Homogeneous Multiplex Genotyping of Hemochromatosis Mutations with Fluorescent Hybridization Probes
1998; Elsevier BV; Volume: 153; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)65650-7
ISSN1525-2191
AutoresPhilip S. Bernard, Richard S. Ajioka, James P. Kushner, Carl T. Wittwer,
Tópico(s)Trace Elements in Health
ResumoMultiplex polymerase chain reaction amplification and genotyping by fluorescent probe melting temperature (Tm) was used to simultaneously detect multiple variants in the hereditary hemochromatosis gene. Homogenous real-time analysis by fluorescent melting curves has previously been used to genotype single base mismatches; however, the current method introduces a new probe design for fluorescence resonance energy transfer and demonstrates allele multiplexing byTm for the first time. The new probe design uses a 3′-fluorescein-labeled probe and a 5′-Cy5-labeled probe that are in fluorescence energy transfer when hybridized to the same strand internal to an unlabeled primer set. Two hundred and fifty samples were genotyped for the C282Y and H63D hemochromatosis causing mutations by fluorescent melting curves. Multiplexing was performed by including two primer sets and two probe sets in a single tube. In clinically defined groups of 117 patients and 56 controls, the C282Y mutation was found in 87% (204/234) of patient chromosomes, and the relative penetrance of the H63D mutation was 2.4% of the homozygous C282Y mutation. Results were confirmed by restriction enzyme digestion and agarose gel electrophoresis. In addition, the probe covering the H63D mutation unexpectedly identified the A193T polymorphism in some samples. This method is amenable to multiplexing and has promise for scanning unknown mutations. Multiplex polymerase chain reaction amplification and genotyping by fluorescent probe melting temperature (Tm) was used to simultaneously detect multiple variants in the hereditary hemochromatosis gene. Homogenous real-time analysis by fluorescent melting curves has previously been used to genotype single base mismatches; however, the current method introduces a new probe design for fluorescence resonance energy transfer and demonstrates allele multiplexing byTm for the first time. The new probe design uses a 3′-fluorescein-labeled probe and a 5′-Cy5-labeled probe that are in fluorescence energy transfer when hybridized to the same strand internal to an unlabeled primer set. Two hundred and fifty samples were genotyped for the C282Y and H63D hemochromatosis causing mutations by fluorescent melting curves. Multiplexing was performed by including two primer sets and two probe sets in a single tube. In clinically defined groups of 117 patients and 56 controls, the C282Y mutation was found in 87% (204/234) of patient chromosomes, and the relative penetrance of the H63D mutation was 2.4% of the homozygous C282Y mutation. Results were confirmed by restriction enzyme digestion and agarose gel electrophoresis. In addition, the probe covering the H63D mutation unexpectedly identified the A193T polymorphism in some samples. This method is amenable to multiplexing and has promise for scanning unknown mutations. As databases for polymorphic markers1Murray JC Buetow KH Weber JL Ludwigsen S Scherpbier-Heddema T Manion F Quillen J Sheffield VC Sunden S Duyk GM Weissenbach J Gyapay G Dib C Morrissette J Lathrop GM Vignal A White R Matsunami N Gerken S Melis R Albertsen H Plaetke R Odelberg S Ward D Dausset J Cohen D Cann H A comprehensive human linkage map with centimorgan density.Science. 1994; 265: 2049-2054Crossref PubMed Scopus (524) Google Scholar and disease-causing mutations2NIH consensus statement: Genetic testing for cystic fibrosis. 1997 Apr 14–16; 15(4):1–37Google Scholar, 3De Vries EMG Ricke DO De Vries TN Hartmann A Blaszyk H Liao D Soussi T Kovach JS Sommer SS Database of mutations in the p53 and APC tumor suppressor genes designed to facilitate molecular epidemiological analyses.Hum Mutat. 