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.

The association of growth differentiation factor-15 levels and osteoporosis in patients with thalassemia.

BACKGROUND: Ineffective erythropoiesis (IE) is a significant risk factor for osteoporosis in individuals with thalassemia. Growth differentiation factor-15 (GDF15), a biomarker of IE, was found to be elevated in thalassemia patients. This study aimed to examine the association between GDF15 levels and osteoporosis in patients with thalassemia. METHODS: A cross-sectional study was conducted in 130 adult patients with thalassemia in Thailand. Bone mineral density (BMD) at the lumbar spine was evaluated by dual-energy X-ray absorptiometry (DXA), and with a Z-score of less than -2.0 SD was defined as osteoporosis. GDF-15 was measured using the enzyme-linked immunosorbent assay (ELISA). Logistic regression analysis was used to examine the associated factors with the development of osteoporosis. Receiver operator characteristic (ROC) curve analysis was used to estimate the threshold of GDF15 in predicting osteoporosis. RESULTS: Osteoporosis was detected in 55.4% (72/130) of the patients. Advanced age and high GDF15 levels were positively associated with osteoporosis, while an increased hemoglobin level was negatively associated with osteoporosis in patients with thalassemia. In this study, the GDF15 level's ROC demonstrated a good performance in predicting osteoporosis (AUC=0.77). CONCLUSIONS: The prevalence of osteoporosis is high among adult thalassemia patients. Age and high GDF15 levels were significantly associated with osteoporosis in this study. A higher hemoglobin level is associated with a lower risk of osteoporosis. This study suggest that GDF15 could be used as a predictive biomarker for osteoporosis in patients with thalassemia. Adequate red blood cell transfusions and suppression of GDF15 function may be beneficial in preventing osteoporosis.

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.

FLT3 Gene Mutations in Acute Myeloid Leukemia Patients in Northeast Thailand.

BACKGROUND Mutations in the FLT3 gene are associated with acute myeloid leukemia (AML). FLT3 mutations have been identified in approximately 30% of de novo AML patients, particularly those with typical karyotype and inferior prognosis. Therefore, we examined the frequencies of an internal tandem duplication (ITD) and missense mutations of the FLT3 gene and their associated clinical characteristics in patients with AML in northeast Thailand. MATERIAL AND METHODS The leftover bone marrow and/or peripheral blood specimens of 65 newly diagnosed AML patients recruited from Srinagarind Hospital, Khon Kaen University, northeast Thailand, between January 2020 and May 2021 were included in this study. FLT3-ITD and FLT3- tyrosine kinase domain (TKD) were amplified using PCR-related techniques. RESULTS The frequencies of FLT3-ITD and TKD were found to be 16.9% (11/65) and 10.8% (7/65), respectively. One patient had ITD and TKD mutations. The white blood cell count and peripheral blast percentage of FLT3-ITD-positive patients were statistically significantly higher than those of the FLT3-wild type patients, while the peripheral blast percentage of FLT3-TKD-positive patients was significantly lower. No other clinical characteristics among FLT3-positive and FLT3-wild-type patients were observed. DNA sequencing identified 4 FLT3-TKD mutations. The c.2504A>T; Asp835Val and c.2503G>C; Asp835His mutations were predicted as pathogenic mutations while the 2 novel mutations, c.2508C>A; Ile836= and c.2508C>G; Ile836Met were predicted as neutral mutations. CONCLUSIONS This study showed for the first time that FLT3-TKD mutation is common among northeast Thai AML patients. The data should prove useful for selecting efficacious targeted treatment plans for the patients.

Phosphatidylserine-exposed red blood cells and ineffective erythropoiesis biomarkers in patients with thalassemia.

OBJECTIVE: The degree of ineffective erythropoiesis is known to be associated with clinical severity among individuals with thalassemia. The association of ineffective erythropoiesis biomarker levels with different thalassemia genotypes, however, remains limited. The aim of this study was to explore the level of phosphatidylserine-exposed red blood cells (PS-exposed RBCs) and ineffective erythropoiesis biomarkers (growth-differentiation factor-15 and soluble transferrin receptors) in patients with different genotypes. METHODS: A cross-sectional study was conducted on 139 patients of age 18 years and above with different genotypes at Srinagarind Hospital, Khon Kaen University, Thailand. The levels of PS-exposed RBCs were determined using flow cytometry. Measurements of growth-differentiation factor-15 (GDF-15) and soluble transferrin receptors (sTfR) were evaluated by the ELISA method. RESULTS: The PS-exposed RBCs levels were found to be significantly higher in splenectomized beta-thalassemia patients. Patients with beta-thalassemia had the highest GDF-15 levels, followed by patients with non-deletional alpha-thalassemia. Patients with non-deletional alpha-thalassemia showed elevated hemoglobin levels and reduced GDF-15 levels after splenectomy. Patients with beta-thalassemia and non-deletional alpha-thalassemia had the highest levels of PS-exposed RBCs and ineffective erythropoiesis biomarkers, which correlated with the clinical severity of thalassemia. CONCLUSIONS: The levels of ineffective erythropoiesis biomarkers were different across thalassemia genotypes. Splenectomy may improve clinical symptoms of patients with non-deletional alpha thalassemia but not of patients with beta-thalassemia. These findings demonstrate differences in the degree of ineffective erythropoiesis in thalassemia, which emphasizes the need for different treatment approaches among patients with different thalassemia genotypes.

