Comprehensive and Rapid Genotyping of Mutations and Haplotypes in Congenital Bilateral Absence of the Vas Deferens and Other Cystic Fibrosis Transmembrane Conductance Regulator-Related Disorders
2007; Elsevier BV; Volume: 9; Issue: 5 Linguagem: Inglês
10.2353/jmoldx.2007.070040
ISSN1943-7811
AutoresCorinne Bareil, Caroline Guittard, Jean-Pierre Altiéri, Carine Templin, Mireille Claustres, Marie des Georges,
Tópico(s)Congenital Ear and Nasal Anomalies
ResumoAvailable commercial kits only screen for the most common cystic fibrosis transmembrane conductance regulator (CFTR) mutations causing classic cystic fibrosis and for the Tn variant in IVS8. However, full scanning of CFTR is needed for the diagnosis of patients with cystic fibrosis or CFTR-related disorders (including congenital bilateral absence of the vas deferens) bearing rare mutations. Standard strategies for detecting point mutations rely on extensive scanning of the gene by denaturing gradient gel electrophoresis or denaturing high performance liquid chromatography, which are time-consuming. Moreover, the haplotyping of IVS8-(TG)m and Tn tracts is still challenging despite several recent improvements. We have optimized both the detection of mutations and the haplotyping of IVS8 polyvariants in developing two methods: i) a rapid and robust direct sequence analysis of all exons/flanking introns of the CFTR gene based on single condition touchdown amplification/sequencing in 96-well plates, and ii) a fluorescent assay that allows haplotyping of IVS8-(TG)mTn even without family linkage study. Combined with search for rare large rearrangements, this strategy detected 87.9% of CFTR defects in congenital bilateral absence of the vas deferens patients, a proportion considerably higher than those usually reported. These highly efficient tests, scanning each sample in a few days, greatly improve the genotyping of patients with CFTR-related symptoms and may be particularly important in emergency situations such as fetus with hyperechogenic bowel suggestive of cystic fibrosis. Available commercial kits only screen for the most common cystic fibrosis transmembrane conductance regulator (CFTR) mutations causing classic cystic fibrosis and for the Tn variant in IVS8. However, full scanning of CFTR is needed for the diagnosis of patients with cystic fibrosis or CFTR-related disorders (including congenital bilateral absence of the vas deferens) bearing rare mutations. Standard strategies for detecting point mutations rely on extensive scanning of the gene by denaturing gradient gel electrophoresis or denaturing high performance liquid chromatography, which are time-consuming. Moreover, the haplotyping of IVS8-(TG)m and Tn tracts is still challenging despite several recent improvements. We have optimized both the detection of mutations and the haplotyping of IVS8 polyvariants in developing two methods: i) a rapid and robust direct sequence analysis of all exons/flanking introns of the CFTR gene based on single condition touchdown amplification/sequencing in 96-well plates, and ii) a fluorescent assay that allows haplotyping of IVS8-(TG)mTn even without family linkage study. Combined with search for rare large rearrangements, this strategy detected 87.9% of CFTR defects in congenital bilateral absence of the vas deferens patients, a proportion considerably higher than those usually reported. These highly efficient tests, scanning each sample in a few days, greatly improve the genotyping of patients with CFTR-related symptoms and may be particularly important in emergency situations such as fetus with hyperechogenic bowel suggestive of cystic fibrosis. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are responsible for cystic fibrosis (CF; Online Mendelian Inheritance of Man no. 219700) and isolated congenital bilateral absence of the vas deferens (CBAVD; Online Mendelian Inheritance of Man no. 277180). More than 96% of the 1500 CFTR defects reported so far are point mutations altering a few bases or only one base (http://www.genet.sickkids.on.ca), whereas an unknown proportion of CFTR dysfunctions are caused by large genomic rearrangements such as large deletions.1Audrézet MP Chen JM Raguenes O Chuzhanova N Giteau K Le Marechal C Quere I Cooper DN Ferec C Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms.Hum Mutat. 