A large cohort of deletional high hemoglobin F determinants in Thailand: A molecular revisited and identification of a novel mutation.

BACKGROUND AND AIMS: High hemoglobin F determinants can be classified into hereditary persistence of fetal hemoglobin (HPFH) and δß-thalassemia with different phenotype. We report the molecular basis and hematological features in a large cohort of deletional high Hb F determinants in Thailand. MATERIALS AND METHODS: Subjects (n = 28,177) encountered during 2015-2022 were reviewed, and those with phenotypically suspected of having high Hb F determinants were selected. Combined PCR, multiplex ligation-dependent probe amplification, next-generation sequencing, and DNA sequencing were used to identify the mutations. RESULTS: Among 28,177 subjects investigated, 300 (1.06 %) were found to carry deletional high Hb F determinants in a total of 302 alleles, including heterozygote, compound heterozygote with ß-hemoglobinopathies, and homozygote. DNA analysis identified eight different DNA deletions, including δß0-thalassemia (12.6 kb deletion) (73.8 %), HPFH-6 (14.9 %), Indian deletion-inversion Aγδß0-thalassemia (3.6 %), Thai deletion-inversion-insertion Aγδß0-thalassemia (3.0 %), SEA-HPFH (3.0 %), Chinese Aγδß0-thalassemia (1.0 %), Thai δß0-thalassemia (11.3 kb deletion) (0.3 %), and a novel δß0-thalassemia (137.1 kb deletion) (0.3 %). In addition, three novel genetic interactions, including Chinese Aγδß0-thalassemia/Hb E, δß0-thalassemia/Indian deletion-inversion Aγδß0-thalassemia, and homozygous δß0-thalassemia were found. Hematological features and Hb analysis results of 20 different genotypes were recorded. Multiplex gap-PCR assays for detection of these genetic determinants were described. CONCLUSIONS: Deletional high Hb F determinants are common and heterogeneous in Thailand. Data on the prevalence, molecular spectrum, phenotypic expression, and complex interactions of these genetic determinants should prove useful in the study and a prevention and control program of hemoglobinopathies in the region.

Prospective screening for δ-hemoglobinopathies associated with decreased hemoglobin A2 levels or hemoglobin A2 variants: A single center experience.

BACKGROUND: δ-hemoglobinopathies may lead to misdiagnosis of several thalassemia syndromes especially ß-thalassaemia carrier, it is important to evaluate the δ-globin gene defects in areas with high prevalence of globin gene disorders. We describe a prospective screening for δ-hemoglobinopathies in a routine setting in Thailand. METHODS: Study was done on a cohort of 8,471 subjects referred for thalassemia screening, 317 (3.7%) were suspected of having δ-globin gene defects due to reduced hemoglobin (Hb) A2 levels and/or appearance of Hb A2-variants on hemoglobin analysis. Hematologic and DNA analysis by PCR and related assays were carried out. RESULTS: DNA analysis of δ-globin gene identified seven different δ-globin mutations in 24 of 317 subjects (7.6%). Both known mutations; δ-77(T>C) (n = 3), δ-68(C>T) (n = 1), δ-44(G>A) (n = 8), Hb A2-Melbourne (n = 5), δIVSII-897(A>C) (n = 5), and Hb A2-Troodos (n = 1) and a novel mutation; the Hb A2-Roi-Et (n = 1) were identified. This Hb A2-Roi-Et, results from a double mutations in-cis, δCD82(AAG>AAT) and δCD133(GTG>ATG), was interestingly found in combination with an in trans, 12.6 kb deletional δß0-thalassemia in an adult Thai woman who had no Hb A2 and elevated Hb F. A multiplex-allele-specific PCR was developed to detect these novel δ-globin gene defects. CONCLUSIONS: The result confirms a diverse heterogeneity of δ-hemoglobinopathies in Thailand which should prove useful in a prevention and control program of thalassemia in the region.

Anemia in an ethnic minority group in lower northern Thailand: A community-based study investigating the prevalence in relation to inherited hemoglobin disorders and iron deficiency.

