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Technical Briefs |
A and I148T Mutations in Cystic Fibrosis
1
Department of Pathology, Molecular Diagnostics Laboratory, Duke University Medical Center, Durham, NC 27710
2
Laboratory Corporation of America, Research Triangle Park, NC 27709
aaddress correspondence to this author at: Box 3712, Department of Pathology, Duke University Medical Center, Durham, NC 27710; fax 919-684-8901, e-mail stenz001{at}mc.duke.edu
Cystic fibrosis [(CF); Online Mendelian Inheritance in Man No. 602421] is an autosomal recessive disorder characterized by chronic broncho-pulmonary disease, pancreatic insufficiency, and increased sweat electrolytes (1). The CF transmembrane conductance regulator (CFTR) gene was identified by three groups (2)(3)(4) and was mapped to the long arm of chromosome 7 (7q31). The CFTR contains 24 exons, encodes a protein of 1480 amino acids, functions as a chloride channel, and regulates other transport pathways. Over 900 disease-causing mutations in the CFTR gene have been identified.
The American College of Medical Genetics has recommended a core mutation panel for general population CF carrier screening (5). This panel contains 25 of the known CF mutations, including 3120+1G
A and I148T. 3120+1GA (intron 16) is the second most common CF mutation in the African American population and accounts for 9–14% of the African American CF chromosomes (6). I148T (exon 4) is the second most common CF mutation in the French Canadian population and accounts for 9.1% of the French Canadian CF chromosomes (7).
Most of the 25 mutations recommended by the American College of Medical Genetics can be detected with commercially available methods [Roche Molecular Biochemicals, Applied Biosystems and Innogenetics (ABI)], but no assays test for both the 3120+1GA and I148T mutations, and two of the three assays do not detect either mutation. Therefore, we developed a multiplex primer-extension assay that rapidly and simultaneously detects these two CF mutations using the ABI Prism® SNaPshotTM ddNTP Primer-Extension assay. We believe this is the first demonstration of the use of this assay in the literature. The method uses a dideoxynucleotide primer-extension reaction (PER) in conjunction with an ABI genetic analyzer to detect the addition of a fluorescently labeled ddNTP to the 3' end of a sequencing primer (Fig. 1E
). Each ddNTP is labeled with a different fluorescent dye, allowing the identification of the specific polymorphism or mutation. Specific PCR products act as templates for the PER.
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Seven samples with known genotypes for 3120+1GA and I148T were used to verify our assay’s ability to identify the wild-type and mutated alleles. All identifiers were removed from those samples that were residual clinical samples. Institutional review board approval was obtained, waiving consent. One sample was heterozygous for 3120+1GA, another was heterozygous for I148T, and the other five contained neither mutation. For each sample, genomic DNA was extracted from blood or transformed lymphocytes according to standard procedures (Puregene System; Gentra). Each DNA sample was diluted to a concentration of 40 ng/µL. Separate PCR reactions were set up to the following primer pairs: 3120F, 5'-TATTTGCTAATTCTTATTTGGGTTCTGAATG-3'; 3120R, 5'-ATAGACAGGACTTCAACCCTCAATCAAATA-3'; and I148TF, 5'-AATCATAGCTTCCTATGACCCGGATA3-', I148TR5'-AGCATTTATCCCTTACTTGTACCAGCAC-3'. Each PCR reaction contained 200 ng of genomic DNA and 100 ng of each primer in a volume of 50 µL, containing 5 µL of 10x PCR buffer (15 mM MgCl2; Qiagen), 5 µL of 10 mM dNTP (Applied Biosystems), and 0.25 µL of Hotstar Taq (5 U/µL; Qiagen).
The PCR amplification was performed in an ABI 9700 thermocycler beginning with 15 min at 94 °C, followed by 35 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for 2 min, and a final cycle of at 72 °C for 10 min. To verify specific amplification, 15 µL of the 3120+1GA (318 bp) and I148T (291 bp) PCR-amplified products were electrophoresed on a 3% agarose gel (I.D.NA agarose; Bio Whittaker Molecular Applications). To remove primers and excess dNTPs from the PCR products, which would interfere with the PER, reactions were set up containing 4 µL of PCR product, 2 µL of shrimp alkaline phosphatase (1 U/µL; USB Corporation), 0.2 µL of Exonuclease (10 U/µL; USB Corporation), and 6 µL of water. The reactions were incubated at 37 °C for 1 h, followed by a 72 °C incubation for 15 min. The samples were then diluted to a concentration 0.15 pmol/µL.
