Department of Molecular Biology, 12 De Octubre University Hospital, Madrid, E-28041, Spain.
BMC Cancer. 2010 Aug 5;10:408. doi: 10.1186/1471-2407-10-408.
MUTYH-associated polyposis (MAP) is a disorder caused by bi-allelic germline MUTYH mutation, characterized by multiple colorectal adenomas. In order to identify mutations in MUTYH 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.
In this study, we analyzed phenotype and genotype data from 82 patients, with multiple (> or = 10) synchronous (19/82) or metachronous (63/82) adenomas and negative APC study (except one case). Analysis was performed by HRM-PCR and direct sequencing, in order to identify mutations in MUTYH exons 7, 12 and 13, where the most prevalent mutations are located. In monoallelic mutation carriers, we evaluated entire MUTYH 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).
MUTYH 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.
Here, we describe a screening method, HRM, for the detection of both heterozygous and homozygous mutations in the gene encoding MUTYH 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.
MUTYH 相关性息肉病（MAP）是一种由双等位基因突变引起的疾病，其特征为多发性结直肠腺瘤。为了鉴定 MUTYH 基因突变，我们采用了高分辨率熔解（HRM）基因分型。HRM 分析广泛应用于检测杂合突变的扫描方法。因此，我们应用 HRM 来显示其在检测这些临床上重要且常见的患者的纯合突变方面的有效性。
在这项研究中，我们分析了 82 名患者的表型和基因型数据，这些患者具有多发性（≥10 个）同步（19/82）或异时性（63/82）腺瘤和 APC 阴性研究（除 1 例外）。分析采用 HRM-PCR 和直接测序进行，以鉴定 MUTYH 外显子 7、12 和 13 中最常见突变的突变。在单等位基因突变携带者中，我们评估了整个 MUTYH 基因，以寻找另一种可能的改变。HRM-PCR 采用严格的条件进行了几轮实验：第一轮是为了区分异源双链体和同源双链体模式，接下来的几轮是为了细化和确认参数。获得的基因型与表型特征（同步或异时性的腺瘤数量、结直肠癌（CRC）和家族史）相关。
在 82 名患者中发现了 MUTYH 种系突变，占 15.8%（13/82）。热点突变 Y179C（外显子 7）和 G396D（外显子 13）很容易被识别，其他突变也被检测到。通过 HRM，每种突变都有可重复的融解曲线，无论是杂合突变还是纯合突变。在我们对 82 名患者的研究中，双等位基因突变与携带≥10 个同步息肉有关（p=0.05），但与 CRC（p=0.39）或家族史（p=0.63）无关。在 82 名患者中发现了 23.1%（19/82）的 G338H 非致病性多态性（外显子 12）。在所有情况下，HRM（第一轮和后续轮次）和测序数据之间均具有一致性。
在这里，我们描述了一种筛选方法，即 HRM，用于检测 MAP 表型患者的 MUTYH 基因编码中杂合和纯合突变。我们改进了 HRM-PCR 的能力，并将其应用于尚未用该工具分析的基因。随着临床决策越来越依赖于分子医学，识别种系突变的能力必须不断得到评估和改进。