BACKGROUND: Anemia is a globally well-known major public health problem. In Southeast Asia where there is ethnic diversity, both iron deficiency (ID) and inherited hemoglobin disorders (IHDs) are prevalent and are considered to be the major factors contributing to anemia. However, little is known about the anemia burden among the ethnic minorities. In this study, we determine the burden of anemia, in relation to ID and IHDs, among the Karen ethnic minorities living in the rural area of lower northern Thailand. METHODS: A cross-sectional community-based study was conducted at Ban Rai district, Uthai Thani province. Study participants included 337 Karen people aged over 18 years. Socio-economic and health-related information were obtained through interviews and recorded by local health staff. Anemia, IHDs and ID were diagnosed according to standard laboratory methods. Multivariate logistic regression analysis was applied to identify risk factors of moderate-to-severe anemia. RESULTS: The prevalence of overall anemia was 27.9% (95% CI = 23.2-33.0). Mild and moderate anemia were detected in 18.7% (95% CI = 14.7-23.3) and 8.9% (95% CI = 6.1-12.5) respectively. Severe anemia was found in one case (0.3%). Various forms of IHDs were identified in 166 participants, constituting 49.3% (95% CI = 43.8-54.7). The most common form of IHDs was α+-thalassemia (32.9%), followed by ß-thalassemia (12.2%), α0-thalassemia (4.2%), hemoglobin E (3.9%), and hemoglobin Constant Spring (0.9%). Among 308 participants who were investigated for ID, the prevalence was discovered to be 6.8% (95% CI = 4.3-10.2). Analysis of risk factors of moderate-to-severe anemia revealed that individuals with ID, ß-thalassemia and age > 65 years were at high risk with adjusted odds ratio of 17 (95% CI = 3.8-75.2), 6.2 (95% CI = 1.4-27.8) and 8.1 (95% CI = 1.6-40.4) respectively. CONCLUSIONS: Anemia among the Karen is of public health significance; and IHDs are the major contributing factors. Because of the high risk of developing moderate-to-severe anemia, special attention should be paid to individuals affected with ID, ß-thalassemia and the elderly. Public awareness of the health burden of severe thalassemia syndromes should also be campaigned.

Molecular basis of a high Hb A2/Hb Fß-thalassemia trait: a retrospective analysis, genotype-phenotype interaction, diagnostic implication, and identification of a novel interaction with α-globin gene triplication.

Background: ß 0-thalassemia deletion removing 5´ß-globin promoter usually presents phenotype with high hemoglobin (Hb) A2 and Hb F levels. We report the molecular characteristics and phenotype-genotype correlation in a large cohort of the ß 0-thalassemia with 3.4 kb deletion. Methods: A total of 148 subjects, including 127 heterozygotes, 20 Hb E-ß-thalassemia patients, and a double heterozygote with α-globin gene triplication, were recruited. Hb and DNA analysis were performed to identify thalassemia mutations and four high Hb F single nucleotide polymorphisms (SNPs) including four base pair deletion (-AGCA) at A γ-globin promoter, rs5006884 on OR51B6 gene, -158 G γ-XmnI, BCL11A binding motifs (TGGTCA) between 3´A γ-globin gene and 5´Î´-globin gene. Results: It was found that heterozygous ß 0-thalassemia and Hb E-ß 0-thalassemia with 3.4 kb deletion had significantly higher Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and Hb F values as compared with those with other mutations. Co-inheritance of heterozygous ß 0-thalassemia with 3.4 kb deletion and α-thalassemia was associated with even higher MCV and MCH values. The Hb E-ß 0-thalassemia patients carried a non-transfusion-dependent thalassemia phenotype with an average Hb of around 10 g/dL without blood transfusion. A hitherto undescribed double heterozygous ß 0-thalassemia with 3.4 kb deletion and α-globin gene triplication presented as a plain ß-thalassemia trait. Most of the subjects had wild-type sequences for the four high Hb F SNPs examined. No significant difference in Hb F was observed between those of subjects with and without these SNPs. Removal of the 5´ß-globin promoter may likely be responsible for this unusual phenotype. Conclusions: The results indicate that ß 0-thalassemia with 3.4 kb deletion is a mild ß-thalassemia allele. This information should be provided at genetic counseling and prenatal thalassemia diagnosis.

Frequency of unnecessary prenatal diagnosis of hemoglobinopathies: A large retrospective analysis and implication to improvement of the control program.

