Multiplex Ligation-Dependent Probe Amplification Identification of Whole Exon and Single Nucleotide Deletions in the CFTR Gene of Hispanic Individuals with Cystic Fibrosis
2008; Elsevier BV; Volume: 10; Issue: 4 Linguagem: Inglês
10.2353/jmoldx.2008.080004
ISSN1943-7811
AutoresIris Schrijver, Krista Rappahahn, Lynn Pique, Martin Kharrazi, Lee‐Jun C. Wong,
Tópico(s)Tracheal and airway disorders
ResumoA disparity between Caucasian and Hispanic mutation detection for cystic fibrosis continues to exist, although the carrier frequency is only moderately lower in Hispanics. We aimed to identify exonic rearrangements that remained undetected by conventional methods. In seven of 32 cystic fibrosis-affected self-identified Hispanics for whom only one or no mutations were identified by extensive molecular testing, exon deletions appeared to be present with a multiplex ligation-dependent probe amplification (MLPA) assay. Two recurrent deletions (of exons 2–3 and exons 22–23) were identified in one and three patients, respectively (12.5%, 11.1% of unidentified alleles). Two apparently novel deletions (exons 6b and 20) were identified in three additional patients. Subsequent sequencing to characterize deletion breakpoints, however, identified single nucleotide deletions at the probe binding sites close to the ligation point. All resulted in false positive MLPA deletion signals. Interestingly, these mutations were not common in Caucasians, and one (935delA) was common in U.S. Hispanics. On examination of all probe binding sites, we identified a total of 76 reported mutations and five silent variants that immediately surrounded the MLPA ligation sites, with 22 occurring in non-Caucasians. These mutations are not all rare. Thus, apparent exon deletions by MLPA may indicate the presence of both large deletions and point mutations, with important implications for pan-ethnic MLPA testing in cystic fibrosis and other genetic conditions. A disparity between Caucasian and Hispanic mutation detection for cystic fibrosis continues to exist, although the carrier frequency is only moderately lower in Hispanics. We aimed to identify exonic rearrangements that remained undetected by conventional methods. In seven of 32 cystic fibrosis-affected self-identified Hispanics for whom only one or no mutations were identified by extensive molecular testing, exon deletions appeared to be present with a multiplex ligation-dependent probe amplification (MLPA) assay. Two recurrent deletions (of exons 2–3 and exons 22–23) were identified in one and three patients, respectively (12.5%, 11.1% of unidentified alleles). Two apparently novel deletions (exons 6b and 20) were identified in three additional patients. Subsequent sequencing to characterize deletion breakpoints, however, identified single nucleotide deletions at the probe binding sites close to the ligation point. All resulted in false positive MLPA deletion signals. Interestingly, these mutations were not common in Caucasians, and one (935delA) was common in U.S. Hispanics. On examination of all probe binding sites, we identified a total of 76 reported mutations and five silent variants that immediately surrounded the MLPA ligation sites, with 22 occurring in non-Caucasians. These mutations are not all rare. Thus, apparent exon deletions by MLPA may indicate the presence of both large deletions and point mutations, with important implications for pan-ethnic MLPA testing in cystic fibrosis and other genetic conditions. Cystic fibrosis (CF; OMIM #219700) is a common and serious condition with autosomal recessive inheritance. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR; OMIM *602421) on chromosome 7q31 and is characterized by malfunction of chloride ion channels and of transport pathway regulation.1Anderson MP Rich DP Gregory RJ Smith AE Welsh MJ Generation of cAMP-activated chloride currents by expression of CFTR.Science. 1991; 251: 679-682Crossref PubMed Scopus (425) Google Scholar,2Schwiebert EM Egan ME Hwang TH Fulmer SB Allen SS Cutting GR Guggino WB CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.Cell. 1995; 81: 1063-1073Abstract Full Text PDF PubMed Scopus (591) Google Scholar CFTR is primarily expressed in the apical membrane of exocrine epithelial cells. Classic CF is characterized by failure to thrive, recurrent bacterial endobronchitis, progressive decline of lung function, exocrine pancreatic dysfunction, and infertility in males.3Welsh MJ Ramsey BW Accurso F Cutting G Cystic fibrosis.in: Scriver CR Beaudet AL Sly WS Valle D The metabolic and molecular basis of inherited diseases. 