globin gene expression and ß(E) -globin pre-mRNA splicing were examined using multiplex quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) and allele-specific RT-qPCR. The α-globin/non α-globin mRNA ratio was demonstrated to be a good indicator for disease severity among different thalassaemia disorders. However, the α-globin/non α-globin mRNA ratio ranged widely in ß-thalassaemia/HbE patients, with no significant difference between mild and severe phenotypes. Interestingly, the correctly to aberrantly spliced ß(E) -globin mRNA ratio in 30% of mild ß-thalassaemia/HbE patients was higher than that of the severe patients. The splicing process of ß(E) -globin pre-mRNA differs among ß-thalassaemia/HbE patients and serves as one of the modifying factors for disease severity.

Quantitative analysis of Hb Bart's in cord blood by capillary electrophoresis system.

It has long been recognized that the presence of hemoglobin (Hb) Bart's in newborn's blood is associated with α-thalassemia. However, the automated high-performance liquid chromatography or low-performance liquid chromatography system is unable to quantify the amount of Hbs Bart's and H, which are eluted at the retention time close to 0 min. This study used automatic capillary electrophoresis (CE) system to diagnose various types of α-thalassemia in 587 cord blood samples, including 429 normal α-globin genotype, 120 cases of thalassemia with one α-globin gene defect, 34 cases with two α-globin genes defect, and four cases with three α-globin genes defect. The result showed that the level of Hb Bart's in cord blood was increased accordingly with the increasing numbers of the defective α-globin genes. In addition, Hb Bart's level at 0.2%, as measured by CE, can be used as a cut-off point for α-thalassemia diagnosis in newborns.

Predictive SNPs for ß0-thalassemia/HbE disease severity.

ß-Thalassemia/HbE disease has a wide spectrum of clinical phenotypes ranging from asymptomatic to dependent on regular blood transfusions. Ability to predict disease severity is helpful for clinical management and treatment decision making. A thalassemia severity score has been developed from Mediterranean ß-thalassemia patients. However, different ethnic groups may have different allele frequency and linkage disequilibrium structures. Here, Thai ß0-thalassemia/HbE disease genome-wild association studies (GWAS) data of 487 patients were analyzed by SNP interaction prioritization algorithm, interacting Loci (iLoci), to find predictive SNPs for disease severity. Three SNPs from two SNP interaction pairs associated with disease severity were identifies. The three-SNP disease severity risk score composed of rs766432 in BCL11A, rs9399137 in HBS1L-MYB and rs72872548 in HBE1 showed more than 85% specificity and 75% accuracy. The three-SNP predictive score was then validated in two independent cohorts of Thai and Malaysian ß0-thalassemia/HbE patients with comparable specificity and accuracy. The SNP risk score could be used for prediction of clinical severity for Southeast Asia ß0-thalassemia/HbE population.

Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at Gγ-globin gene promoter.

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for ß-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from ß-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG - 53, - 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in ß-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

A genome-wide association identified the common genetic variants influence disease severity in beta0-thalassemia/hemoglobin E.

b-Thalassemia/HbE disease is clinically variable. In searching for genetic factors modifying the disease severity, patients were selected based on their disease severities, and a genome-wide association study (GWAS) was performed. Genotyping was conducted with the Illumina Human 610-Quad BeadChips array using DNAs from 618 Thai b0-thalassemia/HbE patients who were classified as 383 severe and 235 mild phenotypes by a validated scoring system. Twenty-three SNPs in three independent genes/regions were identified as being significantly associated with the disease severity. The highest association was observed with SNPs in the b-globin gene cluster (chr.11p15), and rs2071348 of the HBBP1 gene revealed the most significant association [P = 2.96 9 10(-13), odds ratio (OR) = 4.33 (95% confidence interval (CI), 2.74-6.84)]. The second was identified in the intergenic region between the HBS1L and MYB genes (chr.6q23), among which rs9376092 was the most significant [P = 2.36 9 10(-10), OR = 3.07 (95% CI, 2.16-4.38)]. The third region was located in the BCL11A gene (chr.2p16.1), and rs766432 showed the most significant association [P = 5.87 9 10-10, OR = 3.06 (95% CI, 2.15-4.37)]. All three loci were replicated in an independent cohort of 174 Indonesian patients. The associations to fetal hemoglobin levels were also observed with SNPs on these three regions. Our data indicate that several genetic loci act in concert to influence HbF levels of beta(0)-thalassemia/HbE patients. This study revealed that all the three reported loci and the alpha-globin gene locus are the best and common predictors of the disease severity in beta-thalassemia.

