靶向基因扩增揭示了 CFTR 基因型在个体间和个体内的异质性，并与患者来源的肠类器官中的 CFTR 功能相关。
Targeted locus amplification reveals heterogeneity between and within CFTR genotypes and association with CFTR function in patient-derived intestinal organoids.
机构信息
Department of Pediatric Respiratory Medicine, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, the Netherlands; Regenerative Medicine Center, Center for Living Technologies, University Medical Center Utrecht, Utrecht, the Netherlands.
Cergentis BV., Utrecht, the Netherlands.
出版信息
J Cyst Fibros. 2023 May;22(3):538-547. doi: 10.1016/j.jcf.2023.04.003. Epub 2023 Apr 24.
BACKGROUND
Cystic fibrosis (CF) disease severity can be highly variable, even between people with CF (pwCF) with similar genotypes. Here we use patient-derived intestinal organoids to study the influence of genetic variation within the cystic fibrosis transmembrane conductance regulator (CFTR) gene on CFTR function.
METHODS
Organoids of F508del/class I, F508del/S1251N and pwCF with only one detected CF-causing mutation were cultured. Allele-specific CFTR variation was investigated using targeted locus amplification (TLA), CFTR function was measured using the forskolin-induced swelling assay and mRNA levels were quantified using RT-qPCR.
RESULTS
We were able to distinguish CFTR genotypes based on TLA data. Additionally, we observed heterogeneity within genotypes, which we were able to link to CFTR function for S1251N alleles.
CONCLUSIONS
Our results indicate that the paired analysis of CFTR intragenic variation and CFTR function can gain insights in the underlying CFTR defect for individuals where the disease phenotype does not match the CFTR mutations detected during diagnosis.
背景
囊性纤维化（CF）疾病的严重程度可能存在很大差异，即使是具有相似基因型的 CF（pwCF）患者之间也是如此。在这里，我们使用源自患者的肠类器官来研究囊性纤维化跨膜电导调节因子（CFTR）基因内的遗传变异对 CFTR 功能的影响。
方法
培养 F508del/class I、F508del/S1251N 和仅有一个检测到的致病变异体的类器官。使用靶向基因座扩增（TLA）来研究 CFTR 等位基因的特异性变异，使用福司可林诱导的肿胀试验来测量 CFTR 功能，并使用 RT-qPCR 定量 mRNA 水平。
结果
我们能够根据 TLA 数据区分 CFTR 基因型。此外，我们观察到基因型内的异质性，我们能够将其与 S1251N 等位基因的 CFTR 功能联系起来。
结论
我们的结果表明，CFTR 基因内变异与 CFTR 功能的配对分析可以深入了解在疾病表型与诊断期间检测到的 CFTR 突变不匹配的个体中，CFTR 缺陷的潜在原因。
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