Beta-globin gene haplotypes in some minor ethnic groups in Thailand.

In order to provide population genetic data of various ethnic groups in Thailand, we have determined the type of hemoglobin by electrophoresis and the beta-globin gene haplotypes by PCR followed by restriction digestion in five small ethnic groups namely hill tribes, PhuTai, Chong, Lao Song and Sakai inhabiting in the north, northeast, east, central and south of Thailand, respectively. In each group, in addition to HbA and HbA2, the HbE, the most common hemoglobinopathy in Southeast Asia was detected at 2.5%, 51.6%, 84.0%, 8.6% and 11.8%, respectively. Haplotype analysis demonstrated that in all groups the beta A-globin gene was associated with various haplotypes and beta-globin gene frameworks. However, beta E -globin gene was associated with haplotypes ((-)+(-)+ + +(-)) and ((+)-(-)-(-)+(-)) on the beta-globin gene framework 2 in all ethnic groups except in Chong people whose the beta E-globin gene was mostly linked to haplotype ((-)+(-)++(-)+) and beta-globin gene framework 3 which was commonly found among Cambodian. It appears therefore that the Chong population is more related to Cambodian than Thai.

Molecular and hematological characterization of HbE heterozygote with alpha-thalassemia determinant.

Hemoglobin E and alpha-thalassemia are prevalent in Thailand. The chance that an individual heterozygous for HbE also carries an alpha-thalassemia determinant is high. In this individual, the amount of HbE and other hematological parameters may be differed from that of usual observation. In this study, a total of 132 HbE heterozygotes were screened for alpha-thalassemia 1 gene deletion by the polymerase chain reaction. Out of 132 cases, 71 could be completely analyzed for hematologic parameters. Forty-three of 88 cases with HbE less than 25% as measured using microcolumn chromatography were positive for this gene deletion. In twenty of these 43 alpha-thalassemia 1 positive cases, the average values of Hb, Hct, MCV, MCH, MCHC, RDW and HbE were 10.6 g/ dl, 33.1%, 64.8 fl, 21.0 pg, 32.3 pg/dl, 18.6% and 17.4%, respectively. Eight of 9 alpha-thalassemia 1 negative cases were positive for alpha-thalassemia 2 gene deletion in Southern blot analysis. In this later group, hematological parameters were similar to that of the former. Co-inheritance of the Hb Constant Spring gene has no direct effect on the level of HbE. No alpha-thalassemia 1 gene was detected in the remaining 34 cases whose HbE were above 25%. The average amount of Hb, Hct, MCV, MCH, MCHC, RDW and HbE were 12.4 g/dl, 37.7%, 79.7 fl, 26.2 pg, 32.7 pg/dl, 25.8% and 28.5%, respectively. Therefore, screening for HbE level below 25% may be a convenient way of identifying parents of carrying alpha-thalassemia 1 determinant.

Molecular and hematological characterization of Hb Tak and Hb Pyrgos in Thailand.

Two hemoglobin variants that migrate abnormally on gel electrophoresis were found in four unrelated Thai individuals. One variant that migrate faster than HbA but more slowly than Hb Bart's was detected in two heterozygotes. Another abnormal Hb migrating between HbA2 and HbF was found in one heterozygote and one compound heterozygote with HbE. In all cases, no microcytic anemia was observed. PCR amplification and direct DNA sequencing established that the first variant was caused by a missense mutation at codon 83 (GGC-GAC) that leads to Gly to Asp substitution previously described as the Hb Pyrgos in a Greek boy. The second variant was caused by an AC insertion at the termination codon that leads to synthesis of elongated beta-globin chain known as the Hb Tak. Beta globin gene haplotype analysis demonstrated that each variant was found on the same chromosome background in Thai individuals. The simple non-radioactive DNA assays based on allele specific polymerase chain reaction for the detection of these two Hb mutations in a routine laboratory are described.

A simple non radioactive method for detecting beta-thalassemia/hbe disease: application to prenatal diagnosis.

