δ-Hemoglobinopathies in Thailand: screening, molecular basis, genotype-phenotype interaction, and implication for prevention and control of thalassemia.

The δ-globin gene defects are clinically silent but interaction with ß-thalassemia can lead to a misdiagnosis of ß-thalassemia carrier. We report an extensive molecular characterization of δ-hemoglobinopathies in Thailand. Study was done on 32,108 subjects, encountered at the thalassemia screening. Six different approaches based on the reduced Hb A2 or appearance of Hb A2-derivative were established for selective recruitment of subjects. Among 32,108 subjects, a total of 296 subjects were suspected of having δ-globin gene defects. Of these 296 subjects, Hb and DNA analyses identified δ-hemoglobinopathies with 10 different mutations in 34 (0.11%) of them. These included a novel mutation, [δCD30(AGG>GGG) (n = 1)], 5 previously undescribed in Thailand, [δ-44(G>A) (n = 7), Hb A2-Troodos (n = 5), δIVSII-897(A>C) (n = 4), δ-68(C>T) (n = 2), and Hb A2-Indonesia (n = 1)], and 4 mutations previously found in Thailand, [Hb A2-Melbourne (n = 9), δ-77(T>C) (n = 3), Hb A2' (n = 1), and Hb A2-Kiriwong (n = 1)]. Genetic heterogeneities seen included interactions of δ-globin gene defects with heterozygous Hb E, ß-thalassemia, α-thalassemia, and in cis locations of the Hb A2-Troodos and Hb E mutations found for the first time. Rapid identification methods of these δ-globin gene mutations were developed. The results should prove useful to a prevention and control program of hemoglobinopathies in the region.

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.

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.

Factors associated with anaemia and iron deficiency among women of reproductive age in Northeast Thailand: a cross-sectional study.

BACKGROUND: Anaemia and iron deficiency (ID) affect women of reproductive age globally and considered to be a major public health problem in developing countries. This study determines the prevalence of anaemia and ID among women of reproductive age in urban northeast Thailand and examined the relative contribution of various risk factors to anaemia and ID in this population. METHODS: Three hundred ninety-nine non-pregnant women, aged 18-45 years, from three universities in northeast Thailand participated in this cross-sectional study. Selected socio-demographic, history of blood loss, usual consumption of red meat and tea/coffee, and anthropometric data were collected. Complete blood count including haemoglobin (Hb) concentration, serum ferritin (SF), C-reactive protein (CRP), and thalassemia were determined. Multiple logistic regressions were applied to identify the risk factors of anaemia and ID. RESULTS: Overall, 370 participants were included for data analyses after excluding women with severe/intermedia thalassemia diseases and/or those with positive serum CRP. The prevalence of anaemia, ID, and iron deficiency anaemia (IDA) were 28.4, 28.4, and 13.2%, respectively. Women with thalassemia had a higher prevalence of anaemia but a lower prevalence of ID than the women without thalassemia. By multiple regression analysis, ID [adjusted OR (AOR) = 4.9, 95% CI = 2.8-8.3], two α-gene defects (AOR = 8.0, 95% CI = 3.0-21.3) and homozygous Hb E (AOR = 8.5, 95% CI = 3.0-24.3) were identified as the potential risk factors of anaemia. Further, the odds of ID were significantly higher among women who donated blood within the past 3 months (AOR = 6.7, 95% CI = 2.8-16.3), and had moderate to a high amount of blood loss during menstruation (AOR = 2.2, 95% CI = 1.3-3.9). CONCLUSION: This study found a relatively high but differential prevalence of anaemia and ID among women of reproductive age with or without thalassemia. Only homozygous Hb E and two α-gene defects of thalassemia types and ID were the main factors contributing to anaemia. Recent blood donation, and moderate to a high amount of blood loss during menstruation were potential risk factors of ID in this population.

Presentation of Compound Heterozygous Hemoglobin Constant Spring and Hemoglobin Pakse in Neonates.

