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Technical Briefs |
1 Servicio de Bioquímica, Hospital Universitario San Cecilio, Granada, Spain;2 Departamento de Bioquímica y Biología Molecular, Universidad de Granada, Granada, Spain;3 Servicio de Inmunología, Hospital 12 de Octubre, Madrid, Spain;4 Instituto de la Recerca Oncologica (IRO), Hospital Duran I Reynals, Barcelona, Spain;
aaddress correspondence to this author at: Servicio de Bioquímica, Hospital Universitario San Cecilio, Avda. Dr. Oloriz s/n, Granada 18012, Spain; fax 34-958249015, e-mail efarez{at}ugr.es
Since the initial characterization of the predominant mutation (
F508) in cystic fibrosis (CF), more than 1000 pathogenic mutations and numerous polymorphisms have been identified in the CFTR gene (1). The frequencies and types of CFTR mutations vary according to the geographic and ethnic origins of the population under study. A recent worldwide survey revealed great mutational heterogeneity for CF in the Mediterranean region (2). Spain may have the highest heterogeneity of CF mutations among Mediterranean countries, with more than 75 different mutations detected, representing 90.2% of the CF chromosomes (3). On the other hand, only 10 mutations had a frequency higher than 1%, and these accounted for 74.2% of the CF chromosomes studied.
Among the 75 different CFTR mutations detected to date in Spain, 56 are not included in the commercial Applied Biosystems assay, which may account for the numerous uncharacterized CFTR alleles in a previous study in Southern Spain (4). We attempted to solve this analytical problem by developing a PCR method that uses fluorescent detection and capillary electrophoresis to detect 11 of the most frequent CFTR mutations not included in the Applied Biosystems assay. If used in conjunction with the Applied Biosystems assay, our novel PCR technique could increase the rate of CF allele detection among European populations.
Mutations in the CFTR gene were studied in 140 samples from patients diagnosed with CF at the 12 de Octubre Hospital (Madrid, Spain) or the IRO, Hospital Duran I Reynals (Barcelona, Spain), and from patients with suspicion of CF referred to the Hospital Universitario "San Cecilio" (Granada, Spain). Human genomic DNA was extracted from whole-blood samples by use of a QIAamp® Blood Mini Kit (Qiagen). The DNA concentrations in samples were determined spectrophotometrically by use of a UV-1603 Shimadzu spectrophotometer.
Oligonucleotide primers were designed to avoid the formation of primer-dimers, hairpins, and self-complementarity to be compatible in the multiplex PCR. Primers were selected to amplify exons 17b, 15, 13, 10, and 6a and intron 11 of the CF transmembrane regulator gene. Primers for single-nucleotide polymorphism (SNP) reactions differed in length by having poly(dT) tags of variable size at the 5' end. The annealing temperatures of the primers were designed, by Primer Express software, to be similar to enable performance of multiplex analysis. Two multiplex reactions were designed for the analysis of 11 mutations: multiplex 1 (M1) analyzed K710X, R1066C/R1066S, 2869 insG, and Q890X polymorphisms; and multiplex 2 (M2) analyzed L206W, 1609delCA, R1066L/R1066H, R709X, and 1811 + 1.6Kb polymorphisms. The primer sequences are shown in Tables 1 and 2 of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue11/). The CF genotyping assay comprised a PCR amplification and a single base primer extension reaction. PCR amplification was performed in a total volume of 25 µL for the first PCR reaction, containing 50 ng of DNA, 1 U of AmpliTaq Gold® DNA polymerase, 1x PCR Buffer II (Applied Biosystems, Hispania), 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, and primers at 0.2 µM. Cycling conditions were 95 °C for 10 min and 35 cycles of 95 °C for 1 min, 59 °C for 30 s, and 72 °C for 40 s, followed by a step at 72 °C for 7 min. To remove unincorporated primers and deoxynucleotide triphosphates, amplification products were treated with ExoI and shrimp alkaline phosphatase (SAP).
A second round of thermal cycling was carried out for the primer extension reaction: 95 °C for 10 min and 30 cycles of 95 °C for 15 s and 60 °C for 1 min. After the extension reaction, products were treated with SAP. The incorporated dideoxynucleotide triphosphate (ddNTP) was complementary to the nucleotide base of interest (5) and acted as terminator in the extension reaction, producing a single-base difference of the extended products. Individual ddNTPs were fluorescently labeled [A was labeled with dR6G (green); C with dTAMRATM (black); G with dR110 (blue); and T (U) with dROXTM (red)]. Fluorescently labeled products were analyzed on an ABI Prism 310 Genetic Analyzer or ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems) with POP-4 polymer and silica capillaries of 47 cm x 50 µm (i.d.) for the former or a capillary array of 36 cm for the latter.
