Sterrantino Manuela, Fuso Andrea, Pierandrei Silvia, Bruno Sabina Maria, Testino Giancarlo, Cimino Giuseppe, Angeloni Antonio, Lucarelli Marco
Department of Experimental Medicine, Sapienza University of Rome, 00161 Rome, Italy.
Cystic Fibrosis Reference Center of Lazio Region, AOU Policlinico Umberto I, 00161 Rome, Italy.
Diagnostics (Basel). 2021 Jan 25;11(2):168. doi: 10.3390/diagnostics11020168.
Genetic analysis in cystic fibrosis (CF) is a difficult task. Within the many causes of variability and uncertainty, a major determinant is poor knowledge of the functional effect of most DNA variants of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. In turn, knowledge of the effect of a CFTR variant has dramatic diagnostic, prognostic and, in the era of CF precision medicine, also therapeutic consequences. One of the most challenging CFTR variants is the (TG)mTn haplotype, which has variable functional effect and controversial clinical consequences. The exact quantification of the anomalous splicing of CFTR exon 10 (in the HGVS name; exon 9 in the legacy name) and, consequently, of the residual wild-type functional CFTR mRNA, should be mandatory in clinical assessment of patients with potentially pathological haplotype of this tract. Here, we present a real time-based assay for the quantification of the proportion of exon 10+/exon 10- CFTR mRNA, starting from nasal brushing. Our assay proved rapid, economic and easy to perform. Specific primers used for this assay are either disclosed or commercially available, allowing any laboratory to easily perform it. A simplified analysis of the data is provided, facilitating the interpretation of the results. This method helps to enhance the comprehension of the genotype-phenotype relationship in CF and CFTR-related disorders (CFTR-RD), crucial for the diagnosis, prognosis and personalized therapy of CF.
囊性纤维化（CF）的基因分析是一项艰巨的任务。在众多导致变异性和不确定性的原因中，一个主要决定因素是对囊性纤维化跨膜传导调节因子（CFTR）基因的大多数DNA变异的功能效应了解不足。反过来，了解CFTR变异的效应具有重大的诊断、预后意义，并且在CF精准医学时代，还具有治疗意义。最具挑战性的CFTR变异之一是（TG）mTn单倍型，其功能效应可变，临床后果存在争议。在对具有该区域潜在病理单倍型的患者进行临床评估时，应强制准确量化CFTR第10外显子（按照HGVS命名法；传统命名法中的第9外显子）的异常剪接情况，进而量化残余野生型功能CFTR mRNA的情况。在此，我们介绍一种基于实时检测的方法，用于从鼻拭子样本中定量分析第10外显子+/第10外显子-CFTR mRNA的比例。我们的检测方法快速、经济且易于操作。用于该检测的特异性引物已公开或可商购，任何实验室都能轻松进行检测。我们还提供了数据的简化分析方法，便于结果解读。该方法有助于加深对CF及CFTR相关疾病（CFTR-RD）中基因型-表型关系的理解，这对于CF的诊断、预后及个性化治疗至关重要。