BACKGROUND: Mutations causing α thalassemia are divided into deletion and nondeletion groups. In the nondeletion group, hemoglobin constant spring (Hb CS) and hemoglobin Pakse (Hb Pakse) are both caused by a termination codon mutation leading to elongation of the α2 globin gene. In the case of Hb CS, the mutation is TAA→CAA, whereas the mutation causing Hb Pakse is TAA→TAT. Clinical hematologic phenotypes are not significantly different. It is important to identify compound heterozygotes for purposes of genetic counseling. METHODS: We report 5 neonates with compound heterozygous Hb CS/Hb Pakse mutations with respect to clinical courses, hematologic profiles, and management. RESULTS: Among 5 cases (3 male babies and 2 female babies) with mean birth weight 2982 g (range, 2660 to 3440 g), 3 were diagnosed as compound heterozygous Hb CS/Hb Pakse, 1 as homozygous Hb E with compound heterozygous Hb CS/Hb Pakse, and 1 as heterozygous Hb E with compound heterozygous Hb CS/Hb Pakse. Clinical manifestations included fetal anemia (1 case), neonatal hyperbilirubinemia (5), neonatal anemia (2), hepatosplenomegaly (1), and cholestatic jaundice (1). Three cases required a single phototherapy; 2 cases needed double phototherapy for treatment of severe hyperbilirubinemia. During the first few months of life, all cases had mild anemia, slightly low mean corpuscular volume, wide red cell distribution width, and low red cell counts. At 1 to 3 years of age, all patients still had mild microcytic hypochromic anemia with Hb levels around 10 g/dL, increased reticulocyte count, and wide red cell distribution width. CONCLUSIONS: Misdiagnosis of Hb Pakse could occur via Hb typing using Hb electrophoresis, because the band comigrates with that of Hb CS. DNA study is the definitive method for diagnosis.

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.

Thalassemia and Hemoglobinopathies in an Ethnic Minority Group in Northern Vietnam.

This study assessed thalassemia and hemoglobinopathies in a group of the Tay ethnic minority. Participants included 289 women of reproductive-age who enrolled in a pilot screening program for thalassemia conducted at six communities of Thai Nguyen Province, northern Vietnam. Standard procedures including complete blood count (CBC), hemoglobin (Hb) and DNA analyses were performed for all samples. The prevalence of thalassemia in 289 Tay women was 15.6% (gene frequency 0.078) for α0-thalassemia (α0-thal), 10.0% (gene frequency 0.050) for α+-thal, 7.3% (gene frequency 0.036) for ß-thalassemia (ß-thal), 2.4% (gene frequency 0.012) for Hb Constant Spring [Hb CS; α142, Term→Gln, TAA>CAA (α2), HBA2: c.427T>C] and 1.7% (gene frequency 0.009) for Hb E [ß26(B8)Glu→Lys, GAG>AAG; HBB: c.79G>A]. Further analysis of ß-globin gene abnormalities identified four mutations including codons 41/42 (-TCTT) (HBB: c.126_129delCTTT), codon 17 (A>T) (HBB: c.52A>T), codons 71/72 (+A) (HBB: c.216_217insA), and -28 (A>G) (HBB: c.78A>G). The results hint at the remarkably high frequencies of severe forms of thalassemia that indicate a serious public health problem requiring further exploration, and most probably, also intervention within the country.

EE score: an index for simple differentiation of homozygous hemoglobin E and hemoglobin E-ß0-thalassemia.

BACKGROUND: The objective of the study was to describe a formula based on hemoglobin (Hb)A2 and HbF levels for differentiation of homozygous HbE and HbE-ß-thalassemia. METHODS: A total of 1256 subjects suspected for homozygous HbE or HbE-ß0-thalassemia were recruited at the ongoing thalassemia screening program at Khon Kaen University, Thailand. Hb analysis was done using capillary electrophoresis. Genotyping was based on DNA analysis. An arbitrary formula based on HbA2 and HbF was developed statistically for differentiation of the two conditions. Validation was carried out prospectively on another 139 subjects encountered at routine laboratory. RESULTS: Among 1256 subjects, Hb and DNA analyses identified cases with homozygous HbE (n=1076, 85.7%), HbE-ß0-thalassemia (n=140, 11.1%), HbE-δß0-thalassemia (n=30, 2.4%) and unknown HbE-related disorder (n=10, 0.8%). An inverse correlation between the amounts of HbA2 and HbF in HbE-ß0-thalassemia was observed. With differences in the amounts of HbA2 and HbF between the groups, an arbitrary score (7.3 HbA2+HbF) was developed where score above 60 indicated HbE-ß0-thalassemia. Application of this score on another 139 subjects showed accurate prediction of HbE-ß0-thalassemia with 100% sensitivity, 96.5% specificity, 85.7% positive predictive value and 100% negative predictive value. Successful application onto couples at risk was demonstrated. CONCLUSIONS: An established score should prove useful in the differentiation of homozygous HbE and HbE-ß0-thalassemia in routine setting and lead to a significant reduction in number of referring cases for molecular testing.