Five Variable Number of Tandem Repeats Loci (D17S5, APOB, TPO Intron 10, IL-1α Intron 6, and CIAS1) in Thais and Application in the Prenatal Diagnostic Laboratory.

Background: Prenatal diagnosis of genetic disease requires DNA analysis of fetal tissue of a responsible gene. Accurate diagnosis is useful for the appropriate management of pregnancy. However, maternal contamination of fetal specimens poses a high preanalytical risk of prenatal misdiagnosis. We have examined five variable number of tandem repeat (VNTR) polymorphisms for use in monitoring potential maternal contamination. Materials and Methods: A study was conducted to examine the heterozygosities of five VNTR loci including, D17S5, APOB, TPO intron 10, IL-1α intron 6, and CIAS1 in 200 unrelated Thai subjects and applied to the monitoring of maternal contamination in 22 families at risk of having fetuses with severe thalassemia. Results: The heterozygosities of D17S5, APOB, TPO intron 10, IL-1α intron 6, and CIAS1 VNTRs were 59.5, 19.5, 66.0, 35.5, and 42.0%, respectively. Therefore, the TPO intron 10 and D17S5 loci were chosen for prenatal diagnosis of thalassemia in 22 families. Analyses of these VNTRs demonstrated an increase of informative data from 59.1% provided by the routine D1S80 VNTR analysis to 90.9%. Conclusions: The VNTR diagnostic procedure described above is simple, cost-effective, rapid, and does not require the use of sophisticated instruments; it should prove useful in the prenatal diagnosis of thalassemia.

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.

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.

Prevalence and Molecular Characterization of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in Females from Previously Malaria Endemic Regions in Northeastern Thailand and Identification of a Novel G6PD Variant.

INTRODUCTION: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked enzymopathy, highly prevalent in the areas where malaria is or has been endemic. Prevalence of G6PD deficiency and characterization of G6PD variants in females from previously malaria-endemic areas of northeastern Thailand remain unstudied. METHODS: Prevalence of G6PD deficiency was determined by a fluorescent spot test (FST), quantitative G6PD activity assay, and multiplex allele-specific (AS)- and restriction fragment length polymorphic (RFLP)-PCR developed for detection of common G6PD variants in the Thai population. RESULTS: Prevalence of G6PD deficiency in female samples (n = 355) was 18% by FST, 29.6% by quantitation of G6PD activity, and 28.1% by PCR-based genotyping. The most common variant was G6PD Viangchan (54%), followed by G6PD Canton (11%) and G6PD Union (11%); in addition, a novel heterozygous variant, G6PD Khon Kaen (c.305T>C, p.F102S), was identified. The majority of heterozygotes expressed G6PD activity within the intermediate deficiency range (30-70% median of normal enzyme activity). CONCLUSION: High prevalence of G6PD deficiency was present in females from northeastern Thailand, the majority being due to heterozygosity of G6PD variants. The findings will have a bearing on the inclusion of primaquine in antimalarial-based policies for malaria elimination in populations with a high prevalence of G6PD deficiency.

Direct Amplification of Whole Blood and Amniotic Fluid Specimens for Prenatal and Postnatal Diagnosis of Hb E-ß 0-Thalassemia Diseases.

OBJECTIVE: Prenatal and postnatal diagnosis of hemoglobin E-ß 0-thalassemia can be made using polymerase chain reaction (PCR) analysis mostly on purified DNA. We have establihed a direct amplification method without DNA extraction on whole blood (WB) and amniotic fluid (AF) specimens to diagnose the disease. METHODS: Three reactions of WB PCR assays and 7 reactions of AF PCR tests were developed for postnatal and prenatal diagnosis, respectively. Assays were validated against routine tests in a blinded trial. RESULTS: The results showed 100% concordance with routine DNA PCR assays. Among 309 ß-thalassemia carriers, 191 patients (61.8%) carried common ß-thalassemia mutations. Among 448 AF specimens, 116 (25.9%) fetuses were found to be affected, 247 (55.1%) fetuses were carriers, and 85 (19%) fetuses were unaffected. CONCLUSION: We found that WB and AF PCR assays are simple, rapid, and reliable. The developed techniques could be applicable in routine settings.

