Simplifying the detection of MUTYH mutations by high resolution melting analysis

<p>Abstract</p> <p>Background</p> <p><it>MUTYH</it>-associated polyposis (MAP) is a disorder caused by bi-allelic germline <it>MUTYH </it>mutation, characterized by multiple colorectal adenomas. In order to identify mutations in <it>MUTYH </it>gene we applied High Resolution Melting (HRM) genotyping. HRM analysis is extensively employed as a scanning method for the detection of heterozygous mutations. Therefore, we applied HRM to show effectiveness in detecting homozygous mutations for these clinically important and frequent patients.</p> <p>Methods</p> <p>In this study, we analyzed phenotype and genotype data from 82 patients, with multiple (>= 10) synchronous (19/82) or metachronous (63/82) adenomas and negative <it>APC </it>study (except one case). Analysis was performed by HRM-PCR and direct sequencing, in order to identify mutations in <it>MUTYH </it>exons 7, 12 and 13, where the most prevalent mutations are located. In monoallelic mutation carriers, we evaluated entire <it>MUTYH </it>gene in search of another possible alteration. HRM-PCR was performed with strict conditions in several rounds: the first one to discriminate the heteroduplex patterns and homoduplex patterns and the next ones, in order to refine and confirm parameters. The genotypes obtained were correlated to phenotypic features (number of adenomas (synchronous or metachronous), colorectal cancer (CRC) and family history).</p> <p>Results</p> <p><it>MUTYH </it>germline mutations were found in 15.8% (13/82) of patients. The hot spots, Y179C (exon 7) and G396D (exon 13), were readily identified and other mutations were also detected. Each mutation had a reproducible melting profile by HRM, both heterozygous mutations and homozygous mutations. In our study of 82 patients, biallelic mutation is associated with being a carrier of ≥10 synchronous polyps (p = 0.05) and there is no association between biallelic mutation and CRC (p = 0.39) nor family history (p = 0.63). G338H non-pathogenic polymorphism (exon 12) was found in 23.1% (19/82) of patients. In all cases there was concordance between HRM (first and subsequent rounds) and sequencing data.</p> <p>Conclusions</p> <p>Here, we describe a screening method, HRM, for the detection of both heterozygous and homozygous mutations in the gene encoding <it>MUTYH </it>in selected samples of patients with phenotype of MAP. We refine the capabilities of HRM-PCR and apply it to a gene not yet analyzed by this tool. As clinical decisions will increasingly rely on molecular medicine, the power of identifying germline mutations must be continuously evaluated and improved.</p

MUTYH-associated polyposis (MAP): evidence for the origin of the common European mutations p.Tyr179Cys and p.Gly396Asp by founder events.

MUTYH-associated polyposis (MAP) is an autosomal recessive adenomatous polyposis caused by biallelic germline mutations of the base-excision-repair gene MUTYH. In MAP patients of European origin, the combined allele frequency of the mutations p.Tyr179Cys and p.Gly396Asp ranges between 50 and 82%, while these mutations have not been identified in Far Eastern Asian populations, supporting the hypothesis that a founder effect has occurred at some point in European history. To investigate the natural history of the two common European MUTYH alleles, we genotyped six gene-flanking microsatellite markers in 80 unrelated Italian and German MAP patients segregating one or both mutations and calculated their age in generations (g) by using DMLE+2.2 software. Three distinct common haplotypes, one for p.Tyr179Cys and two for p.Gly396Asp, were identified. Estimated mutation ages were 305\u2009g (95% CS: 271-418) for p.Tyr179Cys and 350\u2009g (95% CS: 313-435) for p.Gly396Asp. These results provide evidence for strong founder effects and suggest that the p.Tyr179Cys and p.Gly396Asp mutations derive from ancestors who lived between 5-8 thousand years and 6-9 thousand years B.C., respectivel