Prior-de Castro, CarmenMartínez Gallego, Miguel ÁngelGómez-González, ClaraDe Sancho Martín, RubénRodríguez Antolín, CarlosRodríguez-Jiménez, CarmenDel Pozo Mate, ÁngelaZamarrón de Lucas, EsterRuiz de Balbuena Maiz, MartaDe Manuel Gómez, CristinaAlcolea Batres, SergioPrados Sánchez, María ConcepciónTorres, Rosa J.2025-01-292025-01-292024Prior de Castro, C., Martínez Gallego, M.Á., Gómez-González, C., et al. (2024). Molecular diagnosis of cystic fibrosis by RNA obtained from nasal epithelial cells. Journal of Cystic Fibrosis, 23(4):788-7951569-1993https://hdl.handle.net/10641/5669Background The diagnosis of cystic fibrosis (CF) is established when characteristic clinical signs are coupled with biallelic CFTR pathogenic variants. No previously reported non-canonical splice site variants have to be considered as variants of uncertain significance unless their effect on splicing has been validated. Methods Two variants identified by next-generation sequencing were evaluated. We assayed their effects on splicing employing RNA analysis and real-time expression quantification from RNA obtained from the nasal epithelial cells of a patient with clinically suspected CF and of two patients with milder phenotypes (CFTR-related disorders). Results The variant c.164+2dup causes skipping of exon 2 (p.(Ser18_Glu54del)) and exon 2 plus 3 (p.(Ser18Argfs*16)) in CFTR mRNA. Exon 2 expression in the patient heterozygous for c.164+2dup was decreased to 7 % of the exon 2 expression in the controls. The synonymous variant c.1584G>A causes a partial skipping of exon 11. The exon 11 expression in the two patients heterozygous for this variant was 22 % and 42 % of that of the controls, respectively. Conclusion We conclude that variant c.164+2dup affects mRNA processing and can be considered a CF-causing variant. The results of the functional assay also showed that the p.(Glu528=) variant, usually categorized as a neutral variant based on epidemiological data, partially affects mRNA processing in our patients. This finding would allow us to reclassify the variant as a CFTR-related variant with incomplete penetrance. RNA obtained from nasal epithelial cells is an easy and accurate tool for CFTR functional studies in patients with unclassified splice variants.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/SplicingRNA analysisCF-causing variantCFTR-related variantNasal epithelial cellsjournal articleembargoed access10.1016/j.jcf.2023.12.007