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Technical Briefs |
1
Department of Expression Microarrays, EURAY, and
2
Department of Chemistry, Exiqon A/S, Bygstubben 9, DK-2950 Vedbaek, Denmark
Departments of
3
Clinical Biochemistry and
4
Cardiology, University Hospital of Copenhagen, DK-2650 Hvidovre, Denmark
aauthor for correspondence: fax 45-45-661888, e-mail jacobsen{at}exiqon.com
Hyperlipidemia and coronary heart disease (CHD) are associated with genetic variation in the apolipoprotein B (apoB) gene (1). Nonexchangeable apolipoprotein B-100 (ApoB-100) is an important determinant of LDL-cholesterol in plasma; it plays a central role in cholesterol transport by its association to LDL particles as a ligand for the LDL receptor (2). One of the first mutations in the apoB gene to be discovered was the ApoB-100 R3500Q (apoBR3500Q) mutation (3), a single nucleotide transition, CGG to CAG, in exon 26. This mutation reduces the affinity to the LDL receptor by at least 95% (4) and is the major cause of familial defective ApoB-100 (FDB). The frequency of the mutation is 1:500 to 1:700 in Caucasians (5)(6). Because the cholesterol concentration is often within the reference interval in FDB patients, the only reliable way of detecting FDB is by genotyping. At present, genotyping of the apoBR3500Q mutation is based on PCR (7)(8)(9), but other methods, such as heteroduplex analysis and real-time PCR, have also been applied (10)(11)(12). In general, these methods are time-consuming and need to be optimized. Here we describe a simple, rapid, and sensitive assay for genotyping the apoBR3500Q mutation that is suitable for the 96-well microtiter plate format and relies on hybridization of PCR amplicons to allele-specific locked nucleic acid (LNA) capture probes (13)(14).
The microtiter plates were prepared by covalent photoimmobilization of 10 pmol/well of either wild-type (wt)-LNA8 [AQ-CONH-(CH2)3-TACATGTTATGCTGA-GLALMeCLMeCLGLTLGLTLG] or mutant (m)-LNA8 [AQ-CONH-(CH2)3-TACATGTTATGCTGA-GLALMeCLTLGLTLGLTLG] capture probes using an anthraquinone (AQ) moiety as described by Koch et al. (15). L indicates 2'-O,4'-C-methylene-(D-ribofuranosyl) (LNA) nucleotides, and MeC indicates 5-methylcytosine. After irradiation, the plates were treated as described by Ørum et al. (16).
A plasmid was constructed by cloning the wild-type PCR amplicon to test the specificity of the apoBR3500Q genotyping assay. This was generated from genomic DNA (extracted from 5 mL of EDTA-anticoagulated blood using a Roche DNA Isolation reagent set) with the forward primer 5'-CACCTCTTACTTTTCCATTGAGT-3' and the reverse primer 5'-TTTAGATCATTTAGTTTCAGCCC-3'; the resulting amplicon was then cloned into the pCR®2.1-TOPO plasmid using the TOPOTM TA Cloning® reagent set (Invitrogen). The primers amplify a region within the apoBgene encompassing nucleotide position 9775 (GenBank Accession. No. M19828). A mutant plasmid was then constructed by use of a primer covering the position of interest. The PCR product was generated using the splicing by overlap extension-PCR method (17). Purified plasmids were confirmed by DNA sequencing and used for validating the apoBR3500Q genotyping assay. The specificity of the capture probes was subsequently tested using plasmids where mismatched nucleotides were introduced 5' and 3' of the second base in apoB codon 3500, using site-directed mutagenesis (17). No detectable signals were obtained from either the wild-type or the mutant capture probes when the capture probes were hybridized with DNA targets containing a single mismatch. In addition, measurements of duplex melting temperature (Tm) confirmed the affinity and specificity of the LNA capture probes. Melting curves were constructed without the AQ and the DNA linker moieties. The thermostability of the duplexes was determined as described by Wahlestedt et al. (18). As seen in Table 1
, the Tm values of the LNA capture probes were substantially higher than the Tm values of identical DNA probes.
