2 Next Generation Sequencing genotyping project in Spanish cystic fibrosis patients with uncharacterized CFTR mutations

Objectives: The purpose of this work is to implement non-invasive prenatal diagnosis (NIPD) of cystic fibrosis, based on analysis of cellfree fetal DNA (cffDNA) present in maternal peripheral blood. NIPD of CF was performed by detection of the F508del mutation. Methods: cffDNA occurs in plasma only in form of short fragments. By targeting the shorter DNA molecules, the fractional concentration of cffDNA is enriched. cffDNA was isolated from maternal plasma by the QIAamp DNA Mini Kit and Clean Circulating DNA Kit. Quality of the cffDNA isolation was evaluated by automate capillary electrophoresis on Fragment AnalyzerTM. For confirmation of cffDNA in a sample, we evaluated three approaches: fetal gender determination, comparison of STRs in maternal and fetal genome, and methylation analysis of the maspin gene promoter. In cooperation with TIBMolBiol, we designed HRM-based LightSNiP assays to detect the F508del. PCR amplicons needed to be shorter than 200bp to be able to analyze short fragments of cffDNA. Real-Time PCR is performed by using the DyNamo ColorFlash Probe qPCR Kit, Xceed qPCR Probe 2x Mix No-ROX and SensiFASTTM Probe No-ROX Kit. Results: Based on our results, we concluded that the cffDNA isolation performed by the evaluated kits does not differ in cffDNA yield, but the Clean Circulating DNA Kit separates the larger fragments (predominantly maternal) more effectively. We concluded that there is no significant difference between performances of three Real-Time PCR mastermixes used. Conclusion: It is currently possible to detect the F508del mutation in cffDNA samples; and analysis of more CF mutations for NIPD of CF should be implemented in the future.