Elevations of Thrombotic Biomarkers in Hemoglobin H Disease.

BACKGROUND: Thalassemia is a group of hereditary hemoglobinopathies caused by decreased or absent synthesis of α and/or ß globin chains. Studies have shown that hypercoagulability and thrombosis are common clinical symptoms in ß-thalassemia, especially ß-thalassemia intermedia, but little is known about in α-thalassemia. This study aims to examine phosphatidylserine (PS) levels, platelet activation, and coagulation markers in splenectomized (S) and nonsplenectomy (NS) patients with hemoglobin (Hb) H disease. METHODS: The NS group comprised 20 patients (median age 15.0 years, range, 14-16.5 years), and the S group consisted of 11 patients (median age 16.4 years, range, 14-19.9 years) with Hb H disease; the control group consisted of 20 normal subjects. Hematological parameters were collected. Flow cytometry was used to measure PS exposure on red blood cells. The levels of intercellular adhesive molecule (ICAM)-1, tumor necrosis factor α (TNFα), ß-thromboglobulin (TG) and prothrombin fragment 1 + 2 (F1.2) were determined using ELISA test kits. RESULTS: Significant increases in the levels of PS, ICAM-1, TNFα, ß-TG, and F1.2 were observed in both patient groups compared to normal controls (p < 0.01). CONCLUSION: This observation indicates blood coagulation, endothelial injury, chronic low-grade inflammation, platelet activation, and thrombin generation are present in Hb H disease; these findings merit further assessment in a larger prospective cohort to establish possible links with thrombotic manifestations.

Clinical Course of Homozygous Hemoglobin Constant Spring in Pediatric Patients.

BACKGROUND: Hemoglobin (Hb) Constant Spring is an alpha-globin gene variant due to a mutation of the stop codon resulting in the elongation of the encoded polypeptide from 141 to 172 amino acid residues. Patients with homozygous Hb Constant Spring are usually mildly anemic. METHODS: We retrospectively describe clinical manifestations, diagnosis, laboratory investigations, treatment, and associated findings in pediatric patients with homozygous Hb Constant Spring followed-up at Srinagarind Hospital. RESULTS: Sixteen pediatric cases (5 males and 11 females) were diagnosed in utero (N=6) or postnatal (n=10). Eleven cases were diagnosed with homozygous Hb Constant Spring, 4 with homozygous Hb Constant Spring with heterozygous Hb E, and 1 with homozygous Hb Constant Spring with homozygous Hb E. Three cases were delivered preterm. Six patients had low birth weights. Clinical manifestations included fetal anemia in 6 cases, hepatomegaly in 1 case, hepatosplenomegaly in 2 cases, splenomegaly in 1 case. Twelve cases exhibited early neonatal jaundice, 9 of which required phototherapy. Six cases received red cell transfusions; 1 (3), >1 (3). After the first few months of life, almost all patients had mild microcytic hypochromic anemia and an increased reticulocyte count with a wide red cell distribution (RDW), but no longer required red cell transfusion. At 1 to 2 years of age, some patients still had mild microcytic hypochromic anemia and some had normocytic hypochromic anemia with Hb around 10 g/dL, increased reticulocyte count and wide RDW. Associated findings included hypothyroidism (2), congenital heart diseases (4), genitourinary abnormalities (3), gastrointestinal abnormalities (2), and developmental delay (1). SUMMARY: Pediatric patients with homozygous Hb Constant Spring developed severe anemia in utero and up to the age of 2 to 3 months postnatal, requiring blood transfusions. Subsequently, their anemia was mild with no evidence of hepatosplenomegaly. Their Hb level was above 9 g/dL with hypochromic microcytic blood pictures as well as wide RDW. Blood transfusions have not been necessary since then.