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A selected panel of archival patient samples was analyzed twice by the apoBR3500Q assay without prior knowledge of the genotype. The primers for the apoBR3500Q PCR were as follows: forward primer, 5'-biotin-CTAGTGAGGCCAACACTTACTTGAATTCCAAGAGC-3' (position 9736–9770); reverse primer, 5'-GTTTTTCGTACTGTGCTCCCAGAG-3' (position 9879–9902); these primers produce a 167-bp amplicon from genomic DNA. PCR reactions (50 µL) were prepared by mixing 15 mM Tris-HCl (pH 8.0); 50 mM KCl (GeneAmp Gold buffer; PE Biosystems); 2.5 mM MgCl2; 200 µM each of dATP, dCTP, dGTP, and dTTP (Amersham Pharmacia Biotech); 1 µM forward primer; 1 µM reverse primer; 1.25 U (0.25 µL of a 5 U/µL solution) of AmpliTaq Gold polymerase (PE Biosystems); and 100 ng of genomic DNA as template (purified as described above). After an initial 15-min denaturation step at 95 °C, 30 cycles of PCR were carried out (40 s at 95 °C, 40 s at 65 °C, and 40 s at 72 °C), followed by extension at 72 °C for 10 min.
The microtiter plate assay was performed by mixing 20 µL of the PCR amplicon with 20 µL of denaturation buffer (125 mmol/L NaOH, 8 mmol/L EDTA, 0.2 g/L phenol red) and incubating for 5 min at room temperature. We added 200 µL of hybridization buffer (50 mmol/L sodium phosphate buffer, pH 7.0, 0.1 mL/100 mL Tween 20); 100 µL of this reaction was transferred to the coated microtiter wells containing either the wt-LNA8 or m-LNA8 capture probe and hybridized for 30 min at 37 °C. The wells were washed three times in washing buffer [300 µL/well; 0.5x standard saline citrate buffer (75 mmol/L NaCl, 7.5 mmol/L sodium citrate, pH 7.0, 1 mL/L Tween 20)]. We then added 100 µL of conjugate solution (1 mg/L horseradish peroxidase-streptavidin; Pierce) diluted in the washing buffer to each well and incubated the plate for 15 min at 37 °C. Finally, the wells were washed six times with washing buffer (300 µL/well), and 100 µL of 3,3',5,5'-tetramethylbenzidine substrate (TMB one; KemEnTec) was added. The plate was then incubated in the dark for 10–15 min at room temperature, and the reaction was stopped by the addition of 100 µL/well of 0.5 mol/L H2SO4. The absorbance was measured at 450 nm on a Wallac Victor2 (Perkin-Elmer). Fig. 1
shows that the apoBR3500Q assay yielded an unambiguous response for all archival patient samples, concurring with the results obtained by DNA sequencing.
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We next genotyped DNA samples from 309 patients admitted to the emergency room at the University Hospital of Copenhagen with clinical and biochemical signs of acute myocardial infarction according to the guidelines from the American College of Cardiology (19). Only samples from patients who survived initial treatment were analyzed by the apoBR3500Q assay. The results of 53 randomly selected samples were confirmed by DNA sequencing. All 309 patient samples were genotyped as wild type. Thus, no apoBR3500Q heterozygotes were detected in this population of acutely admitted coronary patients. Considering the impact of the apoBR3500Q mutation on CHD (20)(21), we initially expected the prevalence of the apoBR3500Q mutation to be increased in patients with acute myocardial infarction. In the Danish population, 70% of the mutation carriers suffer from CHD at age 60 compared with 
10% in the general population. It has been shown that 16% of all patients in the 30–74 age group suffering from myocardial infarcts die before admission to the hospital (M. Davidsen et al., submitted for publication), just as the finding of dyslipidemia in adolescents carrying the apoBR3500Q mutation (22) supports the hypothesis that the carriers of the apoBR3500Q mutation are at increased risk of a sudden death. The mutation is codominant and probably leads to development of atherosclerosis and CHD at an early age (5)(23)(24). In Denmark, 5000–10 000 individuals go undetected as carriers of the mutation, and genotyping appears to be the only reliable alternative because determination of fasting cholesterol is an unreliable marker in diagnosing FDB in the young and in middle-aged adults (22)(25)(26)(27)(28). Three persons were found to harbor the apoBR3500Q mutation (unpublished results) when the apoBR3500Q analysis was extended to the Danish MONICA 10 cohort (29), which consists of 2656 persons. This is in accordance with the prevalence of the mutation in the general population (5)(6).
