Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders

Abstract

Patients with cystic fibrosis (CF) manifest a multisystemic disease due to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR); despite extensive testing of coding regions, a proportion of CF alleles remains unidentified. We studied 118 patients with CF and CFTR-related disorders, most with one or both unknown mutations after the scanning of CFTR coding regions, and a non-CF control group (n = 75) by sequencing the 6000-bp region at the 5' of the CFTR gene. We identified 23 mutations, of which 9 were novel. We expressed such mutations in vitro using four cell systems to explore their functional effect, relating the data to the clinical expression of each patient. Some mutations reduced expression of the gene reporter firefly luciferase in various cell lines and may act as disease-causing mutations. Other mutations caused an increase in luciferase expression in some cell lines. One mutation had a different effect in different cells. For other mutations, the expression assay excluded a functional role. Gene variants in the large 5' region may cause altered regulation of CFTR gene expression, acting as disease-causing mutations or modifiers of its clinical phenotype. Studies of in vitro expression in different cell systems may help reveal the effect of such mutations.

An “ex vivo model” contributing to the diagnosis and evaluation of new drugs in cystic fibrosis

“…Also in this case, a simple quantitative RT-PCR analysis of epithelial nasal cells would avoid the complex and expensive procedure of in vitro expression and analysis of mutations in cell lines 19 . However, our study demonstrated that the levels of CFTR gene expression are highly heterogeneous in normal subjects and in CF patients, and it would be necessary to study a larger number of healthy subjects to obtain reference values.…”

“…Quantitative RT-PCR analysis can be performed rapidly on cultured and directly on fresh sampled nasal cells, and can be used to assess the effect of mutations in the promoter region 19 or in other regulatory regions of the gene 20 21 revealing the mutations that may cause a reduced gene expression. Also in this case, a simple quantitative RT-PCR analysis of epithelial nasal cells would avoid the complex and expensive procedure of in vitro expression and analysis of mutations in cell lines 19 .…”

Genotype–phenotype correlation and functional studies in patients with cystic fibrosis bearing CFTR complex alleles

“…Furthermore, the STR analysis40 performed on several patients with CFTR-RD as well as on several asymptomatic subjects excluded the recurrent origin of the p.Leu997Phe mutation and, thus, the possibility that the mutation would be associated—in some cases—with other intragenic mutations that would interfere with its functional impact. We suggest that gene variants within the promoter region7 or at the 3′UTR8 may modulate CFTR expression explaining the variability of the p:Leu997Phe mutation. Of course, factors besides CFTR such as environment and modifier genes contribute to modulate the symptoms of the disease of each patient with CF.…”

“…Moreover, an increasing number of patients is diagnosed as CFTR-related disorders (CFTR-RD)2 generally characterised by a later onset of symptoms involving a single organ (ie, pancreatitis, disseminated bronchiectasis, obstructive azoospermia secondary to congenital bilateral absence of vas deferens (CBAVD)),3 usually associated to borderline SCL and mutations causing a different degree of CFTR protein dysfunction either in coding4–6 and in non-coding CFTR gene regions 7 8. Finally, the growing proportion of newborn screening (NBS) programmes revealed a large number of infants having a Cystic Fibrosis Screen Positive, Inconclusive Diagnosis (CFSPID) including those infants with discordance between immunoreactive trypsinogen at the NBS, SCL, CFTR genotype and clinical phenotype 9 10…”

Genetic Diseases That Predispose to Early Liver Cirrhosis

“…Large gene rearrangements are present in about 2-3% of CF alleles [16]. Finally, pathogenic mutations in noncoding region of the CFTR gene have been described [26, 27], but they are not analyzed for routine purposes. The detection rate of molecular analysis is lower in CFTR -RD [28].…”