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 origin of α0-thalassemia (SEA deletion) in Southeast Asian populations and application to accurate prenatal diagnosis of Hb Bart's hydrops fetalis syndrome.

α0-thalassemia of SEA deletion (-SEA) is common among Southeast Asian and Chinese. Using haplotype and phylogenetic analyses, we examined the origin of this defect in Southeast Asian populations. Study was done on both normal and α0-thalassemia alleles in 3 ethnic groups including 96 Thai, 52 Laotian and 21 Cambodian. Five SNPs encompassing the (-SEA) including (rs3760053 T>G), (rs1211375 A>C), (rs3918352 A>G), (rs1203974 A>G) and (rs11248914 C>T) were examined using high-resolution melting assays. It was found that 94.0% of Thai, 100% of Laotian and 100% of Cambodian α0-thalassemia alleles were linked to the same haplotype: the haplotype H4 (AAGC), representing an Asian specific origin. An G allele of the (rs3760053) was found to be in strong linkage disequilibrium with the α0-thalassemia allele in these populations. A multiplex PCR assay was developed to detect simultaneously the (-SEA) allele and genotyping of a linked (rs3760053) to improve accuracy of prenatal diagnosis of α0-thalassemia. Application of this multiplex PCR assay for routine prenatal diagnosis of α0-thalassemia in 12 families revealed a 100% concordant result with conventional gap-PCR assay. Therefore, a single genetic origin is responsible for the spread and high prevalence of the (-SEA) in the region. The multiplex PCR assay developed should provide a double-check PCR system for more accurate diagnosis and allow the monitoring of possible maternal contamination at prenatal diagnosis of this important genetic disorder.

Molecular analysis of haemoglobin E in Southeast Asian populations.

Haemoglobin (Hb) E is the most common Hb variant in Asia where its gene frequency approaches 0.3 in some areas. We studied genetic background of Hb E genes among Southeast Asian populations. This study examined ß-globin gene haplotypes linked to haemoglobin E (Hb E) in diverse groups of Southeast Asian populations. The study was conducted on southern Thai (22 alleles), Cambodian (84 alleles), Laotian (120 alleles), Vietnamese (87 alleles) and Burmese (one allele) subjects. Results were compared with those of previous studies in northeast Thailand, the Yunnan of China, West India and Europe. Ten different haplotypes were observed. The four most common haplotypes were haplotypes 1 (- + - + + + -) and 2 (+ - - - - + -) on chromosomes with framework 2 and haplotypes 6 (- + - + + - +) and 7 (+ - - - - - +) on chromosomes with framework 3 variety. Phylogenetic analysis indicated that haplotype 1 is a relatively recent haplotype found in all populations, whereas haplotype 6 is found predominately in Cambodians. The results indicate that at least two genetic origins of Hb E are responsible for the high prevalence and spread of Hb E among Southeast Asian populations.

Nine known and five novel mutations in the erythroid transcription factor KLF1 gene and phenotypic expression of fetal hemoglobin in hemoglobin E disorder.

Hemoglobin E is the most common Hb variant found in South East Asia. Variation of Hb F expression in Hb E syndrome is associated with several genetic modifiers. We report several single nucleotide polymorphisms (SNPs), including nine known and five novel mutations of the Krüppel-like factor 1 (KLF1; an erythroid specific transcription factor) gene and determine their associations with phenotypic expression of Hb F in Hb E disorders. KLF1 mutations were examined using high resolution melting (HRM) assay and DNA sequencing in 575 homozygous Hb E, 278 heterozygous Hb E and 100 normal subjects. Fourteen mutations were mostly observed in subjects with elevated Hb F, including nine known mutations (G176AfsX179, T334R, R238H, -154 (C>T), A298P, S270W, R301H, -148 (G>A) and G335R and five novel mutations (Q217X, Q223X, Y290_S293del, K307N, and M358I). None of them, but the -148 (G>A), were observed in normal controls to have Hb F <1%. Combined KLF1 mutations with other SNPs including (G)γ-XmnI, BCL11A and HBS1L-MYB were associated with higher Hb F levels. KLF1 is therefore an important genetic factor associated with increased Hb F and in combination with other modifying factors could explain the phenotypic variation of Hb F expression in this common hemoglobinopathy.

