Scanning for alpha-Hemoglobin Variants by High-Resolution Melting Analysis

作者：Yimniam Walai****; Jindadamrongwech Sumalee^*

来源：Journal of Clinical Laboratory Analysis, 2016, 30(5): 633-640.

DOI：10.1002/jcla.21914

• DNA sequencing
• hemoglobin typing
• hemoglobin variant
• high-resolution melting (HRM) analysis
• quantitative PCR

Background: Definitive detection of hemoglobin (Hb) variants requires DNA sequencing. High-resolution melting (HRM) analysis of polymerase chain reaction (PCR) amplicons was applied to detect and discriminate among uncommon alpha-Hb variants found in Thailand. Methods: Uncommon suspected a-Hb variants observed in Hb typing were identified by sequencing of DNA from whole blood samples. Three pairs of PCR primers covering the mutation regions in the three alpha-globin exons then were used for PCR coupled with difference in HRM analysis to subtract out the concomitant melting profile of the normal allele in the heterozygous state. Results: DNA sequencing identified six heterozygous alpha-Hb variants, namely, Hb G-Waimanalo (HBA2: exon 2, codon 64 G>A), Hb J-Buda (HBA1: exon 2, codon 61 G>T), Hb Kurosaki (HBA2: exon 1; codon 7 A>G), Hb O-Indonesia (HBA1: exon 3 codon 116 G>A), Hb Q-India (HBA1: exon 2, codon 64 G>C), and Hb Q-Thailand (HBA1: exon 2 codon 74 G>C). Difference HRM analysis showed one temperature melting profile using exon 1 primer pair, four different profiles with exon 2 primer pair, and one profile with exon 3 primer pair. Conclusions: PCR-HRM analysis was effective in detecting and discriminating among single point mutations causing six uncommon alpha-Hb variants in heterozygous individuals. The method can be applied for routine screening due to its simplicity and relatively low cost.

• 出版日期2016-9