Four common mutations of the cystathionine [beta]-synthase gene detected by multiplex PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
The CBS gene has been mapped to chromosome 21822.3 (6). It was cloned in 1993 (7), and to date, >60 mutations have been reported (8). The prevalence of the various mutations differs markedly between countries, and screening for homocystinuria should be optimized accordingly (5,9). In a recent study, we found that the most common mutations in Norway are located on exons 7, 8, and 9 (9).
Conventional methods for mutation detection are based on gel or capillary electrophoresis. These methods are time-consuming and provide no direct molecular information. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (10, 11) for the analysis of DNA (12,13) has become an attractive alternative because of advances in both sample preparation and instrument performance. Reduction of cation adduction of oligonucleotides (14-16); minimization of DNA degradation, including depurination (17-19); and selection of proper matrices (20-24) are important to obtain high sensitivity, and the delayed extraction technology (25) has reduced ion fragmentation and increased spectral resolution. Thus, MALDI-TOF mass spectrometry represents a fast method (seconds to minutes) for the analysis of oligonucleotides up to ~2 kbp (26). The spectral data provide information about base sequence (27, 28), and resolution is now at the base level when DNA fragments of several kDa are analyzed. Several methods for mutation detection with MALDI-TOF have been described recently (29-34), but only a few involve multiplex genotyping (35, 36).
We present here a simple and reliable method based on MALDI-TOF mass spectrometry for the determination of common mutations of the CBS gene. The technique involves a primer extension reaction, as originally described by Haff and Smirnov (35), and simultaneously detects four point mutations on three different exons: exon 7, T262M (C785T); exon 8, G307S (G919A) and I278T (T833C); exon 9, V320A (T959C).
Materials and Methods
The QIAAmp Blood Kit was obtained from Qiagen. The Taq polymerase (DyNAzyme[TM]) and reaction buffer for PCR were from Finnzymes; the dNTPs and ddNTPs were from Pharmacia Biotech. The primers were designed with the software Oligo 4.0 (National Biosciences) and synthesized by Eurogentec. PCR products were purified with the QIAquick PCR Purification Kit (cat. no. 28106) from Qiagen. The enzyme and the reaction buffer (ThermoSequenase[TM]) for the primer extension reaction were obtained from Amersham Life Sciences.
The 3-hydroxypicolinic acid, ammonium acetate, and ammonium citrate were purchased from Sigma Aldrich. The cation-exchange beads (AG50W-X8 resin) were from Bio-Rad. The beads were activated by incubation in a 1 mol/L ammonium acetate solution and then washed 5 times with deionized water.
The initial step involved multiplex PCR amplification of exons 7 (T262M), 8 (G307S, I278T), and 9 (V320A) containing the mutation sites (9). Using the amplified DNA as template, the four mutation sites were targeted by site-specific primers (Table 1), which were extended by one matching ddTNP. The four extension primers were designed so that the primers and their extension products spanned a mass range between 4500 and 7500 Da. They were sized to avoid mass interference between primers, extension products, and depurination products.
DNA SAMPLES AND PCR
Blood samples were obtained from 12 patients with known mutations in the CBS gene. DNA was isolated with the QIAAmp Blood Kit. Amplification of exons 7, 8, and 9 of the CBS gene was accomplished by multiplex PCR, using the primers 5'-CCA000AGGGACCCAAGAAT-3' and 5'-CCACTCCGCACTGTCCCTCT-3' for exon 7, 5'-TTTGGCCGGGCTCTGGACTC-3' and 5'-TTTCTGGCCTTGAGCCCTGAA-3' for exon 8, and 5'-CTGACGGGCTGTGGTGGGGTCC-3' and 5'-000ACAGCAGCCCCTCTTGCGC-3' for exon 9. The reaction was performed in a total volume of 100 [micro]L containing 100 ng of template DNA, 1.5 [micro]L of Taq DNA polymerase (1 U/[micro]L), 1.5 mmol/L Mg[Cl.sub.2], 125 [micro]mol/L dNTPs, and 20 pmol of each primer. The cycling conditions were 95[degrees]C for 2 min, followed by 36 cycles at 94[degrees]C for 30 s, 64[degrees]C for 40 s, and a final extension at 72[degrees]C for 6 min.
To remove excess primers and dNTPs, the amplification product was purified with the QIAquick PCR Purification Kit according to the instructions given by the manufacturer. The product was eluted in 45 [micro]L of Tris-EDTA buffer (10 mmol/L Tris,1 mmol/L EDTA, pH 8.0).
