Analysis of the Xmn1‐Gγ polymorphism in β‐thalassemia/hemoglobin E (HbE) and homozygous HbE patients with low and high levels of HbF

Sir, b-Thalassemia is an inherited disease characterized by defects in the rate of b-globin production due to a point mutation or rare gene deletion in the b-globin gene cluster and is associated with increased levels of hemoglobin (Hb) A2 and slight increase in HbF [1]. One of the most common point mutations in the b-globin gene is known as hemoglobin E [HbE, b26 (B8) Glu?Lys, GAG>AAG] [2]. Although the HbE trait has the phenotype of a mild form of b-thalassemia, the combination of b-thalassemia and HbE leads to b-thalassemia/HbE condition, which has a remarkable variability in the phenotype, ranging from thalassemia intermedia to transfusion-dependent thalassemia major [3]. The previous study has indicated that the 158 (C?T) polymorphism of c-globin gene (Xmn1-c polymorphism) is associated with increased production of HbF, which ameliorates the disease severity in b-thalassemia/HbE patients [4]. However, correlations between the Xmn1-c polymorphism, HbF levels, and hematological parameters in b-thalassemia/HbE and homozygous HbE patients with low or high levels of HbF are still unclear. Therefore, in this study, we analyzed the frequency of the Xmn1-c polymorphism and its correlation with HbF and various hematological parameters in these patient groups. The EDTA blood samples were submitted to the Associated Medical Sciences Clinical Service Center, Chiang Mai University, Chiang Mai, Thailand, for thalassemia diagnosis. Hematological parameters were measured with an automated blood counter (Sysmex KX-21; Sysmex Corporation, Kobe, Japan). Quantitation of HbA2 (for detection of b-thalassemia) and identification of hemoglobinopathies, including HbE, were performed using high-performance liquid chromatography (HPLC, VARIANT II, b-thalassemia Short Program; Bio-Rad Laboratories, Hercules, CA, USA). Samples containing HbE >65% and HbF levels varying from 5 to 15% were further discriminated for b-thalassemia/HbE and homozygous HbE by molecular analysis systems. Genomic DNA was extracted from EDTA blood samples using the NucleoSpin kit (Macherey-Nagel, KG, Duren, Germany). The b-thalassemia codons 71/72 (+A), 41/42 ( TCTT), 17 (A>T), and IVSI-nt1 (G>T) mutations were analyzed by the multiplex amplification refractory mutation system (MARMS)-PCR according to the protocol described previously [5]. The amplification refractory mutation system (ARMS)-PCR was performed to provide a rapid molecular diagnosis of HbE as previously described [6]. The Xmn1-c polymorphism analysis was performed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as described by Nemati et al. [7]. In general, the levels of HbA2/E and HbF in b-thalassemia/HbE disease, analyzed by HPLC, were 30–60% and ≥15%, respectively, while those in homozygous HbE were >65% and <5%, respectively [6]. Thus, based on the molecular analysis and the combination with HbF levels, the patients could be divided into 4 groups including 18 and 40 b-thalassemia/HbE patients with HbF <15% and ≥15%, respectively, and 32 and 27 homozygous HbE patients with HbF ≤5% and >5%, respectively (Table 1). The b-thalassemia/HbE patients with HbF ≥15% had significantly lower red blood cell (RBC) counts and significantly higher mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) than those with HbF levels <15%. However, the levels of Hb, hematocrit (Hct), and mean corpuscular hemoglobin concentration (MCHC) between the two groups were not significantly different (Table 1). There were no significant differences in levels of assessed hematological parameters between the groups of homozygous HbE patients with HbF ≤5% and >5%. Furthermore, mean levels of HbF and HbA2/E between each group of patients were significantly different (Table 1). A significant negative correlation of HbF levels with RBC counts (r = 0.564, P < 0.001) and a significant positive correlation of those with MCV (r = 0.598, P < 0.001) and MCH (r = 0.644, P < 0.001) were observed in the group of b-thalassemia/HbE disease, while the group of homozygous HbE patients had a significant negative correlations of HbF levels with RBC counts (r = 0.387, P < 0.002) and a significant positive correlation with MCH (r = 0.288, P = 0.03).