Molecular screening of the Hbs Constant Spring (codon 142, TAA>CAA, α2) and Paksé (codon 142, TAA>TAT, α2) mutations in Thailand.

Hb Constant Spring [Hb CS, α142(H19)Term] and Hb Paksé [α142(H19)Term] occur from the mutation in the termination codon of the α2-globin gene, TAA>CAA (→Gln) and TAA>TAT (→Tyr), respectively. They are the most common nondeletional α-thalassemia (α-thal) variants causing Hb H disease in Southeast Asia. In this study, 587 cord blood samples were screened for the Hb CS and Hb Paksé mutations by a dot-blot hybridization technique using oligonucleotide probes specific for each mutation. The results showed that the prevalence of Hb CS and Hb Paksé in Central Thailand are 5.80 and 0.51%, respectively, which is in concordance with the results from previous studies.

Update in Laboratory Diagnosis of Thalassemia.

Alpha- and ß-thalassemias and abnormal hemoglobin (Hb) are common in tropical countries. These abnormal globin genes in different combinations lead to many thalassemic diseases including three severe thalassemia diseases, i.e., homozygous ß-thalassemia, ß-thalassemia/Hb E, and Hb Bart's hydrops fetalis. Laboratory diagnosis of thalassemia requires a number of tests including red blood cell indices and Hb and DNA analyses. Thalassemic red blood cell analysis with an automated hematology analyzer is a primary screening for thalassemia since microcytosis and decreased Hb content of red blood cells are hallmarks of all thalassemic red blood cells. However, these two red blood cell indices cannot discriminate between thalassemia trait and iron deficiency or between α- and ß-thalassemic conditions. Today, Hb analysis may be carried out by either automatic high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) system. These two systems give both qualitative and quantitative analysis of Hb components and help to do thalassemia prenatal and postnatal diagnoses within a short period. Both systems have a good correlation, but the interpretation under the CE system should be done with caution because Hb A2 is clearly separated from Hb E. In case of α-thalassemia gene interaction, it can affect the amount of Hb A2/E. Thalassemia genotypes can be characterized by the intensities between alpha-/beta-globin chains or alpha-/beta-mRNA ratios. However, those are presumptive diagnoses. Only DNA analysis can be made for specific thalassemia mutation diagnosis. Various molecular techniques have been used for point mutation detection in ß-thalassemia and large-deletion detection in α-thalassemia. All of these techniques have some advantages and disadvantages. Recently, screening for both α- and ß-thalassemia genes by next-generation sequencing (NGS) has been introduced. This technique gives an accurate diagnosis of thalassemia that may be misdiagnosed by other conventional techniques. The major limitation for using NGS in the screening of thalassemia is its cost which is still expensive. All service labs highly recommend to select the technique(s) they are most familiar and most economic one for their routine use.

Development of DNA controls for detection of ß-thalassemia mutations commonly found in Asian.

