Prevalence of Thalassemia and Glucose-6-Phosphate Dehydrogenase Deficiency in Newborns and Adults at the Ramathibodi Hospital, Bangkok, Thailand.

Thalassemias and glucose-6-phosphate dehydrogenase (G6PD) deficiency are the most common inherited blood disorders. They are distributed among populations living in malaria endemic regions resulting in survival advantage from severe malaria disease. The aims of this study were to analyze the prevalence of thalassemias and G6PD deficiency at the Ramathibodi Hospital, Bangkok, Thailand. A total of 616 adult and 174 cord blood samples were collected and analyzed for red blood cell (RBC) parameters, hemoglobin (Hb) typing and DNA analysis for G6PD mutations and α-thalassemia (α-thal). The two most prominent types of thalassemia were heterozygous Hb E (HBB: c.79G>A), (19.5% in newborns and 35.6% in adults) followed by heterozygous α-thal-2 [-α3.7 (rightward) deletion] at 18.7% in newborns and 19.5% in adults. After performing G6PD genotyping using multiplex amplification refractory mutation system-polymerase chain reaction (multiplex ARMS-PCR) for 10 G6PD mutations, the prevalence of G6PD mutation was found in 12.0% of newborns and 11.7% of adults. The G6PD Viangchan [871 (G>A)] is the most common G6PD mutation in newborns (42.9%) and adults (52.8%). In addition, coinheritance of various types of thalassemia with G6PD deficiency were found. The results indicated that heterozygous Hb E and G6PD Viangchan are predominant both in newborns and adults in this study.

Low oxygen saturation and severe anemia in compound heterozygous Hb Louisville [ß42(CD1)Phe→Leu] and Hb La Desirade [ß129(H7)Ala→Val].

OBJECTIVE: To investigate the cause(s) of a Thai male proband presenting low oxygen saturation by pulse oximetry (SpO2) and severe anemia. METHODS: As Hb variant was suspected, Hb typing was determined by high-performance liquid chromatography and capillary electrophoresis, and subsequently Hb variant was identified by DNA sequencing. Complete blood counts were performed using automated blood cell counter and oxygen saturation was measured by pulse oximetry. RESULTS: Proband was compound heterozygous for Hb Louisville [ß42(CD1)Phe→Leu] and Hb La Desirade [ß129(H7)Ala→Val]. Of the proband's two sons, one was compound heterozygous for Hb Louisville and Hb E and the other for Hb La Desirade and Hb E. The former son had similar clinical features and laboratory findings with those of the proband while the latter showed had no abnormal clinical manifestations. CONCLUSION: This the first report of compound heterozygosity of Hb Louisville and Hb La Desirade in an individual of Southeast Asian ethnicity. Hb variant identification is crucial for genetic counseling and appropriate treatment in regions where hemoglobinopathies are common.

Occurrence of the - -(SEA), - -(THAI) and - -(FIL) α-Thalassemia-1 Carriers from a 7-Year Study at Ramathibodi Hospital, Bangkok, Thailand.

α-Thalassemia (α-thal) is one of the most common genetic diseases in Thailand. Homozygosity of α-thal-1 (- -/- -) and compound heterozygosity of α-thal-1/α-thal-2 (- -/-α) leads to Hb Bart's (γ4) hydrops fetalis and Hb H (ß4) disease, respectively. In order to better control and provide prevention of α-thal disease, the prevalence of α-thal-1 carriers and the types of genotypes in the Thai population should be known. A 7-year retrospective study, employing multiplex gap-polymerase chain reaction (gap-PCR) of 31,632 blood samples from Ramathibodi Hospital, Mahidol University, Bangkok, revealed an α-thal-1 carrier rate of 14.40% with the - -(SEA) (NG_000006.1: g.26264_45564del19301), - -(THAI) (NG_000006.1: g.10664_44164del33501) and - -(FIL) (NG_000006.1: g.11684_43534del31851) genotypes, constituting frequencies of 14.21, 0.18 and 0.01%, respectively. Although the - -(FIL) genotype is rare in the Thailand, its detection should be included in future α-thal screening programs.