Molecular Understanding of Non-Transfusion-Dependent Thalassemia Associated with Hemoglobin E-ß-Thalassemia in Northeast Thailand.

Non-transfusion-dependent thalassemia (NTDT) is associated with various forms of thalassemia and genetic modifiers. We report the molecular basis of NTDT in hemoglobin (Hb) E-ß-thalassemia disease. This study was done in 73 adult patients encountered at the prenatal diagnosis center of Khon Kaen University, Northeast Thailand. Hematological parameters and Hb patterns were collected, and α- and ß-globin gene mutations were determined. Multiple single-nucleotide polymorphisms (SNPs) including the rs7482144/Gγ-XmnI polymorphism, rs2297339, rs2838513, rs4895441, and rs9399137 in the HBS1L-MYB gene, rs4671393 and rs11886868 in the BCL11A gene, and G176AfsX179 in the KLF1 gene were examined. Five ß0-thalassemia mutations and a severe ß+-thalassemia mutation in trans to the ßE gene were identified. No significant difference in hematological parameters was observed among ß-thalassemia genotypes. Coinheritance of α-thalassemia was observed in 31 of the 73 subjects (42.5%). Four SNPs including Gγ-XmnI, rs2297339, rs4895441, and rs9399137 of HBS1L-MYB were found to be associated with high Hb F levels in 39 (53.4%) subjects. The molecular basis of NTDT in the remaining 3 (4.1%) cases could not be defined. These results indicate multiple genetic factors in NTDT patients and underline the importance of complete genotyping to provide proper management, make clinical predictions, and improve genetic counseling.

Phenotype and Genotype in a Cohort of 312 Adult Patients with Nontransfusion-Dependent Thalassemia in Northeast Thailand.

Patients with nontransfusion-dependent thalassemia (NTDT) do not require regular blood transfusion for survival but may encounter several complications that contribute to morbidity and mortality. We report the molecular heterogeneity and hematological features of NTDT in 312 adult patients in northeast Thailand. Hemoglobin (Hb) and DNA analyses identified 177 subjects with Hb E-ß-thalassemia, 1 with homozygous ß0-thalassemia and 134 with Hb H, AEBart's and EEBart's diseases. For ß-thalassemia, 12 different mutations including both ß0- and ß+-thalassemias were detected. Coinheritance of α-thalassemia as an ameliorating factor was observed in 18 of 178 cases (10.1%) with ß-thalassemia. The α-globin gene triplicated haplotype (αααanti3.7) was observed in 1 case of Hb E-ß0-thalassemia. The presence of the -158 (Cx2192;T) Gx03B3;-XmnI polymorphism (+/+) was found to be associated with increased Hb F expression, but its frequency in the studied subjects was low. Those with α-thalassemia included 17 with deletional and 51 nondeletional Hb H, and 63 with AEBart's and 3 with EEBart's diseases. The hematological parameters of these NTDT and genotype-phenotype relationships are presented. The diverse molecular heterogeneity of NTDT underlines the importance of complete genotyping of the patient. These results should prove useful for management planning, the prediction of clinical outcome and to improve genetic counseling for NTDT patients.

Molecular Heterogeneity of Thalassemia among Pregnant Laotian Women.

BACKGROUND AND AIMS: A pilot screening program for thalassemia was initiated in the Lao People's Democratic Republic. This study aimed to describe the genotype diversity and hematologic features of thalassemia among the participating pregnant women. METHODS: Blood samples of 411 pregnant Laotian women were collected. Hemoglobin (Hb) profiles were determined using a capillary zone electrophoresis system. Mutations of α- and ß-globin genes were investigated using a polymerase chain reaction and related techniques. RESULTS: As many as 26 different thalassemia genotypes including non-transfusion-dependent thalassemia, i.e. Hb E-ß-thalassemia, Hb H, and EA Bart's diseases, were identified. A variety of phenotypic expressions of hematologic features and Hb profiles were observed, including an unusual phenotype of Hb E-ß0 thalassemia with 89.1% Hb E, 1.6% Hb F, and 9.3% Hb A2. CONCLUSIONS: The remarkable genotype-phenotype diversity indicates a need for careful laboratory interpretation in order to provide appropriate genetic counseling and care to the Laotian population.

