A large cohort of Hb H disease in northeast Thailand: A molecular revisited, diverse genetic interactions and identification of a novel mutation.

BACKGROUND AND AIMS: To update the molecular characteristics of α-thalassemia in northeast Thailand, the molecular basis and genetic interactions of Hb H disease were examined in a large cohort of patients. MATERIALS AND METHODS: A study was done on 1,170 subjects with Hb H disease and various genetic interactions encountered during 2009-2023. Hb and DNA analyses were carried out. RESULTS: As many as 40 genotypes with several known, previously undescribed, and novel mutations were observed. These included 698 subjects (59.8 %) of Hb H disease, 357 (30.6 %) with EABart's disease, 63 (5.4 %) with EEBart's disease, 18 (1.7 %) with abnormal Hbs, 17 (1.5 %) with ß-thalassemia, and 4 (0.4 %) with EFBart's or EFABart's disease. The molecular basis of 13 subjects (1.1 %) remains unknown. The α0-thalassemia included --SEA (n = 1,139, 97.4 %) and --THAI (n = 21, 1.8 %). Two rare mutations were identified in 3 subjects (0.3 %) with --SA and --CR deletions. For α+-thalassemia, -α3.7 kb del (n = 626, 53.5 %), Hb Constant Spring (n = 415, 35.5 %), -α4.2 kb del (n = 44, 3.8 %), Hb Paksé (n = 36, 3.1 %), and Hb Q-Thailand (n = 19, 1.6 %), were detected. Ten rarer α+-thalassemia were identified, including a novel mutation, namely the Hb Chumphae (HBA2:c.32T>A). The Hb H-Lansing-Ramathibodi, Hb H-Jax, and Hb H-Chumphae are hitherto undescribed in this region. PCR-based diagnostic methods for these α-thalassemia defects were described. CONCLUSIONS: This study confirms the diverse heterogeneity and genetic interactions causing Hb H disease in northeast Thailand. The results should prove useful for laboratory diagnosis and genetic counseling of this genetic disorder in the region.

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.

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.

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.

Hemoglobin EE disease in young Laotian children: Hematologic features and the contributions of genetic variations to Hb F expression.

BACKGROUND: The wide variation in hemoglobin (Hb) F levels has been observed in patients with Hb EE disease. This study aimed to describe hematologic features and determine the effect of genetic variants on Hb F expression in young children with Hb EE disease. METHODS: Hematologic features and Hb profiles of Laotian children aged 6-23 months, who originally enrolled in the Lao-Zinc study, were retrospectively reviewed. Only children with Hb EE disease, as indicated by DNA analysis, were included in this current analysis. Genetic variants, including the G γ-XmnI polymorphism (C>T) of the HBG2 gene, the HBS1L-MYB intergenic region on chromosome 6, and the BCL11A on chromosome 2 as well as the mutations occurring on the Krüppel-like factor 1 (KLF1) gene, were examined. RESULTS: In total, 205 children were diagnosed as having Hb EE disease with Hb F ranged from 1.2 to 43.7%. Most of the children had mild to moderate anemia with a remarkable microcytosis. Analysis of the genetic variants revealed an extremely high frequency of the G γ-XmnI (93.7%). Applying multiple regression analysis adjusted for age, sex, and α-thal gene, a positive relation was observed for the rs4671393 (coefficient = 3.87, p = .005) and the rs2297339 (coefficient = 2.48, p = .046), but not the G γ-XmnI. A statistically non-significant relation was noted for the rs9399137 and the -154 (C>T) KLF1 mutation. CONCLUSION: Our findings provide insight into complex situation of Hb F variability in young children with Hb EE disease; and this can guide to appropriate care and counseling to affected families.

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.

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.

Generation of a single-tube quality control material for hemoglobin and DNA analyses of hemoglobinopathies.