GENETIC FACTORS INFLUENCING HEMOGLOBIN F LEVEL IN ß-THALASSEMIA/HB E DISEASE.

Genetic factors influencing Hb F content in adult red blood cells include ß-thalassemia genotypes, co-inheritance of α-thalassemia traits and single nucleotide polymorphisms (SNPs). Genotyping of α- and ß-thalassemia and five SNPs in ß-globin gene cluster previously identified in genome-wide association studies as being markers of elevated Hb F in ß-thalassemia were performed in 81 subjects diagnosed with ß-thalassemia/Hb E. Hb F levels are higher (0.9-7.1 g/dl) in subjects (n = 57) with the severe compared to mild ß-thalassemia (0.8-2.5 g/ dl) (n = 4) genotypes, and are similarly low (0.7-3.5 g/dl) in those (n = 15) with α-thalassemia co-inheritance. Hb F levels in non-thalassemia controls (n = 150) range from 0 to 0.15 g/dl. The presence of homozygous minor alleles of the 5 SNPs are significant indicators of ß-thalassemia/Hb E individuals with high Hb F (> 4 g/dl), independent of their thalassemia genotypes. Given that re-activation of γ-globin genes leads to amelioration of ß-thalassemia severity, understanding how genetic factors up-regulate Hb F production may lead to possible therapeutic interventions, genetically or pharmacologically, of this debilitating disease in the not too distant future.

Scanning for α-Hemoglobin Variants by High-Resolution Melting Analysis.

BACKGROUND: Definitive detection of hemoglobin (Hb) variants requires DNA sequencing. High-resolution melting (HRM) analysis of polymerase chain reaction (PCR) amplicons was applied to detect and discriminate among uncommon α-Hb variants found in Thailand. METHODS: Uncommon suspected α-Hb variants observed in Hb typing were identified by sequencing of DNA from whole blood samples. Three pairs of PCR primers covering the mutation regions in the three α-globin exons then were used for PCR coupled with difference in HRM analysis to subtract out the concomitant melting profile of the normal allele in the heterozygous state. RESULTS: DNA sequencing identified six heterozygous α-Hb variants, namely, Hb G-Waimanalo (HBA2: exon 2, codon 64 G>A), Hb J-Buda (HBA1: exon 2, codon 61 G>T), Hb Kurosaki (HBA2: exon 1; codon 7 A>G), Hb O-Indonesia (HBA1: exon 3 codon 116 G>A), Hb Q-India (HBA1:exon 2, codon 64 G>C), and Hb Q-Thailand (HBA1: exon 2 codon 74 G>C). Difference HRM analysis showed one temperature melting profile using exon 1 primer pair, four different profiles with exon 2 primer pair, and one profile with exon 3 primer pair. CONCLUSIONS: PCR-HRM analysis was effective in detecting and discriminating among single point mutations causing six uncommon α-Hb variants in heterozygous individuals. The method can be applied for routine screening due to its simplicity and relatively low cost.

Identification of Hb Constant Spring (HBA2: c.427T > C) by an Automated High Performance Liquid Chromatography Method.