2004; 23: 343-357Crossref PubMed Scopus (110) Google Scholar2Niel F Martin J Dastot-Le Moal F Costes B Boissier B Delattre V Goossens M Girodon E Rapid detection of CFTR gene rearrangements impacts on genetic counselling in cystic fibrosis.J Med Genet. 2004; 41: e118Crossref PubMed Scopus (63) Google Scholar3Bombieri C Bonizzato A Castellani C Assael BM Pignatti PF Frequency of large CFTR gene rearrangements in Italian CF patients.Eur J Hum Genet. 2005; 13: 687-689Crossref PubMed Scopus (26) Google Scholar4Hantash FM Redman JB Starn K Anderson B Buller A McGinniss MJ Quan F Peng M Sun W Strom CM Novel and recurrent rearrangements in the CFTR gene: clinical and laboratory implications for cystic fibrosis screening.Hum Genet. 2006; 119: 126-136Crossref PubMed Scopus (34) Google Scholar5Hantash FM Milunsky A Wang Z Anderson B Sun W Anguiano A Strom CM A large deletion in the CFTR gene in CBAVD.Genet Med. 2006; 8: 93-95Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar6Niel F Legendre M Bienvenu T Bieth E Lalau G Sermet I Bondeux D Boukari R Derelle J Levy P Ruszniewski P Martin J Costa C Goossens M Girodon E A new large CFTR rearrangement illustrates the importance of searching for complex alleles.Hum Mutat. 2006; 27: 716-717Crossref PubMed Scopus (23) Google Scholar7Ratbi I Legendre M Niel F Martin J Soufir JC Izard V Costes B Costa C Goossens M Girodon E Detection of cystic fibrosis transmembrane conductance regulator (CFTR) gene rearrangements enriches the mutation spectrum in congenital bilateral absence of the vas deferens and impacts on genetic counselling.Hum Reprod. 2007; 22: 1285-1291Crossref PubMed Scopus (66) Google Scholar In CBAVD, 88% of patients found with two CFTR mutations carry a severe mutation (no CFTR function) in trans to a mild mutation (residual function), and 12% carry two mild mutations.8Claustres M Guittard C Bozon D Chevalier F Verlingue C Ferec C Girodon E Cazeneuve C Bienvenu T Lalau G Dumur V Feldmann D Bieth E Blayau M Clavel C Creveaux I Malinge MC Monnier N Malzac P Mittre H Chomel JC Bonnefont JP Iron A Chery M Georges MD Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.Hum Mutat. 2000; 16: 143-156Crossref PubMed Scopus (239) Google Scholar The vast majority of mutations in CBAVD are not detected by routine panels, designed to test up to 30 common severe CF mutations; they are scattered over the whole gene, so that all of the exons and their flanking introns have to be extensively scanned to reach acceptable rates of mutation detection.8Claustres M Guittard C Bozon D Chevalier F Verlingue C Ferec C Girodon E Cazeneuve C Bienvenu T Lalau G Dumur V Feldmann D Bieth E Blayau M Clavel C Creveaux I Malinge MC Monnier N Malzac P Mittre H Chomel JC Bonnefont JP Iron A Chery M Georges MD Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.Hum Mutat. 2000; 16: 143-156Crossref PubMed Scopus (239) Google Scholar,9Claustres M Molecular pathology of the CFTR locus in male infertility.Reprod Biomed Online. 2005; 10: 14-41Abstract Full Text PDF PubMed Scopus (92) Google Scholar The laborious but powerful manual DGGE (denaturing gradient gel electrophoresis) or DHPLC (denaturing high-performance liquid chromatography) techniques represented until recently the most useful approaches for mutation detection in CBAVD. However, these techniques are unable to determine the length variants localized at the polypyrimidine locus upstream to the splice acceptor site of intron 8 (polyTG followed by polyT repeats) that affect the splicing efficiency of exon 9 and act as genetic modifiers of CFTR function. Five variants, (TG)9 to (TG)13, are known in the (TG)m tract, whereas up to seven different alleles have been reported in the Tn tract (common alleles with nine, seven, or five thymidines and rare alleles with three,10Buratti E Baralle FE Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9.J Biol Chem. 2001; 276: 36337-36343Crossref PubMed Scopus (487) Google Scholar six,11Viel M Leroy C Des Georges M Claustres M Bienvenu T Novel length variant of the polypyrimidine tract within the splice acceptor site in intron 8 of the CFTR gene: consequences for genetic testing using standard assays.Eur J Hum Genet. 