PRIMER EXTENSION REACTION
The multiplex extension reaction was performed with a combination of 10 pmol of each of four specific extension primers (Table 1). The total reaction volume of 60 [micro]L contained 10 [micro]mol/L of each ddNTP, 8 U of ThermoSequenase DNA polymerase, 2 [micro]L of ThermoSequenase reaction buffer, and the total multiplex PCR reaction product concentrated down to a volume of 45 [micro]L. The cycling conditions were 40 cycles at 94[degrees]C for 30 s, 64[degrees]C for 30 s, and 72[degrees]C for 1 min. The PCR and extension reactions were performed on a Perkin-Elmer Thermal Cycler 480.
The extension reaction products were lyophilized, dissolved in 10 [micro]L of distilled water, and then incubated for at least 3 h with cation-exchange beads. 3-Hydroxypicolinic acid (0.5 mol/L in water), used as matrix, was incubated separately with beads. To avoid formation of and interference from adducts, the ammonium acetate-treated beads were washed extensively with deionized water before use. The steel surface of the MALDI probe was exposed to 50 mmol/L ammonium citrate for 10 min to avoid sodium contamination of the target. The probe was not exposed to beads. Matrix solution (1 [micro]L) was then applied to the probe and allowed to dry before deposition of 1 [micro]L of primer extension sample. This sequential procedure led to higher ion yields than those observed when a mixture of matrix and sample was deposited on the probe. Because of the inhomogeneous crystallization of the 3-hydroxypicolinic acid matrix, each sample was analyzed at five different positions.
MALDI-TOF MASS SPECTROMETRY
The MALDI-TOF instrument was a Bruker Reflex III (Bruker-Franzen Analytik) with a two-stage gridless reflector. The spectrometer was equipped with a nitrogen laser (Laser Science) and a pulsed-ion extraction source. All analyses were run in the reflector mode with negatively charged ions of 20 kV energy. To avoid detector saturation from matrix ions, masses of <2000 Da were suppressed by an ion deflector. The typical number of shots was 50-100. A synthetic oligonucleotide (31-mer; Eurogentec) was used as external standard.
DETECTION OF FOUR CBS MUTATIONS
Identification of the various alleles was based on the differences in mass shift caused by extension of the primer with two different ddNTPs. The masses of the four ddNTPs were 297.21 (ddA), 273.19 (ddC), 313.21 (ddG), and 288.20 (ddT) Da. Fig. 1 shows the results of three patient samples harboring all eight alleles of the four mutation sites. Fig. 1A shows the mass spectrum of a sample heterozygous for the I278T mutation (extended by ddA and ddG), and wild type for T262M, G307S, and V320A. Fig. 1B shows a sample heterozygous for both G307S and V320A, extended by the pairs ddG/ddA and ddA/ddG, respectively. Fig. 1C shows the extension by both ddC and ddT of a sample heterozygous for the T262M mutation. In all traces in Fig. 1, the extension products were resolved from the primers. With this system, we genotyped blood from 12 homocystinuria patients, and the results (data not shown) were identical to those obtained with reference techniques (9).
[FIGURE 1 OMITTED]
Each blood sample was PCR amplified and processed independently three times, and each preparation deposited on the MALDI probe was analyzed at five different positions. Essentially the same results were obtained. Additional weak signals corresponded to mass peaks of ammonium adducts. There were essentially no sodium adducts. Peaks without labels were minor and could be assigned to depurination (-132 Da) or the presence of an unknown species of 60 Da.
RESOLUTION AND PEAK PATTERN
The peak doublet shown in the inset of Fig. 1C shows the resolution of the MALDI-TOF system in the mass range of 4800-4900 Da. The mass difference between T and C is 15 Da, and the peaks were baseline resolved. The smallest possible mass difference between two bases is 9 Da, which is the difference between T and A. This situation does not exist for the CBS mutation investigated here, but the baseline resolution at a 15-Da difference suggests that an A-T difference of 9 Da would be clearly distinguished.
Although the peak intensities of all extension primers and products varied depending on sample and analysis position, all spectra showed roughly the same peak pattern as shown in Fig. 1. After 40 thermocycles, three of the extension primers showed only weak mass signals, whereas the V320A primer gave a signal equaling that of the extension products.