8th ed. McGraw-Hill, New York2001: 5121-5188Google Scholar,4Robertson MB Choe KA Joseph PM Review of the abdominal manifestations of cystic fibrosis in the adult patient.Radiographics. 2006; 26: 679-690Crossref PubMed Scopus (83) Google Scholar The phenotype is variable, however, and ranges from mild with limited manifestations to rapid deterioration and death within the first year of life. The severity of clinical manifestations depends on the set of CFTR mutations, modifying genes, and other variables.5Mackie AD Thornton SJ Edenborough FP Cystic fibrosis-related diabetes.Diabet Med. 2003; 20: 425-436Crossref PubMed Scopus (91) Google Scholar6The Cystic Fibrosis Genotype-Phenotype Consortium Correlation between genotype and phenotype in patients with cystic fibrosis.N Engl J Med. 1993; 329: 1308-1313Crossref PubMed Scopus (544) Google Scholar7Zielenski J Genotype and phenotype in cystic fibrosis.Respiration. 2000; 67: 117-133Crossref PubMed Scopus (361) Google Scholar To date, more than 1500 sequence variants have been reported to the Cystic Fibrosis Mutation Database (http://www.genet.sickkids.on.ca/cftr/). These include point mutations, deletions, insertions, frameshift mutations, and splice site variants that lead to protein truncation. The incidence of CF (approximately one in 3000 individuals) is highest in Caucasians and Ashkenazi Jews, which are the most extensively studied populations.8Abeliovich D Labon I Cohen T Springer C Avital A Cutting GR Screening for five mutations detects 97% of cystic fibrosis (CF) chromosomes and predicts a carrier frequency of 1:29 in the Jewish Ashkenazi population.Am J Genet. 1992; 51: 951-956PubMed Google Scholar,9Grody WW Cutting GR Klinger KW Richards CS Watson MS Desnick RJ Subcommittee on Cystic Fibrosis Screening Accreditation of Genetic Services Committee, ACMG American College of Medical Genetics Laboratory standards and guidelines for population-based cystic fibrosis carrier screening.Genet Med. 2001; 3: 149-154Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar In other ethnic backgrounds, the incidence is lower and knowledge of mutation spectra is still somewhat limited. It is clear, however, that the range and frequency of individual CFTR mutations varies considerably among different populations, ethnic backgrounds, and geographic locations.10Alper OM Wong LJ Young S Pearl M Graham S Sherwin J Nussbaum E Nielson D Platzker A Davies Z Lieberthal A Chin T Shay G Hardy K Kharrazi M Identification of novel and rare mutations in California Hispanic and African American cystic fibrosis patients.Hum Mutat. 2004; 24 (Erratum: Hum Mutat 2005, 25:223): 353Crossref PubMed Scopus (33) Google Scholar,11Estivill X Bancells C Ramos C Geographic distribution and regional origin of 272 cystic fibrosis mutations in European populations: The Biomed CF Mutation Analysis Consortium.Hum Mutat. 1997; 10: 135-154Crossref PubMed Scopus (266) Google Scholar In Hispanics, the mutation spectrum remains relatively poorly defined despite a carrier frequency that is only moderately lower than that in Caucasians (∼1 in 46 compared with ∼1 in 29, respectively). “Hispanic,” although difficult to strictly define, generally refers to the self-assigned ethnicity by individuals from Latin America, Central America, South America, Spain, and Portugal.12Arzimanoglou II Tuchman A Li Z Gilbert F Denning C Valverde K Zar H Quittell L Arzimanoglou I Cystic fibrosis carrier screening in Hispanics.Am J Hum Genet. 