2005; 13: 136-138Crossref PubMed Scopus (10) Google Scholar 10,12Millson A Pont-Kingdon G Page S Lyon E Direct molecular haplotyping of the IVS-8 poly(TG) and polyT repeat tracts in the cystic fibrosis gene by melting curve analysis of hybridization probes.Clin Chem. 2005; 51: 1619-1623Crossref PubMed Scopus (16) Google Scholar or 1113Kobler D Modi H Goldman B Identification of an 11T allele in the polypyrimidine tract of intron 8 of the CFTR gene.Genet Med. 2006; 8: 125-128Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar thymidines). In Caucasian populations, the frequency of IVS8-T5 allele in CBAVD patients (30%) is six times higher than in the general population (5%), and 34% of men with CBAVD have inherited a CFTR mutation on one gene and IVS8-T5 on the other one, making this combination the most common cause of CBAVD. The T5 variant is associated with high levels of exon 9 skipping, which results in the production of a nonfunctional CFTR protein. However, the splicing efficiency of the IVS8-T5 allele shows inter- and intraindividual variability, and its incomplete penetrance in CBAVD is largely influenced by the IVS8-(TG)m tract.14Cuppens H Lin W Jaspers M Costes B Teng H Vankeerberghen A Jorissen M Droogmans G Reynaert I Goossens M Nilius B Cassiman JJ Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes: the polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation.J Clin Invest. 1998; 101: 487-496Crossref PubMed Scopus (352) Google Scholar,15Disset A Michot C Harris A Buratti E Claustres M Tuffery-Giraud S A T3 allele in the CFTR gene exacerbates exon 9 skipping in vas deferens and epididymal cell lines and is associated with congenital bilateral absence of vas deferens (CBAVD).Hum Mutat. 2005; 25: 72-81Crossref PubMed Scopus (46) Google Scholar The determination of (TG)m repeat number is predictive of pathogenic T5 alleles.16Groman JD Hefferon TW Casals T Bassas L Estivill X Des Georges M Guittard C Koudova M Fallin MD Nemeth K Fekete G Kadasi L Friedman K Schwarz M Bombieri C Pignatti PF Kanavakis E Tzetis M Schwartz M Novelli G D'Apice MR Sobczynska-Tomaszewska A Bal J Stuhrmann M Macek Jr, M Claustres M Cutting GR Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.Am J Hum Genet. 2004; 74: 176-179Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar Longer TG repeats increase exon 9 skipping and raise the proportion of nonfunctional CFTR protein, emphasizing the importance of assessing the length not only of the Tn but also of the (TG)m tract for diagnostic purposes. As the two repeats act in concert when modulating exon 9 inclusion or skipping in the CFTR mRNA, reliable haplotyping is an additional prerequisite for meaningful genetic testing at this locus.We have developed two strategies that improve both the identification of mutations and the haplotyping of IVS8 repeats. First, a method based on single condition touchdown amplification (SiCTA) in a 96-well plate format allows the rapid direct sequence analysis of all CFTR exons and flanking introns together with a portion of IVS11 and IVS19 sequences (which carry common mutations). Second, a fluorescent assay determines the length of the IVS8-(TG)m and Tn tracts of both alleles and their haplotypes even when familial segregation cannot be studied. These two simple, rapid and reliable assays can routinely be performed in a few working days.Materials and MethodsSamplesWe analyzed or reanalyzed a collection of 182 samples (previous CFTR analysis of 85 of these samples had been included as part of a collaborative study8Claustres M Guittard C Bozon D Chevalier F Verlingue C Ferec C Girodon E Cazeneuve C Bienvenu T Lalau G Dumur V Feldmann D Bieth E Blayau M Clavel C Creveaux I Malinge MC Monnier N Malzac P Mittre H Chomel JC Bonnefont JP Iron A Chery M Georges MD Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.Hum Mutat. 