Created: 1st June 2000, last updated: 30th August 2000, © 2000 ABRF
Qi Liang,a P. Ann Davis,a John T. Simpson,a Barry H. Thompson,a Joseph M. Devaney,b,c and James Girardb
aCenter for Medical and Molecular Genetics, Armed Forces Institute of Pathology, Rockville, MD; bDepartment of Chemistry, American University, Washington, DC; ccurrently with Transgenomic, Inc., Gaithersburg, MD
Hereditary hemochromatosis is one of the most common hereditary disorders in Caucasians. The disease is linked to two single-nucleotide polymorphisms (SNPs) in the HFE gene. The two point mutations result in a change of Cys to Tyr at position 282 and His to Asp at position 63 in the resultant protein. We have developed a single-nucleotide extension (SNE) assay for hereditary hemochromatosis genetic testing, which employs capillary electrophoresis to simultaneously detect the SNE products generated from the two SNP sites. An upstream or a downstream primer adjacent to the possible mutation site is designed and extended one nucleotide further at the 3' end, complementary to the nucleotide at the possible mutation site. The extended nucleotide is one of four fluorescently labeled dideoxynucleotide triphosphates that also act as terminators. Analysis of the extended products by laser-induced fluorescence capillary electrophoresis (LIF-CE) directly reflects the identity of the possible mutation site. Using one primer upstream or downstream from the possible mutation site, three genotypes at one mutation site can be distinguished. Using both upstream and downstream primers provides a second level of specificity and increases the accuracy of the genetic test. The protocol can also be applied to the study of other SNP analyses and to simultaneous detection of multiple mutation sites. (J Biomol Tech 2000;11:67-73)
Key Words: hemochromatosis, genetic testing, single-nucleotide extension, single-nucleotide polymorphism (SNP), capillary electrophoresis (CE).
Address correspondence and reprint requests to: Qi Liang, CMMG, 1413 Research Boulevard, Rockville, MD 20850 (email: liangq@afip.osd.mil).
Hemochromatosis is a metabolic disorder resulting in excessive iron accumulation in the liver, heart, and other organs.1 Hereditary hemochromatosis (HH) is an autosomal recessive disorder that occurs in 2 to 5 of every 1000 individuals of Northern European descent.2 Despite the high prevalence of this illness, most cases remain undiagnosed. The many early symptoms, such as fatigue and pain in the joints and abdomen, are nonspecific. These symptoms are often ignored or mistaken for other illnesses.3 Without treatment, irreversible organ damage and dysfunction can develop, including cirrhosis, cardiomyopathy, diabetes, and impotence. Fortunately, hemochromatosis is among a few illnesses that have a simple and effective treatment if diagnosed in its early stages. Treatment consists of removal of the iron burden by regular phlebotomy. Phlebotomy is such an effective treatment that, if implemented before the development of cirrhosis, a patient's life expectancy can be normal.1 Early detection and treatment are therefore essential.
Genetic diagnosis was made possible by identification of a hemochromatosis gene (HFE) by Feder and coworkers in 1996.4 The HFE gene encodes for a 348-amino acid HFE protein, which binds to the transferrin receptor and reduces its affinity for iron-loaded transferrin. Two single-nucleotide polymorphisms (SNPs) in the HFE gene, one from C to T and the other from G to C in the sense strand, are believed to cause HH. These two mutations result in a change of Cys to Tyr at position 282 (C282Y) and His to Asp at position 63 (H63D) in the protein, respectively.4,5 A review of HH has summarized studies from nine research groups.5 A combined total of 935 hemochromatosis patients and 1252 persons with no clinical symptoms of hemochromatosis were included. An average of 83% of these patients had a homozygous C282Y mutation, compared with 0.2% that were homozygous at C282Y in control samples. There was no substantial difference in the percentage of compound heterozygous (heterozygous H63D in association with heterozygous C282Y) or homozygous H63D mutation in patients versus controls. Nonetheless, the occurrence of heterozygous H63D was as high as 21.3% in controls, whereas it was 3.4% in patients. It is agreed that compound heterozygotes are at much less risk for iron overload than homozygous C282Y and that homozygous H63D individuals bear even less risk.6-8
Several methods for SNP detection can be applied for the analysis of point mutations in HH. These methods include general sequencing,4 restriction analysis,6,8,9 primer extension,10-12 polymerase chain reaction (PCR) with sequence-specific primers,13-15 heteroduplex formation,16 and single-strand conformation polymorphism (SSCP) formation.17 Of these methods, analysis of primer extension products offers considerable versatility for SNP detection. The SNE method requires an upstream or downstream primer immediately 5' to the possible mutation site. The primer is extended at the 3' end with one of the four fluorescently labeled dideoxynucleotide triphosphates (ddNTP) complementary to the nucleotide at the possible mutation site. The ddNTPs also act as terminators in the primer extension reactions. Using one primer upstream or downstream from the possible mutation site, three genotypes at one mutation site can be distinguished. The use of both primers provides a second level of specificity and increases the accuracy of the genetic test. Because the extended products have relatively low molecular weights, a one nucleotide difference in sequence can be easily separated and detected by various instrumentation such as high-pressure liquid chromatography (HPLC), mass spectrometry (MS), and slab gel or capillary electrophoresis (CE).