OBJECTIVE: To determine the frequency and etiology of unnecessary prenatal diagnosis for hemoglobinopathies during 12 years of services at a single university center in Thailand. METHODS: We conducted a retrospective cohort analysis of prenatal diagnosis during 2009-2021. A total of 4,932 couples at risk and 4,946 fetal specimens, including fetal blood (5.6%), amniotic fluid (92.3%), and chorionic villus samples (2.2%) were analyzed. Identification of mutations causing hemoglobinopathies was carried out by PCR-based methods. Maternal contamination was monitored by analysis of the D1S80 VNTR locus. RESULTS: Among 4,946 fetal specimens, 12 were excluded because of poor PCR amplification, maternal contamination, non-paternity, and inconsistency of the results of the fetuses and parents. Breakdown of 4,934 fetuses revealed 3,880 (78.6%) at risk for the three severe thalassemia diseases, including ß-thalassemia major, Hb E-ß-thalassemia, and homozygous α0-thalassemia, 58 (1.2%) at risk for other α-thalassemia diseases, 168 (3.4%) at risk for ß+-thalassemia, 109 (2.2%) at risk for high Hb F determinants, 16 (0.3%) at risk for abnormal Hbs, and 294 (6.0%) with no risk of having severe hemoglobinopathies. The parents of 409 (8.3%) fetuses had inadequate data for fetal risk assessment. Overall, we encountered unnecessary prenatal diagnostic requests for 645 (13.1%) fetuses. CONCLUSIONS: The frequency of unnecessary prenatal diagnosis was high. This could lead to unnecessary risk of complications associated with fetal specimen collection, psychological impacts to the pregnant women and their families, as well as laboratory expenses and workload.

Loop-mediated isothermal amplification (LAMP) colorimetric phenol red assay for rapid identification of α0-thalassemia: Application to population screening and prenatal diagnosis.

BACKGROUND: Identification of α0-thalassemia (SEA and THAI deletions) is essential in preventing and controlling of severe thalassemia diseases. We have developed the LAMP colorimetric assays for the detection of these two thalassemia defects and validated them in population screening and prenatal diagnosis. METHODS: Three LAMP colorimetric assays specific for α0-thalassemia (SEA deletion), α0-thalassemia (THAI deletion) and normal DNA sequence were developed. These assays were validated on 341 subjects who had initial thalassemia screening positive and various thalassemia genotypes. Prenatal diagnosis of α0-thalassemia (SEA deletion) was done on 33 fetuses at risk of having Hb Bart's hydrops fetalis syndrome. RESULTS: The LAMP colorimetric assays for α0-thalassemia (SEA and THAI deletions) could be clearly interpreted by naked eyes. The assay for α0-thalassemia (SEA deletion) showed a 100% (62/62 x 100) sensitivity and 98.2% (274/279 x 100) specificity whereas, that of the α0-thalassemia (THAI deletion) showed 100% (1/1 x 100) sensitivity and 99.7% (339/340 x 100) specificity. We obtained a 100% concordant prenatal diagnosis results using LAMP assays of α0-thalassemia (SEA deletion) in 33 fetuses as compared to the conventional PCR analysis. CONCLUSIONS: The LAMP colorimetric assays developed are simple, rapid, and do not require sophisticated equipment. Inclusion of the LAMP tests in the existing screening protocol significantly reduce the screening cost and the molecular analysis workload, which should prove useful in the prevention and control program of hemoglobinopathies in the region.

α0-thalassemia in affected fetuses with hemoglobin E-ß0-thalassemia disease in a high-risk population in Thailand.

OBJECTIVES: A co-inheritance of α0-thalassemia can ameliorate the clinical severity of the hemoglobin (Hb) E-ß-thalassemia disease. This information should be provided at prenatal diagnosis. Identification of α0-thalassemia in an affected fetus is therefore valuable. We have explored this genetic interaction in a large cohort of affected fetuses with hemoglobin (Hb) E-ß-thalassemia in northeast Thailand. METHODS: A study was done retrospectively on 1,592 couples at risk of having fetuses with Hb E-ß0-thalassemia, encountered from January 2011 to December 2019. A total of 415 left-over DNA specimens of the affected fetuses with Hb E-ß0-thalassemia disease were further investigated. Examination of α0-thalassemia was done using gap-PCR or a multiplex PCR assay for simultaneous detection of Hb E and α0-thalassemia mutations. RESULTS: Of the 415 affected fetuses, the two most common ß0-thalassemia genes found were the codons 41/42 (-TTCT) (199/415; 48.0%) and codon 17 (A-T) (115/415; 27.7%). α0-thalassemia was found unexpectedly in 21 (5.1%) fetuses. Hematologic phenotypes of the parents indicated that it was impossible to differentiate a pure ß0-thalassemia carrier from a double ß0-thalassemia/α0-thalassemia heterozygote unless DNA analysis is performed. In contrast, a reduced level of Hb E in the Hb E carrier (<25%) is a valuable marker for predicting double heterozygosity for Hb E/α0-thalassemia. This could be further confirmed using a multiplex PCR assay. CONCLUSIONS: There is a high prevalence of co-inheritance of α0-thalassemia in fetuses with Hb E-ß0-thalassemia disease. In a high-risk population such as Thailand, we recommend screening for α0-thalassemia in all affected fetuses with Hb E-ß0-thalassemia disease and providing complete genetic information to the parents to make appropriate decisions at prenatal diagnosis and genetic counseling.

