Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders
2013; Elsevier BV; Volume: 15; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2013.01.001
ISSN1943-7811
AutoresSonia Giordano, Felice Amato, Ausilia Elce, Maria Monti, Carla Iannone, Piero Pucci, Manuela Seia, Adriano Angioni, Federica Zarrilli, Giuseppe Castaldo, Rossella Tomaiuolo,
Tópico(s)Congenital Ear and Nasal Anomalies
ResumoPatients 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. 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. CME Accreditation Statement: This activity ("JMD 2013 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("JMD 2013 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s)TM. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("JMD 2013 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2013 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s)TM. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Cystic fibrosis (CF) is the most frequent lethal inherited disorder among white people, with an incidence of 1:2500 newborns. It depends on alterations of the chloride channel expressed by most epithelial cells and encoded by the cystic fibrosis transmembrane regulator gene (CFTR).1McIntosh I. Cutting G.R. Cystic fibrosis transmembrane conductance regulator and the etiology and pathogenesis of cystic fibrosis.FASEB J. 1992; 6: 2775-2782PubMed Google Scholar The diagnosis of CF is based on symptoms, sweat chloride levels, and molecular analysis findings. However, causative mutations are identified in 90% to 95% of CF chromosomes using scanning procedures to analyze whole coding regions of CFTR2Castaldo G. Polizzi A. Tomaiuolo R. Cazeneuve C. Girodon E. Santostasi T. Salvatore D. Raia V. Rigillo N. Goossens M. Salvatore F. Comprehensive cystic fibrosis mutation epidemiology and haplotype characterization in a southern Italy population.Ann Hum Genet. 2005; 69: 15-24Crossref PubMed Scopus (45) Google Scholar and large gene rearrangements,3Tomaiuolo R. Sangiuolo F. Bombieri C. Bonizzato A. Cardillo G. Raia V. D'Apice M.R. Bettin M.D. Pignatti P.F. Castaldo G. Novelli G. Epidemiology and a novel procedure for large scale analysis of CFTR rearrangements in classic and atypical CF patients: a multicentric Italian study.J Cyst Fibros. 2008; 7: 347-351Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar and this percentage is lower in CFTR-related disorders (CFTR-RDs).4Amato F. Bellia C. Cardillo G. Castaldo G. Ciaccio M. Elce A. Lembo F. Tomaiuolo R. Extensive molecular analysis of patients bearing CFTR-related disorders.J Mol Diagn. 2012; 14: 81-89Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar It is conceivable that some disease-causing mutations lie in gene regulatory regions. Furthermore, a known feature of CF is the scarce genotype-phenotype correlation and the different expression of the disease in patients with CF bearing the same genotype5Castaldo G. Rippa E. Salvatore D. Sibillo R. Raia V. de Ritis G. Salvatore F. Severe liver impairment in a cystic fibrosis-affected child homozygous for the G542X mutation.Am J Med Genet. 1997; 69: 155-158Crossref PubMed Scopus (13) Google Scholar and in sib-pairs with CF.6Castaldo G. Fuccio A. Salvatore D. Raia V. Santostasi T. Leonardi S. Lizzi N. La Rosa M. Rigillo N. Salvatore F. Liver expression in cystic fibrosis could be modulated by genetic factors different from the cystic fibrosis transmembrane regulator genotype.Am J Med Genet. 2001; 98: 294-297Crossref PubMed Scopus (48) Google Scholar A variety of modifier genes of the CF phenotype7Salvatore F. Scudiero O. Castaldo G. Genotype-phenotype correlation in cystic fibrosis: the role of modifier genes.Am J Med Genet. 