We have developed a MALDI-TOF mass spectrometry procedure for the simultaneous detection of four common mutations in the CBS gene. All four mutations were unambiguously determined for the 12 homocystinuria patients.
To date, the purification of samples to allow successful detection of oligonucleotides has required tedious and lengthy procedures. These approaches involve ethanol precipitation (37) and various solid-phase methods combined indirectly (14,15) or directly (16, 38, 39) with MALDI-MS. In 1992, Nordhoff et al. (14) described a procedure for removing alkali cations from nucleic acid samples based on a treatment of the matrix and the deposited analyte/matrix droplet by cation-exchange beads. We have used cation-exchange beads for separate desalting of the matrix and analytes. Incubation times of 3 h gave high quality and reproducible mass spectra. If salt adducts were detected, incubation overnight was sufficient. No beads were required on the sample stage. Additional procedures to improve stability (17,18) or to optimize the charge state of the DNA (19) were not necessary.
The performance of our system in terms of resolution equals that reported recently for a delayed extraction MALDI-TOF system used for single-nucleotide polymorphism identification (36). A clear separation of oligonucleotides with only an A-T (9 Da) difference in mass should be possible within the mass range investigated (4500-7500 Da). No mass-tagged ddNTPs (34) or primers (35) were required to carry out multiplex detection of the four CBS mutations.
Signal interference usually did not obscure interpretation of spectra. However, peak doublets ddA/ddG and ddC/ddT caused by heterozygous mutations should be evaluated to avoid misinterpretation of ammonium adducts (with a mass increase of 17 Da). In our experience, the doublets related to sample heterozygosity have a balanced height, whereas ammonium adducts usually cause smaller peaks.
The mass spectra usually showed good reproducibility, allowing unequivocal interpretation of data, but small variations between sample preparations or different analyses of the same sample preparation were occasionally observed. This may be the result of unbalanced PCR, variation in primer extension efficiency, inhomogeneous spatial distribution of oligonucleotides and matrix at the MALDI probe, variability in ionization/ desorption, and detection probability of the oligonucleotides.
In conclusion, we used MALDI-TOF mass spectrometry with pulsed ion extraction and in the reflector mode for multiplex genotyping of four common mutations in the CBS gene. The technique is based on primer extension without DNA strand separation or labeling of primers or ddNTPs. The sample purification step is simple and relatively inexpensive and involves treatment of the extension products and the matrix with cation-exchange beads. This procedure ensures sensitive oligonucleotide analysis of high quality without mass interference or sodium adduct formation. Our results demonstrate unambiguous multiplex genotyping by MALDI-TOF mass spectrometry.
This work was sponsored by The Norwegian Research Council on Cardiovascular Diseases, The Advanced Research Program within the Norwegian Research Council, and by the EU Commission, as part of the EU Commission Demonstration Project, Contract BMH4-98-3549. A.H. is a fellow of The Norwegian Research Council on Cardiovascular Diseases. We thank Anne Berit Guttormsen for supplying the clinical material and Arve Ulvik for helpful advice.
Received March 8, 1999; accepted May 18, 1999.
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ANGELA HARKSEN, PER MAGNE UELAND, HELGA REFSUM, and KLAUS MEYER *
Department of Pharmacology, University of Bergen, Armauer Hansens Hus, 5021 Bergen, Norway.
* Author for correspondence. Fax 47-55-974605; e-mail email@example.com.
Table 1. Sequences and sizes of the DNA oligonucleotides used as extension probes. Mass, Da Amino acid substitution Exon Sequence Unextended T262M 7 5'-CGGGCGGCACCATCA-3' 4563 G307S 8 5'-TACGAGGTGGAAGGGATC-3' 5629 1278T 8 5'-ACCCTTCGGGATCCACCCCA-3' 5983 V320A 9 5'-TTTTTGCTGGACAGGACGGTGG-3' 6837 Mass, Da Amino acid Wild type, Mutation type, substitution extended extended T262M 4836 (+ddC) 4851 (+ddT) G307S 5942 (+ddG) 5926 (+ddA) 1278T 6280 (+ddA) 6296 (+ddG) V320A 7135 (+ddA) 7151 (+ddG)
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|Title Annotation:||Molecular Diagnostic and Genetics|
|Author:||Harksen, Angela; Ueland, Per Magne; Refsum, Helga; Meyer, Klaus|
|Date:||Aug 1, 1999|
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