Gene Mutation in MicroRNA Target Sites of CFTR Gene: A Novel Pathogenetic Mechanism in Cystic Fibrosis?

Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on alterations of a chloride channel expressed by most epithelial cells and encoded by CFTR gene. Also using scanning techniques to analyze the whole coding regions of CFTR gene, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTR-related disorders (CFTR-RD). Other gene regions may be the site of causing-disease mutations. We searched for genetic variants in the 1500 bp of CFTR 3′ untranslated region, typical target of microRNA (miRNA) posttranscriptional gene regulation, in either CF patients with the F508del homozygous genotype and different clinical expression (n = 20), CF (n = 32) and CFTR-RD (n = 43) patients with one or none mutation after CFTR scanning and in controls (n = 50). We identified three SNPs, one of which, the c.*1043A>C, was located in a region predicted to bind miR-433 and miR-509-3p. Such mutation was peculiar of a CFTR-RD patient that had Congenital Bilateral Absence of Vas Deferens (CBAVD), diffuse bronchiectasis, a borderline sweat chloride test and the heterozygous severe F508del mutation on the other allele. The expression analysis demonstrated that the c.*1043A>C increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Both miRNAs cause in vitro a reduced expression of CFTR protein. Thus, the c.*1043A>C may act as a mild CFTR mutation enhancing the affinity for inhibitory miRNAs as a novel pathogenetic mechanism in CF.囊性纤维化（Cystic Fibrosis, CF）是高加索人群中最常见的致死性遗传性疾病。其发病机制与多数上皮细胞表达的、由CFTR基因编码的氯离子通道异常密切相关。当前通过扫描技术分析CFTR基因全编码区后，仍有至多10%的囊性纤维化等位基因无法检出致病突变，且这一比例在CFTR相关疾病（CFTR-RD）中进一步升高，提示其他基因区域可能存在致病突变位点。本研究针对CFTR基因3'端非翻译区（该区域是微小RNA（miRNA）进行转录后基因调控的典型靶标）的1500bp序列开展遗传变异筛查，纳入研究对象包括：20例携带F508del纯合基因型但临床表型各异的囊性纤维化患者、32例经CFTR扫描后仅检出1个或未检出突变的CF患者、43例CFTR-RD患者，以及50例健康对照。本次研究共检出3个单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs），其中c.*1043A>C位点位于预测可结合miR-433与miR-509-3p的区域内。该变异仅见于1例CFTR-RD患者：该患者表现为先天性双侧输精管缺如（Congenital Bilateral Absence of Vas Deferens, CBAVD）、弥漫性支气管扩张，汗液氯离子检测结果处于临界值，且另一等位基因携带重度F508del杂合突变。表达分析结果显示，c.*1043A>C变异可增强其与miR-509-3p的结合亲和力，同时略微降低其与miR-433的结合能力。两种miRNA均可在体外环境中下调CFTR蛋白的表达水平。综上，c.*1043A>C可作为一种轻度CFTR突变，通过增强与抑制性miRNA的结合亲和力发挥作用，这为囊性纤维化的发病机制提供了一种全新的阐释路径。