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Division of Transfusion Medicine, University Hospital of Cologne, Joseph-Stelzmann Strasse 9, 50924 Cologne, Germany
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NAXCOR, 4600 Bohannon Dr., Suite 220, Menlo Park, CA 94025
a author for correspondence: fax 49-0221-478-6179, e-mail Birgit.Gathof{at}medizin.uni-koeln.de
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Introduction |
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Current HH genotyping techniques include restriction fragment length polymorphism (RFLP) analysis (5) and heteroduplex analysis (6), both PCR based. We evaluated a nucleic acid-based test with cross-linkable DNA probes to screen for the Cys282Tyr mutation in a total of 101 presumably healthy blood donors. The assay uses oligonucleotide probes modified with photo-activatable cross-linker molecules (7) and has been used to detect the factor V Leiden mutation (8). Two sets of allele-specific cross-linkable DNA probes were prepared that detect either the wild-type or mutant Cys282Tyr gene sequences. Samples prepared from donor blood were assayed with each probe set, and the genotype of each individual was determined by comparison of the fluorescent signals obtained. All blood samples were also assayed by a PCR-RFLP test.
Two capture probes that hybridized preferentially to the wild-type or mutant HFE gene sequence, respectively, were synthesized for the cross-linking assay: (5'-3') AXATACGTGCCAGGTG and AXATACGTACCAGGTGG (the underlined bases represent the position of the mutation site in the target). The coumarin-based cross-linking nucleotide is denoted as X; both probes were biotinylated at the 3' end (8). In addition, 24 reporter probes were synthesized, each containing two fluorescein residues at the 5' terminus and a cross-linker molecule in place of a nucleotide one position from the 3' terminus, the 5' terminus, or both. The reporter probes were designed from HFE gene sequence flanking the mutation, corresponding to the following nucleotide positions (9): 6443–6462, 6472–6493, 6495–6518, 6574–6593, 6597–6618, 6632–6653, 6661–6682, 6686–6707, 6716–6739, 6780–6799, 6801–6822, 6838–6861, 6877–6900, 6905–6928, 6951–6973, 6984–7006, 7060–7083, 7089–7108, 7128–7151, 7163–7186, 7208–7231, 7235–7256, 7301–7322, and 7381–7402.
Blood specimens were obtained from 101 blood donors with informed consent under an institutional review board-approved protocol (University of Cologne). Leukocytes were isolated from blood samples as described (8), resuspended in leukocyte lysis reagent (0.28 mol/L NaOH), and either boiled at 100 °C for 30 min immediately before the assay or stored at -20 °C for up to 14 days before boiling.
Processed samples were placed into two wells each of a 96-well polypropylene microtiter plate. Each assay plate also contained four negative controls (leukocyte lysis reagent that had not been boiled) and two positive controls (50 amol/well of a PCR amplicon covering the assay locus amplified from a Cys282Tyr and wild-type heterozygote in leukocyte lysis reagent that had not been boiled). Two different probe solutions were prepared, each containing the same set of 24 reporter probes and 1 of the 2 allele-specific capture probes. Aliquots of each probe solution were added to one of each pair of sample wells, as well as two negative and one positive control wells. Neutralization of the solutions, photo cross-linking, and addition of the streptavidin-coated magnetic beads have been described (8). The beads were then washed twice with wash reagent (0.15 mol/L NaCl, 0.015 mol/L sodium citrate, 1 mL/L Tween-20). The beads were incubated in the presence of anti-fluorescein antibody-alkaline phosphatase conjugate (Dako), washed four times, and resuspended in AttophosTM (Promega) as described (8). The fluorescence signal was determined by reading the plate in a microplate fluorometer (Packard Instrument).
Genomic DNA was extracted from whole blood by the QIAquick Blood reagent set (QIAGEN). A sequence flanking the variant codon 282 was amplified by PCR (DyNAzyme PCR reagent set; Biometra), and the amplicons were digested with RsaI (Roche), size-fractionated by agarose gel electrophoresis, and genotyped as described (5).
