Targeted locus amplification reveals heterogeneity between and within CFTR genotypes and association with CFTR function in patient-derived intestinal organoids

J W Lefferts; V Boersma; N D A Nieuwenhuijze; S W F Suen; K Hajo; N Sanchez Collantes; C Vermeulen; T Groeneweg; M C Hagemeijer; H R de Jonge; C K van der Ent; E Splinter; J M Beekman

doi:10.1016/j.jcf.2023.04.003

Targeted locus amplification reveals heterogeneity between and within CFTR genotypes and association with CFTR function in patient-derived intestinal organoids

J Cyst Fibros. 2023 May;22(3):538-547. doi: 10.1016/j.jcf.2023.04.003. Epub 2023 Apr 24.

Authors

J W Lefferts¹, V Boersma², N D A Nieuwenhuijze³, S W F Suen⁴, K Hajo², N Sanchez Collantes⁵, C Vermeulen⁵, T Groeneweg⁶, M C Hagemeijer⁷, H R de Jonge⁶, C K van der Ent⁸, E Splinter², J M Beekman⁹

Affiliations

¹ 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.
² Cergentis BV., Utrecht, the Netherlands.
³ 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; Gastroenterology & Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands.
⁴ 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; Xilis BV, Utrecht, the Netherlands.
⁵ Oncode Institute, Utrecht, the Netherlands; Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.
⁶ Gastroenterology & Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands.
⁷ Current affiliation: Center for Lysosomal and Metabolic Diseases, Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, the Netherlands.
⁸ Department of Pediatric Respiratory Medicine, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, the Netherlands.
⁹ 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; Center for Living Technologies, Eindhoven-Wageningen-Utrecht Alliance, the Netherlands. Electronic address: j.beekman@umcutrecht.nl.

PMID: 37100706
DOI: 10.1016/j.jcf.2023.04.003

Abstract

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.

Keywords: CFTR; Haplotyping; Organoids; Phenotype-genotype relation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cystic Fibrosis Transmembrane Conductance Regulator* / genetics
Cystic Fibrosis* / genetics
Genotype
Humans
Intestines
Mutation
Organoids

Substances

Cystic Fibrosis Transmembrane Conductance Regulator
CFTR protein, human