Comparison of the mismatch-specific endonuclease method and denaturing high-performance liquid chromatography for the identification of HBB gene mutations

Abstract

Background: Beta-thalassemia is a common autosomal recessive hereditary disease in the Meditertanean, Asia and African areas. Over 600 mutations have been described in the beta-globin (HBB), of which more than 200 are associated with a beta-thalassemia phenotype.

Results: We used two highly-specific mutation screening methods, mismatch-specific endonuclease and denaturing high-performance liquid chromatography, to identify mutations in the HBB gene. The sensitivity and specificity of these two methods were compared. We successfully distinguished mutations in the HBB gene by the mismatch-specific endonuclease method without need for further assay. This technique had 100% sensitivity and specificity for the study sample.

Conclusion: Compared to the DHPLC approach, the mismatch-specific endonuclease method allows mutational screening of a large number of samples because of its speed, sensitivity and adaptability to semi-automated systems. These findings demonstrate the feasibility of using the mismatch-specific endonuclease method as a tool for mutation screening.

Figure 1

Principles of the mismatch-specific endonuclease cleavage of the heteroduplex formation using primers labeled with two different fluorescent dyes.

Figure 2

Capillary electrophoresis analysis of HBB genes for PCR products cleaved by mismatch-specific endonuclease fromA) an individual with a genotype of c.-78 A>G/WT, B) an individual with a genotype of c.2 T>G/WT, C) an individual with a genotype of c.9 C>T/WT, D) an individual with a genotype of c.52 A>T/WT, E) an individual with a genotype of c.125_128 delTCTT/WT, F) an individual with a genotype of c.316-197 C>T/WT, G) an individual with a genotype of c.316-185 T>C/WT, H) an individual with a genotype of compound heterozygous mutation c.-78 A>G/c.2 T>G, I) an individual with a genotype of c.9 C>T/c.52 A>T, and J) an individual with a genotype of c.316-197 C>T/c.316-185 T>C.

Figure 3

Human beta-globin gene sequence (NG_000007), primer locations and the variation sites. The primers are labeled in boldface letters and blue font, the mutation sites are labeled in boldface letters and red font, and the polymorphism are marked in boldface letters and green font. The overlap sequence is labeled in pink font.

Figure 4

DHPLC chromatography analyses of HBB genes for PCR products. (amplicon B1B2 for a, b, c, d, e, f ; B3B4 for g and Y3Y4 for h, i, j). a) an individual with a genotype of c.-78 A>G/WT, b) an individual with a genotype of c.2 T>G/WT, c) an individual with a genotype of c.9 C>T/WT, d) an individual with a genotype of c.52 A>T/WT, e) an individual with a compound heterozygous mutation c.-78 A>G/c.2 T>G, f) an individual with a genotype of c.9 C>T/c.52 A>T, g) an individual with a genotype of c.125_128 delTCTT/WT, h) an individual with a genotype of c.316-197 C>T/WT, i) an individual with a genotype of c.316-185 T>C/WT, and j) an individual with a genotype of c.316-197 C>T/c.316-185 T>C.