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• Exonic variants may have various molecular mechanisms of pathogenicity. • The impact on splicing of exonic sequence variations should be systemically assessed. • Splicing default of exonic variants may hamper the efficiency of targeted pharmacotherapy. • Functional in vitro experiments are key tools to classify CFTR rare variants in order to offer personalized therapies. The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR -France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments. [Display omitted] [ABSTRACT FROM AUTHOR]

The most common form of hereditary haemochromatosis is an adult-onset condition usually associated with the HFE C282Y/C282Y genotype. The phenotypic expression of this genotype is heterogeneous and depends on a complex interplay of genetic and non-genetic factors. The aim of the present study was to determine if mutations in the recently identified HJV gene were associated with more severe iron overload phenotypes in C282Y homozygous patients. From a cohort of 310 C282Y homozygous patients, we found nine (six males and three females) with an additional HJV missense mutation in the heterozygous state (S105L, E302K, N372D, R335Q or the previously described L101P and G320V). The iron indices of eight patients appeared to be more severe than those observed in C282Y homozygous patients of identical sex and similar age ranges. The mean serum ferritin concentration of the six males with an HJV mutation was significantly higher than that of C282Y homozygous males without an additional mutation [2350.3 (sigma=1429.9) versus 1227.2 (sigma=1130.1) microg/l; P=0.0233, Student's t-test]. We have recently reported that mutations in the gene that encodes hepcidin (HAMP) could explain one part of the C282Y/C282Y-related phenotypic heterogeneity by accentuating the iron burden. Our new data reveal that mutations in the HJV gene could be associated with a similar effect. Taken together, these results emphasize that a search for modifier genes could enable us to more precisely distinguish those C282Y homozygous patients with a higher risk to develop a severe iron overload and, consequently, clinical complications., (Copyright 2004 Oxford University Press)