2000; 16: 143-156Crossref PubMed Scopus (239) Google Scholar). Clinical diagnosis of CBAVD was based on clinical examination with impalpable vas deferens, transrectal ultrasonography, semen analysis (volume, pH, and sperm count in accordance with the World Health Organization guidelines; World Health Organization, 1992), and low concentrations of fructose and citrate. Patients with renal abnormalities were excluded. Informed consent was obtained from all patients. DNA was extracted from peripheral blood samples by using standard procedures.Classic Protocols for Analysis of CFTR Mutations and IVS8-Tn AllelesA complete scan of the 27 coding/flanking sequences of the CFTR gene was performed either by DGGE or by DHPLC. In addition, two intronic mutations, 1811+1.6kbA>G in IVS11 and 3849+10kbC>T in IVS19, and variations at locus IVS8-Tn were screened by specific PCR restriction tests. Samples showing abnormal profiles were reamplified from genomic DNA and directly sequenced with the BigDye Terminator v1.1 cycle sequencing kit from Applied Biosystems (Warrington, UK). Samples found with only one or no CFTR disease-causing mutation were further investigated for large rearrangements such as large deletions by a semiquantitative fluorescent PCR assay previously developed in our laboratory that uses three multiplex PCRs covering the entire gene.17Taulan M Girardet A Guittard C Altieri JP Templin C Beroud C des Georges M Claustres M Large genomic rearrangements in the CFTR gene contribute to CBAVD.BMC Med Genet. 2007; 8: 22Crossref PubMed Scopus (38) Google Scholar In most cases, the cis versus trans status of the alterations was obtained by familial segregation.New Protocol for Haplotyping IVS8-(TG)mTn RepeatsA fluorescent assay based on three PCRs was designed. Exon 9 was first amplified using primers 9i5/9i3 described by Zielenski et al.18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar and purified according to the manufacturer's recommendations by QIAquick PCR purification kit (Qiagen, Hilden, Germany) to remove the unincorporated primers. An aliquot of this amplicon was then used as a template for three internal fluorescent PCRs amplifying the (TG)m and the Tn tracts separately and the (TG)mTn tracts simultaneously (Table 1 and Figure 1). Each amplification was performed in a final volume of 100 μl containing PCR buffer 10×, 20 mmol/L of each dNTP, 10 pmol of each primer, 1 U of Taq Polymerase (Applied Biosystems, Branchburg, NJ), and 1 μl of PCR1. After an initial denaturation step at 94°C for 2 minutes, 22 cycles were performed with denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, followed by a final extension step at 72°C for 30 minutes. Aliquots of the three amplicons [(TG)m or Tn repeats, and (TG)mTn haplotypes] were pooled and separated by multicapillary electrophoresis on an ABI 3130xl Genetic Analyzer running GeneMapper v4.0 for allele identification. A control DNA with fully characterized IVS8-(TG)mTn haplotypes was added to the run.Table 1Conditions for PCR Amplification of CFTR Exon 9, IVS8-(TG)m or -Tn Repeats and IVS8-(TG)mTn HaplotypesPrimer nameSequenceReferenceAmplicon lengthAnnealing temperature (°C/no. of cycles)Exon 99i55′-TAATGGATCATGGGCCATGT-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar558 bp for (TG)11T756/209i35′-ACAGTGTTGAATGTGGTGCA-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar(TG)mCF95′-TGAAAATATCTGACAAACTC-3′19Fanen P Ghanem N Vidaud M Besmond C Martin J Costes B Plassa F Goossens M Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions.Genomics. 