Thalassemia and erythroid transcription factor KLF1 mutations associated with borderline hemoglobin A2 in the Thai population.

INTRODUCTION: Elevated hemoglobin (Hb) A2 is an important diagnostic marker for ß-thalassemia carriers. However, diagnosis of cases with borderline Hb A2 may be problematic. We described the molecular characteristics found in a large cohort of Thai subjects with borderline Hb A2. MATERIAL AND METHODS: Examination was done on 21,657 Thai subjects investigated for thalassemia at Khon Kaen University, Thailand. A total of 202 subjects with borderline Hb A2 (3.5-4.0%) were selectively recruited and hematological parameters were recorded. DNA variants in α-, ß-, δ-globin, and Krüppel-like factor 1 (KLF1) genes were examined using PCR. RESULTS: Among 202 subjects, DNA analysis identified carriers of α+-thalassemia (n = 48; 23.8%), ß-thalassemia (n = 22; 10.9%) and KLF1 mutations (n = 48; 23.8%). No molecular defect was observed in the remaining 84 (41.5%) subjects. Interaction of KLF1 and α-thalassemia was observed in 10 cases. Of the 22 ß-thalassemia carriers, five ß+-thalassemia mutations were identified with lower MCV and higher Hb A2. Seven KLF1 mutations were detected in 10 genotypes in subjects with higher MCV and Hb F. No ß0-thalassemia, α-globin gene triplication or δ-globin gene mutation was detected. CONCLUSIONS: A large proportion of subjects with borderline Hb A2 are not ß-thalassemia carriers and for those with ß-thalassemia, only mild ß+-thalassemia mutations were detected. Evaluation of the patients using Hb A2, Hb F and MCV values will help in selecting cases for further molecular analysis. The results should explain the unusual phenotype of the cases and facilitate a thalassemia screening program in the region.

Diagnostic value of fetal hemoglobin Bart's for evaluation of fetal α-thalassemia syndromes: application to prenatal characterization of fetal anemia caused by undiagnosed α-hemoglobinopathy.

BACKGROUND: To evaluate whether the quantification of fetal hemoglobin (Hb) Bart's is useful for differentiation of α-thalassemia syndromes in the fetus and to characterize the fetal anemia associated with fetal α-hemoglobinopathy. METHODS: A total of 332 fetal blood specimens collected by cordocentesis were analyzed using capillary electrophoresis and the amount of Hb Bart's was recorded. The result was evaluated against thalassemia genotypes determined based on Hb and DNA analyses. Prenatal Hb and DNA characterization of the fetal anemia observed in two families was done. RESULTS: Among 332 fetuses investigated, Hb and DNA analyses identified 152 fetuses with normal genotypes. The remaining 180 fetuses carried α-thalassemia with several genotypes. Variable amounts of Hb Bart's were identified in all fetuses with α-thalassemia, which could be used for simple differentiation of fetal α-thalassemia genotypes. These included α+- and α0-thalassemia traits, homozygous α+-thalassemia and Hb Constant Spring (CS), Hb H disease, Hb H-CS and Hb H-Quong Sze diseases, homozygous α0-thalassemia causing the Hb Bart's hydrops fetalis and a remain uncharacterized α-thalassemia defect. The previously undescribed interactions of Hb Queens Park and Hb Amsterdam A1 with Hb E were detected in two fetuses with Hb Bart's of 0.5%. The Hb Queens Park-AEBart's disease was also noted in one pregnant woman. Prenatal analysis of the fetuses with severe fetal anemia and cardiomegaly with Hb Bart's of 9.0% and 13.6% revealed unexpectedly the homozygous Hb CS and a compound heterozygosity of Hb CS/Hb Pakse' with Hb E heterozygote, respectively. CONCLUSIONS: The usefulness of detecting and differentiation of fetal α-thalassemia syndromes by quantifying of Hb Bart's was demonstrated. Apart from the fatal condition of Hb Bart's hydrops fetalis associated with homozygous α0-thalassemia, homozygous Hb CS and a compound Hb CS/Hb Pakse' could result in severe fetal anemia and fetal complications, prenatal diagnosis is highly recommended. The simple Hb Bart's quantification of fetal blood should prove helpful in this matter.

Direct PCR assays without DNA extraction for rapid detection of hemoglobin Constant Spring and Pakse' genes: application for carrier screening and prenatal diagnosis.

