Molecular confirmatory testing of hemoglobin Constant Spring by real‐time polymerase chain reaction SYBR Green1 with high‐resolution melting analysis

To the Editor: Hemoglobin Constant Spring (Hb CS) is the most prevalent non-deletional a-thalassemia in the southeast Asian population. This abnormal hemoglobin results from a point mutation at the stop codon of a2-globin gene (TAA fi CAA), which leads to the addition of 31 amino acids to normal a-globin sequence (1, 2). The Hb CS mRNA is unstable and therefore the rate of a-globin chain synthesis is decreased (3). Although the heterozygote of Hb CS is clinically and hematologically normal, the homozygote shows a clinical picture as thalassemia intermedia with mild anemia, jaundice and hepatosplenomegaly. Moreover, the interaction of Hb CS with a-thalassemia leads to Hb H disease () ⁄ aa), which tends to be more severe than Hb H disease caused by a triple a gene deletion () ⁄ -a) (4). Hb CS diagnosis is often missed by routine laboratory testing, especially in the heterozygote as the Hb CS is unstable and presented at a low level in peripheral blood (5, 6). The present study used the real-time polymerase chain reaction (PCR) with SYBR Green1 and high-resolution melting (HRM) analysis for molecular confirmatory testing of Hb CS. Blood samples were collected from three patients who were diagnosed as heterozygote of Hb CS by using capillary electrophoresis (CAPILLARYS 2; Sebia, Norcross, GA, USA) together with clinical and hematological parameters as shown in Table 1. However, the hemoglobin analysis by high-performance liquid chromatography (HPLC; BIO-RAD, Variant b-thalassemia short program, Hercules, CA, France) of three blood samples showed no peak of Hb CS. The DNA was extracted from 200 lL whole blood sample using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). The amplification was carried out in a reaction volume of 25 lL containing 12.5 lL of 2· SYBR Green1 PCR master mix (QuantiTect SYBR Green PCR Kits; Qiagen), 0.1 lm of internal control forward primer (aG17; 5¢-AGATGGCGCCTTCCTCTCAGG-3¢), 1.2 lm of Hb CS forward primer (aG2; 5¢-GCTGACCTC CAAATACCGTC-3¢) and common reward primer (C3; 5¢-CCATTGTTGGCACATTCCGG-3¢), 0.6 m betaine, 5% DMSO and 5 lL of DNA sample. The multiplex SYBR Green1-PCR was performed on Rotor-Gene 6000 (Corbett Research, Mortlake, NSW, Australia). The mixture was preheated at 95 C for 5 min and then the PCR was cycled 40 times at 94 C for 1 min and 65 C for 1 min 30 s. The amplification cycles were then followed by a HRM cycle from 80 to 95 C at a rate of 0.1 C per 2 s. The DNA amplification fragments of internal control and Hb CS were 391 and 180, respectively, as a previous study reported (7). The amplified internal control fragment had a specific peak height at melting temperature (Tm) of 86 ± 1 C, whereas the amplified fragments of Hb CS had a specific peak height at Tm of 83 ± 1 C (Fig. 1). Both peaks were observed in all three blood samples in which Hb CS could be detected by the capillary electrophoresis method. One DNA sample of three heterozygotes of Hb CS showed a lower peak was because of a lower copy number of the target sequence than the other two. However, when analyzing separately by using the auto analysis program, the two-specific peak heights were clearly observed in this sample (data not shown). Only a specific peak of amplified internal control fragment was found in the blood sample of a normal individual (Fig. 1). This technique was tested further with five DNA samples of normal individuals and five from heterozygotes of Hb CS, and we found that the similar oneand two-specific peak