Association of Hb Q-Thailand with homozygous Hb E and heterozygous Hb Constant Spring in pregnancy

Hemoglobin (Hb) Q-Thailand [alpha74(EF3): Asp-->His] is an abnormal Hb found mainly in China and South-east Asian countries. Association of the alpha(Q-Thailand) allele with alpha-thalassemia has important implications in diagnosis. We report the hitherto undescribed conditions of this variant in two unrelated pregnant Thai women. Routine Hb analyses using high-performance liquid chromatography identified abnormal Hb migrating after Hb A(2) in addition to a homozygous Hb E in the proband 1 and to a heterozygous Hb Constant Spring (Hb CS) in the proband 2. Further alpha-globin gene analysis identified that the variant was caused by the GAC to CAC mutation at codon 74 of the alpha1-globin gene corresponding to the Hb Q-Thailand, detected in cis to the 4.2 kb deletional alpha-thalassemia 2 in both cases. Interaction of the alpha(Q-Thailand) with the beta(E) globin chains in the proband 1 leads to a Hb variant, namely the Hb QE. Family study of the proband 1 showed that her non-pregnant sister had the same genotype but her father was a double heterozygote for Hb E and Hb Q-Thailand in whom both Hb Q-Thailand and Hb QE were detected. Genotype-phenotype relationships observed in these families with complex hemoglobinopathies are presented and compared with those of simple homozygote for Hb E, heterozygote for Hb CS and heterozygote for Hb Q-Thailand found in other unrelated subjects. A simple DNA assay based on allele-specific polymerase chain reaction for simultaneous detection of the Hb Q-Thailand mutation and the 4.2 kb deletional alpha-thalassemia 2 determinant was developed and validated.     

Molecular characterization of thalassemia intermedia associated with HPFH-6/beta-thalassemia and HPFH-6/Hb E in Thai patients

We report the molecular and hematological characterizations of thalassemia caused by interactions of the hereditary persistence of fetal hemoglobin (HPFH)-6 with beta-thalassemia in 2 Thai patients and the HPFH-6 with Hb E in another Thai patient. Marked hypochromic microcytosis, characteristics of thalassemia intermedia, were obvious in the former 2 cases but the latter had much milder clinical phenotype with normal Hb and a slightly reduced mean corpuscular volume (MCV) value. Hb analysis revealed no Hb A but Hb A(2)F patterns in the compound HPFH-6/beta-thalassemia patients and the EF pattern in the HPFH-6/Hb E patient. The (G)gamma-globin chain predominated in all cases. Globin gene analyses demonstrated that all patients carried the 101-kb HPFH-6 deletion in trans to the beta-thalassemia genes with the IVS1#5 G-C mutation and the G insertion between codons 8/9 and the beta(E)-gene, respectively. Hematologic data of the patients were compared to those of the HPFH-6 heterozygotes found in their family members and different genotype-phenotype interactions of this HPFH determinant in these Thai patients are illustrated.     

Hb Paksé [(alpha2) codon 142 (TAA-->TAT or Term-->Tyr)J in Thai patients with EAbart's disease and Hb H Disease

Hb Paksé is caused by an alpha2-globin gene termination codon mutation, TAA-->TAT or Term-->Tyr, initially described in a Laotian family. We now report for the first time that the same mutation has been found in 14 Thai patients, seven with EABart's disease, four with Hb H disease, and three with alpha-thalassemia trait who were initially diagnosed as having Hb Constant Spring (Hb CS; alpha2-globin gene termination codon mutation TAA-->CAA or Term-->Gln). Co-inheritance of this mutation with alpha-thalassemia-1 (SEA type) leads to Hb H disease (hereafter designated as Hb H-Paksé disease) and to a complex thalassemia syndrome, namely EABart's-Paksé disease. Hematological data of these patients were compared with those of classical Hb H-CS and the EABart's patients. To facilitate epidemiological and diagnostic screening of Hb Paksé, a simple assay procedure based on allele specific polymerase chain reaction (PCR) amplifications was developed and validated. Using this allele specific PCR as a screening method, five additional individuals with Hb Paksé were found among 71 Thai subjects previously thought to have Hb CS.     

Multiplex allele-specific PCR assay for differential diagnosis of Hb S, Hb D-Punjab and Hb Tak

BACKGROUND: Apart from hemoglobin (Hb) E, Hb D-Punjab [beta121(GH4)Glu-Gln] and Hb Tak [beta147Term-Thr] are the two most common beta-chain variants among the Asian population. These two Hb variants have similar alkaline electrophoretic mobilities and HPLC profiles as those of the Hb S [beta6(A3)Glu-Val]. Differential diagnosis of these clinically relevant hemoglobinopathies is therefore problematic. Direct detection of the beta-globin gene mutations would be another diagnostic alternative. METHODS: A simultaneous DNA diagnosis of the three Hb variants was developed based on the multiplex allele-specific polymerase chain reaction (PCR) approach. The method was validated on 10 carriers of Hb D-Punjab, 5 carriers of Hb Tak, 2 carriers of Hb S and 50 normal individuals of Thai origin. RESULTS: The three abnormal Hbs could be correctly diagnosed with the simultaneous PCR approach, and a complete concordance with results using other established methods was obtained. CONCLUSIONS: The multiplex allele-specific PCR approach developed should prove useful in complementing routine Hb analysis for differential diagnosis of these three common Hb variants and should facilitate a program of hemoglobinopathy screening in the region.     