We present a protocol for the simultaneous analysis of both SNPs in HH, based on laser-induced fluorescent capillary electrophoresis (LIF-CE) detection of SNE products with different fluorescently labeled ddNTPs.
DNA was extracted from FTA923 bloodstain cards (Fitzco, Maple Plain, MN) as previously described.18 Hemochromatosis-positive control samples were provided by Dr. Susan Leitman, Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD.
Primers F1, R1, SE18, and SE23 were synthesized on an Expedite Nucleic Acid Synthesis System (PerSeptive Biosystems, Inc., Framingham, MA). Primers SE16 and SE21 were synthesized and HPLC purified by Synthetic Genetics (San Diego, CA). The 282 mutation site can be amplified by either of two sets of primers: F1 (5'-CCACTGATGACTCCAATGACTA-3') and R1 (5'-AAGCAGCCAATGGATGCCAAG-3'), which bracket the 282 site and amplify a 367-bp DNA fragment, or SE16 and SE21 (Fig. 1), which are upstream and downstream adjacent to the possible 282 mutation site and amplify a 38-bp product. Primers SE18 and SE23 (Fig. 1) are upstream and downstream adjacent to the possible 63 mutation site and amplify a 42-bp product. All four SE primers (SE16, SE21, SE18, and SE23) were used in the same PCR reaction to co-amplify both sites if subsequent multiplex SNE was to be performed.
FIGURE 1. Primers designed for PCR amplification and single-nucleotide extension for multiplex detection of hereditary hemochromatosis. (A) Primers for the 282 site. The bold C/G base pair is the possible mutation site that, when replaced by T/A, causes the Cys-->Tyr mutation at the 282 site in the protein. (B) Primers for the 63 site. The bold G/C base pair is the possible mutation site that, when replaced by C/G, causes the His-->Asp mutation at the 63 site in the protein.
PCR reactions were performed using the GeneAmp PCR system 9600 thermal reaction cycler (PE Biosystems, Foster City, CA). A 20-µL total PCR reaction mixture contained extracted DNA (about 10 ng), 2 µL of 10X PCR reaction buffer (PE Biosystems), 0.4 µL of dNTP mix (10 mM each; Boehringer Mannheim Corp., Indianapolis, IN), 0.2 µL of bovine serum albumin (4 µg/mL; Boehringer Mannheim), 2 µL of the appropriate primers (10 µM F1/R1 or SE16/SE21 for the 282 site and SE18/SE23 for the 63 site), and 0.2 µL of AmpliTaq Gold polymerase (PE Biosystems). Amplification was performed with an initial denaturation at 96°C for 10 minutes, followed by 32 cycles of denaturation at 94°C for 10 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 20 seconds.
Unincorporated primers and dNTPs were removed from the amplified PCR products by passing through a Centricon column filter (Millipore Corp., Bedford, MA) or by enzyme treatment. Enzyme treatment was performed by adding 1 µL each of shrimp alkaline phosphatase and Exonuclease I (both from USB Corp., Cleveland, OH) to the PCR mixture. The samples were incubated at 37°C for 30 minutes, followed by heat inactivation of enzymes at 80°C for 15 minutes.
