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Technical Advance |
From the Departments of Pathology*
and Internal
Medicine,
University of Utah Medical School,
Salt Lake City, Utah
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hereditary hemochromatosis is the most common genetic illness known in the Northern Hemisphere with more than 1 million Americans estimated to be at risk for the disease.5 This autosomal recessive disorder of iron metabolism occurs with a frequency of approximately 0.5% in Caucasian populations.6,7 The dysregulation of intestinal iron absorption can eventually lead to parenchymal cell damage and end-organ dysfunction. Long-term complications of iron overload include arthritis, cardiomyopathy, diabetes, cirrhosis, and hepatocellular cancer. The morbidity associated with iron overload is preventable through early diagnosis and treatment by phlebotomy.
A cysteine-to-tyrosine amino acid substitution, caused by a G845A transition at codon 282 (C282Y), is found on 85 to 100% of disease chromosomes from patients of northern European ancestry who meet well defined clinical criteria for iron overload.8-11 Another mutation (H63D) is created by a C187G transversion. This substitution has an estimated penetrance between 0.44 and 1.5% of the homozygous C282Y genotype.8-10
Current methods for genotyping the C282Y and H63D hemochromatosis-causing mutations include oligonucleotide ligation,8 allele-specific oligonucleotide hybridization,9 and PCR restriction fragment length analysis.10-12 All of these methods require multiple manual steps and are time consuming. An alternative is the use of fluorescent hybridization probes and rapid-cycle PCR, a technique that provides homogeneous amplification and genotyping in approximately 45 minutes.13,14 This technique previously used an internally labeled Cy5 primer to asymmetrically amplify excess Cy5-labeled strand.13,14 Biallelic variations at a single site were genotyped by a complementary 3'-fluorescein-labeled probe. This method requires manual synthesis of the Cy5 primer, and multiplex analysis is difficult because the primers must be near the mutation sites.
In this paper, a more versatile fluorescence energy transfer probe design is introduced that uses adjacent fluorescent hybridization probes and allows multiple variants to be analyzed simultaneously. The 5'-Cy5-labeled and 3'-fluorescein-labeled probes can be made on automated synthesizers and are designed to hybridize to the same strand between unlabeled primers. Monitoring the fluorescence energy transfer between adjacent hybridization probes allows real-time detection of specific PCR product.15 When one of the probes is positioned over an allele variant, the melting curve profile allows homogeneous genotyping in the same instrument.
The thermal stability of a DNA duplex relies on duplex length, GC content, and Watson-Crick base pairing.16 Changes from Watson-Crick pairing destabilize a duplex by varying degrees depending on the length of the mismatched duplex, the particular mismatch, the position of the mismatch, and neighboring base pairs.17,18 Considering these factors, two probe sets were designed for multiplex analysis of the two mutation sites in the hemochromatosis gene. Four different alleles could be identified simultaneously during melting curve analysis. In addition, a single probe identified three alleles, including an unexpected polymorphism (A193T), demonstrating the potential of adjacent fluorescent hybridization probes for scanning unknown mutations.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genomic DNA from Caucasian individuals was collected over a 10-year period for studying hemochromatosis pedigrees in Utah and neighboring states.19 Methods of collection were approved by the institutional review board at the University of Utah. Two clinically defined subject groups consisting of 117 patients and 56 controls were selected from 250 genotyped samples to determine the prevalence of the C282Y and H63D mutations in the HFE gene. Family-based controls had either married into a pedigree or had no HLA identity with the proband. Genotyped patients with ambiguous clinical histories were excluded from the subject group. Patients were selected through laboratory evidence of iron overload (transferrin saturation >55% and serum ferritin >600 µg/L), and liver biopsies were performed on most of these patients to determine the grade of liver siderosis. All controls had normal values for serum ferritin and percent transferrin saturation.
Genotyping
All samples were genotyped at the C282Y and H63D sites with adjacent fluorescent hybridization probes. Genotyping both sites simultaneously by multiplexing was performed on 70 samples. Reagent concentrations for multiplexing and single-site analysis were the same. However, multiplexed reactions contained two primer sets and two fluorescently labeled probe sets. Each 10-µl reaction contained 50 mmol/L Tris, pH 8.3 (25°C), 500 µg/ml bovine serum albumin, 0.2 mmol/L each deoxyribonucleoside triphosphate, 4 mmol/L MgCl2, 0.5 µmol/L each primer, 0.1 µmol/L site-specific 3'-fluorescein-labeled probe, 0.2 µmol/L site-specific 5'-Cy5-labeled probe, 50 ng of genomic DNA, and 0.4 U of native Taq DNA polymerase. Samples were loaded into separate plastic/glass composite cuvettes, centrifuged, and capped. Homogeneous PCR and melting curve acquisition used a 24-sample rapid fluorescent thermal cycler (LightCycler LC24, Idaho Technology, Idaho Falls, ID).
