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

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.

Targeted Locus Amplification and Next-Generation Sequencing.

Despite developments in targeted and whole-genome sequencing, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Targeted locus amplification (TLA) is a cross-linking-based technique that generates complex DNA libraries covering >100 kb of contiguous sequence surrounding one primer pair complementary to a short locus-specific sequence. In combination with next-generation sequencing, TLA enables the complete sequencing and haplotyping of targeted regions of interest. Here we outline the basis of TLA, together with a detailed protocol of the technique.

Absence of habituation of airway dilation following lung inflation.

In order to test the possibility that airway dilation following lung inflation (ADFLI) could exhibit habituation, partial and complete maximum expiratory flow-volume curves were obtained in 12 healthy individuals before and after inhaling methacholine aerosol in a dose that reduced in each individual the partial expiratory flows to approximately 50% of baseline values. Following methacholine inhalation, the difference in flows between complete and partial flow-volume curves at 40% VC was 64 +/- 22% (mean +/- SD) of partial curves after a single full lung inflation and 79 +/- 36% after 12 consecutive full inflations (p = NS). The absence of demonstrable habituation (tachyphylaxis) of ADFLI makes it unlike that ADFLI is due to a release of mediators and supports the view that it may be due to the intrinsic properties of the airway smooth muscle.