Electrophoresis was conducted with a GS STR POP4 (1 mL) E module 5 and GS Matrix E 5 color as matrix file. Before the analysis, 0.5 µL of SNP product was added to 8.5 µL of deionized Hi-Di formamide and 1 µL of GenescanTM-120 LIZTM (15, 20, 25, 35, 50, 62, 80, 110, and 120 bp; internal size standard). After denaturation, samples were injected at 15 kV for 5 s. Separation was performed at 15 kV and 60 °C for 24 min, using filter set E.
After the electrophoresis, the data were analyzed by GeneScan Analysis software, Ver. 3.7 (Applied Biosystems), with a GeneScan E matrix (allowing simultaneous analysis of five colors), and GeneMapper Software, Ver. 3.0 (Applied Biosystems).
Before we performed the SNP genotyping multiplex reactions, we determined whether two oligonucleotides would produce overlapping signals when analyzed simultaneously, enabling the setting of tight loci windows for the GeneMapper 3.0 analysis software. We performed this analysis by adding 1 µL of primer (2 µM) to a master mixture containing ddNTPs, 10x reaction buffer, cofactor, enzyme, and distilled, deionized H2O. Amplification was at 37 °C for 15 min and 70 °C for 10 min. After SAP treatment, samples were analyzed on the ABI Prism Genetic analyzer as described above. (Fig. 1 of the online Data Supplement shows mutation 2869 insG as an example, with the positions of the differently colored peaks corresponding to the different nucleotides incorporated for this oligonucleotide.)
We determined the mobility of oligonucleotides subjected to capillary electrophoresis based on size, nucleotide composition, and fluorophores. Because the sizes of the oligonucleotide probes were different, products analyzed by capillary electrophoresis showed reproducible and specific electrophoreses for each mutation. The electropherograms of heterozygous samples contained two peaks (different color for each incorporated base), whereas those for homozygous samples had a single peak, with the color depending on the allele (wild-type or mutant). Thus, although some nonspecific incorporation was observed, these peaks were not at positions under investigation and were therefore not considered in the analysis. Fig. 1A
shows M1 multiplex analysis of a sample heterozygous for the Q890X mutation; the colored peaks correspond to the following wild-type alleles: 2869 insG (green), Q890X (black), K710X (green), and R1066C/S (black). Fig. 1B
shows M2 multiplex analysis of a sample heterozygous for the 1609delCA mutation; the colored peaks correspond to the following wild-type alleles: L206W (red), 1609delCA (black), R1066L/H (blue), R709X (black), and 1811 + 1.6Kb (green). The peak sizes exactly matched those expected for the amplified fragments (sizes varied by <1 nucleotide for each set of analyses). [Results for individuals carrying the K710X (M1), R1066C/S (M1), 2869 insG (M1), L206W (M2), R1066 L/H (M2), R709X (M2), and 1811 + 1.6 Kb (M2) mutations, respectively, are shown in Figs. 2–8 of the online Data Supplement.]
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Our approach is based on (a) single-base differences between alleles; (b) different capillary electrophoresis mobilities of DNA fragments based on their size and on the presence of different fluorescently labeled ddNTPs; (c) peak size linearity; (d) analysis of five colors for each sample by GeneScan software, Ver. 3.5; and (e) the reproducibility of the capillary electrophoresis system.
To achieve the benefits (6)(7)(8) of early diagnosis of CF will require carefully planned trypsinogen/CFTR multimutational analysis (6) that includes the alleles occurring in the population being screened. In 1997, analysis of the geographic distribution of more than 200 CFTR mutations in several European and Middle Eastern populations revealed that the Mediterranean region had the highest mutation heterogeneity for CF (9). None of the mutation detection assays available to date have been satisfactory for the Mediterranean area because they detect <70% of the CF alleles, impeding effective molecular CF screening programs.
Use of the 11 new mutations in the multiplex assay described here could increase the detection rate in the Spanish population by 
8%, estimated from the incidence of each mutation published previously for the Spanish population (3). In the south of Spain, where the F508 mutation has an incidence of 43.5% (4), lower than in the rest of Spain (53.2%) (3), the incidence of these 11 mutations may be higher.
Used in conjunction with the 31 mutations analyzed with the CF Genetic Analysis assay from Applied Biosystems, the multimutational analysis of the 11 mutations presented here could enhance the detection rate in Spanish and Mediterranean populations to 80%.
Acknowledgments
We thank Drs. M. Asuncion Ortega and Marivi Carretero (Applied Biosystems, Hispania) for their collaboration and comments on this report. M.E.F-V. is recipient of a grant from the Spanish Fondo de Investigación Sanitaria (FIS) del Instituto de Salud Carlos III. C.G.L.L. is recipient of a Predoctoral FIS fellowship. We are grateful to Richard Davies for assistance with the English version.
References
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