We have developed allele specific polymerase chain reaction (ASPCR) that allows rapid screening of the beta E-globin and common beta-thalassemia genes in Thailand. These non-radioactive methods are based on the amplification by the polymerase chain reaction of the beta E and beta-thalassemia specific DNA fragments using specific primers. With this approach, both heterozygote and homozygote for the disease could readily be identified on agarose gel electrophoresis of the amplified DNA. We have applied the method for a prenatal diagnosis of beta-thalassemia/HbE disease in a Thai family at the second trimester of pregnancy. The result obtained was comparable to that of conventional dot blot hybridization using radioactive probes. The simplicity, accuracy and non isotopic of the approach make it a highly promising method for a carrier screening and a prenatal diagnosis of this common disorder.

Expression of hemoglobin E in newborn.

Hemoglobin E(HbE) is an abnormal hemoglobin resulted from a point mutation in codon 26 (GAG-AAG) of beta-globin gene. The mutation causes an amino acid substitution (Glu-Lys) and abnormal splicing in exon 1 that reduce the amount of beta E chain synthesis. While in adult, the HbE can easily be diagnosed, its level in newborn is usually underrepresent. In this study, we examined a relationship between genotype of HbE and the amount of HbE in cord blood. 145 Cord blood specimens were analysed by starch gel electrophoresis and the amounts of HbE were determined by microcolumn chromatography. The zygosity of beta E globin gene was determined by the polymerase chain reaction. The levels of HbE were 3.17 +/- 1.79% for 59 heterozygotes, 8.55 +/- 2.52% for 3 homozygotes and 0.48 +/- 0.22% in 83 normal newborns. This result provides useful data for a neonatal screening program and implies that expression of HbE in newborn dependent on a copy number of beta E globin gene in each individual.

Molecular basis of alpha (0)-thalassemia in northeast of Thailand.

The molecular basis of alpha(0) thalassemia were studied in 95 patients who attended at Srinagarind Hospital, Khon Kaen University during September 1993-January 1994. From these 95 patients, hemoglobin electrophoresis indicated that there were 14 cases with A2AH, 21 cases with A2ABart'sH, 6 cases with ConSpA2AH, 31 cases with ConSpA2ABart'sH, 6 cases with EABart's, 3 case with EFBart's, 4 cases with ConSpEABart's, 5 cases with Portland Bart's and 5 cases with A2A. White blood cell lysate was prepared from peripheral blood leukocytes of these patients and was subjected to the polymerase chain reactions for detection of the SEA type alpha thalassemia gene deletion. Altogether 100 chromosomes with the SEA deletion were detected from all patients examined, the result indicated that this type of alpha(0) thalassemia gene deletion is the most common among these Thai population. These data will be useful for a carrier screening and a prenatal diagnosis program of homozygous alpha (0) thalassemia which is a lethal condition in northeast of Thailand.

Molecular heterogeneity of beta-thalassemia in Thailand.

beta-Globin genes in 294 chromosomes of beta-thalassemia homozygotes and patients of beta-thalassemia/HbE in the northeast, the middle and the south of Thailand were analyzed by the PCR related techniques: dot blot hybridization, direct restriction assay, direct cloning and direct sequencing of the amplified DNA fragments. Twelve different mutations were detected at various frequencies. They are an A-G at-28, codon 19 (AAC-AGC), a G-T at IVS-1 nt1,a G-C at IVS-1 nt5, a C-T at IVS-2 nt654, a G addition in codons 8/9, a C deletion in codon 41, a 4 bp deletion in codons 41/42, an A addition in codons 71/72, an AAG-TAG in codon 17, a CAG-TAG in codon 26, a TAC-TAA in codon 35 and a 8 bp deletion in codons 123-125. We also developed allele specific-polymerase chain reaction to facilitate non-radioactive detection of the mutation. Origins and spread of mutations are speculated based on the results of determination of haplotypes and frameworks that are linked to the thalassemia alleles.