2002; 111: 88-95Crossref PubMed Scopus (148) Google Scholar can explain the phenotypic heterogeneity, but each acts in a small percentage of patients with CF.8Tomaiuolo R. Degiorgio D. Coviello D.A. Baccarelli A. Elce A. Raia V. Motta V. Seia M. Castaldo G. Colombo C. An MBL2 haplotype and ABCB4 variants modulate the risk of liver disease in cystic fibrosis patients: a multicentric study.Digest Liver Dis. 2009; 41: 817-822Abstract Full Text Full Text PDF Scopus (28) Google Scholar, 9Bartlett J.R. Friedman K.J. Ling S.C. Pace R.G. Bell S.C. Bourke B. Castaldo G. Castellani C. Cipolli M. Colombo C. Colombo J.L. Debray D. Fernandez A. Lacaille F. Macek Jr., M. Rowland M. Salvatore F. Taylor C.J. Wainwright C. Wilschanski M. Zemková D. Hannah W.B. Phillips M.J. Corey M. Zielenski J. Dorfman R. Wang Y. Zou F. Silverman L.M. Drumm M.L. Wright F.A. Lange E.M. Durie P.R. Knowles M.R. Gene Modifier Study GroupGenetic modifiers of liver disease in cystic fibrosis.JAMA. 2009; 302: 1076-1083Crossref PubMed Scopus (219) Google Scholar These results prompted studies of noncoding regions of CFTR, including intronic10Elce A. Boccia A. Cardillo G. Giordano S. Tomaiuolo R. Paolella G. Castaldo G. Three novel CFTR polymorphic repeats improve segregation analysis for cystic fibrosis.Clin Chem. 2009; 55: 1372-1379Crossref PubMed Scopus (33) Google Scholar and flanking regions, potentially involved in the regulation of gene expression.11McCarthy V.A. Harris A. The CFTR gene and regulation of its expression.Pediatr Pulmonol. 2005; 40: 1-8Crossref PubMed Scopus (58) Google Scholar The CFTR gene shows clear temporal and developmental regulation of its expression, but the molecular mechanisms underlying the transcriptional control in different tissues and organs are still poorly understood.11McCarthy V.A. Harris A. The CFTR gene and regulation of its expression.Pediatr Pulmonol. 2005; 40: 1-8Crossref PubMed Scopus (58) Google Scholar In fact, few regulatory elements have been detected so far. Analysis of 3.8 kb of genomic sequence upstream of exon 1 of the CFTR gene revealed high GC content (65%), no TATA box, multiple transcriptional start sites, and several potential Sp1 and AP-1 protein binding sites.11McCarthy V.A. Harris A. The CFTR gene and regulation of its expression.Pediatr Pulmonol. 2005; 40: 1-8Crossref PubMed Scopus (58) Google Scholar Moreover, the presence of conserved sequence tags12Boccia A. Petrillo M. di Bernardo D. Guffanti A. Mignone F. Confalonieri S. Luzi L. Pesole G. Paolella G. Ballabio A. Banfi S. DG-CST (Disease Gene Conserved Sequence Tags), a database of human-mouse conserved elements associated to disease genes.Nucleic Acids Res. 2005; 33: D505-D510Crossref PubMed Scopus (13) Google Scholar confirms that the region at the 5′ of CFTR may have a relevant role in the regulation of CFTR expression. Mutations in this region may impair the interaction between the gene and regulatory factors and act as disease-causing mutations; moreover, they may modulate expression of the gene (and thus of the disease) at different organs and tissue levels. Some examples of mutations that interfere with CFTR transcriptional activity13Taulan M. Lopez E. Guittard C. René C. Baux D. Altieri J.P. DesGeorges M. Claustres M. Romey M.C. First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding.Biochem Biophys Res Commun. 2007; 361: 775-781Crossref PubMed Scopus (23) Google Scholar have been described in the more proximal 5′ region of CFTR in patients with CF14Romey M.C. Guittard C. Carles S. Demaille J. Claustres M. Ramsay M. First putative sequence alterations in the minimal CFTR promoter region.J Med Genet. 