Thalassemia Screening Using Different Automated Blood Cell Counters: Consideration of Appropriate Cutoff Values.

BACKGROUND: To evaluate the use of MCV and MCH cutoffs for thalassemia screening in areas with a high frequency of thalassemia and hemoglobinopathies using different hematology analyzers. METHODS: Blood samples of known α(0)-thalassemia and ß-thalassemia carriers were analyzed to establish the appropriate cutoffs for each analyzer. These selected cutoffs were validated prospectively for screening of α-thalassemia, ß-thalassemia in combination with the dichlorophenolindophenol test for hemoglobin E on another 288 Thai and 325 Laotian students. Genotypes were defined by standard hemoglobin and DNA analyses. RESULTS: The appropriate cutoffs for the Sysmex XS-800i and Sysmex XN-1000, Coulter LH 780, and Pentra ES-60 were found to be 78 fL for MCV and 25 pg for MCH. These were 82 fL and 25 pg for the Cell-Dyn Ruby. Further validation on Thai and Laotian students revealed 100% sensitivity and specificity of higher than 80% for all analyzers. CONCLUSIONS: While using MCV in screening requires establishment of appropriate cutoffs, MCH appears to be applicable to all analyzers. Each analyzer should be evaluated and appropriate cutoffs should be established before application in the field.

PILOT SCREENING PROGRAM FOR THALASSEMIA IN A COUNTRY WITH LIMITED RESOURCES: A COLLABORATION MODEL BETWEEN CLOSE NEIGHBORING COUNTRIES.

Screening for thalassemia carriers should not only be conducted in middle-income countries but also can be possible in low-middle income countries, through cooperation of experienced professionals from middle income countries. We describe a collaborating model between two close neighboring countries in establishing such a screening program for thalassemia. After training and setting up of facilities, a total of 152 out of 187 hospital staff were screened as a pilot activity to encourage community participation. Referring system for sending blood samples to a reference center in Thailand was also established. Among 152 health staff, 12.5% α0-thal, 2% ß-thal and 13% Hb E carriers were found. Applying thalassemia screening to 411 pregnant women and 71 spouses, 5 couples at risk of bearing a child of thalassemia disease were identified. The thalassemia screening program has a sensitivity of 99.5%, specificity of 77%, positive predictive value of 73%, and negative predictive value of 99.5%. Thus, it is possible to operate a thalassemia screening program with acceptable performance in a low-middle income country (Lao People's Democratic Republic) with the cooperation of a referral center located within close proximity in a middle income country (Thailand).

A large cohort of ß(+)-thalassemia in Thailand: molecular, hematological and diagnostic considerations.

We report the molecular and hematological characteristics associated with a large cohort of ß(+)-thalassemia in Thailand. Study was done on 21,068 unrelated subjects referred to our center in northeast Thailand for hemoglobinopathies investigation. Among 21,068 subjects, 2637 (12.5%) were found to carry ß-thalassemia. Of these 2637 cases, 705 (26.7%) carried ß(+)-thalassemia with eight different mutations including 6 promoter mutations; NT-28 (A-G), NT-31 (A-G), NT-50 (G-A), NT-86 (C-G), NT-87 (C-A) and NT-90 (C-T) and two missense mutations; Hb Malay (codon 19; AAC-AGC) and Hb Dhonburi (codon 126; GTG-GGG). Hematological features of carriers with these ß(+)-thalassemia (n=528) were compared with those with ß(0)-thalassemia (n=309). Data for Hb E-ß(+)-thalassemia (n=177) were also presented along with Hb E-ß(0)-thalassemia in our series (n=94). All patients with Hb E-ß(+)-thalassemia were associated with mild thalassemia intermedia phenotypes. Most of the ß(+)-thalassemia carriers had elevated Hb A2 and mild hypochromic microcytosis, some demonstrated borderline MCV and MCH values which, could compromise carrier screening. Analysis of α/ß-globin mRNA ratio in representative cases with normal, Hb E trait, ß(+)-thalassemia trait, Hb Dhonburi trait and ß(0)-thalassemia trait demonstrated the average values of 1.1, 1.7, 2.1, 1.7 and 3.1, respectively which is helpful in identification and differentiation of the cases.