Hemoglobin Constant Spring (Hb CS) and Hb Pakse' (PS) are the common non-deletional α+-thalassemia found in Thailand. These two variants can cause severe thalassemia syndromes, especially in fetus and neonate. Molecular diagnosis is the only confirmatory method because Hb CS and Hb PS are usually missed by routine screening and Hb analysis. Therefore, we aimed to develop rapid direct PCR for the diagnosis of Hb CS and PS genes. Multiplex direct PCR assays for identifying the Hb CS and PS genes in whole blood (WB) and amniotic fluid (AF) specimens were developed. The assays were firstly validated on 290 unrelated whole blood specimens. Hb CS and PS carriers were identified in 67 (23.1%) and 6 (2.1%) cases, respectively. A 100% concordant result as compared to routine PCR assay was observed. The direct PCR assays have been applied successfully for prenatal diagnosis in two families. The result showed that the fetuses were affected by homozygous Hb CS and compound heterozygous Hb CS/Hb PS. Accurate prenatal diagnosis of these families was observed using the newly developed assays. These assays should be applicable in routine thalassemia diagnostics as well as in the large-scale screening of Hb CS and PS in the region.

ß-Hemoglobinopathies in the Lao People's Democratic Republic: Molecular diagnostics and implication for a prevention and control program.

INTRODUCTION: A high frequency of ß-thalassemia in Lao People's Democratic Republic necessitates the importance of complete molecular data before a prevention and control program could be established. Limited data are available for Lao PDR. We have now reported an extended information on the molecular basis of ß-hemoglobinopathies in this population. METHODS: The study was done on 519 unrelated Laos subjects requested for thalassemia investigation. Hematological data were recorded. Hb profiles were obtained using a capillary electrophoresis system. α-And ß-globin genotyping was performed using PCR and related techniques. RESULTS: Among the 519 subjects, 287 (55.3%) were found to carry ß-hemoglobinopathies based on Hb and DNA analyses. These included Hb E carriers (n = 135), homozygous Hb E (n = 47), ß-thalassemia carriers (n = 70), Hb E-ß-thalassemia (n = 25), homozygous ß-thalassemia (n = 4), heterozygous δß0 -thalassemia (n = 2), and carriers of the ß-Hb variant (n = 3). Mutation analysis identified in addition to the Hb E, 8 different ß-thalassemia mutations including codon 17 (A-T), codons 41/42 (-TTCT), NT-28 (A-G), codons 71/72 (+A), IVS1-1 (G-T), 3.4 kb deletion, an initiation codon (T-G) and IVS2-654 (C-T). Two δß0 -thalassemia carriers (12.6 kb deletion) and three subjects with Hb Hope (ß136GGT-GAT ) were identified. Hematological features associated with these ß-hemoglobinopathies were presented. CONCLUSION: ß-hemoglobinopathies in the Laos population is heterogeneous. This information is relevant for setting up a molecular diagnostics and can provide a basis for genetic counseling and enable prenatal diagnosis.

A novel SNP rs11759328 on Rho GTPase-activating protein 18 gene is associated with the expression of Hb F in hemoglobin E-related disorders.

Hemoglobin (Hb) F has a modulatory effect on the clinical phenotype of ß-thalassemia disease. High expression of Hb F in Hb E-related disorders has been noted, but the mechanism is not well understood. We have examined the association of a novel SNP rs11759328 on ARHGAP 18 gene and other known modulators with a variability of Hb F in Hb E-related disorders. Genotyping of SNP rs11759328 (G/A) was performed based on high-resolution melting analysis. The rs11759328 (A allele) was shown to be significantly associated with Hb F levels (p < 0.05) in heterozygous and homozygous Hb E. High levels of Hb F in both heterozygous and homozygous Hb E were also found to be associated with SNPs in the study of other modifying genes including KLF 1 mutation, rs7482144 (Gγ-XmnI), rs4895441, rs9399137 of (HBS1L-MYB), and rs4671393 (BCL11A). Multivariate analysis showed that KLF1 mutation and SNP rs11759328 (GA) (ARHGAP18) modulated Hb F expression in heterozygous Hb E. For homozygous Hb E, this was found to be related to five modifying factors, i.e., KLF1 mutation, rs4895441 (GG), rs9399137 (CC), rs4671393 (AA), and rs4671393 (GA). These results indicate that a novel SNP rs11759328 is a genetically modifying factor associated with increased Hb F in Hb E disorder.

Molecular basis of Hb H and AEBart's diseases in the Lao People's Democratic Republic.