1992; 13: 770-776Crossref PubMed Scopus (245) Google Scholar67 bp for (TG)1155/22RI8TG85′-(6FAM)GCGGCGGAAACACACACACACACACA-3′This studyTnI9D3T5′-CCGCCGCTGTGTGTGTGTGTGTGTTT-3′Adapted from 20Chillón M Casals T Mercier B Bassas L Lissens W Silber S Romey MC Ruiz-Romero J Verlingue C Claustres M Nunes V Férec C Estivill X Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens.N Engl J Med. 1995; 332: 1475-1480Crossref PubMed Scopus (808) Google Scholar118 bp for T755/22IVS8Rm5′-(HEX)CTGAAGAAGAGGCTGTCATC-3′This studyUnderline, stabilizing tail. IVS8-(TG)mTn, (CF9-IVS8Rm) = 152 bp for (TG)11T7; 55°C/28 cycles. Open table in a new tab New Protocol for CFTR Analysis: Single Condition Touchdown Amplification/SequencingThe 27 coding/flanking sequences and the portions of introns 11 and 19 that contain the common mutations 1811+1.6kbA>G and 3849+10kbC>T, respectively, were amplified in 30 separate amplicons obtained under single PCR cycling conditions in a 96-well plate and sequenced. For exons 6b and 9, two sequencing reactions differing at the 5′ end were necessary to read both strands and avoid two polymorphic regions [IVS6a-(GATT)n and IVS8-(TG)mTn repeats, respectively]. Because of its large size, exon 13 was analyzed by use of two overlapping amplicons. The sequences of the primers were derived from Zielenski et al,18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar or Fanen et al,19Fanen P Ghanem N Vidaud M Besmond C Martin J Costes B Plassa F Goossens M Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions.Genomics. 1992; 13: 770-776Crossref PubMed Scopus (245) Google Scholar with the exception of IVS1121Chillón M Dork T Casals T Gimenez J Fonknechten N Will K Ramos D Nunes V Estivill X A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA→G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.Am J Hum Genet. 1995; 56: 623-629PubMed Google Scholar and IVS1922Highsmith WE Burch LH Zhou Z Olsen JC Boat TE Spock A Gorvoy JD Quittel L Friedman KJ Silverman LM Boucher RC Knowles MR A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations.N Engl J Med. 1994; 331: 974-980Crossref PubMed Scopus (352) Google Scholar; reverse primers for exons 1, 2, and 14b and forward primers for exons 2, 17a, and IVS11 had to be slightly modified to ensure specific amplification. For all studied regions except exons 6b and 9, PCR and sequencing reactions were performed using the same primers. The primer stocks for PCR (a mixture of reverse and forward primers; Table 2) and for sequencing reactions were stored at −20°C in 96-well plates so that both PCRs and sequencing reaction set-up can be done with pipetting robot or multichannel pipettors. PCRs were performed in 96-well plates in a 25-μl final volume containing 1× PCR Master Mix (Promega, Madison, WI), 3.2 pmol of each PCR primer and 10 ng of genomic DNA. The use of a touchdown PCR protocol24Don RH Cox PT Wainwright BJ Baker K Mattick JS 'Touchdown' PCR to circumvent spurious priming during gene amplification.Nucleic Acids Res. 1991; 19: 4008Crossref PubMed Scopus (2236) Google Scholar allows a single amplification condition for all of the exons: a denaturation step at 94°C for 5 minutes, 10 touchdown cycles with annealing temperature decreasing 1°C per cycle (denaturation 94°C for 30 seconds, annealing 60°C for 40 seconds, primer extension 72°C for 1 minute), 30 cycles at the final touchdown temperature (50°C), and a final extension step at 72°C for 8 minutes. To validate specific size and quantity of amplicons, 5 μl of the PCR products were checked by 2% agarose gel electrophoresis. Five μl of each amplicon were then transferred to a 96-well plate and treated with ExoSAP-IT (USB Corporation, Cleveland, OH) for unused nucleotides and primers removal, according to the manufacturer's recommendations. Depending on the number of patients to be sequenced, a single 96-well plate can be used to analyze the CFTR gene either of one patient in both directions (64 sequences) or of three patients in one direction (96 sequences). Sequencing reactions (5-μl final volume) were assembled by transfer of 1.6 pmol of sequencing primer in a 96-well plate along with a 2.5-μl mixture of 1 μl of ABI Prism BigDye terminators (version 1.1), 0.5 μl of 5× buffer mix, and 1 μl of purified PCR product. Cycling conditions followed manufacturer's recommendations. Sequencing products were purified on a 96-well Montage SEQ96 Sequencing Reaction Cleanup Kit (Millipore, Billerica, MA). The samples were run on an ABI 3130xl Genetic Analyzer (Applied Biosystems). The resulting sequences (10,254 bases in total) were analyzed for mutations, independently by two reviewers, using ABI SeqScape v2.5 automated assembly