1995; 56: 544-547PubMed Google Scholar The population of Hispanics continues to grow both in absolute terms and relative to other subpopulations. In California, one of every two children born in 2001–2005 was Hispanic (http://ww2.cdph.ca.gov/data/statistics/Pages/default.aspx), and it is expected that approximately one-third of all new CF patients in this state will be of Hispanic origin (Martin Kharrazi, California Department of Public Health, September 14, 2007, personal communication). Identification of CFTR gene mutations in Hispanic CF patients can facilitate diagnosis, improve screening and management, and add value to genetic counseling. After screening for 30 to 70 known mutations with commercially available DNA testing panels, temporal temperature gradient electrophoresis and direct DNA sequencing have been used to detect unknown mutations.13Wong LJ Wang J Zhang YH Hsu E Heim RA Bowman CM Woo MS Improved detection of CFTR mutations in Southern California Hispanic CF patients.Hum Mutat. 2001; 18: 296-307Crossref PubMed Scopus (40) Google Scholar,14Schrijver I Ramalingam S Sankaran R Swanson S Dunlop CL Keiles S Moss RB Oehlert J Gardner P Wassman ER Kammesheidt A Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics.J Mol Diagn. 2005; 7: 289-299Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar By using a commercial panel of 87 mutations combined with temporal temperature gradient electrophoresis mutation analysis, the overall mutation detection rate for Hispanic individuals with CF in California could be raised to 94.5%.10Alper OM Wong LJ Young S Pearl M Graham S Sherwin J Nussbaum E Nielson D Platzker A Davies Z Lieberthal A Chin T Shay G Hardy K Kharrazi M Identification of novel and rare mutations in California Hispanic and African American cystic fibrosis patients.Hum Mutat. 2004; 24 (Erratum: Hum Mutat 2005, 25:223): 353Crossref PubMed Scopus (33) Google Scholar With other methods, a limited number of exonic rearrangements of CFTR have been identified (Table 1), suggesting that exon deletions and duplications are of clinical importance in the etiology of CF, albeit in a minority of cases. The overall frequency of large rearrangements is probably underestimated because of the testing methods commonly used, but is likely to account for several percentages of all affected alleles. Until now, large exonic rearrangements have not been studied specifically among Hispanics. To better characterize the mutation spectrum in Hispanics, we aimed to identify large deletions or duplications in the CFTR gene of Hispanic CF patients whose mutations remained undetected after conventional DNA sequence testing.Table 1CFTR Rearrangements Involving ExonsExonsType of rearrangmentReported in HispanicsReferences1–24 (all exons)Del40Niel 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 counseling in cystic fibrosis.J Med Genet. 2004; 41: e118Crossref PubMed Scopus (62) Google Scholar47Ratbi 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 ScholarPromoter-1Del32Hantash 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 ScholarPromoter-2Del32Hantash 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 Scholar1Del19Audrézet MP Chen JM Raguénès O Chuzhanova N Giteau K Le Maréchal C Quéré I Cooper DN Férec C Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms.Hum Mutat. 2004; 23: 343-357Crossref PubMed Scopus (110) Google Scholar40Niel 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 counseling in cystic fibrosis.J Med Genet. 2004; 41: e118Crossref PubMed Scopus (62) Google Scholar2Del/Indel40Niel 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 counseling in cystic fibrosis.J Med Genet. 2004; 41: e118Crossref PubMed Scopus (62) Google Scholar45Schneider M Hirt C Casaulta C Barben J Spinas R Buhlmann U Spalinger J Schwizer B Chevalier-Porst F Gallati S Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF.Clin Genet. 2007; 72: 30-38Crossref PubMed Scopus (9) Google Scholar46Taulan 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 Scholar2–3DelX17Dörk T Macek Jr, M Mekus F Tümmler B Tzountzouris J Casals T Krebsová A Koudová M Sakmaryová I Macek Sr, M Vávrová V Zemková D Ginter E Petrova NV Ivaschenko T Baranov V Witt M Pogorzelski A Bal J Zékanowsky C Wagner K Stuhrmann M Bauer I Seydewitz HH Neumann T Jakubiczka S Characterization of a novel 21-kb deletion, CFTRdele2,3 (21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in central and east Europe.Hum Genet. 2000; 106: 259-268Crossref PubMed Scopus (118) Google Scholar32Hantash 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 Scholar39Kilinc MO Ninis VN Dagli E Demirkol M Ozkinay F Arikan Z Cogulu O Huner G Karakoc F Tolun A Highest heterogeneity for cystic fibrosis: 36 mutations account for 75% of all CF chromosomes in Turkish patients.Am J Med Genet. 