Single-nucleotide extension was performed on PCR products according to the protocols accompanying the ABI PRISM dye terminator cycle sequencing core kit (PE Biosystems). For the detection of the 282 site only, the SE16/SE21 pair was used as SNE primers on the F1/R1 or SE16/SE21 PCR product. For detection of the 63 site only, the SE18/SE23 pair can be used as SNE primers on the SE18/SE23 PCR product. For multiplex detection, SE16, SE18, SE21, and SE23 were used together as SNE primers on the co-amplified PCR product produced by those same primers. The reaction mixture of a total of 20 µL contained 4 µL of 5X reaction buffer, 1 to 2 µL of purified PCR products (about 10 ng), 1 pmol of detection primer, 0.2 µL of each ddNTP terminator, and 0.3 µL of AmpliTaq polymerase FS.
The mixture underwent the following thermal cycling: 94°C for 1 minute followed by 20 cycles of denaturation at 92°C for 30 seconds and annealing and extension at 70°C for 40 seconds with -0.5°C per cycle. This protocol covers a range of annealing temperatures to allow several primers to be annealed and extended.
The reaction mixture was then passed through an AGTC column (Advanced Genetic Technologies Corp., Gaithersburg, MD) to remove the unincorporated terminators and salts. Then 1 to 2 µL of the purified reaction mixture was added to 12 µL of the Template Suppression Reagent (a denaturing buffer; PE Biosystems), heated at 95°C for 2 minutes, cooled on ice for 5 minutes, and loaded onto the autosampler of the 310 Genetic Analyzer.
Analysis of the single-nucleotide extension was performed on a PE ABI PRISM 310 Genetic Analyzer. All materials for the 310 Genetic Analyzer were obtained from PE Biosystems. A fused silica capillary (50 µm inside diameter X 47 cm, with an effective length of 36 cm) was installed for the separation of DNA fragments. The capillary was filled with Performance Optimized Polymer 4. The running buffer was a 1:10 dilution from 10X Genescan Buffer with EDTA. The sample was injected at 15 kV for 5 seconds and run at 15 kV for 15 minutes at 60°C using filter set F. After the run, the raw data were analyzed with a matrix generated from [F]dNTP Matrix Standards.
DNA was extracted from the blood samples and amplified by PCR. For the amplification of the DNA, any set of primers bracketing the possible mutation sites can be applied. Initially, we chose F1/R1 as PCR primers for the amplification of DNA containing the 282 site, where F1 is 248 bp upstream from the 282 site and R1 is 119 bp downstream from the 282 site. F1/R1 generated a 367-bp DNA fragment. Later, we found that the primer set SE16/SE21 produced PCR products of a shorter DNA fragment (38 bp, see Fig. 1) containing the 282 site as efficiently as with F1/R1. We recommend using SE16/SE21 for the amplification of DNA containing the 282 site, because these primers are also used for the single-nucleotide extension reactions. In this manner, we only need one set of primers for the initial DNA amplification and for the SNE. For the detection of the 282 site only, we used SE16/SE21 pair as SNE primers on the F1/R1 PCR products. For multiplex detection, SE16, SE18, SE21, and SE23 were used together as SNE primers on the co-amplified PCR product produced by those same primers.
DNA fragments containing the Cys282Tyr locus of hemochromatosis were amplified by PCR with primers F1 and R1 (see the Materials and Methods section). Complete removal of unincorporated primers and dNTPs from the PCR products was necessary so that they would not otherwise interfere with the subsequent single-nucleotide extension, resulting in high background and peaks from the remaining primers. The unincorporated primers and dNTPs were successfully removed by column purification or inactivated by Exonuclease I and shrimp alkaline phosphatase directly after the PCR (Fig. 2). After completion of the PCR reactions, a PCR product peak (367 bp, Fig. 2B-D) appeared with a retention time close to the 400-bp standard (Fig. 2E). The excess primers and dNTPs that eluted at about 10 minutes were substantially decreased after enzyme treatment or column purification (Fig. 2C, D). The PCR product and primer peaks were more prominent after column purification because of removal of salts and other low-molecular-weight impurities. The relatively small amount of remaining impurities did not affect the subsequent primer extension reactions.
FIGURE 2. Monitoring the PCR products and purification processes by laser-induced fluorescence capillary electrophoresis. (A) Reaction mixture before PCR hot start. (B) Unpurified reaction mixture after PCR. (C) Enzyme-treated PCR products. (D) Column-purified PCR products. (E) DNA mass ladder (100, 200, 400, 800, 1200, and 2000 bp).