1999; 36: 263-264PubMed Google Scholar and CFTR-RDs,15Lopez E. Viart V. Guittard C. Templin C. René C. Méchin D. Des Georges M. Claustres M. Romey-Chatelain M.C. Taulan M. Variants in CFTR untranslated regions are associated with congenital bilateral absence of the vas deferens.J Med Genet. 2011; 48: 152-159Crossref PubMed Scopus (25) Google Scholar but for other mutations identified in single patients (such as those reported in the Cystic Fibrosis Mutation Database, http://www.genet.sickkids.on.ca/Home.html, last accessed March 13, 2013.), the lack of expression studies did not permit definition of the functional role. We studied the 6000-bp region at the 5′ of CFTR in a large group of patients with CF and CFTR-RDs, most with one or both unknown mutations after scanning of the CFTR coding region, and in non-CF controls. We expressed some mutations in four cell systems to define their effect. This study was performed on 118 unrelated Italian patients (Table 1) affected by CF (58 cases) or CFTR-RDs (60 cases). In detail, we studied i) 20 patients with CF homozygous for the F508del mutation, aged >18 years, and classified as having severe pulmonary and liver expression (n = 10) or mild pulmonary and no liver expression (n = 10) as previously described7Salvatore F. Scudiero O. Castaldo G. Genotype-phenotype correlation in cystic fibrosis: the role of modifier genes.Am J Med Genet. 2002; 111: 88-95Crossref PubMed Scopus (148) Google Scholar, 8Tomaiuolo R. Degiorgio D. Coviello D.A. Baccarelli A. Elce A. Raia V. Motta V. Seia M. Castaldo G. Colombo C. An MBL2 haplotype and ABCB4 variants modulate the risk of liver disease in cystic fibrosis patients: a multicentric study.Digest Liver Dis. 2009; 41: 817-822Abstract Full Text Full Text PDF Scopus (28) Google Scholar; ii) 38 patients with CF with one (n = 32) or both (n = 6) undetected mutations after analysis of the most frequent CFTR mutations in this population,16Castaldo G. Fuccio A. Cazeneuve C. Picci L. Salvatore D. Raia V. Scarpa M. Goossens M. Salvatore F. Detection of five rare cystic fibrosis mutations peculiar to Southern Italy: implications in screening for the disease and phenotype characterization for patients with homozygote mutations.Clin Chem. 1999; 45: 957-962PubMed Google Scholar CFTR gene scanning,2Castaldo G. Polizzi A. Tomaiuolo R. Cazeneuve C. Girodon E. Santostasi T. Salvatore D. Raia V. Rigillo N. Goossens M. Salvatore F. Comprehensive cystic fibrosis mutation epidemiology and haplotype characterization in a southern Italy population.Ann Hum Genet. 2005; 69: 15-24Crossref PubMed Scopus (45) Google Scholar and the search for large gene rearrangements3Tomaiuolo R. Sangiuolo F. Bombieri C. Bonizzato A. Cardillo G. Raia V. D'Apice M.R. Bettin M.D. Pignatti P.F. Castaldo G. Novelli G. Epidemiology and a novel procedure for large scale analysis of CFTR rearrangements in classic and atypical CF patients: a multicentric Italian study.J Cyst Fibros. 2008; 7: 347-351Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar; and iii) 60 patients with CFTR-RDs4Amato F. Bellia C. Cardillo G. Castaldo G. Ciaccio M. Elce A. Lembo F. Tomaiuolo R. Extensive molecular analysis of patients bearing CFTR-related disorders.J Mol Diagn. 2012; 14: 81-89Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar who had normal sweat chloride levels (ie, chloride <60 mEq/L) on two test occasions, no familiarity for CF, and the absence of colonization by Pseudomonas aeruginosa and other pathogens typically identified in sputum from patients with CF. None of these patients met the criteria for CF diagnosis.17Rosenstein B.J. Cutting G.R. Cystic Fibrosis Foundation Consensus PanelThe diagnosis of cystic fibrosis: a consensus statement.J Pediatr. 