Denaturing high performance liquid chromatogrpahy (DHPLC) used in the detection of mutation in hMLH1 and hMSH2 genes. S. Pawar¹, S. Ciocci², A. Markowitz², S. Jhanwar², N. Ellis². 1) IMPATH, New York, NY; 2) Memorial Sloan-Kettering Cancer Center New York, NY.
   Hereditory nonpolyposis colorectal cancer (HNPCC) is the most common genetic condition that determines susceptibility to colorectal cancer. Studies for this disease have revealed it to be a simple mendelian disease involving five mismatch repair genes: hMLH1 and hMSH2 being the major ones. Rapid and accurate identification of DNA sequence heterogeneity is being recognized as of major importance in disease management. In this study we have used Denaturing high performance liquid chromatography (DHPLC) as a method of screening for germ line mutation in the hMSH2 and hMLH1 genes. The method is based on differential separation and detection of mismatched heteroduplexes formed after re-annealing of normal and mutant DNA strands. Analysis of all the exons and the exon-intron borders of hMSH2 and hMLH1 genes in 16 unrelated HNPCC kindreds was done. Optimization of DHPLC analysis of each exon was carried out by design of primers with minimum variation in the melting temperature of the amplicon, and titration of both elution gradient and temperature. To confirm the validity of DHPLC analysis all the 560 amplicons corresponding to 16 fragments for hMSH2 gene and 19 fragments for hMLH1 gene for each DNA sample were directly sequenced. All but 2 patients tested showed at least one heterozygous DHPLC profile in either hMLH1 or hMSH2 gene. A total of 18 unique DNA sequence variants were identified. Sequencing of the variant amplicons detected one novel and four previously reported pathogenic mutations, as well as seven novel and six known polymorphisms or unique sequence variants that are probably of no clinical significance. The amplicons that were observed to have a normal homozygous DHPLC profile detected no sequence variation in cycle sequencing. Therefore under our DHPLC conditions 100% of the sequence variation in hMLH1 and hMSH2 genes for the 16 individuals were correctly identified. Our results demonstrate that DHPLC is a highly sensitive, specific and inexpensive method to screen for mutations.