Phenotypic expression of Hb F in common high Hb F determinants in Thailand: roles of α-thalassemia, 5' δ-globin BCL11A binding region and 3' ß-globin enhancer.

BACKGROUND: Deletions of δ- and ß-globin genes are associated with different Hb F levels. To address this, we have examined hematological and molecular characteristics in a large cohort of high Hb F determinants in Thailand. METHODS: A total of 160 unrelated adult subjects with heterozygous trait for high Hb F determinants and another 10 patients with compound heterozygous trait for Hb E were selectively recruited. Hematological parameters and Hb analysis were recorded, and α-thalassemia mutations were investigated. DNA deletions causing δß(0) -thalassemia and hereditary persistence of fetal hemoglobin (HPFH) were identified using multiplex PCR and denaturing high-performance liquid chromatography (HPLC) assays developed. RESULTS: Four different DNA deletions were detected including the 12.6 kb deletion δß(0) -thalassemia (n = 79), 79 kb deletion hereditary persistence of fetal Hb (HPFH)-6 (n = 65), Indian deletion-inversion (G) γ((A) γδß)-thalassemia (n = 15) and 78 kb deletion Chinese (G) γ((A) γδß)-thalassemia (n = 1). Eighteen cases were found to carry α-thalassemia with 10 different genotypes. All 10 patients who had similar hematological phenotype with that of Hb E-ß(0) -thalassemia were found to be compound Hb E-δß(0) -thalassemia. Differences in hematological features as well as Hb F levels were noted and are presented comparatively. CONCLUSION: Comparison of phenotypes, genotypes, and the deletion breakpoints of these Thai high Hb F determinants indicates that differences in Hb F expression are correlated with the existence of α-thalassemia, the loss of BCL11A binding region located 5' to the δ-globin gene and the 3' ß-globin enhancer, which confirms their important roles in fetal Hb expression.

Prenatal and post-natal screening of ß-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography.

We have developed methods based on PCR and denaturing high performance liquid chromatography (DHPLC) for rapid identifications of common ß-thalassemia mutations found in Thailand. The ß-globin gene was separately amplified by PCR on four different fragments covering eight most common ß-thalassemia mutations including nucleotide -28 A-G, codon 17 (A-T), IVSI-1 (G-T), IVSI-5 (G-C), codon 26 (G-A or Hb E), codons 41/42 (-TTCT), codons 71/72 (+A) and IVSII-654 (C-T). After PCR amplification, heteroduplex was generated by denaturation at 95 °C for 5 min followed by a slow reduction in temperature to 25 °C at 0.03 °C/s. Analysis of heteroduplex was done on an automated WAVE Nucleic Acid Fragment Analysis System. Specific DHPLC profile for each mutation was demonstrated which could be used in screening for all eight ß-thalassemia mutations. Further validation was done on 42 pre- and post-natal DNA samples which demonstrated 100 % accuracy as compared to the result obtained with conventional PCR assays. In a remaining case with an unknown mutation, a different DHPLC profile was noted on one of the amplified fragment. Further DNA sequencing of this fragment revealed a T-G transversion at the IVSI-116, a previously un-described mutation in Thai population. The DHPLC assay developed should prove useful for rapid screening of known and unknown ß-thalassemia mutations during carrier screening and pre-natal diagnosis which would facilitate an ongoing prevention and control program of thalassemia.

Complex interaction of Hb E [beta26(B8)Glu-->Lys], Hb Korle-Bu [beta73(E17)Asp-->Asn] and a deletional alpha-thalassemia-1 in pregnancy.