Laboratory investigation of hemoglobinopathies includes complete blood count (CBC), hemoglobin (Hb) typing by high performance liquid chromatography (HPLC) and DNA analysis. DNA analysis is the most reliable method but requires a manually laborious procedure and is time consuming. A more practical method of detecting abnormal Hbs is the HPLC technique, because it is more rapid and easier to interpret. Hb Constant Spring (Hb CS; HBA2: c.427T > C) is an abnormal variant that is labile and difficult to detect using conventional methods. To evaluate the efficiency of Hb CS determination by HPLC, blood samples from 578 subjects were analyzed using an automated cell analyzer for hematological parameters, automated HPLC for Hb identification, and polymerase chain reaction (PCR) for α-thalassemia (α-thal) and Hb CS confirmation. These included 169 normal, 119 heterozygous α-thal-2, 30 homozygous α-thal-2, 177 heterozygous α-thal-1, 59 heterozygous Hb CS, seven homozygous Hb CS and 17 compound heterozygous α-thal-2 and Hb CS subjects. The results showed that sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Hb CS by HPLC were 93.78, 99.80, 98.73 and 99.00%, respectively. The mean of misdiagnosis value of the three groups of Hb CS subjects (total 83) was 6.02% (n = 5), with percentages for heterozygous Hb CS, homozygous Hb CS, and compound heterozygous α-thal-2 and Hb CS being 6.8, 0.0 and 5.9%, respectively. The HPLC method yielded good results, although it may also lead to misdiagnosis of Hb CS due to the relatively small amount and lability.

Elevated erythropoietin and cytokines levels are related to impaired reticulocyte maturation in thalassemic patients.

Serum EPO concentration is related primarily to the rate of erythrocyte production and, under the stimulation of hypoxia, increases exponentially as hemoglobin (Hb) decreased. The level of EPO was determined in 141 subjects including 43 normal, 44 thalassemic patients and 54 thalassemic trait subjects. The EPO level was significantly higher in the thalassemic patients (54.8mU/ml in HbH disease [α thal1/α thal2;], 78.1mU/ml in HbH with Hb CS [α thal 1/CS]; 95.6mU/ml in ß-thal/HbE splenectomized [BE(S)]; and 114.8mU/ml in ß-thal/HbE non-splenectomized [BE(NS)]as compared with 12.0mU/ml in normal subjects. No significant differences were detected in thalassemic trait subjects. In addition, the levels of EPO in thalassemic patients is correlated significantly with the number of reticulocytes and the reticulocyte fractions especially the fraction of immature reticulocytes. Interestingly, the highest level of EPO/% retic ratio as indicated for EPO non-responder was detected in BE(NS) patients. However, the impaired reticulocytes maturation was found to be related significantly with the levels of TNF-α,IFN-γ,IL-10, and VEGF. Since, TNF-α, IFN-γ, IL-10 and VEGF are reported as the cytokines with erythropoietic inhibitory mediators, the variation of these cytokines in thalassemic environments may be associated to the anemic crisis in these patients.

Implication of globin gene expression, hemoglobin F and hemoglobin E levels on ß-thalassemia/Hb E disease severity.

One of the factors affecting the degree of severity in ß-thalassemia disease is the presence of unmatched α-hemoglobin chains. Thus, the expression levels of globin genes in reticulocytes of ß-thalassemia subjects were measured using quantitative RT-PCR, demonstrating that α/ß globin mRNA ratio, as well as levels of γ-globin mRNA and Hb F, increased with progressing degree of ß globin synthesis defect. The levels of γ-globin mRNA and Hb F could not be directly correlated with severity of ß-thalassemia/Hb E disease due to a low statistical power of this analysis. Higher levels of Hb E were present, however, in clinically mild patients, as compared to moderately severe ß-thalassemia/Hb E subjects. This suggests that in ß-thalassemia/Hb E disease, elevation of Hb E level through enhancing correctly spliced ß(E)-globin mRNA offers another approach in ameliorating disease severity. In addition, co-inheritance of α-thalassemia 2 trait in ß-thalassemia/Hb E subjects was associated with milder outcome compared with those with the same ß-thalassemia genotypes, confirming the notion of the beneficial effect of a more balanced α:ß-globin chain ratio.

Prevalence and hematological parameters of thalassemia in Tha Kradarn subdistrict Chachoengsao Province, Thailand.