INTRODUCTION: Several DNA-based approaches including a reverse dot-blot hybridization (RDB) have been established for detection of ß-thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe ß-thalassemia. However, one of major concerns of these techniques is a risk of misdiagnosis due to a lack of DNA controls. Here, we constructed positive DNA controls for ß-thalassemia genotyping in order to ensure that all steps in the analysis are performed properly. METHODS: Four recombinant ß-globin plasmids, including a normal sequence and three different mutant panels covering 10 common ß-thalassemia mutations in Asia, were constructed by a conventional cloning method followed by sequential rounds of site-directed mutagenesis. These positive DNA controls were further validated by RDB analysis. RESULTS: We demonstrated the applicability of established positive DNA controls for ß-thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant ß-globin plasmids into a single positive control, which showed positive signals for both normal and mutant probes of all tested mutations. Therefore, only two positive DNA controls, normal and combined mutant ß-globin plasmids, are required for detecting 10 common ß-thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis. CONCLUSION: The ß-globin DNA controls established here provide efficient alternatives to a conventional DNA source from peripheral blood, which is more difficult to obtain. They also provide a platform for future development of ß-globin plasmid controls with other mutations, which can also be suitable for other DNA-based approaches.

Molecular prevalence of thalassemia and hemoglobinopathies among the Lao Loum Group in the Lao People's Democratic Republic.

INTRODUCTION: Thalassemias and hemoglobinopathies are the most prevalent inherited anemias detected in South East Asians. These disorders represent not only a clinical health problem but also a socioeconomic problem for this region. Regarding the prevention and control of thalassemias and hemoglobinopathies in the Lao PDR, screening and diagnostic strategies should be strongly considered. The knowledge about the prevalence and molecular genotyping of thalassemias and hemoglobinopathies among the Lao Loum group, which includes the majority of Lao people, is now limited, making the prevention and control of thalassemias difficult. METHODS: This study aimed to determine the prevalence of thalassemia among Lao Loum subjects of reproductive age. Multiplex gap PCR and direct sequencing were used to investigate the mutations of α-globin and ß-globin genes. RESULTS: Thalassemias and hemoglobinopathies were detected in 154 of 354 (43.50%) patients, and 22 different genotypes were identified in this cohort. Remarkably, high frequencies of hemoglobin E, α0 -thalassemia (--SEA ), and α+ -thalassemia (-α3.7 ) were noted. A variety of hematologic features was observed, including co-inheritance of heterozygous HbE and heterozygous α-thalassemia, which was associated with significantly lower levels of MCV and MCH values than those observed in typical HbE heterozygotes. Female participants who were heterozygous for ß0 or co-inheritance of heterozygous ßE with heterozygous α-thalassemia exhibited mild anemia. CONCLUSION: Our data show that thalassemias and hemoglobinopathies have become health problems imposing a serious burden in the Lao PDR. Prevention programs aimed at decreasing the incidence of severe thalassemia diseases should be designed and initiated.

Impact of the detection of ζ-globin chains and hemoglobin Bart's using immunochromatographic strip tests for α0-thalassemia (--SEA) differential diagnosis.

α0-Thalassemia is an inherited hematological disorder caused by the deletion of α-globin genes. The Southeast Asian deletion (--SEA) is the most common type of α0-thalassemia observed in Southeast Asian countries. Regarding WHO health policy, an effective α0-thalassemia screening strategy is needed to control new severe α-thalassemia cases. In this study, a monoclonal antibody panel was used to develop immunochromatographic (IC) strip tests for detecting the Hb Bart's and ζ-globin chain. Among 195 samples, all α0-thalassemia traits (78 α0-thalassemia (--SEA) and 4 α0-thalassemia (--THAI)) had low MCV or MCH values. The sensitivity, specificity, PPV and NPV of the IC strip tests for ζ-globin and Hb Bart's for screening α0-thalassemia (--SEA) within the low MCV or MCH samples were 100%, 65.2%, 90.7%, 100% and 96.2%, 47.8%, 86.6%, 78.6%, respectively. All 4 α0-thalassemia (--THAI) traits were negative for ζ-globin chains but positive for Hb Bart's using the IC strip tests. These results led to a α0-thalassemia screening being proposed in which blood samples are first evaluated by MCV, MCH and Hb typing. Samples with high MCV and MCH values are excluded for the presence of the α0-thalassemia gene. Samples with low MCV or MCH values are assayed using the developed IC strip tests, where only samples testing positive are further assayed for α0-thalassemia by PCR. Patients with Hb H, EA Bart's or EF Bart's diseases do not need to use this IC strip assay. Thus, in this study, a simple and cost effective α0-thalassemia point of care test was developed.