2002; 113: 250-257Crossref PubMed Scopus (52) Google Scholar2–9Del45Schneider M Hirt C Casaulta C Barben J Spinas R Buhlmann U Spalinger J Schwizer B Chevalier-Porst F Gallati S Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF.Clin Genet. 2007; 72: 30-38Crossref PubMed Scopus (9) Google Scholar2–10Del41Chevalier-Porst F Souche G Bozon D Identification and characterization of three large deletions and a deletion/polymorphism in the CFTR gene.Hum Mutat. 2005; 25: 504Crossref PubMed Scopus (20) Google Scholar3–10, 14b–16Del40Niel 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 counseling in cystic fibrosis.J Med Genet. 2004; 41: e118Crossref PubMed Scopus (62) Google Scholar44Niel 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 (22) Google Scholar4Del19Audrézet MP Chen JM Raguénès O Chuzhanova N Giteau K Le Maréchal C Quéré I Cooper DN Férec C Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms.Hum Mutat. 2004; 23: 343-357Crossref PubMed Scopus (110) Google Scholar4–6aDel19Audrézet MP Chen JM Raguénès O Chuzhanova N Giteau K Le Maréchal C Quéré I Cooper DN Férec C Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms.Hum Mutat. 2004; 23: 343-357Crossref PubMed Scopus (110) Google Scholar32Hantash 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 Scholar4–7, 11–18Del34Morral N Nunes V Casals T Cobos N Asensio O Dapena J Estivill X Uniparental inheritance of microsatellite alleles of the cystic fibrosis gene (CFTR): identification of a 50 kilobase deletion.Hum Mol Genet. 1993; 2: 677-681Crossref PubMed Scopus (27) Google Scholar4–8Dup40Niel 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 counseling in cystic fibrosis.J Med Genet. 2004; 41: e118Crossref PubMed Scopus (62) Google Scholar4–10Del41Chevalier-Porst F Souche G Bozon D Identification and characterization of three large deletions and a deletion/polymorphism in the CFTR gene.Hum Mutat. 2005; 25: 504Crossref PubMed Scopus (20) Google Scholar6b-10Dup48Hantash FM Redman JB Goos D Kammesheidt A McGinniss MJ Sun W Strom CM Consultations in Molecular Diagnostics. Characterization of a recurrent novel large duplication in the cystic fibrosis transmembrane conductance regulator gene.J Mol Diagn. 2007; 9: 556-560Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar11–13Dup47Ratbi 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 Scholar11–16Del19Audrézet MP Chen JM Raguénès O Chuzhanova N Giteau K Le Maréchal C Quéré I Cooper DN Férec C Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms.Hum Mutat. 2004; 23: 343-357Crossref PubMed Scopus (110) Google Scholar14aDel36Mickle JE Macek Jr, M Fulmer-Smentek SB Egan MM Schwiebert E Guggino W Moss R Cutting GR A mutation in the cystic fibrosis transmembrane conductance regulator gene associated with elevated sweat chloride concentrations in the absence of cystic fibrosis.Hum Mol Genet. 1998; 7: 729-735Crossref PubMed Scopus (80) Google Scholar14b–17bDel40Niel 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 counseling in cystic fibrosis.J Med Genet. 2004; 41: e118Crossref PubMed Scopus (62) Google Scholar41Chevalier-Porst F Souche G Bozon D Identification and characterization of three large deletions and a deletion/polymorphism in the CFTR gene.Hum Mutat. 2005; 25: 504Crossref PubMed Scopus (20) Google Scholar45Schneider M Hirt C Casaulta C Barben J Spinas R Buhlmann U Spalinger J Schwizer B Chevalier-Porst F Gallati S Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF.Clin Genet. 2007; 72: 30-38Crossref PubMed Scopus (9) Google Scholar16–17bDel41Chevalier-Porst F Souche G Bozon D Identification and characterization of three large deletions and a deletion/polymorphism in the CFTR gene.Hum Mutat. 2005; 25: 504Crossref PubMed Scopus (20) Google Scholar17a–17bDel32Hantash 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 Scholar45Schneider M Hirt C Casaulta C Barben J Spinas R Buhlmann U Spalinger J Schwizer B Chevalier-Porst F Gallati S Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF.Clin Genet. 