Abstract
BackgroundAmong the members of the ATP binding cassette transporter superfamily, MRPs share the closest homology with the CFTR protein, which is defective in CF disease. MRP1 has been proposed as a potential modifier gene and/or as novel target for pharmacotherapy of CF to explain the clinical benefits observed in some CF patients treated with the macrolide AZM. The 5'UTR of the MRP1 gene contains a GCC triplet repeat that could represent a polymorphic site and affect the activity of the promoter.MethodsThe MRP1 5' flanking region was amplified by PCR from 36 CF patients and 100 non-CF subjects and the number of GCC triplets of each allele was determined by sequence and electrophoretic analysis. We performed gene reporter studies in CF airway epithelial cells 16HBE14o-AS3, in basal conditions and in the presence of AZM.ResultsWe found that the GCC repeat is polymorphic, ranging from 7 to 14 triplets either in CF or in non-CF subjects. Our data are preliminary and have to be confirmed on a larger population of CF subjects. The transcriptional activity of the proximal MRP1 5' regulatory region revealed no statistically significant correlations between the number of repeats and treatment with AZM.ConclusionWe identified a novel polymorphism in the 5'UTR of MRP1 gene that provides multiple alleles in a gene relevant for multidrug resistance as well as for CF, determining that this region is transcriptionally active and that this activity does not appear to be influenced by AZM treatment.
Highlights
Among the members of the ATP binding cassette transporter superfamily, MRPs share the closest homology with the CFTR protein, which is defective in CF disease
We searched a genetic marker for MRP1 with potential functional relevance aiming to establish: 1) whether the GCC repeat in the MRP1 5'UTR is polymorphic, 2) if the GCC triplets length could affect the activity of the promoter in CF airways epithelial cells either in basal conditions or upon AZM treatment
The CF airway epithelial cell line 16HBE14o-AS3 was transiently co-transfected using FUGENE (Roche) with the reporter vector MRP1(GCC)7-luc or Assessment of transcriptional activity In order to establish whether the length of the GCC repeat can affect the transcriptional activity we performed gene reporter studies in CF airway epithelial cells 16HBE14oAS3
Summary
Among the members of the ATP binding cassette transporter superfamily, MRPs share the closest homology with the CFTR protein, which is defective in CF disease. CF is caused by mutations of the CFTR gene whose product acts as a cAMP-activated chloride channel that is permeable to organic anions, including GSH [1,2]. A wide range of disease severities has been described, even among CF patients harbouring the same mutation. Endogenous factors, that modulate or complement CFTR function, must exist. Because of the structural homology between CFTR and MRP1, a functional complementation of CFTR defect by MRP1 has been hypothesized [3]. Induction of MRP1 expression has been suggested to be responsible for improvement in lung function in a CF (page number not for citation purposes)
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