Determination of the genotype of an individual with the cross-linking assay was based on the relative signals obtained with the two allele-specific capture probe preparations. The net sample signal (NSS) was derived for each sample and each probe set by subtracting the mean of the negative control values from the sample signal. The NSS ratio was defined for each sample as the NSS for the mutation divided by the NSS for the wild type. The NSS ratio intervals that define a particular genotype were set before the donor samples were tested by assaying PCR amplicons derived from individuals who were wild type, heterozygous, or mutant homozygous for the Cys282Tyr allele (20 determinations for each genotype). These experiments yielded the following mean NSS ratios: wild type, 0.05 (range, 0.01–0.45); heterozygous, 1.22 (range, 0.95–2.15); and mutant homozygous, 6.13 (range, 2.95–15.00). On the basis of these results, the following NSS ratio intervals were used to assign a sample genotype: wild type, NSS ratio = 0–0.75; heterozygous, NSS ratio = 0.76–2.5; and homozygous mutant, NSS ratio >2.5.
The sample data fell into two groups (Fig. 1
). The first group of 93 samples had NSS ratios of 0.12–0.66
(mean = 0.36; SD = 0.11) and was assigned a wild-type
genotype. The second group (eight samples) had NSS ratios of
1.02–1.55 (mean = 1.30; SD = 0.18), compatible with
heterozygosity. No individuals homozygous for the Cys282Tyr mutation
were identified. To validate the method for detection of the homozygous
mutant genotype, the assay was performed on a blood sample from a known
homozygote. The NSS ratios for this individual, in two evaluations,
were 9.1 and 7.4, within the predicted range.
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The results of PCR-RFLP testing were in complete agreement with those obtained with the cross-linking assay for all 101 samples.
The cross-linking assay has several advantages. It allows detection of the Cys282Tyr mutation without the laborious steps of DNA purification, PCR, and RFLP analysis, and it eliminates problems of sample inhibition of polymerases and sample contamination by amplicons. An additional advantage is the large-scale simultaneous processing of DNA samples, using the microtiter plate format. With automated detection, the cross-linking assay can be finished within 4 h.
Further work is needed to fully define the set of NSS ratio ranges that determine the three genotypes. Data from the blood sample assays showed wider variation among samples of the same genotype than was seen with the PCR samples. Presumably, this indicates that signal intensity is influenced by factors such as the efficiency of the overall sample preparation procedure and variation in blood volume and leukocyte concentration. Further sample data will allow us to set finer intervals for genotype assignment and to set "gray zone" values for repeat testing.
Large-scale, presymptomatic screening of blood donors for the Cys282Tyr mutation could identify individuals at risk for HH, who are then candidates for prophylactic phlebotomy, which increases the life expectancy to that of the general population. If such a screening regimen was to be implemented, the tests needed to perform genotype analysis will have to be accurate, inexpensive, and automatable. The cross-linking assay used here is an efficient, simple, and rapid method of genotyping HFE mutations that, with automation, would be suitable for routine genetic analysis in a large-scale manner.
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References |
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The following articles in journals at HighWire Press have cited this article:
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  C. French, C. Li, C. Strom, W. Sun, R. Van Atta, B. Gonzalez, and M. Wood Detection of the Factor V Leiden Mutation by a Modified Photo-Cross-Linking Oligonucleotide Hybridization Assay Clin. Chem., February 1, 2004; 50(2): 296 - 305. [Abstract] [Full Text] [PDF]
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R. Peoples, H. Weltman, R. Van Atta, J. Wang, M. Wood, M. Ferrante-Raimondi, P. Cheng, and B. Huan High-Throughput Detection of Submicroscopic Deletions and Methylation Status at 15q11-q13 by a Photo-Cross-Linking Oligonucleotide Hybridization Assay Clin. Chem., October 1, 2002; 48(10): 1844 - 1850. [Abstract] [Full Text] [PDF]
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