According to reports on systematic mutation analysis of the entire MUTYH gene (2, 3, 6,10,11, 21, 22), screening for these 6 mutations would identify at least 85% of patients with biallelic MUTYH mutations.
To carry out even more rapid genotyping, we designed a set of 3 multiplex T-ARMS-PCRs for the detection of these 6 frequent MUTYH mutations: (a) the relatively frequent Y165C and G382D mutations; (b) the 1103de1C and 1395 7de1GGA mutations; and (c) the Y90X and R231H mutations (Table 1).
We designed the primers on the basis of the published MUTYH genomic sequence (GenBank accession no.
5%-29% of patients were expected to carry MUTYH mutations (2,4-6), and we identified 3 Y165C homozygotes, 4 compound heterozygotes (2 Y165C/G382D, 1 Y165C/1395 7de1GGA, and 1 R231H/G382D),1 Y90X heterozygote, and 1 G382D heterozygote.
The multiplex T-ARMS-PCR procedures were validated by the analysis of DNA samples from a previously characterized group of 22 patients without and 31 patients with MUTYH mutations.
This method provides rapid, reproducible, and costeffective detection of common MUTYH mutations without the use of any special equipment.
This simple, inexpensive, and accurate method could be used to genotype relatives of patients with known MUTYH mutations, to optimize the strategy for identification of MUTYH mutations in a diagnostic setting, and to carry out the large population-based epidemiologic studies needed to investigate the possible role of mono-allelic MUTYH mutations in predisposing to colorectal cancer.