1996; 7: 202-213Crossref PubMed Scopus (37) Google Scholar continue to grow, the ability to simultaneously interrogate multiple DNA sites that may be physically separated by great distances becomes increasingly useful. Multiplex polymerase chain reaction (PCR) is a rapid, versatile, and cost-effective method for this type of analysis. Since the introduction of multiplex PCR in 1988 for identifying specific exon deletions,4Chamberlain JS Gibbs RA Ranier JE Nguyen PN Caskey CT Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification.Nucleic Acids Res. 1988; 16: 11141-11156Crossref PubMed Scopus (1100) Google Scholar the concept has continued to develop in both application and technique. We have developed a new homogeneous multiplex genotyping method and applied it to the simultaneous analysis of multiple mutations within two exons of the hemochromatosis gene (HFE). 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.5McLaren CE Gordeuk VR Looker AC Hasselblad V Edwards CQ Griffen LM Kushner JP Brittenham GM Prevalence of heterozygotes for hemochromatosis in the white population of the United States.Blood. 1995; 86: 2021-2027PubMed Google Scholar This autosomal recessive disorder of iron metabolism occurs with a frequency of approximately 0.5% in Caucasian populations.6Simon S Bourel M Genetet B Fauchet R Idiopathic hemochromatosis: demonstration of recessive inheritance and early detection by family HLA typing.N Engl J Med. 1977; 297: 1017-1021Crossref PubMed Scopus (312) Google Scholar, 7Edwards CQ Griffen LM Goldgar D Drummond C Skolnick MH Kushner JP Prevalence of hemochromatosis among 11,065 presumably healthy blood donors.N Engl J Med. 1988; 318: 1355-1362Crossref PubMed Scopus (578) Google Scholar 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.8Feder JN Gnirke A Thomas W Tsuchihashi Z Ruddy DA Basava A Dormishian F Domingo Jr, R Ellis MC Fullan A Hinton LM Jones NL Kimmel BE Kronmal GS Lauer P Lee VK Loeb DB Mapa FA McClelland E Meyer NC Mintier GA Moeller N Moore T Morikang E Prass CE Quintana L Starnes SM Schatzman RC Brunke KJ Drayna DT Risch NJ Bacon BR Wolff RK A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nature Genet. 1996; 13: 399-408Crossref PubMed Scopus (3402) Google Scholar, 9Beutler E Gelbart T West C Lee P Adams M Blackstone R Pockros P Kosty M Venditti CP Phatak PD Seese NK Chorney KA Ten Elshof AE Gerhard GS Chorney M Mutation analysis in hereditary hemochromatosis.Blood Cells Mol Dis. 1996; 22: 187-194Crossref PubMed Scopus (387) Google Scholar, 10Jouanolle AM Fergelot P Gandon G Yaouanq J Le Gall JY David V A candidate gene for hemochromatosis: frequency of the C282Y and H63D mutations.Hum Genet. 1997; 100: 544-547Crossref PubMed Scopus (119) Google Scholar, 11Jazwinska EC Cullen LM Busfield F Pyper WR Webb SI Powell LW Morris CP Walsh TP Haemochromatosis and HLA-H.Nature Genet. 1996; 14: 249-251Crossref PubMed Scopus (418) Google Scholar 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.8Feder JN Gnirke A Thomas W Tsuchihashi Z Ruddy DA Basava A Dormishian F Domingo Jr, R Ellis MC Fullan A Hinton LM Jones NL Kimmel BE Kronmal GS Lauer P Lee VK Loeb DB Mapa FA McClelland E Meyer NC Mintier GA Moeller N Moore T Morikang E Prass CE Quintana L Starnes SM Schatzman RC Brunke KJ Drayna DT Risch NJ Bacon BR Wolff RK A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nature Genet. 1996; 13: 399-408Crossref PubMed Scopus (3402) Google Scholar, 9Beutler E Gelbart T West C Lee P Adams M Blackstone R Pockros P Kosty M Venditti CP Phatak PD Seese NK Chorney KA Ten Elshof AE Gerhard GS Chorney M Mutation analysis in hereditary hemochromatosis.Blood Cells Mol Dis. 1996; 22: 187-194Crossref PubMed Scopus (387) Google Scholar, 10Jouanolle AM Fergelot P Gandon G Yaouanq J Le Gall JY David V A candidate gene for hemochromatosis: frequency of the C282Y and H63D mutations.Hum Genet. 