INTRODUCTION: As compared to other neighboring countries, limited information on α-thalassemia diseases is available for Lao PDR. We reported for the first time a genetic diversity associated with Hb H and AEBart's diseases in Laos patients. METHODS: Study was done on Laos patients with Hb H disease (n = 14) and AEBart's disease (n = 14) whose blood specimens were transferred to our laboratory for the investigation of thalassemia. Hematological parameters were recorded. Hb analysis was done using a capillary electrophoresis system. α- and ß-globin genotypes were determined using PCR and related techniques. RESULTS: Hb and DNA analyses identified Hb H disease resulted from [--SEA /-α3.7 , ßA /ßA ] (n = 7), [--THAI /-α3.7 , ßA /ßA ] (n = 1), Hb H-Constant Spring (CS) disease (--SEA /αCS α, ßA /ßA ; n = 5), and Hb H-IVSI-117 (--SEA /ααIVSI-117G>A , ßA /ßA ; n = 1). For those of the AEBart's disease (n = 14), five were found to be AEBart's-CS disease [--SEA /αCS α, ßE /ßA ], two had [--THAI /αCS α, ßE /ßA ] genotype, six had AEBart's disease with (--SEA /-α3.7 , ßE /ßA ) genotype, and the remaining one was a patient with AEBart's-Pakse' [--SEA /αPS α, ßE /ßA ] disease. These --THAI and αIVSI-117G>A mutations are reported herein for the first time in Laos population. Accurate diagnosis in most cases was obtained after DNA analysis. CONCLUSIONS: This study demonstrates the diverse heterogeneity and highlights the importance of molecular diagnosis of α-thalassemia diseases in Laos population. Data on the molecular basis of α-thalassemia should prove useful for setting up a molecular diagnostic testing for thalassemia in Laos and further hemoglobin genetic study in the region.

Molecular Survey of Hemoglobinopathies in Myanmar Workers in Northeast Thailand Revealed an Unexpectedly High Prevalence of α+-Thalassemia.

To provide the molecular information on hemoglobinopathies in the Myanmar population, the study was carried out on Myanmar workers in Khon Kaen Province in northeast Thailand. A total of 300 anonymous Myanmar factory workers were randomly recruited during their annual medical checkup. Hemoglobinopathies were identified using hemoglobin (Hb) and DNA analyses. These identified heterozygous α0-thalassemia (α0-thal) [- -SEA (Southeast Asian) deletion] (n = 5, 1.7%), heterozygous α+-thal (n = 103, 34.3%), homozygous α+-thal (n = 12, 4.0%), heterozygous ß-thalassemia (ß-thal) (n = 3, 1.0%), heterozygous ß-thal with homozygous α+-thal (n = 2, 0.7%), double heterozygous ß-thal/α0-thal (n = 1, 0.3%)], heterozygous Hb E (HBB: c.79G>A) with α0-thal/α+-thal (n = 1, 0.3%), heterozygous Hb E (n = 27, 9.0%), heterozygous Hb E with α+-thal (n = 24, 8.0%), homozygous Hb E with α0-thal/α+-thal (n = 1, 0.3%), homozygous Hb E (n = 3, 1.0%) and homozygous Hb E with heterozygous α+-thal (n = 3, 1.0%). No thalassemia defect was found in the remaining 115 subjects (38.4%). Haplotypes associated with Hb E and Hb Dhonburi (or Hb Neapolis) [ß126(H4)Val→Gly, codon 126 (T>G), HBB: c.380T>G] are reported. While the proportions of α0-thal, ß-thal and Hb E are comparable to those described in neighboring countries, a markedly high prevalence of α+-thal (48.6% in total) is unexpected. The molecular information obtained should provide necessary information for diagnostic improvement and planning of a prevention and control program of severe thalassemia in the Myanmar population.

Molecular Characteristics of Hb New York [ß113(G15)Val→Glu, HBB: c.341T>A] in Thailand.

Hb New York or Hb Kaohsiung [ß113(G15)Val→Glu (GTG>GAG), HBB: c.341T>A] has been considered a rare ß hemoglobin (Hb) variant found originally in an Iranian woman and later in diverse populations but its genetic origin has not been elucidated. Here we report molecular and hematological descriptions of this variant found in the Thai population. Among 5643 subjects referred for hemoglobinopathy investigation during January 2015 to September 2017, 183 (3.2%) were found to carry several Hb variants, including ß chain variants (n = 135, 2.4%), α chain variants (n = 33, 0.6%), Hb Lepore-Hollandia (NG_000007.3: g.63290_70702del) and Hb Lepore-Boston-Washington (NG_000007.3: g.63632_71046del) (뫧 hybrid Hb) (n = 12, 0.2%) and δ chain variants (n = 3, 0.05%). Of patients with ß chain variants, six with normal high performance liquid chromatography (HPLC) patterns, had an abnormal Hb in zone 11 of capillary electrophoresis (CE), the amounts of which ranged from 29.6-45.4% with normal levels of Hb A2 and Hb F. DNA analysis identified a heterozygous Hb New York mutation in all cases. Further screening of α-thalassemia (α-thal) identified coinheritance of α+- and α0-thal in two of them who had reduced levels of Hb New York. Haplotype analysis suggested that the Thai Hb New York was likely associated with a single ß-globin haplotype [+ - - - - + +], indicating that it was of the same origin. Hematological findings and simple DNA assay based on allele-specific polymerase chain reaction (PCR) for rapid detection of Hb New York are presented.