1998; 132: 589-595Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar Of these 60 patients with CFTR-RDs, 11 had recurrent pancreatitis, 11 had disseminated bronchiectasis, and 38 had congenital bilateral absence of vasa deferentia (CBAVD) characterized by azoospermia with a low semen plasma volume (<1.5 mL) and low pH ( A/unknown; during the present study, we revealed the second mutation (ie, Y849X).PositiveSevere1CFMutation/TG12-T5-470VBorderlineMild1CFU/UPositiveVariable6CBAVDMutation/UNegativeCBAVD alone32CBAVDU/UNegativeCBAVD alone6Recurrent pancreatitisMutation/UNegativePS, no L, no (or mild) P11Disseminated bronchiectasisMutation/UNegativePS, no L, no (or mild) P11CF, cystic fibrosis; CBAVD, congenital bilateral absence of vasa deferents; L, liver expression; P, pulmonary expression.∗ The patient originally had the genotype 2789 + 5G>A/unknown; during the present study, we revealed the second mutation (ie, Y849X). Open table in a new tab CF, cystic fibrosis; CBAVD, congenital bilateral absence of vasa deferents; L, liver expression; P, pulmonary expression. Genomic DNA was extracted from whole blood samples collected on EDTA (Nucleon BACC3; Amersham Biosciences, Little Chalfont, UK). To detect CFTR gene mutations, we analyzed a panel of the most frequent mutations in Italy and then scanned all 27 exons and the intronic boundaries by denaturing gradient gel electrophoresis and gene sequencing.2Castaldo G. Polizzi A. Tomaiuolo R. Cazeneuve C. Girodon E. Santostasi T. Salvatore D. Raia V. Rigillo N. Goossens M. Salvatore F. Comprehensive cystic fibrosis mutation epidemiology and haplotype characterization in a southern Italy population.Ann Hum Genet. 2005; 69: 15-24Crossref PubMed Scopus (45) Google Scholar Large CFTR rearrangements were tested by scanning all the CFTR exons using a commercial kit based on quantitative PCR followed by capillary electrophoresis (MLPA SALSA kit; MRC-Holland, Amsterdam, The Netherlands). Molecular analysis of the 6000-bp region at the 5′ of the gene was performed by direct sequencing. DNA was amplified as seven fragments of various lengths (400 to 900 bp) by PCR using the Veriti thermal cycler (Applied Biosystems, Foster City, CA) and the HotStarTaq DNA polymerase kit (Qiagen Inc., Valencia, CA); the sequences of the PCR primers are listed in Table 2. The PCR was standardized according to the manufacturer's instructions. The assay was performed in a 30-μL reaction mixture containing 2 μL of genomic DNA (120 ng), 8 μmol/L of each primer, 3 μL of the 10X buffer, 10 mmol/L dNTPs, HotStarTaq DNA polymerase (0.75 U per reaction), and autoclaved water. The cycling conditions were as follows: HotStarTaq DNA polymerase activation at 95°C for 15 minutes, 50 cycles at 94°C for 30 seconds, 54° to 58°C for 30 seconds, and 72°C for 1 minute, with a final extension at 72°C for 10 minutes. All amplification products were checked by electrophoresis in agarose gel (20 g/L) with the Tris-borate-EDTA buffer and then were sequenced.Table 2Sequences of OligonucleotidesPrimer namePrimer sequenceCFTR promoter sequencing primersFragment from −263 to +97 forward5′-CCGGTAATTACGCAAAGCAT-3′Fragment from −263 to +97 reverse5′-CTGGGCTCAAGCTCCTAATG-3′Fragment from −1276 to −333 forward5′-TTTGGGTGACCACAAGTCAA-3′Fragment from −1276 to −333 reverse5′-AACGCTGGAGGACAGAAGAA-3′Fragment from −2219 to −1256 forward5′-TTTCTGCTTTCCTGTTTCATTG-3′Fragment from −2219 to −1256 