Noel Sabrina, Servel Nathalie, Hatton Aurélie, Golec Anita, Rodrat Mayuree, Ng Demi R S, Li Hongyu, Pranke Iwona, Hinzpeter Alexandre, Edelman Aleksander, Sheppard David N, Sermet-Gaudelus Isabelle
INSERM U1151, Institut Necker-Enfants Malades, Paris, France.
Université de Paris, Paris, France.
J Physiol. 2022 Mar;600(6):1515-1531. doi: 10.1113/JP282143. Epub 2021 Dec 8.
Dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes a wide spectrum of disease, including cystic fibrosis (CF) and CFTR-related diseases (CFTR-RDs). Here, we investigate genotype-phenotype-CFTR function relationships using human nasal epithelial (hNE) cells from a small cohort of non-CF subjects and individuals with CF and CFTR-RDs and genotypes associated with either residual or minimal CFTR function using electrophysiological techniques. Collected hNE cells were either studied directly with the whole-cell patch-clamp technique or grown as primary cultures at an air-liquid interface after conditional reprogramming. The properties of cAMP-activated whole-cell Cl currents in freshly isolated hNE cells identified them as CFTR-mediated. Their magnitude varied between hNE cells from individuals within the same genotype and decreased in the rank order: non-CF > CFTR residual function > CFTR minimal function. CFTR-mediated whole-cell Cl currents in hNE cells isolated from fully differentiated primary cultures were identical to those in freshly isolated hNE cells in both magnitude and behaviour, demonstrating that conditional reprogramming culture is without effect on CFTR expression and function. For the cohort of subjects studied, CFTR-mediated whole-cell Cl currents in hNE cells correlated well with CFTR-mediated transepithelial Cl currents measured in vitro with the Ussing chamber technique, but not with those determined in vivo with the nasal potential difference assay. Nevertheless, they did correlate with the sweat Cl concentration of study subjects. Thus, this study highlights the complexity of genotype-phenotype-CFTR function relationships, but emphasises the value of conditionally reprogrammed hNE cells in CFTR research and therapeutic testing. KEY POINTS: The genetic disease cystic fibrosis is caused by pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel, which controls anion flow across epithelia lining ducts and tubes in the body. This study investigated CFTR function in nasal epithelial cells from people with cystic fibrosis and CFTR variants with a range of disease severity. CFTR function varied widely in nasal epithelial cells depending on the identity of CFTR variants, but was unaffected by conditional reprogramming culture, a cell culture technique used to grow large numbers of patient-derived cells. Assessment of CFTR function in vitro in nasal epithelial cells and epithelia, and in vivo in the nasal epithelium and sweat gland highlights the complexity of genotype-phenotype-CFTR function relationships.
上皮阴离子通道囊性纤维化跨膜传导调节因子（CFTR）功能障碍会引发多种疾病，包括囊性纤维化（CF）和CFTR相关疾病（CFTR-RDs）。在此，我们使用来自一小群非CF受试者、CF患者以及CFTR-RDs患者的人鼻上皮（hNE）细胞，运用电生理技术研究基因型-表型-CFTR功能关系，这些个体具有与残余或最小CFTR功能相关的基因型。收集的hNE细胞要么直接采用全细胞膜片钳技术进行研究，要么在条件重编程后作为原代培养物在气液界面生长。新鲜分离的hNE细胞中cAMP激活的全细胞Cl电流特性表明它们是由CFTR介导的。其幅度在同一基因型个体的hNE细胞之间存在差异，且按以下顺序降低：非CF＞CFTR残余功能＞CFTR最小功能。从完全分化的原代培养物中分离的hNE细胞中CFTR介导的全细胞Cl电流在幅度和行为上与新鲜分离的hNE细胞中的相同，表明条件重编程培养对CFTR表达和功能没有影响。对于所研究的受试者队列，hNE细胞中CFTR介导的全细胞Cl电流与使用尤斯灌流小室技术在体外测量的CFTR介导的跨上皮Cl电流密切相关，但与使用鼻电位差测定法在体内测定的电流无关。然而，它们确实与研究受试者的汗液Cl浓度相关。因此，本研究突出了基因型-表型-CFTR功能关系的复杂性，但强调了条件重编程的hNE细胞在CFTR研究和治疗测试中的价值。要点：遗传性疾病囊性纤维化由囊性纤维化跨膜传导调节因子（CFTR）的致病变异引起，CFTR是一种离子通道，控制阴离子穿过体内管道和导管内衬上皮的流动。本研究调查了患有囊性纤维化的人和具有一系列疾病严重程度的CFTR变体的鼻上皮细胞中的CFTR功能。根据CFTR变体的类型，鼻上皮细胞中的CFTR功能差异很大，但不受条件重编程培养的影响，条件重编程培养是一种用于培养大量患者来源细胞的细胞培养技术。在体外鼻上皮细胞和上皮组织中以及在体内鼻上皮和汗腺中对CFTR功能的评估突出了基因型-表型-CFTR功能关系的复杂性。
