A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology
2017; Elsevier BV; Volume: 19; Issue: 5 Linguagem: Inglês
10.1016/j.jmoldx.2017.06.002
ISSN1943-7811
AutoresMarco Lucarelli, Luigi Porcaro, Alice Biffignandi, Lucy Costantino, Valentina Giannone, Luisella Alberti, Sabina Maria Bruno, Carlo Corbetta, Erminio Torresani, Carla Colombo, Manuela Seia,
Tópico(s)Respiratory viral infections research
ResumoSearching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing–based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling. Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing–based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling. Cystic fibrosis (CF) is a chronic, life-threatening genetic disease caused by loss-of-function mutations in the CF transmembrane conductance regulator gene (CFTR).1Lucarelli M, Pierandrei S, Bruno SM, Strom R: The genetics of CFTR: genotype – phenotype relationship, diagnostic challenge and therapeutic implications. Cystic Fibrosis - Renewed Hopes Through Research. Edited by Sriramulu D. Rijeka, Croatia: Intech, 2012. pp. 91–122.Google Scholar, 2Bombieri C. Seia M. Castellani C. Genotypes and phenotypes in cystic fibrosis and cystic fibrosis transmembrane regulator-related disorders.Semin Respir Crit Care Med. 2015; 36: 180-193Crossref PubMed Scopus (42) Google Scholar Notwithstanding the considerable ethnic and geographic variability in the frequency of CF, the mean incidence of 1 in 2500 live births makes CF the most frequent severe autosomal recessive disease in the white population.3Rowntree R.K. Harris A. The phenotypic consequences of CFTR mutations.Ann Hum Genet. 2003; 67: 471-485Crossref PubMed Scopus (272) Google Scholar CF is characterized by wide genetic and clinical heterogeneity, which complicates diagnosis, prognosis, and therapy. From birth to adulthood, there is considerable variability in the severity and rate of disease progression in CF, with varying clinical presentations and different organs involved at different ages.4Castellani C. Cuppens H. Macek Jr., M. Cassiman J.J. Kerem E. Durie P. Tullis E. Assael B.M. Bombieri C. Brown A. Casals T. Claustres M. Cutting G.R. Dequeker E. Dodge J. Doull I. Farrell P. Ferec C. Girodon E. Johannesson M. Kerem B. Knowles M. Munck A. Pignatti P.F. Radojkovic D. Rizzotti P. Schwarz M. Stuhrmann M. Tzetis M. Zielenski J. Elborn J.S. 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For example, the DRs of the 23-mutation panel established by the American Congress of Obstetrician and Gynecologist and the American College of Medical Genetics differ substantially between various geographic regions, ranging from 49% to 94%.13American College of Obstetricians and Gynecologists Committee on GeneticsACOG Committee Opinion No. 486: update on carrier screening for cystic fibrosis.Obstet Gynecol. 2011; 117: 1028-1031Crossref PubMed Scopus (152) Google Scholar, 14Lao O. Andres A.M. Mateu E. Bertranpetit J. Calafell F. Spatial patterns of cystic fibrosis mutation spectra in European populations.Eur J Hum Genet. 2003; 11: 385-394Crossref PubMed Scopus (38) Google Scholar This genetic heterogeneity is further enhanced if various well-recognized clinical forms of CF, such as classic CF, CFTR-related disorders, and congenital bilateral absence of vas deferens, are taken into consideration.15Bombieri C. Claustres M. De B.K. Derichs N. Dodge J. Girodon E. Sermet I. Schwarz M. 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The impact on genetic testing of mutational patterns of CFTR gene in different clinical macrocategories of cystic fibrosis.J Mol Diagn. 2016; 18: 554-565Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar In this article, CF refers to the classic form of the disease and excludes CFTR-related disorders and congenital bilateral absence of vas deferens, which will be specifically mentioned when appropriate. Limiting the scope to CF, but considering geographic areas with high genetic heterogeneity, an extended Sanger sequencing protocol of the 5′-flanking region, 27 exons and proximal intronic flanking regions, plus selected deep intronic zones for specific intron mutations in CFTR, have shown DRs of up to 97%.5Lucarelli M. Bruno S.M. Pierandrei S. Ferraguti G. Stamato A. Narzi F. Amato A. Cimino G. Bertasi S. Quattrucci S. Strom R. 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A comprehensive assay for CFTR mutational analysis using next-generation sequencing.Clin Chem. 2013; 59: 1481-1488Crossref PubMed Scopus (40) Google Scholar NGS has been used mainly in validation studies and has been performed on a limited number of samples in comparisons of the performance of NGS to those of classic Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Although the NGS approach provides an obvious reduction in cost per base, and an actual possibility of multiple-gene analysis for multigenic disorders, the suitability of this approach in monogenic disorders is still debated. In particular, the classic sequencing approach and MLPA have shown excellent laboratory operative characteristics for CFTR.5Lucarelli M. Bruno S.M. Pierandrei S. Ferraguti G. Stamato A. Narzi F. Amato A. Cimino G. Bertasi S. Quattrucci S. Strom R. 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Stevens C.F. Saavedra-Matiz C.A. Tavakoli N.P. Krein L.M. Parker A. Zhang Z. Maloney B. Vogel B. DeCelie-Germana J. Kier C. Anbar R.D. Berdella M.N. Comber P.G. Dozor A.J. Goetz D.M. Guida Jr., L. Kattan M. Ting A. Voter K.Z. van Roey P. Caggana M. Kay D.M. Clinical sensitivity of cystic fibrosis mutation panels in a diverse population.Hum Mutat. 