reverse5′-TTGACTTGTGGTCACCCAAA-3′Fragment from −3138 to −2197 forward5′-TGTAAGAAGCACCCAGCACA-3′Fragment from −3138 to −2197 reverse5′-CAATGAAACAGGAAAGCAGAAA-3′Fragment from −4078 to −3118 forward5′-GCTAAGTGTGGTGCCAGGAT-3′Fragment from −4078 to −3118 reverse5′-TGTCTGGGTCTTCTTACA-3′Fragment from −5049 to −4058 forward5′-GCAAAGGGACATTTTCACCA-3′Fragment from −5049 to −4058 reverse5′-ATCCTGGCACCACACTTAGC-3′Fragment from −6000 to −5029 forward5′-GTGACTTCATGTCCCGTCCT-3′Fragment from −6000 to −5029 reverse5′-TGGTGAAAATGTCCCTTTGC-3′Expression vector construct primersConstruct 1 forward5′-TTTCTGCTTTCCTGTTTCATTG-3′Construct 1 reverse5′-GACCCGAGTTCGAGGATTAC-3′Construct 2 forward5′-GTGACTTCATGTCCCGTCCT-3′Construct 2 reverse5′-ACCACTTTTACAGGGAAACG-3′Construct 3 forward5′-TGTTAGTGCCCATGTGCAAT-3′Construct 3 reverse5′-AACTGAACACCAGTGGGTTT-3′Site-directed mutagenesis primers−542pGL3B forward5′-ATACGAAAGGCACACTTTCCTTCCCTTTTC-3′−542pGL3B reverse5′-GGGAAGGAAAGTGTGCCTTTCGTATATCAA-3′−680pGL3B forward5′-TTGGAGTTCACGCACCTAAACCTGAAACTA-3′−680pGL3B reverse5′-TCAGGTTTAGGTGCGTGAACTCCAAGGGTG-3′−1176pGL3B forward5′-TACTTTCCTTTGAGTTTTTCAATTCAAACACAATGTATGCTTGC-3′−1176pGL3B reverse5′-CATACATTGTGTTTGAATTGAAAAACTCAAAGGAAAGTAAAAAT-3′c.-1773_-1772delATpGL3B forward5′-GAGTTCAATCACGTCTGGGAAAAGTCAATAG-3′c.-1773_-1772delATpGL3B reverse5′-CTTTTCCCAGACGTGATTGAACTCACCACAT-3′−2068pGL3B forward5′-ACACAGTGATAGGAATAATGGTTTAGAACT-3′−2068pGL3B reverse5′-TAAACCATTATTCCTATCACTGTGTAATAC-3′−3500pGL3B forward5′-CACTGTTGAATAGCTGTGGCTGTTCTTACC-3′−3500pGL3B reverse5′-AACAGCCACAGCTATTCAACAGTGGGCCAC-3′−5183pGL3B forward5′-TAAGACTTCCTTAATAAGAAACTACCTTTA-3′−5183pGL3B reverse5′-GGTAGTTTCTTATTAAGGAAGTCTTAAAGA-3′−5782pGL3B forward5′-TATTGCCTTTTCTCAGATATCAGGTTATGAGAATAATA-3′−5782pGL3B reverse5′-ATTCTCATAACCTGATATCTGAGAAAAGGCAATATGTA-3′ Open table in a new tab The recommendations of the Human Genome Variation Society18den Dunnen J.T. Antonarakis S.E. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion.Hum Mutat. 2000; 15: 7-12Crossref PubMed Scopus (1503) Google Scholar were followed in naming the mutations, using + 1 as the A of the initiation ATG codon in the reference sequence NM_000492.3. Table 3 reports the official nomenclature and the current legacy name for each mutation.Table 3Gene Variants Identified in the Region of 6000 bp at the 5′ of CFTRVariant (cDNA name)Variant (legacy name)No. (%) of allelesControl subjects (150 alleles)Patients with CF (116 alleles)Patients with CFTR-RDs (120 alleles)c.-274C>A-142C>A5 (3.3)00c.-275G>A∗Novel variants.-143G>A2 (1.3)01 (0.8)c.-461A>G-329A/G3 (2.0)01 (0.6)c.-593A>G-461A->G2 (1.3)2 (1.7)2 (1.7)c.-674T>C∗Novel variants.-542T>C003 (2.5)c.-737G>A5 (3.3)00c.-751A>G2 (1.3)2 (1.7)2 (1.7)c.-812T>G-680T>G2 (1.3)02 (1.7)c.-869T[8_9]-790T9/84 (2.7)1 (0.9)0c.-887C>T-816C->T4 (2.7)1 (0.9)0c.-966T>G-834T>G9 (6.0)2 (1.7)1 (0.8)c.-1043dupT-911dupT6 (4.0)00c.-1043delT-911delT1 (0.6)01 (0.8)c.-1308A>G-1176A>G03 (2.6)0c.-1773_-1772delAT∗Novel variants.-1641-1640delAT02 (1.7)0c.-2200G>A∗Novel variants.-2068G>A2 (1.3)1 (0.9)0c.-3136T>G∗Novel variants.-3004T>G4 (2.7)00c.-3632G>T∗Novel variants.-3500G>T2 (1.3)1 (0.9)0c.-3966T>C-3834T>C40 (26.7)83 (71.6)24 (20.0)c.-5315G>A∗Novel variants.-5183G>A1 (0.6)01 (0.8)c.-5671C>T-5539C>T41 (27.3)88 (75.9)29 (24.2)c.-5914A>G∗Novel variants.-5782A>G001 (0.8)c.-5947T>G∗Novel variants.