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.

Molecular basis of non-deletional HPFH in Thailand and identification of two novel mutations at the binding sites of CCAAT and GATA-1 transcription factors.

High Hb F determinants are genetic defects associated with increased expression of hemoglobin F in adult life, classified as deletional and non-deletional forms. We report the first description of non-deletional hereditary persistence of fetal hemoglobin (HFPH) in Thailand. Study was done on 388 subjects suspected of non-deletional HPFH with elevated Hb F expression. Mutations in the Gγ- and Aγ-globin genes were examined by DNA analysis and rapid diagnosis of HPFH mutations were developed by PCR-based methods. Twenty subjects with five different mutations were identified including three known mutations, - 202 Aγ (C>T) (n = 3), - 196 Aγ (C>T) (n = 3), and - 158 Aγ (C>T) (n = 12), and two novel mutations, - 117 Aγ (G>C) (n = 1) and - 530 Gγ (A>G) (n = 1). Interaction of the - 117 Aγ (G>C) and Hb E (HBB:c.79G>A) resulted in elevation of Hb F to the level of 13.5%. Two plain heterozygous subjects with - 530 Gγ (A>G) had marginally elevated Hb F with 1.9% and 3.0%, whereas the proband with homozygous - 530 Gγ (A>G) had elevated Hb F of 11.5%. Functional prediction indicated that the - 117 Aγ (G>C) and - 530 Gγ (A>G) mutations dramatically alter the binding of transcription factors to respective γ-globin gene promotors, especially the CCAAT and GATA-1 transcription factors. Diverse heterogeneity of non-deletional HFPH with both known and new mutations, and complex interactions of them with other forms of thalassemia are encountered in Thai population.

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.

Results from 8 years of the proficiency testing program for diagnosis of hemoglobinopathies under the prevention and control program of thalassemia in Thailand.

INTRODUCTION: Hemoglobin (Hb) analysis is a key testing for diagnosis of hemoglobinopathies. Accurate analysis, interpretation of results, and genetic risk assessment are important. We report on 8 years of the proficiency testing (PT) program for hemoglobinopathies in Thailand. METHODS: Laboratory participants were required to test two simulated PT items in each cycle using capillary electrophoresis, one was a husband and another was his pregnant wife. Related hematological parameters were provided. The participants also provide interpretation and evaluate the risk of having three severe thalassemia diseases in an expected fetus. Three cycles were operated per year in accordance with the ISO17043 and ISO13528 guidelines. A total of 84 laboratories throughout Thailand were participated. RESULTS: A total of 24 PT cycles were performed during 2012-2019. Most participants had Excellent performance for the PT items with normal, ß-thalassemia trait, hemoglobin E trait, hemoglobin E trait with α-thalassemia, and Hb H disease. However, when the PT items with homozygous Hb E and Hb E-ß-thalassemia were tested, an increase in a Needs improvement performance was noted. From 24 PT cycles, the performance with Excellent, Good, Fair, and Needs improvement was ranging from 10.5%-95.8%, 0%-11.3%, 0%-77.2%, and 2.3%-37.0%, respectively. CONCLUSION: Most participants have proven their performance to be reliable and demonstrated their abilities to provide interpretation and genetic risk assessment on most of the PT items. For complex thalassemia however, a need to improve the interpretation and risk assessment skills is required which is essential for effective prevention and control of severe thalassemia diseases in Thailand.

A proficiency testing program of hemoglobin analysis in prevention and control of severe hemoglobinopathies in Thailand.

BACKGROUND: No external quality assessment program for hemoglobin (Hb) analysis in the prevention and control of thalassemia has been established in Thailand. To improve the first line provisional diagnostics, the first proficiency testing (PT) program has been established. METHODS: External Hb controls prepared at our center were sent to Hb analysis laboratories all over the country. Three cycles per year were performed in 2010 and 2011. In each cycle, two control samples with corresponding hematological parameters, designated as husband and his pregnant wife were supplied for Hb analysis. Each member analyzed the control samples in their routine practices. The results of Hb analysis, laboratory interpretation and risk assessment of the expected fetus for severe thalassemia diseases targeted for prevention and control were entered into the report form and sent back to our center. Participants reports were analyzed and classified into four different quality groups; Excellent (when all the three parameters are correct), Good (correct Hb analysis and interpretation but incorrect risk assessment), Fair (correct Hb analysis but incorrect interpretation and risk assessment) and Needs improvement (incorrect Hb analysis). RESULTS: It was found that most participants could report correct Hb types and quantifications but some misinterpretations and risk assessments were noted. These were clearly seen when control samples with more complexity were supplied. CONCLUSIONS: These results indicate a further improvement is required in the laboratory interpretation and knowledge of the laboratory diagnosis of thalassemia. The established system should facilitate the prevention and control program of thalassemia in the region.

Evaluation of the URIT-2900 automated hematology analyzer for screening of thalassemia and hemoglobinopathies in Southeast Asian populations.

BACKGROUND: The effectiveness of the URIT-2900 Hematology Analyzer for screening of hemoglobinopathies commonly found in Southeast Asian populations was examined. METHODS: Appropriate cut-off values of MCV and MCH for screening of α(0) and ß thalassemias were derived from the receiver operator characteristic curve conducted initially on 279 subjects with various thalassemia genotypes. Validation was performed additionally in a cohort of another unrelated 313 subjects. RESULTS: The best cut off values of MCV and MCH were found to be 78fL and 27pg, respectively. Using these cut off values in combination with the dichlorophenolindophenol test in screening of α(0) thalassemia, ß thalassemia and Hb E in a cohort study revealed 100% sensitivity, 79.6% specificity, 80.0% positive predictive value and 100% negative predictive value. CONCLUSION: The combined blood cell counting using the URIT-2900 Automated Hematology Analyzer and dichlorophenolindophenol test is suitable for population screening of thalassemia and hemoglobinopathies in Southeast Asia.

Anemia, iron deficiency and thalassemia among adolescents in Northeast Thailand: results from two independent surveys.

The prevalence of adolescent anemia, iron deficiency and thalassemia were examined in 2 provinces of northeast Thailand. Blood specimens were collected from adolescent subjects aged 15-17 years in 2 areas; 185 (85 males and 100 females) in Mukdahan province and 313 (116 males and 197 females) in Roi-Et. RBC parameters, serum ferritin levels, Hb and DNA analyses for the identification of common thalassemia genes in Thailand were investigated. The prevalences of anemia were found to be 21.1% (8.1 in male and 13.0 in female) and 16.6% (8.9 in male and 7.7 in female) in Mukdahan and Roi-Et province, respectively. Iron deficiency was observed to be 24.3% in Mukdahan and 14.7% in Roi-Et. Various types of thalassemia were identified in 62.2 and 58.8% of the subject populations, respectively. The proportions of iron deficiency, thalassemia and combined thalassemia and iron deficiency among anemic subjects were 10.2, 53.8 and 30.8% in Mukdahan, and 7.7, 67.3 and 9.6% in Roi-Et. Hematological characteristics were analyzed and are presented. It is concluded that thalassemia and hemoglobinopathies rather than iron deficiency are major causes of adolescent anemia which should be taken into account in public health strategies for the control of anemia in the region.