and basecalling software. Setting of the basecaller was according to the manufacturer and recorded any base with a second peak of >5% as mixed. All mutations were confirmed by DGGE, DHPLC, or restriction analysis from a PCR using a different primer pair and a new DNA dilution. For convenience to the clinicians, mutations and polymorphisms were named according to the nomenclature that is used by the members of the CF Genetic Analysis Consortium (http://www.genet.sickkids.on.ca).Table 2Primers Used for PCR Amplification and Sequencing of the CFTR GeneLocationForward sequenceReferenceReverse sequenceReferenceExon 15′-CGTAGTGGGTGGAGAAAGC-3′19Fanen P Ghanem N Vidaud M Besmond C Martin J Costes B Plassa F Goossens M Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions.Genomics. 1992; 13: 770-776Crossref PubMed Scopus (245) Google Scholar5′-AGCGCATCTTTTTAAAAACTGCTTA-3′This studyExon 25′-CAAATCTGTATGGAGACC-3′*The forward primers of exon 2 and IVS11 were adapted from Zielenski et al18 and Chillon et al21, respectively.This study5′-TGTTTGCTTTCTCTTCTCTAAAT-3′This studyExon 35′-CTTGGGTTAATCTCCTTGGA-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar5′-ATTCACCAGATTTCGTAGTC-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google ScholarExon 45′-TCACATATGGTATGACCCTC-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar5′-TTGTACCAGCTCACTACCTA-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google ScholarExon 55′-ATTTCTGCCTAGATGCTGGG-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar5′-AACTCCGCCTTTCCAGTTGT-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google ScholarExon 6a5′-TTAGTGTGCTCAGAACCACG-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar5′-CTATGCATAGAGCAGTCCTG-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google ScholarExon 6b5′-TGGAATGAGTCTGTACAGCG-3′†The 5′ end of exons 6b and 9 were sequenced using two different forward primers to avoid the polymorphic regions [IVS6a-(GATT)n and IVS8-(TG)mTn tracts, respectively]. 5′-GATTTACAGAGATCAGAG-3′†The 5′ end of exons 6b and 9 were sequenced using two different forward primers to avoid the polymorphic regions [IVS6a-(GATT)n and IVS8-(TG)mTn tracts, respectively].18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar5′-GAGGTGGAAGTCTACCATGA-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google ScholarExon 75′-AGACCATGCTCAGATCTTCCAT-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar5′-GCAAAGTTCATTAGAACTGATC-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google ScholarExon 85′-TGAATCCTAGTGCTTGGCAA-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar5′-TCGCCATTAGGATGAAATCC-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google ScholarExon 95′-TAATGGATCATGGGCCATGT-3′†The 5′ end of exons 6b and 9 were sequenced using two different forward primers to avoid the polymorphic regions [IVS6a-(GATT)n and IVS8-(TG)mTn tracts, respectively]. 5′-TTTTTAACAGGGATTTGGGG-3′†The 5′ end of exons 6b and 9 were sequenced using two different forward primers to avoid the polymorphic regions [IVS6a-(GATT)n and IVS8-(TG)mTn tracts, respectively].18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar5′-ACAGTGTTGAATGTGGTGCA-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google ScholarExon 105′-GCAGAGTACCTGAAACAGGA-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar5′-CATTCACAGTAGCTTACCCA-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google ScholarExon 115′-CAACTGTGGTTAAAGCAATAGTGT-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google Scholar5′-GCACAGATTCTGAGTAACCATAAT-3′18Zielenski J Rozmahel R Bozon D Kerem B Grzelczak Z Riordan JR Rommens J Tsui LC Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (496) Google ScholarIVS115′-TTTCTTAATTGTGTGCTGAATAC-3′*The forward primers of exon 2 and IVS11 were adapted from Zielenski et al18 and Chillon et al21, respectively.This study5′-CAGTTCCCATATTAAATAGAAATGA-3′21Chillón M Dork T Casals T Gimenez J Fonknechten N Will K Ramos D Nunes V Estivill X A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA→G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.Am J Hum Genet.
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