2007; 72: 30-38Crossref PubMed Scopus (9) Google Scholar17a–18*Two different types of deletions involving exons 17a and 18 have been reported.DelX32Hantash 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 Scholar37Lerer I Laufer-Cahana A Rivlin JR Augarten A Abeliovich D A large deletion mutation in the CFTR gene (3120+1Kbdel8.6Kb): a founder mutation in the Palestinian Arabs (mutation in brief no. 231).Online Hum Mutat. 1999; 13: 337Crossref PubMed Scopus (43) Google Scholar43Nectoux J Audrezet MP Viel M Leroy C Raguenes O Ferec C Lesure JF Davy N Renouil M Cartault F Bienvenu T A frequent large rearrangement in the CFTR gene in cystic fibrosis patients from Reunion Island.Genet Test. 2006; 10: 208-214Crossref PubMed Scopus (6) Google Scholar47Ratbi 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 Scholar17bDel35Magnani C Cremonesi L Giunta A Magnaghi P Taramelli R Ferrari M Short direct repeats at the breakpoints of a novel large deletion in the CFTR gene suggest a likely slipped mispairing mechanism.Hum Genet. 1996; 98: 102-108Crossref PubMed Scopus (20) Google Scholar20DelX33Ferec C Casals T Chuzhanova N Macek Jr, M Bienvenu T Holubova A King C McDevitt T Castellani C Farrell PM Sheridan M Pantaleo SJ Loumi O Messaoud T Cuppens H Torricelli F Cutting GR Williamson R Ramos MJ Pignatti PF Raguenes O Cooper DN Audrezet MP Chen JM Gross genomic rearrangements involving deletions in the CFTR gene: characterization of six new events from a large cohort of hitherto unidentified cystic fibrosis chromosomes and meta-analysis of the underlying mechanisms.Eur J Hum Genet. 2006; 14: 567-576Crossref PubMed Scopus (81) Google Scholar22–23DelX19Audrézet MP Chen JM Raguénès O Chuzhanova N Giteau K Le Maréchal C Quéré I Cooper DN Férec C Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms.Hum Mutat. 2004; 23: 343-357Crossref PubMed Scopus (110) Google Scholar32Hantash 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 Scholar47Ratbi 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 Scholar22–24DelX31Hantash 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 Scholar32Hantash 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 Scholar41Chevalier-Porst F Souche G Bozon D Identification and characterization of three large deletions and a deletion/polymorphism in the CFTR gene.Hum Mutat. 2005; 25: 504Crossref PubMed Scopus (20) Google Scholar46Taulan 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 Scholar24DelX33Ferec C Casals T Chuzhanova N Macek Jr, M Bienvenu T Holubova A King C McDevitt T Castellani C Farrell PM Sheridan M Pantaleo SJ Loumi O Messaoud T Cuppens H Torricelli F Cutting GR Williamson R Ramos MJ Pignatti PF Raguenes O Cooper DN Audrezet MP Chen JM Gross genomic rearrangements involving deletions in the CFTR gene: characterization of six new events from a large cohort of hitherto unidentified cystic fibrosis chromosomes and meta-analysis of the underlying mechanisms.Eur J Hum Genet. 2006; 14: 567-576Crossref PubMed Scopus (81) Google ScholarDel, genomic rearrangement involves a deletion; Dup, genomic rearrangement involves a duplication; Indel, genomic rearrangement involves an insertion and deletion.* Two different types of deletions involving exons 17a and 18 have been reported. Open table in a new tab Del, genomic rearrangement involves a deletion; Dup, genomic rearrangement involves a duplication; Indel, genomic rearrangement involves an insertion and deletion. The study consisted of 32 Hispanic probands with classic CF who had at least one unidentified mutation each. In a previous study,10Alper OM Wong LJ Young S Pearl M Graham S Sherwin J Nussbaum E Nielson D Platzker A Davies Z Lieberthal A Chin T Shay G Hardy K Kharrazi M Identification of novel and rare mutations in California Hispanic and African American cystic fibrosis patients.Hum Mutat. 