1997; 100: 544-547Crossref PubMed Scopus (119) Google Scholar Current methods for genotyping the C282Y and H63D hemochromatosis-causing mutations include oligonucleotide ligation,8Feder JN Gnirke A Thomas W Tsuchihashi Z Ruddy DA Basava A Dormishian F Domingo Jr, R Ellis MC Fullan A Hinton LM Jones NL Kimmel BE Kronmal GS Lauer P Lee VK Loeb DB Mapa FA McClelland E Meyer NC Mintier GA Moeller N Moore T Morikang E Prass CE Quintana L Starnes SM Schatzman RC Brunke KJ Drayna DT Risch NJ Bacon BR Wolff RK A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nature Genet. 1996; 13: 399-408Crossref PubMed Scopus (3402) Google Scholar allele-specific oligonucleotide hybridization,9Beutler E Gelbart T West C Lee P Adams M Blackstone R Pockros P Kosty M Venditti CP Phatak PD Seese NK Chorney KA Ten Elshof AE Gerhard GS Chorney M Mutation analysis in hereditary hemochromatosis.Blood Cells Mol Dis. 1996; 22: 187-194Crossref PubMed Scopus (387) Google Scholar and PCR restriction fragment length analysis.10Jouanolle AM Fergelot P Gandon G Yaouanq J Le Gall JY David V A candidate gene for hemochromatosis: frequency of the C282Y and H63D mutations.Hum Genet. 1997; 100: 544-547Crossref PubMed Scopus (119) Google Scholar, 11Jazwinska EC Cullen LM Busfield F Pyper WR Webb SI Powell LW Morris CP Walsh TP Haemochromatosis and HLA-H.Nature Genet. 1996; 14: 249-251Crossref PubMed Scopus (418) Google Scholar, 12Lynas C A cheaper and more rapid polymerase chain reaction-restriction fragment length polymorphism method for the detection of the HLA-H gene mutations occurring in hereditary hemochromatosis.Blood. 1997; 90: 4235-4237PubMed Google Scholar 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.13Lay MJ Wittwer CT Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR.Clin Chem. 1997; 43: 2262-2267PubMed Google Scholar, 14Bernard PS Lay MJ Wittwer CT Integrated amplification and detection of the C677T point mutation in the methylenetetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves.Anal Biochem. 1998; 255: 101-107Crossref PubMed Scopus (108) Google Scholar This technique previously used an internally labeled Cy5 primer to asymmetrically amplify excess Cy5-labeled strand.13Lay MJ Wittwer CT Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR.Clin Chem. 1997; 43: 2262-2267PubMed Google Scholar, 14Bernard PS Lay MJ Wittwer CT Integrated amplification and detection of the C677T point mutation in the methylenetetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves.Anal Biochem. 1998; 255: 101-107Crossref PubMed Scopus (108) Google Scholar 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.15Wittwer CT Herrmann MG Moss AA Rasmussen RP Continuous fluorescence monitoring of rapid cycle DNA amplification.BioTechniques. 1997; 22: 130-138Crossref PubMed Scopus (1148) Google Scholar 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.16Wetmur JG DNA probes: applications of the principles of nucleic acid hybridization.Crit Rev Biochem Mol Biol. 1991; 26: 227-259Crossref PubMed Scopus (352) Google Scholar 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.17Ke SH Wartell RM Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA: determination by temperature-gradient gel electrophoresis.Nucleic Acids Res. 1993; 21: 5137-5143Crossref PubMed Scopus (106) Google Scholar, 18Guo Z Liu Q Smith LM Enhanced discrimination of single nucleotide polymorphisms by artificial mismatch hybridization.Nature Biotechnol. 1997; 15: 331-335Crossref Scopus (115) Google Scholar 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. Genomic DNA from Caucasian individuals was collected over a 10-year period for studying hemochromatosis pedigrees in Utah and neighboring states.19Thomas SM Moreno RF Tilzer LL DNA extraction with organic solvents in gel barrier tubes.Nucleic Acids Res. 1989; 17: 5411Crossref PubMed Scopus (43) Google Scholar 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. 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. 