Menéndez Mireia, González Sara, Obrador-Hevia Antònia, Domínguez Ana, Pujol Maria Jesús, Valls Joan, Canela Núria, Blanco Ignacio, Torres Asunción, Pineda-Lucena Antonio, Moreno Víctor, Bachs Oriol, Capellá Gabriel
Laboratori de Recerca Translacional, Departament de Prevenció i Control del Càncer, Servei d'Epidemiologia i Registre del Càncer, Institut d'Investigació Biomèdica de Bellvitge-Institut Català d'Oncologia, L'Hospitalet de Llobregat, Barcelona, Spain.
Gastroenterology. 2008 Jan;134(1):56-64. doi: 10.1053/j.gastro.2007.10.009. Epub 2007 Oct 10.
BACKGROUND & AIMS: We identified the APC N1026S variant of unknown malignant potential in the adenomatous polyposis coli (APC) gene in a Spanish attenuated familial adenomatous polyposis (AFAP) family. The variant was located in the first of the 4 highly conserved 15-amino acid (AA) repeats within the beta-catenin union domain. Our aim was to determine its functional relevance to establish its pathogenicity.
N1026S variant was analyzed in 22 members of the AFAP family studied, in 236 sporadic colorectal cancer cases, 203 matched controls, and 205 unrelated familial colorectal cancer cases. To assess its effects on beta-catenin binding, beta-catenin/Tcf-4-mediated transcription and beta-catenin subcellular distribution we performed affinity chromatography experiments, BIAcore 1000 (BIAcore AB, Uppsala, Sweden) assays, luciferase reporter assays, assessment of c-myc messenger RNA levels, and cell fractionation.
N1026S variant cosegregated with the disease in the AFAP family studied. None of the sporadic or familial cases as well as the controls analyzed was positive for the variant. N1026S variant completely precluded beta-catenin binding to the first 15-AA repeat and diminished it when all four 15-AA repeats were present. Expression of APC N1026S in SW480 and DLD-1 cells did not diminish beta-catenin/Tcf-4-mediated transcription as effectively as APC wild-type. N1026S did not decrease c-myc transcription in DLD1 cells and nuclear beta-catenin in SW480 cells as effectively as WT.
These findings strongly support a pathogenic role of the APC N1026S variant in the AFAP phenotype, reinforcing the importance of functional characterization of APC variants for genetic counseling.
我们在一个西班牙的 attenuated 家族性腺瘤性息肉病（AFAP）家族中，鉴定出腺瘤性息肉病 coli（APC）基因中具有未知恶性潜能的 APC N1026S 变体。该变体位于β-连环蛋白结合域内 4 个高度保守的 15 个氨基酸（AA）重复序列中的第一个。我们的目的是确定其功能相关性以确定其致病性。
在研究的 AFAP 家族的 22 名成员、236 例散发性结直肠癌病例、203 例匹配对照以及 205 例无关的家族性结直肠癌病例中分析 N1026S 变体。为了评估其对β-连环蛋白结合、β-连环蛋白/Tcf-4 介导的转录以及β-连环蛋白亚细胞分布的影响，我们进行了亲和色谱实验、BIAcore 1000（BIAcore AB，瑞典乌普萨拉）分析、荧光素酶报告基因分析、c-myc 信使核糖核酸水平评估以及细胞分级分离。
在研究的 AFAP 家族中，N1026S 变体与疾病共分离。所分析的散发性或家族性病例以及对照中均无该变体呈阳性。N1026S 变体完全阻止β-连环蛋白与第一个 15-AA 重复序列结合，并且当所有四个 15-AA 重复序列都存在时会减少这种结合。APC N1026S 在 SW480 和 DLD-1 细胞中的表达不像 APC 野生型那样有效地减少β-连环蛋白/Tcf-4 介导的转录。N1026S 在 DLD1 细胞中不像 WT 那样有效地降低 c-myc 转录，在 SW480 细胞中也不像 WT 那样有效地降低核β-连环蛋白。
这些发现有力地支持了 APC N1026S 变体在 AFAP 表型中的致病作用，强化了对 APC 变体进行功能表征以用于遗传咨询的重要性。