OBJECTIVE: To determine the prevalence, molecular characteristics and hematological study of thalassemia in Tha Kradarn Subdistrict Chachoengsao Province. MATERIAL AND METHOD: The present study population consisted of266 participants from Moo 19 Baan Na-Ngam, Chachoengsao Province, Thailand. After blood collection, all samples were screened for thalassemia by initial screening with the OF and DCIP tests and additional testing by CBC, RBC indices, hemoglobin typing and determination of Hb A2 and Hb E. All common alpha-thalassemia mutations were determined using the PCR with allele specific primers and Gap PCR for common deletions. RESULTS: The prevalence of alpha-thal 1, alpha-thal 2 and beta-thal were found as 2.72%, 11.26% and 0.97%, respectively. Regarding the abnormal hemoglobins, the prevalence of Hb E, Hb Constant Spring and Hb Pakse was 38.45%, 3.69% and 0.78%, respectively. MCV and MCH were significantly different between P-thalassemia as well as a-thal 1 carriers and normal subjects. In all alpha-thal 1 traits, it was found that the MCV and MCH were less than 75 fL and 25 pg, therefore, these parameters can be used for alpha-thal 1 screening. CONCLUSION: In the present study, the prevalence of thalassemia was similar to previous studies. Moreover, using the combination of OF and DCIP tests compared with MCV, MCH and DCIP tests for the initial thalassemia screening, it was found that the OF and DCIP tests gave more false positive results, which increased the need for further Hb typing. Hence, the MCV and MCH combined with DCIP tests provide cost minimization and practical for a large population-based screening program.

A rare Hb H disease due to the - -(SEA) and 16.6 kb α-thalassemia-2 deletions.

A large deletional α-thalassemia-2 (α-thal-2) allele was identified in a Thai woman with Hb H disease. The proband has α-thal-1 (SEA type) in conjunction with a 16.6 kb deletion affecting the α2-globin allele. The proband had severe anemia and required a blood transfusion during puerperium.

Nondeletional Hb Queens Park [α32(B13)Met→Lys]/Hb H (ß4) disease.

A rare nondeletional α-thalassemia-2 (α-thal-2) allele was identified in a Thai boy with Hb H (ß4) disease. The proband has α-thal-1 (- -(SEA) type) together with a non productive Hb Queens Park (HBA1:c.98T>A) [α32(B13)Met→Lys] α1-globin variant. No abnormal hemoglobin (Hb) fraction was detected by high performance liquid chromatography (HPLC). The clinical effect of this mutation in the proband was comparable to that of deletional α-thal-2 present in Hb H disease.

Effect of genetic alterations of cytarabine- metabolizing enzymes in childhood acute lymphoblastic leukemia.

BACKGROUND: Single nucleotide polymorphisms (SNPs) of deoxycytidine kinase (dCK) and cytidine deaminase (CDA) are known to alter their enzymatic activities, which affect the metabolism of cytarabine. Currently, treatment of childhood acute lymphoblastic leukemia (ALL) includes cytarabine, especially in high-risk patients. Therefore, we hypothesized that a genetic variation of dCK and CDA genes may influence the risk of cytarabine-related toxicities and early response to treatment. PATIENTS AND METHODS: We included children diagnosed with ALL and lymphoblastic lymphoma (LL) stage III and IV. The patients received a modified St Jude Total Therapy Study XV protocol. Cytarabine was used during induction remission (low-dose cytarabine) and reinduction II (high-dose cytarabine) phases. Genotyping of dCK-360C>G and -201C>t and CDA 79A>C and 208G>A was performed. Minimal residual disease (MRD) at the end of the induction phase was measured using flow cytometry. RESULTS: Ninety-four children with ALL (n=90) and LL (n=4) were analyzed. The median age at diagnosis was 5.8 years (range, 0.4-15 years). All four SNPs showed predominant wild type alleles. There was no CDA-208A allele in our population. Children with dCK-360G allele were at risk of mucositis after receiving low-dose cytarabine (OR=3.7; 95%CI, 1.2--11.3). neither dCK nor CDA polymorphisms affected the MRD status at the end of the induction phase. CONCLUSION: The dCK-360G allele was found to increase the risk of mucositis after exposure to low-dose cytarabine in childhood ALL therapy.