A simplified screening strategy for thalassaemia and haemoglobin E in rural communities in south-east Asia

OBJECTIVE: To evaluate a simple screening strategy for thalassaemia and haemoglobin (Hb) E in a prevention and control programme for thalassaemia in rural communities with limited resources. METHODS: Blood samples from 301 Thai-Khmer participants were screened for thalassaemia and Hb E using a combined modified one-tube osmotic fragility (OF) test and a modified dichlorophenolindophenol (DCIP) precipitation test. Results were evaluated with standard haematological analyses including erythrocyte indices, Hb typing and quantification and polymerase chain reaction (PCR) analysis of alpha-globin and beta-globin genes. FINDINGS: Participants were divided into four groups according to the results of the combined tests. Altogether, 104 of 301 participants (34.6%) had negative results on both tests; 48 (15.9%) were positive on the OF test but not the DCIP test; 40 (13.3%) were negative on the OF test but positive on DCIP test; and 109 (36.2%) were positive on both tests. No carrier of clinically significant forms of thalassaemia (alpha(o)-thalassaemia, beta-thalassaemia) or Hb E was found among the group that had negative results for both tests. All participants with Hb E had positive DCIP tests. Carriers of alpha+-thalassaemia or Hb Constant Spring could generate either positive or negative OF test results but they all had negative DCIP tests. Using both tests as a preliminary screening for the three important groups of carriers gave a sensitivity of 100% and a specificity of 69.8%. The positive predictive value of the combined test was 77.2%. The negative predictive value was 100%. Further evaluation of the screening system by local staff at three community hospitals found a sensitivity of 98.1-100% and a specificity of 65.4-88.4% with positive predictive values of 75.0-86.9% and negative predictive values of 98.1-100%. CONCLUSION: A combined test using OF and DCIP could be used as an effective preliminary screening alternative to an electronic blood cell count for identifying carriers with alpha(o)-thalassaemia, beta-thalassaemia and Hb E. The strategy should prove useful for population screening in prevention and control programmes in rural communities in south-east Asia where laboratory facilities and economic resources are limited.     

Prenatal detection of fetal hemoglobin E gene from maternal plasma

In order to provide a noninvasive prenatal diagnosis of the hemoglobin E (Hb E) related disorder, we have evaluated the possibility of identifying the fetal beta(E)-globin gene in maternal plasma. The analysis was performed during 8 to 18 weeks of gestation using DNA extracted from 200 micro L of plasma from pregnant women whose husbands carried Hb E. The beta(E)-globin mutation in maternal plasma was detected by a nested PCR amplification followed by the Mnl I restriction analysis. The result was compared with that of routine analysis of the CVS specimens. Among the five pregnant women examined, the fetal beta(E)-globin gene was identified in maternal plasma in three of them and the result was completely concordant with the conventional CVS analysis. This simple noninvasive prenatal detection of the fetal beta(E)-globin gene should prove useful in a prevention and control program of Hb E/beta-thalassemia in countries where the beta(E)-globin gene is prevalent.     

Rapid molecular characterization of Hb Queens and Hb Siam: two variants easily misidentified as sickle Hb

OBJECTIVE: To establish a rapid differential diagnosis of hemoglobin (Hb) Queens and Hb Siam from other clinically relevant variants. DESIGN AND METHODS: Molecular and hematological features associated with two pregnant Thai women who were mistaken for Hb S were investigated. A simultaneous DNA diagnosis based on multiplex allele specific PCR approach was developed and tested with other common variants. RESULTS: Apart from mild anemia, the two subjects were generally healthy. DNA analysis identified that they were respectively carriers of Hb Siam [alpha15(A13)Gly-Arg] and Hb Queens [alpha34(B15)Leu-Arg]. A successful application of the multiplex allele specific PCR for differential diagnosis was demonstrated. CONCLUSION: Diagnosis of these clinically relevant hemoglobinopathies is problematic in the routine setting, and the method developed should prove useful in complementing routine Hb analysis for providing accurate diagnosis.     

Interactions of hemoglobin Lepore (δβ hybrid hemoglobin) with various hemoglobinopathies: A molecular and hematological characteristics and differential diagnosis

Hemoglobin (Hb) Lepore is a variant consisting of two α-globin and two δβ-globin chains. In heterozygote, it is associated with clinical findings of thalassemia minor but interactions with other hemoglobinopathies can lead to various clinical phenotypes. Using a combination of Hb-HPLC, Hb-capillary electrophoresis and DNA analyses, we have identified 14 patients with Hb Lepore–Hollandia including eight heterozygotes, two double heterozygotes with α⁺-thalassemia, two compound heterozygotes with Hb E (initially diagnosed as Hb E-β-thalassemia) and two previously undescribed conditions of double heterozygote for Hb Lepore/Hb Constant Spring and Hb Lepore/α⁰-thalassemia, both associated with higher levels of Hb F and lower levels of Hb Lepore. Hematological and molecular features of these patients are presented along with those observed in four other Thai individuals encountered with heterozygous Hb Lepore–Washington–Boston. Haplotype analysis of the β-globin gene cluster showed that all Hb Lepore–Hollandia genes were associated with a single haplotype not described previously in other populations, (? + ? + + ? +) whereas the four Hb Lepore–Washington–Boston genes were associated with haplotypes (+ ? ? ? ? + ?/+) (N = 1) and (+ ? ? ? ? ? +) (N = 3), data indicating multiple origins of these two variants. Hb Lepore may not be uncommon in the Thai and other Asian populations and both hematological and molecular studies are required for accurate diagnosis. To facilitate rapid epidemiological, diagnostic screening and differentiation of the two Hb Lepore defects, a simple assay based on multiplex PCR has been developed.