Novel interactions of two α-Hb variants with SEA deletion α0-thalassemia: hematological and molecular analyses.

OBJECTIVES: To report the hematological and molecular features as well as diagnostic aspects of the hitherto un-described interactions of two rare α-globin chain variants with α0-thalassemia commonly found among Southeast Asian populations. METHODS: The study was done on two adult Thai patients (P1 and P2) who had hypochromic microcytic anemia. Hb analysis was carried out using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Mutations were identified by PCR and related techniques. RESULTS: Hb analysis of P1 using HPLC showed a normal Hb pattern, but CE demonstrated an abnormal peak at zone 7. DNA sequencing identified a CCG-CTG mutation at codon 95 of the α2 globin gene corresponding to the Hb G-Georgia [α95(G2)Pro → Leu(α2)] previously undescribed in the Thai population. In contrast, Hb analysis of P2 demonstrated an abnormal peak not fully separated from Hb A on HPLC, but not on CE. DNA analysis identified the rarely described Hb Nakhon Ratchasima [α63(E12)Ala → Val(α2)] mutation. Routine DNA analysis detected the SEA deletion α0-thalassemia in trans to the Hb variants in both cases. Hematological parameters were compared with those of patients with compound heterozygote for other α-globin variants and α0-thalassemia previously documented. CONCLUSIONS: Identification of the patients confirmed that interaction of these rare Hb variants with α0-thalassemia does not lead to the Hb H disease. Differentiation of these two Hb variants from other clinically relevant hemoglobinopathies in a routine setting is, however, necessary. This can be accomplished using a combined Hb-HPLC and CE analysis followed by PCR-RFLP assays.

ARKRAY ADAMS A1c HA-8180T Analyzer for Diagnosis of Thalassemia and Hemoglobinopathies Common in Southeast Asia.

OBJECTIVE: To evaluate the ARKRAY ADAMS A1c HA-8180T analyzer for diagnosis of thalassemias and hemoglobinopathies commonly found in the Southeast Asian population. METHODS: Our cohort consisted of 557 specimens from adults referred for thalassemia diagnosis. From these, we selected 457 specimens and subjected them to DNA analysis to determine various thalassemia genotypes. Also, to confirm the reference range for HbA2, we obtained an additional 48 specimens from healthy individuals. We estimated the diagnostic range for Hb E from specimens from another 52 subject individuals previously diagnosed with heterozygous HbE. All of these individuals had negative results in DNA testing for all common α-thalassemia alleles found in Thailand. We performed hemoglobin (Hb) analysis and compared the results with those we derived from testing the CAPILLARYS 2 Flex Piercing device. We defined genotypes via by DNA analysis. RESULTS: Performance evaluation revealed the within- and between-run precision for analysis of HbA2 and HbE, with coefficients of variation (CVs) ranging from 0.6% to 2.5%. We determined the reference ranges of HbA2 and HbE in the HbE heterozygote to be 2.2% to 3.4 % and 25.7% to 31.0%, respectively. We were able to identify all cases of ß-thalassemia and HbE disorders. We coeluted HbH and Hb Bart and interfered with acetylated HbF. CONCLUSION: The ARKRAY ADAMS A1c HA-8180T analyzer could accurately identify which individuals had ß-thalassemia and HbE disorders. However, compared with other high-performance liquid chromatography instruments in diagnosing α-thalassemia disease with HbH and Hb Bart, this analyzer is relatively difficult to use.

A large cohort of hemoglobin variants in Thailand: molecular epidemiological study and diagnostic consideration.