Hb Tak and Hb Q-Thailand in Thai patients are S-window hemoglobin variants revealed by high performance liquid chromatography.

The S-window hemoglobin (Hb) variants revealed by high performance liquid chromatography (HPLC) were studied in 12 Thai individuals. The variants were identified, using DNA sequencing and multiplex amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), to be six cases of Hb Tak [beta147 (+AC)], and six cases of Hb Q-Thailand [alpha74(EF3)Asp-->His], respectively. By using the Capillarys 2-capillary zone electrophoresis (CE), Hb Tak and Hb Q-Thailand co-migrated with Hb F in zone 7. This might pose a problem as the high Hb F conditions suggest a differential diagnosis. The S-window Hb variants are mostly Hb Tak and Hb Q-Thailand in the Thai population rather than Hb S [beta6(A3)Glu-->Val]. The definite identification of Hb variants detected by HPLC or capillary electrophoresis (CE) requires DNA analysis.

Virus Overlay Protein Binding Assay (VOPBA) reveals serotype specific heterogeneity of dengue virus binding proteins on HepG2 human liver cells.

OBJECTIVE: This study sought to investigate the presence of dengue virus binding proteins expressed on the surface of HepG2 cells and to determine if there were serotype specific differences in binding. METHODS: HepG2 cell membrane proteins were extracted and separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with dengue virus serotypes 2, 3 and 4 under varying hybridization conditions. The positions of dengue virus binding proteins were established with a pan-specific anti-dengue virus monoclonal antibody. RESULTS: Dengue virus binding proteins were seen at approximately 78-80, 90, 98, and 102 kD for dengue serotype 2, 90, 130 and 182 kD for dengue serotype 3, and 90 and 130 kD for dengue serotype 4. Binding of the serotypes 3 and 4 was significantly altered by the hybridization conditions, while serotype 2 was affected to a lesser extent. CONCLUSIONS: The virus overlay assay used here provides further evidence that there is a serotype specific component regulating the entry of the dengue virus into cells. Given that several virus binding proteins are seen for each serotype, multiple proteins may be required to facilitate the entry of the virus into some cell types.

Increased circulating activated endothelial cells, vascular endothelial growth factor, and tumor necrosis factor in thalassemia.

An increased number of circulating endothelial cells (CECs) was demonstrated in alpha- and beta-thalassemic patients, beta-thalassemia/hemoglobin E (BE), both splenectomized (BE[S]) and non-splenectomized (BE[NS]), had higher numbers of CECs than alpha-thalassemia, both HbH (alpha-thal l/alpha-thal 2; H) and HbH with hemoglobin Constant Spring (alpha-thal 1/CS; H/CS). CECs were also increased in heterozygous HbE (EA) and homozygous HbE (EE). The highest level of tumor necrosis factor-alpha (TNF-alpha) was found in HbH/CS patients, whereas the highest levels of vascular endothelial growth factor (VEGF) was observed in BE[S] patients. Significant decreases, in protein C and protein S levels were found in both alpha- and beta-thalassemia compared with normal. Good correlations between the numbers of CEC and TNF-alpha, VEGF, protein C, and protein S levels were demonstrated in this study. In addition, markers for endothelial cell activation and injury (intercellular adhesion molecule-1, ICAM-1/CD54; vascular cell adhesion molecule-1, VCAM-1/CD106; and E-selectin, ELAM-1/CD62E) were detected on the surface of isolated CECs using immunofluorescence technique. Appearance of CECs with markers for endothelial cell activation, together with increased levels of TNF-alpha and VEGF and decreased levels of protein C and protein S in the circulation, may account for the propensity of vascular perturbation in thalassemic subjects.