Southeast Asian Ovalocytosis and Hemoglobinopathies in Newborns: Prevalence, Molecular, and Hematologic Analyses.

OBJECTIVES: Southeast Asian ovalocytosis (SAO) is an inherited red blood cell (RBC) membrane disorder, whereas hemoglobinopathies are inherited globin gene disorders. In an area where both diseases are prevalent, the interaction between them resulting in variable hematologic parameters can be encountered. However, little is known about the genetic interaction of SAO and thalassemia. We investigated the prevalence of SAO and hemoglobinopathy genotypes among newborns in southern Thailand. PATIENTS AND METHODS: This study was carried out on 297 newborns recruited consecutively at Naradhiwas Rajanagarindra Hospital in the south of Thailand. The SAO was identified on blood smear examination and polymerase chain reaction analysis. Thalassemia genotypes were defined. Hematologic parameters and hemoglobin (Hb) profiles were recorded and analyzed. RESULTS: Among 297 newborns, 15 (5.1%) carried SAO, whereas 70 (23.6%) had thalassemia with 15 different thalassemia genotypes. Abnormal Hb including Hb C, Hb Q-Thailand, and Hb D-Punjab were observed in 5 newborns. It was found in the nonthalassemic newborns that RBC count, Hb, and hematocrit of the nonthalassemic newborns with SAO were significantly lower than those without SAO. The same finding was also observed in the thalassemic newborns; RBC count, Hb, and hematocrit of the thalassemic newborns with SAO were significantly lower than those without SAO. However, the mean corpuscular volume, mean corpuscular Hb, and RBC distribution width of the SAO-newborns were significantly higher. CONCLUSIONS: Both SAO and hemoglobinopathy genotypes are common in southern Thailand. One should take this into consideration when evaluating neonatal anemia and other hematologic abnormalities. Identification of both genetic defects and long-term monitoring on the clinical outcome of this genetic interaction should be essential to understand the pathogenesis of these common genetic disorders 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 Analysis of Non-Transfusion Dependent Thalassemia Associated with Hemoglobin E-ß-Thalassemia Disease without α-Thalassemia.

BACKGROUND: The finding of many Thai Hb E-ß0-thalassemia patients with non-transfusion dependent thalassemia (NTDT) phenotype without co-inheritance of α-thalassemia has prompted us to investigate the existence of other genetic modifying factors. METHODS: Study was done on 122 adult Thai patients with NTDT Hb E-ß-thalassemia patients without co-inheritance of α-thalassemia. Multiple single-nucleotide polymorphisms (SNPs) associated with γ-globin gene expression including the Gγ-XmnI of HBG2 gene, rs2297339, rs4895441, and rs9399137 of the HBS1L-MYB gene, rs4671393 in the BCL11A gene, and G176AfsX179, T334R, R238H and -154 (C-T) in the KLF1 gene were investigated using PCR and related techniques. RESULTS: Heterozygous and homozygous for Gγ-XmnI of HBG2 gene were detected at 70.5% and 7.4%, respectively. Further DNA analysis identified the rs2297339 (C-T), rs4895441 (A-G), and rs9399137 (T-C) of HBS1L-MYB gene in 86.9%, 25.4%, and 23.0%, respectively. The rs4671393 (G-A) of the BCL11A gene was found at 31.2%. For the KLF1 gene, only T334R was detected at 9.0%. CONCLUSIONS: It was found that these SNPs, when analyzed in combination, could explain the mild phenotypic expression of all cases. These results underline the importance of these informative SNPs on phenotypic expression of Hb E-ß-thalassemia patients.

Whole Blood PCR for Rapid Screening of α0-Thalassemia.

Hemoglobin Bart's hydrops fetalis (homozygous α0-thalassemia) is the most severe form of thalassemia in the Southeast Asian population. Fetuses with this disorder almost always die in utero or shortly after birth. Screening of α0-thalassemia carrier is therefore crucial. Currently, diagnosis of α0-thalassemia genes is done by DNA-based analysis which relies on DNA extraction. We have developed a simple screening format based on whole blood PCR assay. The method was validated on 198 specimens and the results show 100% concordance with a conventional gap-PCR on DNA specimens. The protocol could also be applied to amniotic fluid specimens in prenatal diagnostic testing. The assay developed should facilitate carrier screening and prenatal diagnosis of Hb Bart's hydrops fetalis syndrome in the region.