-5815T>G4 (2.7)00∗ Novel variants. Open table in a new tab Total RNA was isolated from frozen pellets of the HepG2, HeLa, PanC-1, and A549 cell lines using TRIzol reagent (Invitrogen, Carlsband, CA) according to the manufacturer's protocol. RNA concentration and purity were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE); reverse transcription was performed on 1 μg of total RNA resuspended in diethyl-pyrocarbonate–treated Nanopure water using a QuantiTect reverse transcription kit (Qiagen Inc.) and the protocol supplied by the manufacturer. To check levels of CFTR transcripts in the HepG2, HeLa, PanC-1, and A549 cell lines, relative quantification of real-time PCR was performed in duplicate using the LightCycler 480 Probes Master containing CFTR primers and a TaqMan CFTR probe (CFTR and H. sapiens, ID. Assay 102716; Roche Diagnostics GmbH, Mannheim, Germany). Amplification was performed using the LightCycler 480 systems for real-time PCR (Roche) with a two-step PCR protocol (preincubation of 10 minutes at 95°C followed by 45 cycles of amplification at 95°C for 10 seconds, 60°C for 10 seconds, and 72°C for 1 second). mRNA quantification results were normalized using the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (ID. Assay 101128; Roche) as an endogenous control. In all the studies, each sample was run in triplicate, and the CT method (ΔΔCT method) was used to examine the relative quantification of the samples. Fold expression changes were calculated using the following equation: 2 − ΔΔCT. To facilitate the promoter cloning, we divided the region of 6000 bp into three fragments that were cloned progressively. Construct 1 extends from positions −2219 to 97 (referred to as the major transcription initiation site), construct 2 extends from nucleotides −6000 to −5030, and construct 3 extends from nucleotides −5727 to −1256. Segments were amplified using specific primers (Table 2), and these primers were flanked at the 5′ end by a synthetic restriction enzyme site; the reaction was performed using a PfuUltra high-fidelity DNA polymerase (Stratagene, an Agilent Technologies company, La Jolla, CA). Construct 1 had at the 5′ end an XhoI restriction site and at the 3′ end an XbaI restriction site; construct 2 had at the 5′ end a KpnI restriction site and at the 3′ end an XbaI restriction site; and the last fragment had at both ends an XbaI restriction site. The integrity and accuracy of the wild-type (WT) cloned promoter regions in the constructs were checked by DNA sequencing. After this step, they were inserted in sequence in a temporary expression vector (pGEM-T vector; Promega Corp., Madison, WI) using the T4 DNA ligase kit (Promega Corp.). Then, the entire WT 6-kb construct was excised from the pGEM-T vector by a restriction reaction with KpnI and XhoI endonucleases and then was ligated into a pGL3-Basic vector (Promega Corp.); this vector contains a modified firefly luciferase coding sequence upstream of the SV40 late poly(A) signal and no eukaryotic promoter or enhancer sequences. Mutagenesis of the WT–pGL3-Basic 6-kb construct was achieved by using a QuikChange II XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions; all the primers used are listed in Table 2. Subsequently, we checked for each genetic variant by direct sequencing. Plasmids were sequenced after site-directed mutagenesis to confirm the changes and to rule out additional nonspecific changes. Cell lines were purchased from ATCC (Manassas, VA). Human hepatocellular carcinoma cells (HepG2, ATCC number HB-8065), human cervical carcinoma cells (HeLa, ATCC number HB-8065), human lung carcinoma cells (A549, ATCC number CCL-185), and epithelioid carcinoma pancreatic duct cells (PanC-1, ATCC number CRL-1469) were maintained in Dulbecco's modified Eagle's medium (Gibco Invitrogen, Grand Island, NY) with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT) without the addition