To demonstrate that the SNE method was able to detect different genotypes, samples of known genotypes were first studied. The genotypes of these samples were confirmed by a restriction-enzyme reaction method9 and by general sequencing. CE peak colors of the SNE products can be predicted for known site point mutations once we know the genotype, the primers, and the fluorescent tag of the four dideoxynucleotide triphosphate (Table 1). Each dideoxynucleotide triphosphate--ddATP, ddCTP, ddGTP, and ddTTP--is labeled with a different fluorescent tag--R6G, ROX, R110, and TAMRA, respectively (ABI Prism Terminator Core Kit). When excited with an argon laser beam (488 nm), each tag emits light in the range of green, red, blue, or yellow, respectively (yellow is converted to black on the screen and in the hardcopy). In the CE electropherogram, a shorter electrophoresis time indicates a shorter DNA fragment, and the color of a peak indicates which nucleotide has been added to the primer. As predicted from Figure 1A, DNA coding for a wild-type or a mutant 282 site in HFE results in extending a C or a T to primer SE16 (Table 1), which generates a red peak or a black peak in CE (Fig. 3). CE profiles (the relative elution time and peak colors) of the SNE products from known genotypes, as shown in Figure 3, completely confirmed the predictions in Table 1. A homozygote normal sample generates a red peak from SE16 and a blue peak from SE21; a homozygote mutant sample generates a black peak from SE16 and a green peak from SE21; and a heterozygote sample generates both red and black peaks from SE16 and green and blue peaks from SE21. With this knowledge, we can determine the genotypes for the 282 site for the detection of HH.
TABLE 1
Predicted Capillary Electrophoresis Peak Colors Generated From the Single-Nucleotide Extension Products of Primers SE16 and SE21, Deduced From the Three Genotypes of the 282 Site Associated With Hemochromatosis
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| Genotype | ||||||
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| Primer | Normal/ normal |
Normal/ mutant |
Mutant/ mutant |
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| SE16 | ||||||
| Extended by | ddCTP | ddCTP, ddTTP | ddTTP | |||
| Peak color | Red | Red, black | Black | |||
| SE21 | ||||||
| Extended by | ddGTP | ddATP, ddGTP | ddATP | |||
| Peak color | Blue | Green, blue | Green | |||
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FIGURE 3. Capillary electrophoresis peak profiles of three genotypes for the 282 site. The single-nucleotide extension products are (A) primer SE16, (B) primer SE21, and (C) primers SE16 and SE21. The small amount of bleed-through in the mutant samples probably results from background contamination and is under investigation. H, heterozygote; M, homozygote mutant; N, homozygote normal.
After investigating the feasibility of detecting genotypes for the 282 site, we applied the SNE method for the detection of the 282 site and the 63 site simultaneously. For multiplex detection of both sites, the same primers were used for amplification and detection; however, amplification and detection were carried out separately. The two DNA fragments, a 42-bp fragment bracketing the 63 site and a 38-bp fragment bracketing the 282 site, were amplified simultaneously using SE18 and SE23 for the 63 site and using SE16 and SE21 for the 282 site (Fig. 1). After enzyme treatment, the PCR mixture was combined with these same primers and fluorescently labeled ddNTPs to perform SNE reactions. The SNE products were analyzed by LIF-CE, and the genotypes at both possible mutations were determined.
Theoretically, as shown in Tables 1 and 2, one primer from the primer set SE16/SE21 and SE18/SE23, upstream or downstream at the detection site, is sufficient to distinguish three genotypes: homozygous normal, homozygous mutant, and heterozygous. This is the case for detection of the 282 site as shown in Figure 3. In the multiplex detection, SE16 was chosen for the detection of the site 282 site rather than SE21, because it provided better peak separation from the SNE product peaks generated from primers SE18 or SE23 (Fig. 4C). For detection of the 63 site, both SE18 and SE23 primers were necessary. An example shown in Figure 4B demonstrates that SE23 alone cannot clearly distinguish the heterozygote from homozygous normal for the 63 site. In the red/blue pair of peaks indicating heterozygosity, the blue peak is very small compared with the red peak, which can lead to questionable interpretation of the data. The bleed-through of the adjacent high-intensity red peak tends to lower the signal-to-noise ratio of the blue peak. From our studies of four samples with a heterozygous 63 site, the blue peak generated from SE18 or SE23 may be too small for genotype determination. In this case, as indicated in Table 2, using both SE18 and SE23 can clearly distinguish the heterozygote from the normal or the mutant at the 63 site. Although the blue peak may not be a good candidate as a primary criterion for the heterozygous 63 site, it is certainly useful as a secondary criterion.