In summary, we have established a genotyping method based on LNA technology for the detection of the apoBR3500Q mutation in a microtiter plate format. The assay consists of a single hybridization step in which the DNA target is captured, followed by detection of the hybrids. It demonstrates that LNA oligonucleotides are uniquely suited as probes in mutation and single-nucleotide polymorphism detection assays. Conformational fixation of the sugar moiety in the LNA nucleotide enables more specific hybridization with DNA targets compared with the corresponding DNA-based capture probes (Table 1
). The apoBR3500Q assay includes capture probes for both the wild-type and the apoBR3500Q mutation and detects both homozygous and heterozygous patient samples. The design ensures that no false-negative results occur in the genotyping of the apoBR3500Q mutation. The assay is also suited for automation and high-throughput screening and can be adapted to genotyping microarrays.
Acknowledgments
This stuy was supported by Grant 1998-503/0002-1 from The Danish Agency for Trade and Industry.
References
The following articles in journals at HighWire Press have cited this article:
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  K. Kubota, A. Ohashi, H. Imachi, and H. Harada Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes Appl. Envir. Microbiol., August 1, 2006; 72(8): 5311 - 5317. [Abstract] [Full Text] [PDF]
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J. Rupp, W. Solbach, and J. Gieffers Single-Nucleotide-Polymorphism-Specific PCR for Quantification and Discrimination of Chlamydia pneumoniae Genotypes by Use of a "Locked" Nucleic Acid. Appl. Envir. Microbiol., May 1, 2006; 72(5): 3785 - 3787. [Abstract] [Full Text] [PDF]
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 A. Valoczi, C. Hornyik, N. Varga, J. Burgyan, S. Kauppinen, and Z. Havelda Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes Nucleic Acids Res., December 14, 2004; 32(22): e175 - e175. [Abstract] [Full Text] [PDF]
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M. P. Johnson, L. M. Haupt, and L. R. Griffiths Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR Nucleic Acids Res., March 26, 2004; 32(6): e55 - e55. [Abstract] [Full Text] [PDF]
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D. A. Di Giusto and G. C. King Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays Nucleic Acids Res., February 18, 2004; 32(3): e32 - e32. [Abstract] [Full Text] [PDF]
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N. Tolstrup, P. S. Nielsen, J. G. Kolberg, A. M. Frankel, H. Vissing, and S. Kauppinen OligoDesign: optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling Nucleic Acids Res., July 1, 2003; 31(13): 3758 - 3762. [Abstract] [Full Text] [PDF]
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N. Jacobsen, J. Bentzen, M. Meldgaard, M. H. Jakobsen, M. Fenger, S. Kauppinen, and J. Skouv LNA-enhanced detection of single nucleotide polymorphisms in the apolipoprotein E Nucleic Acids Res., October 1, 2002; 30(19): e100 - e100. [Abstract] [Full Text] [PDF]
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A. Simeonov and T. T. Nikiforov Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection Nucleic Acids Res., September 1, 2002; 30(17): e91 - e91. [Abstract] [Full Text] [PDF]
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, signal from wells containing wt-LNA8 capture probe. 
, signals from wells containing m-LNA8 capture probe. Absorbance values (y axis) are relative to sample 8.