Molecular characterization of thalassemia intermedia with homozygous Hb Malay and Hb Malay/HbE in Thai patients.

Patients with thalassemia intermedia have a mild anemia and survive without needing regular blood transfusions.1 Here we report two cases of Thai patients with β-thalassemia intermedia caused by homozygosity of hemoglobin Malay (Hb Malay; α2β219Asn-Ser)2 and compound heterozygosity of Hb Malay and hemoglobin E (Hb E; α2β226Glu-Lys). Both patients presented with a history of anemia with marked microcytosis and hypochromia for years. They had normal growth and development and were not blood transfusion-dependent. The results of other blood examinations are listed in Table 1. DNA analysis with polymerase chain reaction3 detected homozygosity for the β19; AAC-AGC mutation in patient #1 and a compound β19; AAC-AGC and β26; GAG-AAG for Hb E in patient #2. This β19 mutation leads to a substitution of serine for asparagine at codon 19 of the β-globin chain known previously as Hb Malay.2 The mutation increases the homology of a cryptic splice site in exon 1 of the β-globin gene to the donor splice site that leads to a β+-thalassemia phenotype.4 No α–globin gene deletion causing α-thalassemia 1 or α-thalassemia 2 was found in either case. These findings confirm that, unlike the compound Hb Malay /βO-thalassemias which are associated with severe thalassemia symptoms,5,6 homozygosity for Hb Malay and compound Hb Malay/Hb E produces features of thalassemia intermedia. Hb Malay accounts for 15% of Malaysian β-thalassemia genes and 16% of those in Southern Thailand.2,3,5 It has also been found among Indonesians7 and Chinese.8 Identification of Thai βMalay genes on the Malaysian haplotype; (+ + + + )2 (Table 1) indicates that Hb Malay appears to have originated only once in the Asian population. Since Hb Malay is indistinguishable from Hb A on routine hemoglobin electrophoresis and chromatography, it is usually identified as Hb A. It is conceivable that the incidence data observed could be underestimated and Hb Malay needs to be included in the differential diagnosis for patients of Asian descent with β-thalassemia major or intermedia. In order to provide a rapid method of diagnosing Hb Malay, we applied the allele-specific PCR shown in Figure 1. The βMalay specific primer, G28R (5’ ACC ACC AAC TTC ATC CAC GC 3’) and the βA specific primer, G27 (5’ GCC CTG TGG GGC AAG GTG AA 3’) were used with primers S1 (5’ TGT CAT CAC TTA GAC CTC AC 3’) and S3 (5’ T CCC ATA GAC TCA CCC TGA A 3’) to produce the 230 bp βMalay specific and the 420 bp normal specific fragments, respectively. In each reaction tube, as an internal control, two additional primers with the sequences (5’ G GCC TAA AAC CAC AGA GAG T 3’) and (5’ C CAG AAG CGA GTG TGT GGA A 3’) for amplification of the 578 bp Gγ–globin gene promotor fragment10 were also included. The allele-specific PCR reaction mixture (50 mL) contains 0.1 mg DNA, 15 pmol of each primer, 200 mM dNTPs and 1 unit of Taq DNA polymerase (Promega Co., USA) in 10 mM TrisHCl ( pH 8.3), 50 mM KCl, 0.01% gelatin and 3.0 mM MgCl2. The amplification reactions were carried out in a DNA Thermal Cycler 480 ( Perkin-Elmer Cetus Co., USA). After initial heating at 94°C for 3 min, 30 cycles were performed under the following PCR conditions: 94°C for 1 min and 68°C for 1.5 min. The amplified product was analyzed by 1.5% agarose gel electrophoresis and Table 1. Results of hematologic and globin genes analyses of the two Thai patients with β-thalassemia intermedia.