TABLE 2
Predicted Capillary Electrophoresis Peak Colors Generated From the Single-Nucleotide Extension Products From Primers SE18 and SE23, Deduced From the Three Genotypes of the
63 Site Associated With Hemochromatosis
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| Genotype | ||||||
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| Primer | Normal/ normal |
Normal/ mutant |
Mutant/ mutant |
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| SE18 | ||||||
| Extended by | ddGTP | ddCTP, ddGTP | DdCTP | |||
| Peak color | Blue | Red, (blue)a | Red | |||
| SE23 | ||||||
| Extended by | ddCTP | ddCTP, ddGTP | ddGTP | |||
| Peak color | Red | Red, (blue)a | Blue | |||
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aParentheses indicate that, during observation, the intensity of that peak is relatively low and may not serve as a primary criterion. In this case, use of both primers is necessary for the genetic determination of the 63 site.
FIGURE 4. Multiplex detection of the genotypes of hemochromatosis at the 282 site and the 63 site. (A) Three detection primers (SE16, SE18, and SE23) are sufficient for simultaneous determination of both sites. The peak labeled N-16 denotes a normal individual, with the SNE product from primer SE16; the peak labeled H-18 denotes a heterozygote individual, with the SNE product from primer SE18; the peak labeled M-16 denotes a mutant individual, with the SNE product from primer SE16, and so forth. (B) Two primers (SE16 and SE23) are not sufficient to distinguish the heterozygous from the normal individual at the 63 site. (C) Four primers (SE16, SE18, SE21, and SE23) are sufficient but crowded. Genotype is 282 site/63 site. H, heterozygote; M, homozygote mutant; N, homozygote normal.
Figure 4A demonstrates that primers SE16, SE18, and SE23 generated specific and unambiguous peak profiles for simultaneous genotype detection for the 282 site and the 63 site. In all cases, detection for the 282 site is unambiguous and is determined by the color of the earliest eluting peak: red for homozygous normal, black for homozygous mutant, red and black for heterozygote. For the 63 site, the SNE products from primer SE18 elute early, and the SNE products from primer SE23 elute later. The peak colors correspond with the genotypes listed in Table 2. For example, in a normal 63 site sample, a single red peak from SE23 products is seen (Fig. 4A, peak N-23). Figure 4C further demonstrates that by using all four primers, two for each site, genetic determination for both sites is accomplished. However, the use of all four primers made the interpretation of the results somewhat complicated. Given these results, the use of three primers--SE16, SE18, and SE23--for the testing of HH associated with mutations at the 282 site and the 63 site yields the clearest and most consistent analysis.
We applied an SNE method for the detection of Cys282Tyr and His63Asp mutations in HH. A primer set upstream and downstream adjacent to each of the possible mutation sites was designed for the PCR amplification and for SNE reaction. Fluorescently labeled dideoxynucleotides were added to the 3' end of the primers so that extended products contained appropriate fluorescent tags, which correspond to the genotype of the detection site. The extended DNA fragments were then analyzed by LIF-CE to determine the genotypes of the samples. CE easily separated the short, primer-extended products, and laser-induced fluorescence provided sensitive and specific detection of these products. The use of a fluorescent-labeled dideoxynucleotide aids in differentiation of the DNA fragments with one nucleotide difference in sequence and provides a specific tag to determine the identity of the extended nucleotide. This SNE method therefore can be used for multiplex detection of multiple point mutations, with the only requirement being that the primer lengths are different from one another. Using one primer for each mutation allows three genotypes to be distinguished simultaneously. By using two primers for each mutation site, unambiguous detection of each SNP is possible, which is critical for screening HH or other SNP diseases.
The authors thank Dr. Susan Leitman, Department of Transfusion Medicine, National Institutes of Health, for access to hemochromatosis control samples. Q.L. would like to thank Dr. Michael Marino for informative suggestions and discussions. The views stated here are the opinions of the authors and in no way reflect the position of the U.S. Army, U.S. Air Force, or U.S. Department of Defense.
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