High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene

MUTYH 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.The 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.The 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.Colorectal carcinoma (CRC) is the second most common cause of cancer-related mortality in developed countries. 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). MAP is transmitted as an autosomal recessive trait due to biallelic mutations of the MUTYH gene, whose product is a DNA glycosylase that removes adenine from A？8-oxoG as part of the base excision repair process [1].MAP patients are homozygotes or compound heterozygotes for mutations of the MUTYH gene, that is comprised of 16 exons. Two missense base substitutions, c.536A > G (p.Tyr179Cys) and c.1187G > A (p.Gly396Asp), located in exons 7 and 13, respectively, account for about 75% of pathogenic MUTYH allelic variants reported among Caucasians [2]. In addition, other recurrent MUTYH mutations have been identified in Pakistani, Dutch, Portuguese and Japanese patients [3,4]. The remaining fraction of MUTYH variants identified in MAP patients is highly heterogeneous and can be located along the whole coding sequence.The identification of MUTYH g