Abstract
BackgroundMUTYH-associated polyposis (MAP) is an autosomal recessive form of intestinal polyposis predisposing to colorectal carcinoma. High resolution melting analysis (HRMA) is a mutation scanning method that allows detection of heterozygous sequence changes with high sensitivity, whereas homozygosity for a nucleotide change may not lead to significant curve shape or melting temperature changes compared to homozygous wild-type samples. Therefore, HRMA has been mainly applied to the detection of mutations associated with autosomal dominant or X-linked disorders, while applications to autosomal recessive conditions are less common.MethodsMUTYH coding sequence and UTRs were analyzed by both HRMA and sequencing on 88 leukocyte genomic DNA samples. Twenty-six samples were also examined by SSCP. Experiments were performed both with and without mixing the test samples with wild-type DNA.ResultsThe results show that all MUTYH sequence variations, including G > C and A > T homozygous changes, can be reliably identified by HRMA when a condition of artificial heterozygosity is created by mixing test and reference DNA. HRMA had a sensitivity comparable to sequencing and higher than SSCP.ConclusionsThe availability of a rapid and inexpensive method for the identification of MUTYH sequence variants is relevant for the diagnosis of colorectal cancer susceptibility, since the MAP phenotype is highly variable.
Highlights
MUTYH-associated polyposis (MAP) is an autosomal recessive form of intestinal polyposis predisposing to colorectal carcinoma
In order to verify whether High resolution melting analysis (HRMA) allowed detection of homozygous sequence changes based on Tm differences alone, the 26 samples were examined without prior mixing with the reference sample
In all samples harbouring MUTYH variants previously identified by single strand conformational polymorphism (SSCP), the shapes of the melting curves were altered compared to wild-type samples
Summary
MUTYH-associated polyposis (MAP) is an autosomal recessive form of intestinal polyposis predisposing to colorectal carcinoma. HRMA has been mainly applied to the detection of mutations associated with autosomal dominant or X-linked disorders, while applications to autosomal recessive conditions are less common. About 1% of CRCs arise in individuals affected with familial adenomatous polyposis. This condition can be caused by mutations in at least 2 distinct genes: APC, implicated in the autosomal dominant form, and MUTYH, which is involved in MUTYH-associated polyposis (MAP: MIM#608456). The remaining fraction of MUTYH variants identified in MAP patients is highly heterogeneous and can be located along the whole coding sequence
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