INTRODUCTION: Hemoglobinopathies are major public health problems worldwide. Accurate laboratory diagnosis of the carrier is essential, which includes initial screening, Hb analysis, and DNA analysis. For the first time, we have developed a single-tube quality control (QC) sample for these laboratory tests. METHODS: The QC sample was made from a lyophilized mixture of the stabilized hemolysate with carbon monoxide saturation and the white blood cells of known thalassemia mutations. Homogeneity and stability were examined by Hb and DNA analyses on day 0 and every month for 12 months, at room temperature, 4°C, and -20°C. A preliminary proficiency testing (PT) program for hemoglobinopathies using this single QC material was developed. RESULTS: Hemoglobin (Hb) and DNA analyses of a single-tube QC sample demonstrated satisfactory results of Hb analysis for at least five months and DNA analysis for at least one year of storage at -20°C. The results obtained from a preliminary PT program on five expert laboratories confirmed that a single tube QC sample prepared could be used as a PT item with various Hb and DNA analyses methods. CONCLUSION: A single lyophilized control sample has been generated for use in hemoglobinopathies' internal and external quality control program. Unlike other available control materials, which are used for individual testing, a single-tube QC sample generated can be used to control the pre-analytical and analytical processes of both Hb and DNA analyses and is suitable for use in the PT program of hemoglobinopathies.

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.

Hemoglobins F, A2 , and E levels in Laotian children aged 6-23 months with Hb E disorders: Effect of age, sex, and thalassemia types.

INTRODUCTION: Determination of hemoglobins (Hbs) F, A2, and E is crucial for diagnosis of thalassemia. This study determined the levels of Hbs F, A2, and E in children aged 6-23 months and investigated the effect of age, sex, and types of thalassemia on the expression of these Hbs. METHODS: A total of 698 blood samples of Laotian children including 272 non-Hb E, 271 Hb E heterozygotes, and 155 Hb E homozygotes were collected. Hb profiles were determined using the capillary zone electrophoresis. Coinheritance of α-thalassemia and the homozygosity for Hb E mutation were checked by PCR-based assay. RESULTS: Children heterozygous and homozygous for Hb E had significantly higher Hb F and A2 levels than non-Hb E children (median Hb F = 1.1% for non-Hb E group, 2.7% for Hb E heterozygotes, and 9.4% for Hb E homozygotes; median Hb A2  = 2.6% for non-Hb E group, 3.8% for Hb E heterozygotes, and 5.2% for Hb E homozygotes). The median Hb E levels were 21.9% for Hb E heterozygotes and 85.3% for Hb E homozygotes. Comparing within group, there was a statistically significant difference between children with and without an α-gene defect for Hb A2 and E, but not Hb F. Based on a multiple regression analysis, age and sex were significantly associated with the expression of Hb F and A2 but not Hb E. CONCLUSIONS: Our findings can guide the development of a diagnostic approach to thalassemia in children aged 6-23 months.

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.

Prevalence of Thalassemia among Newborns: A Re-visited after 20 Years of a Prevention and Control Program in Northeast Thailand.

BACKGROUND: To provide accurate prevalence information of thalassemia in northeast Thailand, authors performed thalassemia screening in newborns after 20 years implementation of a prevention and control program. METHODS: Study was done on 350 cord blood specimens collected consecutively at Maternal and Child Hospital, Regional Health Promotion Center 7, Khon Kaen, Thailand. All kinds of α- and ß-thalassemias were identified using combined hemoglobin (Hb) and DNA analyses. RESULTS: Among 350 newborns examined, subjects with thalassemia genes were identified in 184 (52.6%) cases with as many as 22 different genotypes. The most prevalent one was Hb E (39.1%). The incidence of 3.1% α0-thalassemia, 25.9% α+-thalassemia, 5.4% Hb Constant Spring and 1.4% of Hb Paksé were encountered. Heterozygous ß-thalassemia was found in 2 cases (0.6%). Hb capillary electrophoresis could demonstrate Hb E in all cases with Hb E and detected different levels of Hb Bart's for different α-thalassemia genotypes but not in all cases with α-thalassemia. No newborn with severe thalassemia diseases was encountered. CONCLUSION: This study reveals that α-thalassemia, ß-thalassemia, and Hb E carriers as well as complex thalassemia syndromes are still prevalence and indicates a need for continuing a prevention and control program in the region.

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.

