a-Thalassemia is one of the most serious genetically transmitted diseases creating health problems in many countries, with gene frequencies varying between 1% and 98% throughout the tropics and subtropics. More than 95% of recognized a-thalassemia involved deletion of 1 or both a-globin genes from chromosome 16p13.3 (1, 2). These gene deletions caused mild a-thalassemia-2 and severe thalassemia-1, respectively. The most common type of thalassemia-1 in the Asian population is the Southeast Asian type (SEA) (3). Even thought carriers of the a-thalassemia-1 with SEA type do not manifest any clinical symptoms, couples who are both carriers have a 25% chance of conceiving a homozygous fetus, which manifests as Bart's hydrops fetalis, the most severe thalassemic syndrome. All of these fetuses die either in utero or soon after birth (4-6). In addition, approximately 75% of mothers carrying fetuses with homozygous for the a-thalassemia-1 SEA type will develop toxemia of pregnancy (7). An investigation of a-thalassemia-1 SEA type is therefore essential for carrier couples and for prenatal diagnosis of fetus conceived by couples who are both carriers of this type of gene deletion.The gap-PCR analysis currently used to diagnose athalassemia-1 SEA type is based on multiplex amplification at the breakpoint area of thalassemia-1 with SEA type and the wide type a-globin gene allele. The technique requires labor intensive, time-consuming, and post-PCR processing steps (8). In an effort to develop a more straightforward diagnostic test, quantitative realtime PCR with specific probes (9, 10) has been used for detection of a-thalassemia-1 SEA type. Probe-based assays are generally used for multiplex real-time PCR analysis. However, they are relatively expensive. Realtime PCR with SYBR Green1 followed by melting curve analysis has also been use to enhance the speed Abstract a-Thalassemia-1 Southeast Asian (SEA) type is the most common genetic disorder in the Asian population. Couples who are both carriers have a 25% chance of conceiving Bart's hydrops fetalis. Therefore, results from carrier screening and prenatal diagnosis frequently need to be available rapidly. A rapid technique for diagnosis of a-thalassemia-1 SEA type was implemented. The technique used is based on real-time gap-PCR and high resolution melting (HRM) analysis of the amplified fragment using the Rotor-Gene 6000Ô. The DNA samples used for amplification were obtained from whole blood, cord blood, and chorionic villus sampling (CVS). With this method, the a-thalassemia-1 SEA allele can be easily distinguished from wild type a-globin gene allele. The real-time gap-PCR and HRM analysis offers additional benefits including minimal labor, rapid turnaround time, and a decreased risk of PCR carryover contamination. It is cost-effective and safe, does not require fluorescently labeled probe and hazardous chemicals. Moreover, it is accurate showing 100% concordance with conventional gap-PCR analysis.

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Mutations in the KLF1 gene, which encodes a transcription factor playing a role in erythropoiesis, have recently been demonstrated to be a rare cause of hereditary haemolytic anaemia. We described the genotypic and phenotypic spectra of four unrelated families with compound heterozygous class 2/class 3 KLF1 mutations. All patients had p.G176RfsX179 on one allele and either p.A298P, p.R301H or p.G335R on the other allele. All presented on the first day of life with severe haemolytic anaemia with abnormal red blood cell morphology, markedly increased nucleated red blood cells and hyperbilirubinaemia. Three patients later became transfusion-dependent. All parents with heterozygous KLF1 mutation without co-inherited thalassaemia had normal to borderline mean corpuscular volume (MCV) and normal to slightly elevated Hb F. Fifteen previously reported cases of biallelic KLF1 mutations were identified from a literature review. All except one presented with severe haemolytic anaemia in the neonatal period. Our finding substantiates that compound heterozygous KLF1 mutations are associated with severe neonatal haemolytic anaemia and expands the haematologic phenotypic spectrum. In carriers, the previously suggested findings of low MCV, high Hb A 2 and high Hb F are inconsistent; thus this necessitates molecular studies for the identification of carriers.

High-resolution melting (HRM) analysis is a rapid mutation analysis which assesses the pattern of reduction of fluorescence signal after subjecting the amplified PCR product with saturated fluorescence dye to an increasing temperature. We used HRM analysis for prenatal diagnosis of beta-thalassemia disease in northern Thailand. Five PCR-HRM protocols were used to detect point mutations in five different segments of the beta-globin gene, and one protocol to detect the 3.4 kb beta-globin deletion. We sought to characterize the mutations in carriers and to enable prenatal diagnosis in 126 couples at risk of having a fetus with beta-thalassemia disease. The protocols identified 18 common mutations causing beta-thalassemia, including the rare codon 132 (A-T) mutation. Each mutation showed a specific HRM pattern and all results were in concordance with those from direct DNA sequencing or gap-PCR methods. In cases of beta-thalassemia disease resulting from homozygosity for a mutation or compound heterozygosity for two mutations on the same amplified segment, the HRM patterns were different to those of a single mutation and were specific for each combination. HRM analysis is a simple and useful method for mutation identification in beta-thalassemia carriers and prenatal diagnosis of beta-thalassemia in northern Thailand.

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