2016; 37: 201-208Crossref PubMed Scopus (31) Google Scholar In this work, we developed, validated, and tested a high-throughput, NGS-based approach using a customized mutation panel containing 188 CF-causing mutations in CFTR. We analyzed 1828 subjects referred for diagnosis and a case series of 1138 patients with CF (646 patients from northern Italy and 492 patients from central Italy). We called this assay 188-CF-NGS. This approach revealed DRs of 95.0% and 95.6% in CF patients from central and northern Italy, respectively. Also in geographic regions with high genetic heterogeneity (such as Italy), a suitably customized and reasonably large panel of CFTR mutations, together with an NGS-based approach, allows the attainment of a high DR that is definitely suited for diagnostic purposes, at least in CF, and for neonatal and carrier screening. We designed a specific, customized panel of 188 CF-causing mutations, described in Supplemental Table S1. The mutations were included based on their high frequency in the Italian population and their documented associations with CF. Frequency information was obtained using the data on the frequency of CF mutations in Italian patients in the literature,5Lucarelli M. Bruno S.M. Pierandrei S. Ferraguti G. Stamato A. Narzi F. Amato A. Cimino G. Bertasi S. Quattrucci S. Strom R. 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Comprehensive cystic fibrosis mutation epidemiology and haplotype characterization in a southern Italian population.Ann Hum Genet. 2005; 69: 15-24Crossref PubMed Scopus (45) Google Scholar, 35Bombieri C. Bonizzato A. Castellani C. Assael B.M. Pignatti P.F. Frequency of large CFTR gene rearrangements in Italian CF patients.Eur J Hum Genet. 2005; 13: 687-689Crossref PubMed Scopus (26) Google Scholar, 41Dequeker E. Stuhrmann M. Morris M.A. Casals T. Castellani C. Claustres M. Cuppens H. Des G.M. Ferec C. Macek M. Pignatti P.F. Scheffer H. Schwartz M. Witt M. Schwarz M. Girodon E. Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders–updated European recommendations.Eur J Hum Genet. 2009; 17: 51-65Crossref PubMed Scopus (190) Google Scholar With this panel of CFTR mutations, we optimized an NGS-based assay we called 188-CF-NGS. Our assay underwent six steps of validation (Figure 1). If allowed by the overall number of mutant alleles, each mutation was validated in at least three independent samples. Also, possible differences in quality, analytical sensitivity, and analytical specificity of the 188-CF-NGS assay between analysis of dried blood spots (DBSs) and analysis of genomic DNA were evaluated. The first validation step was the analysis of 48 selected DBSs (referred to the Newborn Screening Laboratory, ASST Fatebenefratelli Sacco—PO Ospedale dei Bambini "V. Buzzi," Milan, Italy) that were previously investigated by mass spectrometry assay (Mass Spectrometry Assay). The DBSs were obtained from neonates screened as positive through the immunoreactive trypsinogen assay. The composition of this subset of DBS samples was as follows: eight homozygotes, 26 compound heterozygotes, four heterozygotes, and 10 with no CFTR mutation. The second validation step was the analysis of 24 genomic DNA samples extracted from peripheral blood (referred to the Medical Genetics Laboratory, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy), which had been genotyped by confirmatory methods (Confirmatory Methods). The subset of 24 genomic DNA samples consisted of nine compound heterozygotes, eight heterozygotes, and seven with no CFTR mutation. In the third validation step, 1378 DBSs from neonates (referred to the Newborn Screening Laboratory, Azienda Socio Sanitaria Territoriale Fatebenefratelli Sacco—PO Ospedale dei Bambini "V. Buzzi," Milan, Italy) with elevated levels of immunoreactive trypsinogen at neonatal screening were analyzed for mutations using our 188-CF-NGS assay. All of the identified mutations were subsequently confirmed by confirmatory methods (Confirmatory Methods). In the fourth validation step, 378 genomic DNA samples from subjects referred for genetic analysis (to the Medical Genetics Laboratory, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy) were analyzed using the 188-CF-NGS assay. A subset of 325 subjects underwent carrier screening or carrier testing (211 for family history, 98 as partners of carriers, and 16 for consanguinity), and another subset (53 subjects) were analyzed for hyperechogenic bowels. All of the identified mutations were subsequently confirmed by confirmatory methods (Confirmatory Methods). To complete the experimental validation of all of the mutations available in our case series, we applied a fifth validation step. We used samples from a case series comprising 1138 patients affected by CF and diagnosed according to the present guidelines41Dequeker E. Stuhrmann M. Morris M.A. Casals T. Castellani C. Claustres M. Cuppens H. Des G.M. Ferec C. Macek M. Pignatti P.F. Scheffer H. Schwartz M. Witt M. Schwarz M. Girodon E. Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders–updated European recommendations.Eur J Hum Genet. 2009; 17: 51-65Crossref PubMed Scopus (190) Google Scholar: 646 from northern Italy (referred to the Lombardia Regional Reference CF Center) and 492 from central Italy (referred to the Lazio Regional Reference CF Center). Patients with clinical manifestations associated with CFTR dysfunction but whose conditions did not fulfill the diagnostic criteria for CF (namely, CFTR–related disorders or congenital bilateral absence of vas deferens15Bombieri C. Claustres M. De B.K. Derichs N. Dodge J. Girodon E. Sermet I. Schwarz M. Tzetis M. Wilschanski M. Bareil C. Bilton D. Castellani C. Cuppens H. Cutting G.R. Drevinek P. Farrell P. Elborn J.S. Jarvi K. Kerem B. Kerem E. Knowles M. Macek Jr., M. Munck A. Radojkovic D. Seia M. Sheppard D.N. Southern K.W. Stuhrmann M. Tullis E. Zielenski J. Pignatti P.F. Ferec C. Recommendations for the classi
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