of antibiotics; cell cultures were maintained in a 5% (v/v) CO2–humidified atmosphere at 37°C. For HeLa and A549 cells, transient transfection was performed using Attractene reagent (Qiagen Inc.), whereas PanC-1 and HepG2 cells were transfected using Lipofectamine 2000 CD and Lipofectamine LTX with Plus reagent (Invitrogen), respectively, according to the manufacturer's specifications. All the cells were plated in a 24-well culture dish 24 hours before the experiments. HeLa cells were seeded at a density of approximately 8 × 104 per well; A549 at 5 × 104 per well; PanC-1 at 2 × 104 per well; and HepG2 at 1.2 × 105 per well. When cells reached an appropriate confluence (approximately 50% to 60% for HepG2 and PanC-1 cells and 70% to 80% for HeLa and A549 cells), they were cotransfected with 700 ng per well of plasmid reporter and 50 ng per well of internal control pRL-CMV containing Renilla luciferase (Promega Corp.) driven by a cytomegalovirus promoter to normalize transfection efficiency. Because pGL3-Basic vector lacks any eukaryotic promoter or enhancer sequences, it was used as a negative control. Cells were grown for an additional 24 hours, washed in phosphate-buffered saline, lysed in 100 μL of passive lysis buffer (Promega Corp.), collected, and centrifuged for 10 minutes at 12,000 rpm to remove cell debris. Ten microliters of cleared lysate was assayed sequentially for firefly and Renilla luciferase activity using the Dual-Luciferase Reporter Assay system (Promega Corp.) according to the manufacturer's recommendations. Luminescence measurements were performed using a VICTOR3-1420 multilabel counter (PerkinElmer, Waltham, MA) with a 5-second measurement period; firefly luciferase activity was normalized to Renilla luciferase activity. Experiments were made at least in duplicate, and each set of transfections was repeated four times for each cell line with independently purified plasmid DNA preparations and reaction mix. Comparisons of promoter activity between WT and variant CFTR promoter constructs from luciferase assays were conducted by using the unpaired t-test; data were considered statistically significant at P < 0.001; data are expressed as means ± SE of four different assays. Molecular analysis of the 6000-bp region at the 5′ of the CFTR gene was performed in all patients with CF and CFTR-RDs and in control subjects. We identified 23 mutations, 9 of which are novel (Figure 1). A summary of these gene variants appears in Table 3. Two mutations have a high frequency: c.-3966T>C, with an allele frequency ranging from 20.0% in patients with CFTR-RDs to 71.6% in patients with CF, and c.-5671C>T, with an allele frequency ranging from 24.2% in patients with CFTR-RDs to 75.9% in patients with CF. The high frequency of such mutations in patients depends on the linkage disequilibrium of both the mutations with the F508del, which is the most frequent mutation in this series of patients. c.-274C>A, c.-737G>A, c.-966T>G, and c.-1043dupT have allelic frequencies of 3.3%, 3.3%, 6.0%, and 4.0%, respectively, in control subjects. The other 17 mutations have allelic frequencies <3.0% and were expressed in vitro. To study the functional effect of CFTR mutations, we used four cell systems with different baseline levels of CFTR expression because such cells would produce different (levels of) CFTR interactors. We tested CFTR expression by real time-PCR and reported the result as a ratio to GAPDH mRNA. As shown in Figure 2, no basal CFTR expression was found in HepG2 cells, HeLa cells expressed low basal levels of CFT
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