Forty repeats of a two-temperature cycle were performed (94°C for 0 seconds and 62°C for 20 seconds with programmed transitions of 20°C/second). Fluorescence was acquired once each cycle for 50 milliseconds per sample at the end of the combined annealing/extension step. An appended analytical cycle after amplification allowed immediate genotyping by derivative melting curves. The genotyping protocol included denaturation at 94°C for 20 seconds; annealing for 20 seconds each at 65°C, 55°C, and 45°C (C282Y, S65C, and multiplexing) or 75°C, 65°C, and 55°C (H63D); and a high-resolution melting transition to 75°C at a rate of 0.1°C/second. Cy5 (655 to 695 nm) and fluorescein (520 to 560 nm) fluorescence were monitored for 50 milliseconds per sample at each 0.1°C temperature increment.
The data collected during the melting phase were used to genotype each sample. Melting curves were generated by plotting Cy5 fluorescence (F) versus temperature (T). Easily discriminated melting peaks were obtained by plotting the same data as -dF/dT versus temperature.
Genotyping performed in the LightCycler was compared with conventional PCR restriction fragment length analysis. PCR restriction fragment length analysis was performed on all samples for the C282Y mutation and on 40 random samples for the H63D mutation. Amplification for the PCR restriction fragment length method was performed in an air thermal cycler (RapidCycler, Idaho Technology) using the same primers, MgCl2 concentration, and temperature parameters as that used in the LightCycler. Probes were not added for PCR restriction fragment length analysis. Samples were restriction digested at C282Y or H63D by adding 1 µl of either SnaBI (4 U/µl; New England Biolabs, Beverly, MA) or BclI (10 U/µl; New England Biolabs), respectively, to 1 µl of the recommended digestion buffer and 8 µl of amplicon. The samples were incubated for 2 hours at either 37°C (SnaBI) or 50°C (BclI), and products were visualized by ethidium bromide staining after separation on a 1.5% agarose gel at 5 V/cm for 60 minutes.
Sequencing
Four samples were sequenced for identification of the A193T polymorphism. PCR products were sequenced (model 377, Perkin-Elmer, Foster City, CA) from TOPO TA plasmid vectors (TOPO TA Cloning, Invitrogen, San Diego, CA).
Primer/Probe Synthesis
Primers for the C282Y codon8 and the H63D codon were synthesized by standard phosphoramidite chemistry (Pharmacia Biotech Gene Assembler Plus, Piscataway, NJ). The 3'-fluorescein-labeled probes were synthesized on fluorescein-controlled pore glass cassettes (BioGenex, San Ramon, CA). A 5'-trityl group was retained on the fluorescein-labeled probes for purification of full-length sequences. Detritylation was performed on a Polypack column (Glen Research, Sterling, VA), and the labeled oligo was eluted with 50% acetonitrile. The 5'-Cy5 probes were synthesized using a Cy5 phosphoramidite (Pharmacia Biotech) and a chemical phosphorylation reagent (Glen Research) to prevent extension from the 3' end of the probe.
Purity of probe synthesis was determined by calculating the ratio of fluorophore concentration to oligonucleotide concentration.20 Probes with ratios outside 0.8 to 1.2 were further purified by reverse-phase C18 high-pressure liquid chromatography. Labeled oligonucleotides were passed through a 4x 250-mm Hypersil ODS column (Hewlett Packard, Fullerton, CA) using 0.1 mol/L triethylammonium acetate, pH 7.0, and a 20 to 60% (fluorescein probe) or 40 to 80% (Cy5 probe) gradient of acetonitrile (1 ml/minute). The eluate was monitored with tandem absorbance and fluorescence detectors (Waters 486 and 474, Milford, MA). Fractions with both A260 and fluorescence peaks were collected.
