This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Cystic fibrosis (CF) is caused by mutations in the CFTR gene. The hypothesis for this study is that reduced mRNA expression from one CFTR allele causes 'non-classic' forms of CF and investigation of these alleles will identify sequence elements necessary for regulation of CFTR transcription. This hypothesis will be studied by addressing the following three specific aims: (1) To evaluate CFTR function in non-classic CF patients with one 'severe' CFTR mutation. (2) To determine whether CFTR mRNA is full-length in patients with one 'severe' CFTR mutation and CFTR dysfunction in vivo. (3) To identify patients with reduced CFTR mRNA transcription .Patients with CF and one defined CFTR mutation will be evaluated to determine if the CF phenotype is due a decrease in CFTR mRNA from the other allele. A diagnosis of non-classic CF will be confirmed in patients with CF-like disease in at least two organ systems (lungs, gastrointestinal tract, reproductive tract and/or sweat gland). CFTR dysfunction will be confirmed by sweat chloride, nasal potential difference (NPD) and sweat rate measurements. CFTR mRNA expression will be determined in respiratory epithelia will be obtained from the inferior turbinate of both nostrils of non-classic CF patients with CFTR dysfunction. Total RNA will be extracted from the nasal epithelial cells. CFTR mRNA will be evaluated by reverse transcriptase (RT)-PCR to confirm that it is full length. CFTR mRNA levels will be quantified using three independent but complementary methods. CFTR mRNA expression from study patients will be compared to normal subjects and CF patients with homozygous ?F508 CFTR mutations. We anticipate that this project will identify one or more patients with one 'severe' CFTR mutation who have CF because CFTR expression from the other allele is diminished. We anticipate that analysis of these patients will identify cis-acting elements and transcription factors that are important in the regulation of CFTR expression.
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