Genetic heterogeneity of hemoglobin AEBart's disease: a large cohort data from a single referral center in northeast Thailand.

AEBart's disease is a thalassemia intermedia usually characterized by the interaction of α(0)-thalassemia with either deletional or non-deletional α(+)-thalassemia in Hb E heterozygote. Genotypic and phenotypic features are heterogeneous. We studied the hematologic and molecular characteristics of this disease in a cohort of 173 Thai patients encountered at our center in northeast Thailand. Hemoglobin and DNA analyses identified patients with deletional AEBart's disease (n=84), Hb Constant Spring AEBart's disease (n=81), Hb Paksé-AEBart's disease (n=5), AEBart's disease with codon 30 mutation (n=1) and two hitherto un-described forms of AEBart's disease due to interaction of Hb E heterozygote and α(0)-thalassemia with the -α(16.6)kb deletional α(+)-thalassemia (n=1) and Hb Q-Thailand (n=1). Different phenotypic expression of these AEBart's diseases with low Hb, Hct and MCV and increased RDW values with marked reduction in Hb E levels were observed. It was found that all these forms of AEBart's disease showed similar thalassemia intermedia phenotypes but those with non-deletional forms were relatively more anemic. Our data confirm that in such area with high prevalence of hemoglobinopathies such as Southeast Asia, identification of rare thalassemia alleles in a thalassemia intermedia patient should not be ignored. Careful consideration of different phenotypic expression may help in providing presumptive diagnosis of this disease where access to molecular testing is limited. However, molecular diagnostic is useful for predicting the clinical outcome and improving genetic counseling of these complex hemoglobinopathies.

A newly modified hemoglobin H inclusion test as a secondary screening for α(0)-thalassemia in Southeast Asian populations.

Screening for α(0)-thalassemia is usually associated with a high false-positive rate, leading to an unnecessary PCR workload for accurate diagnosis. We have developed a modified Hb H inclusion test for use as a secondary screening. This test was performed on young red blood cell enriched fractions using dextran sedimentation. The study was performed in 100 subjects positive on initial screening. Confirmatory tests included Hb analysis and a multiplex PCR assay to identify α(0)-thalassemia deletions. A modified Hb H inclusion test was positive in 31 cases, 30 of whom were α(0)-thalassemia carriers (97%). The remaining case (3.0%) was homozygous for α(+)-thalassemia. The remaining 69 cases with a negative Hb H inclusion test included normal subjects, α(+)-thalassemia carriers and ß-thalassemia carriers. Two of them (2/69, 3.0%) were found to be double heterozygotes for ß(0)-thalassemia and α(0)-thalassemia. The overall sensitivity and specificity of the modified Hb H inclusion test for screening of α(0)-thalassemia were 94.0 and 99.0%, respectively. Therefore, we recommend the use of this test in combination with Hb analysis to exclude cases with αß-thalassemia. This should lead to a significant reduction in the number of cases referred for PCR analysis of α(0)-thalassemia by about 50.0%.

Molecular and hematological studies in a large cohort of α(0)-thalassemia in northeast Thailand: data from a single referral center.

α(0)-thalassemia is the most severe form of α-thalassemia alleles found among Southeast Asian and Chinese populations and can cause a fatal condition known as hemoglobin Bart's hydrops fetalis and hemoglobin H disease. In order to provide the molecular epidemiological characteristic of α(0)-thalassemia in northeast Thailand, a total of 12,525 blood specimens referred to our center at Khon Kaen University in northeast Thailand during October 2008 to January 2012 were studied. Hematological parameters were recorded and DNA deletions causing α(0)-thalassemia were examined by PCR related techniques. Among 12,525 samples examined, α(0)-thalassemia alleles were identified in 1,873 (15.0%) samples, including 1855 (14.8%) cases with Southeast Asian (--(SEA)) deletion and 18 cases (0.2%) with THAI deletion (--(THAI)). As many as twenty genotypes were encountered. Hb profiles and hematological parameters were comparatively presented. Data on prevalence, molecular features and phenotypic expression of α(0)-thalassemia should prove useful in a carrier screening and a prevention and control program of this common genetic disorder in the region.