BACKGROUND: Hemoglobin (Hb) variants are structurally inherited changes of globin chains. Accurate diagnoses of these variants are important for planning of appropriate management and genetic counseling. Since no epidemiological study has been conducted before, we have investigated frequencies, molecular and hematological features of Hb variants found in a large cohort of Thai subjects. MATERIALS AND METHODS: Study was conducted on 26,013 unrelated subjects, inhabiting in all geographical parts of Thailand over a period of 11 years from January 2002-December 2012. Hb analysis was done on high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Mutations causing Hb variants were identified using PCR and related techniques. RESULTS: Among 26,013 subjects investigated, 636 (2.4%) were found to carry Hb variants. Of these 636 subjects, 142 (22.4%) carried α-chain variants with 13 different mutations. The remaining included 451 (70.9%) cases with 16 ß-chain variants, 37 (5.8%) cases with Hb Lepore (δß-hybrid Hb) and 6 (0.9%) cases with a single δ-chain variant. The most common α-globin chain variant was the Hb Q-Thailand (α74GAC-CAC, Asp-His) which was found in 101 cases (15.8%). For ß-globin chain variants, Hb Hope (ß¹³6GGT-GAT, Gly-Asp) and Hb Tak (ß¹46+AC, Ter-Thr) are the two most common ones, found in 121 (19.0%) and 90 (14.2%) cases, respectively. Seven Hb variants have never been found in Thai population. Hb analysis profiles on HPLC or CE of these variants were illustrated to guide presumptive diagnostics. CONCLUSIONS: Hb variants are common and heterogeneous in Thai population. With varieties of thalassemias and hemoglobinopathies in the population, interactions between them leading to complex syndromes are common and render their diagnoses difficult in routine practices. Knowledge of the spectrum, molecular basis, genotype-phenotype correlation and diagnostic features should prove useful for prevention and control of the diseases in the region.

Association of Hb Thailand [α56(E5)Lys→Thr] and Hb Phnom Penh [α117(GH5)-Ile-α118(H1)] with α(0)-thalassemia: molecular and hematological features and differential diagnosis.

We report the molecular and hematological characteristics of two rare hemoglobin (Hb) variants found in associations with a common α(0)-thalassemia (α(0)-thal) in Thai patients. The first case (P1) was a generally healthy 27-year-old man discovered during our ongoing thalassemia screening program. Hemoglobin and DNA analyses identified a previously undescribed condition of compound heterozygosity for Hb Thailand [α56(E5)Lys→Thr] and α(0)-thal (SEA deletion). The second case (P2) was a 4-year-old boy with hypochromic microcytic anemia. Hemoglobin and DNA analyses also identified a compound heterozygosity for Hb Phnom Penh [α117(GH5)-Ile-α118(H1)] in association with α(0)-thal (SEA deletion). Although Hb H (ß(4)) inclusion bodies were observed in both cases, Hb analysis using both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) did not show Hb H. While the two Hb variants could be recognized on Hb HPLC analysis, their corresponding Hb A(2) derivatives: the Hb A(2)-Thailand and Hb A(2)-Phnom Penh, were clearly observed on CE. Apparently, combination of these two Hb variants with α(0)-thal are not expressed as Hb H disease. The two Hb variants could be confirmed by polymerase chain restriction-restriction fragment length polymorphism (PCR-RFLP) and allele-specific PCR assays.

Phenotypic expression of hemoglobins A2, E and F in various hemoglobin E related disorders.

Study on the phenotypic expression of hemoglobin (Hb) A(2) and Hb E in Hb E disorders has been difficult due to the co-separation of Hb A(2) and Hb E in most Hb analysis assays. Because these two Hbs are separated on capillary electrophoresis, we studied phenotypic expression of Hbs A(2), E and F in various Hb E disorders using this system. This was done on 362 subjects with several Hb E disorders including heterozygous Hb E, homozygous Hb E, ß-thalassemia/Hb E, δß-thalassemia/Hb E, and Hb Lepore/Hb E and those of these disorders with several forms of α-thalassemia. Normal controls showed Hb A(2) of 2.7 ± 0.3%. Heterozygous Hb E and homozygous Hb E had elevated Hb A(2) i.e. 3.8 ± 0.3% and 4.8 ± 0.5%, respectively. Further elevations were observed for ß(0)-thalassemia/Hb E (6.1 ± 1.9%) and ß(+)-thalassemia/Hb E (7.1 ± 1.2%). Interestingly, no elevation of Hb A(2) was found in the δß-thalassemia/Hb E, and Hb Lepore/Hb E (2.3 ± 0.3%) but higher Hb F levels were noted which could be useful diagnostic markers. The levels of Hb E were variable. Co-inheritance of these Hb E disorders with α-thalassemia were associated with lower outputs of Hb E and Hb F but the levels of Hb A(2) were not altered. Different phenotypic expression of Hb A(2), Hb E and Hb F could help in differential diagnosis of these Hb E related disorders commonly encountered in the regions where access to molecular techniques is limited.