Primer/Probe Design
The HFE cDNA sequence was used for selection of primers and probes
(Genbank accession M31944). Primers and probes were chosen using Primer
Designer for Windows (Scientific and Educational Software, State Line,
PA). Primers for both mutation sites were selected with similar melting
temperature (Tm) values and GC content to
allow multiplexing. The longer 5'-Cy5-labeled probes were designed with
at least a 15°C higher Tm than the
3'-fluorescein-labeled probes that span the area targeted for mutation
detection. In this way a Cy5-labeled probe acts as an anchor and
remains annealed to the single-stranded amplicon while the
fluorescein-labeled probe is heated through the characteristic
Tm for that allele. The fluorescein-labeled
probes were designed to have Tm values that
would allow differentiation of all four alleles by melting peak
analysis. The primer and probe sequences are shown in Table 1
. Empirical melting temperatures for the
3'-fluorescein probe/allele duplexes are shown in Table 2
.
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The statistical significance of the H63D mutation among the non-ancestral chromosomes was determined using a one-tailed Z test.21
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A schematic representation of the adjacent fluorescent
hybridization probes used for genotyping the HFE locus is shown in
Figure 1
. The 3'-fluorescein-labeled
probes spanning the C282Y and H63D sites were 21 and 27 bp long,
respectively. These probes were designed so that during hybridization
each probe formed a mismatch with the wild-type allele extended by the
downstream primer. Table 1
shows the positions of the potential probe
mismatches.
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. The 21-mer fluorescein probe formed an
A:C mismatch with the wild-type sequence, lowering the
Tm of the probe by 7°C from the completely
complementary duplex. The wild-type allele shows no rise in
fluorescence above background during amplification as the annealing
temperature of the A:C mismatched duplex was below the temperature at
which fluorescence was acquired each cycle.
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. The G:G mismatch formed at the
center of the 27-mer fluorescein probe created a
Tm of 5.5°C from the completely
Watson-Crick paired duplex. During amplification, both the wild-type
allele and the allele with the H63D mutation were annealed to the probe
at the fluorescence acquisition temperature of 62°C.
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and Table 1
). When the probe hybridizes
to an allele that is wild type at the H63D site but contains the S65C
polymorphism, two mismatches are present. This destabilizes the probe
by 10°C compared with the completely complementary duplex. Samples
heterozygous at the C282Y, H63D, and S65C sites are shown in Figure 4
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All cases genotyped by adjacent fluorescent hybridization probes agreed with PCR restriction fragment length analysis. Primers used for analysis of the C282Y site produced a 389-bp amplicon that was cleaved by SnaBI into fragments of 276 and 113 bp in the presence of the C282Y mutation. The PCR product for the H63D site was 241 bp long. The H63D mutation destroyed a BclI restriction site that upon restriction digestion of the wild-type allele yielded fragments of 138 and 103 bp. The run time alone required for PCR and genotype analysis by restriction digestion and gel electrophoresis was approximately 3 hours and 30 minutes.
In comparison, genotyping with adjacent fluorescent hybridization
probes was faster and easier. Amplification and analysis of 24 samples
independently at both sites required 45 minutes. No manual manipulation
between amplification and genotyping was required. Analysis by
multiplexing allowed twice as many samples to be genotyped at both
sites in the same amount of time. Genotype analysis by multiplexing
with adjacent fluorescent hybridization probes is shown in Figure 5
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One hundred and seventeen patients who met clinical criteria for
iron overload and fifty-six normal controls were selected for analysis
of the C282Y and H63D mutations in the HFE gene. Both groups were
Caucasian Americans from Utah and neighboring states. The results of
this study are summarized in Table 3
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The S65C polymorphism was found in 4 of 50 samples. Three of these samples were from the normal control group. The allelic frequency for the S65C variant was 5.5% among the control chromosomes and 2.8% among the patient chromosomes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although many PCR-based fluorescent multiplex techniques are being developed,28-32 few are homogeneous and none genotype by allele/probe duplex Tm. Several methods multiplex with target-specific oligonucleotides labeled with different colored fluorophores.28-30 For example, hairpin and exonuclease probes use oligonucleotides dually labeled with quencher and reporter dyes to genotype during real-time PCR. Targets are distinguished by multiplexing allele-specific probes each labeled with a different colored reporter. The probe that is completely complementary to the template shows a rise in reporter fluorescence as it is cleaved during the extension of Taq polymerase. Multiplexing using a completely complementary probe for each allele provides a positive control for amplification, and variants can be genotyped in a homogeneous reaction.