A pregnant Thai woman with mild hypochromic microcytic anemia caused by alpha- and beta- globin defects is described. The proband was a 26-year-old pregnant woman discovered through our ongoing thalassemia screening program. Initial hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis revealed a homozygosity for an unknown variant at the D window, inconsistent with results of family analyses. Further Hb analysis using automated capillary zone electrophoresis identified that the proband was in fact a compound heterozygote for Hb E [beta26(B8)Glu-->Lys, GAG>AAG] and another beta chain variant. DNA analysis demonstrated that she carried the Hb Korle-Bu mutation [beta73(E17)Asp-->Asn (GAT>AAT)] in trans to the Hb E and an alpha-thalassemia-1 (alpha-thal-1) with the Southeast Asian (- -(SEA)) deletion. Family studies identified that her father and sister were double heterozygotes for Hb Korle-Bu and alpha-thal-1, whereas her mother was a double heterozygote for Hb E/Hb Constant Spring [Hb CS; alpha142, Term-->Gln (TAA>CAA in alpha2)]. The genotype-phenotype relationship observed in this Thai family with complex hemoglobinopathies and methods for characterization are presented.

Hemoglobin profiles and hematologic features of thalassemic newborns: application to screening of alpha-thalassemia 1 and hemoglobin E.

CONTEXT: Thalassemia and hemoglobinopathies are major public health problems worldwide. To establish a cost-effective screening tool for newborns in regions where the incidence of these disorders is significant, study of the hemoglobin and hematologic features of normal and thalassemic newborns is necessary. OBJECTIVE: To study hemoglobin and hematologic characteristics of normal and various thalassemic newborns and to assess the effectiveness of simple screening methods for alpha-thalassemia 1 and hemoglobin E. DESIGN: Study was made of 402 cord blood specimens collected from unrelated Thai individuals. Hematologic parameters and hemoglobin profiles were determined. Thalassemia mutations were identified using polymerase chain reaction-related techniques. RESULTS: As many as 178 subjects (44.3%) were found to carry thalassemia genes with 18 different genotypes. All forms of alpha-thalassemia including double heterozygote for hemoglobin E and alpha-thalassemia showed significant reduction in hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin with increasing trend of red blood cell as compared with a non-alpha-thalassemic group. Although heterozygous hemoglobin E and beta-thalassemia showed no hematologic difference from nonthalassemic group, heterozygous alpha-thalassemia 1 including those with hemoglobin E showed significant increase in hemoglobin Bart level. CONCLUSIONS: Based on these findings, effective primary screening with 100% accuracy for alpha-thalassemia 1 and hemoglobin E in newborns in the region could be carried out using mean corpuscular volume less than 95 fL, mean corpuscular hemoglobin less than 30 pg, or hemoglobin Bart greater than 8.0% and hemoglobin E greater than 0.5%, respectively.

Prenatal diagnosis of Hb Bart's hydrops fetalis caused by a genetic compound heterozygosity for two different alpha-thalassemia determinants.

OBJECTIVE: To describe prenatal diagnosis of hemoglobin (Hb) Bart's hydrops fetalis caused by a previously undescribed condition in Thailand of the interaction of alpha(o)-thalassemia with the Southeast Asian (-(SEA)) and the THAI (-(THAI)) deletions in a Thai family. METHODS: Molecular and hematological features associated with alpha(o)-thalassemia carriers found in a pregnant Thai woman and her husband were investigated. Prenatal diagnosis was performed at her third pregnancy on amniotic fluid using a multiplex PCR for simultaneous detection of the (-(SEA)) and (-(THAI)) alpha(o)-thalassemia determinants. RESULTS: Initial DNA analysis for the common form of alpha(o)-thalassemia (-(SEA)) identified that the pregnant woman was a carrier whereas her husband was negative for this mutation. They had a healthy non-thalassemic daughter from the first pregnancy but the second pregnancy ended unexpectedly with Hb Bart's hydrops fetalis. Analysis of the archived DNA specimen of the husband demonstrated that he was in fact a carrier of the (-(THAI)) alpha(o)-thalassemia determinant. A successful application of a multiplex PCR for simultaneous detection of these two forms of alpha(o)-thalassemia in prenatal diagnosis at the third pregnancy was demonstrated and identified that the fetus was a carrier of the (-(SEA)) alpha(o)-thalassemia. CONCLUSION: Although relatively rare, the (-(THAI)) alpha(o)-thalassemia should be included in a routine screening to prevent a Hb Bart's hydrops fetalis syndrome. A simple DNA assay based on a multiplex PCR approach would therefore provide an effective means for alpha(o)-thalassemia screening in the region.