Before developing the multiplex primer-extension assay, separate PERs were set up for the 3120+1GA and I148T mutations. Each single PER contained 5 µL of SNaPshot Ready Reaction Mix, 1 µL of either the 3120+1GA or the I148T diluted PCR amplified products (0.15 pmol/µL), 1 µL of either the 3120+1GA singleplex sequencing primer (0.15 pmol/µL; 5'-TTACCATATTTGACTTCATCCAG-3') or the I148T sequencing primer (0.15 pmol/µL; 5'-CATTTTTGGCCTTCATCACA-3'), and then 3 µL of water. Thermal cycling for the PERs was performed in an ABI 9700 thermocycler with 25 cycles at 96 °C for 10 s, 50 °C for 5s, and 60 °C for 30 s. To prevent the unincorporated fluorescent ddNTPs from comigrating with the fragments of interest, it is necessary to remove the 5' phosphoryl groups. To do this, we added 0.5 µL of shrimp alkaline phosphatase to each PER tube (3120+1GA or I148T) and incubated them at 37 °C for 1 h, followed by 72 °C for 15 min. Final sample preparation was performed by adding 1 µL of the post-PER to 9 µL of formamide. We loaded each sample on the ABI 310 genetic analyzer and analyzed the samples using Pop4 polymer, a 47-cm capillary column, and the ABI GeneScan E Run Module. The results obtained from the single primer-extension analyses (data not shown) were in complete concordance with the known genotypes of the samples.
During development of the multiplex assay, we discovered that sequencing primers that have a 2–3 bp difference cannot be clearly resolved (overlapping peaks) on the ABI Genotyper electropherogram. This effect was attributable to the primer size and the specific dyes (causing mobility shifts) attached to the ddNTPs. For the ABI 310 genetic analyzer to clearly resolve overlapping peaks, the 3120+1GA singleplex sequencing primer was increased in length by 10 bp on the 5' end (and named the 3120+1GA multiplex sequencing primer). This enabled distinct peak separations on the ABI Genotyper electropherogram (Fig. 1, A–C
). Multiplexing the singleplex assays was then achieved by combining both sequencing primers and the diluted PCR amplified products into a single PER. Each multiplex PER was in a total volume of 10 µL containing 5 µL of SNaPshot Ready Reaction Mix, 1 µL of the 3120+1GA multiplex sequencing primer (0.15 pmol/µL; 5'-CTTCTGCCTCTTACCATATTTGACTTCATCCAG-3'), 1 µL of the I148T sequencing primer (0.15 pmol/µL; 5'-CATTTTTGGCCTTCATCACA3'), 1 µL (0.15 pmol/µL) of each of the 3120+1GA and I148T diluted PCR-amplified products, and 1 µL of water. The conditions for thermal cycling and post-primer-extension treatment was performed as previously stated in the singleplex PER. The multiplex samples were prepared for analysis on the ABI genetic analyzer by adding 2 µL of the post-PER to 9 µL of formamide. Multiplex analysis performed on the ABI 310 capillary electrophoresis unit detected five wild-type samples (Fig. 1C
) and two heterozygote samples (Fig. 1, A and B
). The I148T heterozygote sample exhibited one peak for the two wild-type 3120+1G alleles, one peak for the wild-type I148 allele, and one peak for the mutated I148T allele (Fig. 1A
). The 3120+1GA heterozygote sample exhibited one peak for the two wild-type I148 alleles, one peak for the wild-type 3120+1G allele, and one peak for the mutated 3120+1GA allele (Fig. 1B
). The five wild-type samples all showed one peak for the two wild-type I148 alleles and one peak for the two wild-type 3120+1G alleles (representative sample; Fig. 1C
). The negative water control sample showed no peaks (Fig. 1D
). Multiplexing with the ABI Prism SNaPshot ddNTP primer-extension assay allows rapid identification of both wild-type and mutated alleles at 3120+1G and I148 in the CFTR gene. The successful development of this multiplex assay has enabled us to speculate that further manipulation of the assay could allow more than two mutations to be analyzed, thereby leading to a cost savings. We believe this technique can be used to easily develop multiplex assays for additional CF mutations and for other genes.
Acknowledgments
This work was supported by the CDC (no. 200-2000-10050). We received as a gift one 20-assay SNaPshotTM reagent set from Applied Biosystems. We thank Dr. Karen Snow at The Mayo Clinic for help with this work. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
References
The following articles in journals at HighWire Press have cited this article:
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  S. H. Bernacki, J. C. Beck, K. Muralidharan, F. V. Schaefer, A. E. Shrimpton, K. L. Richie, B. C. Levin, G. Pont-Kingdon, and T. T. Stenzel Characterization of Publicly Available Lymphoblastoid Cell Lines for Disease-Associated Mutations in 11 Genes Clin. Chem., November 1, 2005; 51(11): 2156 - 2159. [Full Text] [PDF]
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