Assay validation for identification of hereditary nonpolyposis colon cancer-causing mutations in mismatch repair genes MLH1, MSH2, and MSH6

Abstract

Hereditary nonpolyposis colon cancer (HNPCC, Online Mendelian Inheritance in Man (OMIM) 114500) is an autosomal dominant disorder that is genetically heterogeneous because of underlying mutations in mismatch repair genes, primarily MLH1, MSH2, and MSH6. One challenge to correctly diagnosing HNPCC is that the large size of the causative genes makes identification of mutations both labor intensive and expensive. We evaluated the usefulness of denaturing high performance liquid chromatography (DHPLC) for scanning mismatch repair genes (MLH1, MSH2, and MSH6) for point mutations, small deletions, and insertions. Our assay consisted of 51 sets of primers designed to amplify all exons of these genes. All polymerase chain reaction reactions were amplified simultaneously using the same reaction conditions in a 96-well format. The amplified products were analyzed by DHPLC across a range of optimum temperatures for partial fragment denaturation based on the melting profile of each specific fragment. DNA specimens from 23 previously studied HNPCC patients were analyzed by DHPLC, and all mutations were correctly identified and confirmed by sequence analysis. Here, we present our validation studies of the DHPLC platform for HNPCC mutation analysis and compare its merits with other scanning technologies. This approach provides greater sensitivity and more directed molecular analysis for clinical testing in HNPCC.

Figure 1

DHPLC profiles for MLH1, MSH2, and MSH6 mutations. The DHPLC profiles of HNPCC patients with mutations in mismatch repair genes are shown for all of the mutations in MLH1, MSH2, and MSH6 used for assay validation compared with the wild-type profiles, respectively. The wild-type profile is shown on the top and mutant profile at the bottom of individual profiles.

Figure 2

DHPLC profiles for MLH1, MSH2, and MSH6 polymorphisms. The DHPLC profiles of HNPCC patients with polymorphisms in mismatch repair genes are shown for all of the mutations in MLH1, MSH2, and MSH6 used for assay validation compared with the wild-type profiles, respectively.

Figure 3

DHPLC profiles for MSH6 intron 6 variable poly(T) region. Note the nonspecific heteroduplexes immediately before the main peak, indicating a highly polymorphic region. The wild-type profile is on the top and the poly(T) profile is shown below it.

Figure 4

Proposed strategy for genetic testing in colorectal cancer patients. A comprehensive strategy incorporating 1) family history and clinical data; 2) microsatellite instability; and 3) immunohistochemistry as screening tools mutation for targeted analysis. Methylation testing further focuses the process on samples most likely to carry a germline mutation (ie, microsatellite unstable, unmethylated, and IHC-positive tumors).

Figure 5

Algorithm for DHPLC screening for identification of mutations in mismatch repair genes in colorectal cancer patients.