A scoring system for the classification of beta-thalassemia/Hb E disease severity.

Beta-thalassemia intermediate patients show a remarkable clinical heterogeneity. We examined the phenotypic diversity of 950 beta-thalassemia/Hb E patients in an attempt to construct a system for classifying disease severity. A novel scoring system based on six independent parameters, hemoglobin level, age at disease presentation, age at receiving first blood transfusion, requirement for transfusion, spleen size, and growth and development, was able to separate patients into three distinctive severity categories: mild, moderate, and severe courses. This system, therefore, can increase the accuracy of studies of genotype-phenotype interactions and facilitate decisions for appropriate patient management.

Genetic variation of Krüppel-like factor 1 (KLF1) and fetal hemoglobin (HbF) levels in ß0-thalassemia/HbE disease.

Heterogeneity of HbF levels in ß0-thalassemia/HbE disease has been reported to be associated with variations in clinical manifestations of the disease, and several genetic-modifying factors beyond the ß-globin gene cluster have been identified as HbF regulators. Down-regulation or heterozygous mutations of Krüppel-like factor 1 (KLF1) is associated with elevated HbF levels in non-thalassemia subjects. This study confirms that experimental down-regulation of KLF1 in ß0-thalassemia/HbE-derived erythroblasts significantly increases HbF production (up to 52.3 ± 2.4%), albeit with slightly delayed erythroid terminal differentiation. KLF1 exome sequencing of 130 Thai ß0-thalassemia/HbE patients without co-inheritance of α-thalassemia found six patients with KLF1 heterozygous mutations including rs2072596 (p.F182L; n = 5) and rs745347362 (p.P284L; n = 1) missense mutations. However, while these patients had high HbF levels (38.1 ± 7.5%), they were all associated with a severe clinical phenotype. These results suggest that while reduction of KLF1 expression in ß0-thalassemia/HbE erythroblasts can increase HbF levels, it is not sufficient to alleviate the clinical phenotype.

A comprehensive ethnic-based analysis of alpha thalassaemia allelle frequency in northern Thailand.

Alpha (α)-thalassaemia is one of the most prevalent hereditary blood disorders, commonly affecting Southeast Asian people, with the highest incidence (30-40%) being seen in northern Thailand. However, this high incidence was estimated without consideration of the variations between ethnic populations and the geographical location of the populations. To address this issue, a total of 688 samples from 13 different northern Thai ethnic groups (30 villages) categorized into three linguistic groups were genotyped for deletional alpha-thalassaemia (-α3.7, -α4.2, --SEA and --THAI) and/or non-deletional alpha-thalassaemia (αCS and αPS) via multiplex gap-PCR and dot-blot hybridization, respectively. Alpha+(-α3.7, -α4.2, αCS and αPS) and alpha°-thalassaemia (--SEA and --THAI) allele frequencies (with 95% Confidence Interval) were the highest in the Sino-Tibetan group [0.13 (0.08-0.18)] and the Tai-Kadai group [0.03 (0.02-0.05)], respectively. With regards to ethnicity, the varying allele frequency of α+ and α°-thalassaemia amongst a variety of ethnic groups was observed. The highest α+-thalassaemia allele frequency was found in the Paluang [0.21 (0.10-0.37)] while α°-thalassaemia allele frequency was the highest in the Yuan [0.04 (0.01-0.10)]. These detailed results of alpha thalassaemia allele frequency and genetic diversity amongst the northern Thai ethnic groups demonstrate the need for ethnicity based thalassaemia prevention programs.