Genotyping of Macrophage Migration Inhibitory Factor (MIF) CATT5–8 Repeat Polymorphism by Denaturing High-Performance Liquid Chromatography (DHPLC)

Abstract

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine expressed in many different cell types and implicated in the pathogenesis of numerous acute and chronic inflammatory diseases. Variable Number of Tandem Repeat (VNTR) CATT5-8 at position -794 in the promoter of the MIF gene has been associated with several human pathological conditions. Different methods for genotyping the CATT tetranucleotide repeats have been described. Here, we report, for the first time, the complete characterization of the CATT5-8 repeat polymorphism using exclusively the denaturing high-performance liquid chromatography (DHPLC) technique under partially denaturing conditions. This approach, based on a step-by-step DHPLC protocol, allowed the accurate determination of all the homozygous and heterozygous genotypes in 350 DNA samples from control subjects. The results were validated by comparison to DNA sequencing, and the DHPLC approach was accurate, sensitive, and highly reproducible. Data from the current study demonstrate that this method of analysis by DHPLC may represent a powerful and sensitive alternative tool for a rapid and efficient genotyping of short tandem repeats presenting a limited number of alleles.