In a similar way, hybridization probes can be used to genotype in real time as shown by the amplification of the C282Y site. When fluorescence is acquired above the Tm for the mismatched probe/allele duplex, only the allele that is completely complementary to the probe shows a change in fluorescence during amplification. Heterozygotes yield one-half the maximal fluorescence compared with the homozygous perfect match. Similar to exonuclease probes, multiplexing with a probe set of a different color would be necessary for a positive control. However, multiple colors are not necessary for homogeneous genotyping with melting curves because multiple alleles can be distinguished with a single probe. Moreover, melting curves prevent false negative results as all alleles are represented and are equally effected by inhibitors within the reaction.
Multiplexing with fluorescent hybridization probes provides rapid and sensitive analysis of multiple alleles. Amplification and genotyping of the HFE gene mutations were performed in approximately 45 minutes. The current rate-limiting step in this assay is DNA extraction. Although the organic extraction method used here provided reliable amplification and fluorescent genotyping, more recent DNA extraction methods yield equivalent amounts of DNA from whole blood while being safer, cheaper, and requiring less time.19,24 For example, several DNA extraction kits now available can provide approximately 2 µg of DNA from 25 µl of whole blood within 1 to 2 hours.24 Thus, a small blood donation obtained by only a finger or heel stick can be rapidly processed and analyzed multiple times.
Genotyping with multiplexed hybridization probes has technical challenges and limitations in addition to the optimizations often necessary for multiplexing primer sets.33 For example, the melting temperatures of the probe/template duplexes must allow differentiation of all alleles by derivative melting curve analysis. The number of mutations that can be analyzed within a single reaction is limited by the number of melting peaks that can be distinguished over a range of probe melting temperatures. The HFE assay demonstrated here showed four different alleles differentiated over a 15°C temperature range.
Another limitation of multiplexing hybridization probes is that unexpected variants may cause erroneous interpretation. When the A193T (S65C) variant was analyzed with the probe designed for the H63D site, the duplex Tm was shifted near the Tm of the probe covering the C282Y mutation. Hence, there is some risk of a C282Y false positive when multiplexing. However, the 1.5°C difference in Tm between S65C and C282Y is distinguishable, and the samples were correctly genotyped at the C282Y codon as verified by restriction enzyme digestion.
The C282Y substitution is a founding mutation for hemochromatosis.34 It has been known for over 20 years to be associated with the HLA A3, B7 haplotype, and it appears to have occurred relatively recently in Celtic history.34-37 Accordingly, studies from populations of northern European descent, including this study, show that between 82 and 100% of hemochromatosis patients are homozygous for this ancestral mutation.8-11 However, an increase in the heterogeneity of the disease is observed in populations outside of northern Europe.38,39
In comparison, the role that the H63D substitution plays in the development of iron overload disease has been controversial. Concerns include 1) that the mutation occurs with similar frequencies among patients and controls, 2) that homozygotes for the H63D mutation are rare among patients, and 3) that compound heterozygotes (C282Y/H63D) within patient populations are found at a lower frequency than predicted.40
As the H63D mutation has never been found on the same chromosome as the C282Y mutation, only the non-ancestral (non-C282Y) haplotypes are at risk for the H63D mutation. In light of this observation, a compelling review of the data has shown that 74% of chromosomes from iron overloaded patients heterozygous at the C282Y site carried the H63D mutation.40 This suggests that the C282Y/H63D genotype confers a risk for the development of iron overload disease.8,9 Our study found that the penetrance of the H63D mutation was slightly higher than previously reported values.8-10
Adjacent fluorescent hybridization probes are versatile and amenable to multiplexing. The C282Y and H63D mutation sites were co-amplified with different primer sets and simultaneously genotyped by the melting temperatures of multiplexed fluorescent probes. Moreover, the probe spanning the C187G (H63D) mutation could also be used for genotyping the A193T (S65C) polymorphism after the variant was detected by an aberrant melting curve and confirmed by sequencing. The identification of this polymorphism demonstrates genotyping of multiple alleles by a single probe and suggests a potential for fluorescent hybridization probes in scanning for unknown variants.
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Acknowledgements |
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Footnotes |
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P.S. Bernard and C.T. Wittwer are supported by NIH Grant GM51647, a Biomedical Engineering grant from the Whitaker Foundation, Idaho Technology, and Associated and Regional University Pathologists. R.S. Ajioka and J.P. Kushner are supported by NIH Grants DK20630, RR00064, and CA42014.
Accepted for publication July 18, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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