2004; 24 (Erratum: Hum Mutat 2005, 25:223): 353Crossref PubMed Scopus (33) Google Scholar 25 of the samples underwent comprehensive mutation analysis using a commercial screening panel of 87 mutations. If only one mutation or no mutations were identified, the DNA was subsequently used for mutation analysis by temporal temperature gradient electrophoresis. The 25 samples were enrolled in the present deletion study because molecular testing thus far had failed to identify the two expected mutations. At the time of this hemi-blinded study, it was unknown to the investigators who performed mutation analysis by multiplex ligation-dependent probe amplification (MLPA) of mutations previously identified. An additional seven samples collected from self-identified Hispanics at Stanford University were previously studied by complete DNA sequencing of the 27 CFTR exons but in none of these affected individuals were two mutations identified. Human subject protocols were approved by the California Health and Human Services Agency Committee for the Protection of Human Subjects (project 99-08-11) and by the Institutional Review Boards of the medical centers of study subjects. First described by Schouten et al,15Schouten JP McElgunn CJ Waaijer R Zwijnenburg D Diepvens F Pals G Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 2002; 30: e57Crossref PubMed Scopus (2067) Google Scholar MLPA enables detection of deletions and duplications up to several kilobases in size by screening for the loss or gain of up to 45 target sequences in a single reaction. Because MLPA is a screening method, however, changes should be confirmed with another independent assay.16Sellner L Taylor G MLPA and MAPH. New techniques for detection of gene deletions.Hum Mutat. 2004; 23: 413-419Crossref PubMed Scopus (242) Google Scholar MLPA is an efficient method to target all 27 exons of the CFTR gene, and we applied this method (MRC-Holland, Amsterdam, The Netherlands) to investigate deletions and duplications in the gene. Because we obtained consistent results with half the volume of polymerase chain reaction (PCR) reagents outlined in the MLPA procedure, these reagents were halved in our protocol. After completion of the MLPA reaction, amplified products were separated by capillary electrophoresis performed on an ABI-310 Genetic Analyzer (Applied Biosystems). PCR fragments were then quantified with ABI Gene Scan Analysis software. The relative peak size of the product from the probe recognition sequence was compared with the relative peak size of the product from a control; a 35 to 50% reduction indicated an exon deletion. The MLPA interpretation was facilitated by GeneMarker genotyping software (Softgenetics). The validity of deletions or duplications detected by MLPA needs to be demonstrated by confirmation with an independent method. An exception to this recommendation could perhaps be made for well characterized exon deletions identified with a validated diagnostic assay in a molecular diagnostic laboratory. The frequently contiguous exon deletions observed in Duchenne muscular dystrophy could be an example of such a scenario. The same deletions may be seen repeatedly, and the additional information from flanking exons may support the deletion findings. The MLPA assay indicates which exons may be missing but does not specifically identify the breakpoints of a deletion. We initially applied two approaches to support and subsequently confirm the presence of deletions identified by MLPA. First, the Cystic Fibrosis Mutation Database (http://www.genet.sickkids.on.ca/cftr/) was searched for known single-nucleotide polymorphisms (SNPs) in the CFTR gene. Primer pairs were designed around the sites of SNPs located within the exons suspected to be deleted (Table 2). After optimization and amplification, the PCR products that encompassed these SNPs were sequenced. Homozygosity present at the SNP site(s) initially supports the notion that an exon may have been deleted but does not confirm it. The second and more direct approach involved the characterization of the actual deletion breakpoints. For each suspected deletion, forward and reverse primers were designed in the flanking introns. For the deletion of exons 2–3, a reported primer pair was used.17Dörk T Macek Jr, M Mekus F Tümmler B Tzountzouris J Casals T Krebsová A Koudová M Sakmaryová I Macek Sr, M Vávrová V Zemková D Ginter E Petrova NV Ivaschenko T Baranov V Witt M Pogorzelski A Bal J Zékanowsky C Wagner K Stuhrmann M Bauer I Seydewitz HH Neumann T Jakubiczka S Characterization of a novel 21-kb deletion, CFTRdele2,3 (21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in central and east Europe.Hum Genet. 2000; 106: 259-268Crossref PubMed Scopus (118) Google Scholar For confirmation of the deletion of exon
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