A spurious haemoglobin A(1c) result associated with double heterozygote for haemoglobin Raleigh (ß1[NA1]Val → Ala) and α(+)-thalassaemia.

BACKGROUND: Accurate measurement of haemoglobin A(1c) (HbA(1c)) is useful for long-term glycaemic control in patients with diabetes. Many Hb variants can interfere with HbA(1c) measurement and cause inaccurate results. METHODS: The subject was a 31-year-old Thai man who was discovered because of an unexpected HbA(1c) result; other diabetic parameters were within the normal range. Abnormal Hb was investigated using automated high-pressure liquid chromatography (HPLC) and a capillary electrophoresis system. Mutation analysis was done by cDNA sequencing, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex allele-specific PCR assays. RESULTS: Evaluation of HbA(1c) by cation-exchange HPLC showed a value of 34.9% (reference interval, 4.0-6.0%), but a value of only 4.0% (reference value, 4.8-5.9%) was found with a turbidimetric immunoassay. Haematological analysis revealed a mild anaemia but other parameters were within the normal range. Hb-HPLC analysis demonstrated an unknown Hb variant (47.0%) separating from HbA (46.7%), but capillary electrophoresis identified no abnormal peaks. Mutation analysis identified the Hb Raleigh (ß1[NA1]Val → Ala [GTG → GCG]) mutation in combination with an α(+)-thalassaemia, a hitherto undescribed association. The Hb Raleigh mutation could be detected by PCR-RFLP or a multiplex allele-specific PCR assay. CONCLUSIONS: Hb Raleigh can cause falsely increased HbA(1c) values on cation-exchange HPLC. Definitive diagnosis of this variant using combined Hb and DNA analyses is therefore essential.

First description of a Hb A2 variant in Thailand. Identification of Hb A2-Melbourne [δ43(CD2)Glu→Lys] in Thai individuals.

We report the molecular and hematological identifications of a Hb A(2) variant found for the first time in Thailand. The subjects were two unrelated Thai women who had normal hematological features. In the first case, hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis identified, in addition to Hb A, Hb A(2) (1.6%) and a minor Hb variant (0.9%) separating after Hb A(2). Analysis using capillary electrophoresis (CE) demonstrated 1.3% Hb A(2) and 1.0% minor variant in zone 1. DNA analysis showed a single nucleotide mutation at codon 43 of the δ-globin gene (HBD:c.130G>A) causing a glutamic acid to lysine substitution corresponding to Hb A(2)-Melbourne, originally documented in an Italian subject, but not previously described in Thailand. The mutation could be confirmed by allele specific polymerase chain reaction (PCR) assay. Further screening using this technique in our series identified another case of a Thai woman with a double heterozygosity for Hb A(2)-Melbourne and α(+)-thalassemia (α(+)-thal). A ß-globin gene haplotype associated with this Thai Hb A(2)-Melbourne gene was also established.

Fine-tiling array CGH to improve diagnostics for α- and ß-thalassemia rearrangements.

Implementation of multiplex ligation-dependent probe amplification (MLPA) for thalassemia causing deletions has lead to the detection of new rearrangements. Knowledge of the exact breakpoint sequences should give more insight into the molecular mechanisms underlying these rearrangements, and would facilitate the design of gap-PCRs. We have designed a custom fine-tiling array with oligonucleotides covering the complete globin gene clusters. We hybridized 27 DNA samples containing newly identified deletions and nine positive controls. We designed specific primers to amplify relatively short fragments containing the breakpoint sequence and analyzed these by direct sequencing. Results from nine positive controls showed that array comparative genomic hybridization (aCGH) is suitable to detect small and large rearrangements. We were able to locate all breakpoints to a region of approximately 2 kb. We designed breakpoint primers for 22 cases and amplification was successful in 19 cases. For 12 of these, the exact locations of the breakpoints were determined. Seven of these deletions have not been reported before. aCGH is a valuable tool for high-resolution breakpoint characterization. The combination of MLPA and aCGH has lead to relatively cheap and easy to perform PCR assays, which might be of use for laboratories as an alternative for MLPA in populations where only a limited number of specific deletions occur with high frequency.