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). Primers for the C282Y codon8Feder JN Gnirke A Thomas W Tsuchihashi Z Ruddy DA Basava A Dormishian F Domingo Jr, R Ellis MC Fullan A Hinton LM Jones NL Kimmel BE Kronmal GS Lauer P Lee VK Loeb DB Mapa FA McClelland E Meyer NC Mintier GA Moeller N Moore T Morikang E Prass CE Quintana L Starnes SM Schatzman RC Brunke KJ Drayna DT Risch NJ Bacon BR Wolff RK A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nature Genet. 1996; 13: 399-408Crossref PubMed Scopus (3402) Google Scholar 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.20Morrison LE Detection of energy transfer and fluorescence quenching.in: Kricka LJ In Nonisotopic DNA Probe Techniques. Academic Press, San Diego1997: 311-352Google Scholar 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 4× 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. 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 characteristicTm 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.Table 1Primer and Probe Sequences Used for Genotyping the HFE GeneCodonForward primersReverse primersFluorescent probesC282YTGGCAAGGGTAAACAGATCCCTCAGGCACTCCTCTCAACCAGATATACGTACCAGGTGGAG-fluoresceinCy5-CCCAGGCCTGGATCAGCCCCTCATTGT- GATCTGGG-PH63DCACATGGTTAAGGCCTGTTGGATCCCACCCTTTCAGACTCCGTGTTCTATGATGATGAGAGTCGCCG-fluoresceinCy5-GGAGCCCCGAACTCCATGGGTTTCCAG- TAGAATTTCAAGCCAGAT-PNucleotide bases involved in duplex mismatch formation are underlined. P indicates the addition of a phosphate group. Open table in a new tab Table 2Empirical Melting Temperatures for Fluorescein Probe/Allele DuplexesCodonAlleleDuplex TmC282YG84553°C845A60°CH63DC18763°C187G68.5°CC187/193T58.5°C Open table in a new tab Nucleotide bases involved in duplex mismatch formation are underlined. P indicates the addition of a phosphate group. The statistical significance of the H63D mutation among the non-ancestral chromosomes was determined using a one-tailedZ test.21Devore JL Tests of hypotheses based on a single sample.in: Kimmel J In Probability and Statistics for Engineering and the Sciences. Brooks/Cole, California1987: 289-302Google Scholar 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. Amplification and genotyping of the C282Y site is illustrated in Figure 2. The 21-mer fluorescein probe formed an A:C mismatch with the wild-type sequence, lowering theTm 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. Amplification and genotype analysis for the H63D site is shown in Figure 3. The G:G mismatch formed at the center of the 27-mer fluorescein probe created a ΔTm 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. Genotyping most samples for the H63D mutation was done with a melting protocol that began at 55°C to observe melting transitions at 63°C and 68.5°C. However, 50 samples were analyzed for the H63D mutation with melting curves beginning 10°C lower at 45°C. Using this melting protocol, four samples were identified that had a melting peak at 58.5°C, 4.5°C lower than the melting peak for the wild-type allele. Sequencing all four samples revealed an A-to-T transversion at nucleotide 193 of the open reading frame. This transversion is a recently reported polymorphism that results in a serine-to-cysteine amino acid substitution at codon 65 (S65C).22Douabin V, Deugnier Y, Jouanolle AM, Moirand R, Macqueron G, Gireau A, Le Gall JY, David V: Polymorphisms in the haemochromatosis gene. International Symposium on Iron in Biology and Medicine. Saint-Malo France, 1997, p 267Google Scholar The S65C polymorphism creates an A:A mismatch located 8 bp in from the 3′ end of the 27-mer probe (see Figure 1 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. 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. 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.Table 3Analysis of the C282Y and H63D Mutations within the Utah PopulationGenotype*hh designates homozygosity for the mutation; Hh designates heterozygosity; HH designates homozygous wild type.Patients with iron overloadControlsC282YH63Dn%n%hhHH9883.800HhHH43.4712.5HhHh43.400HHHH65.13867.9HHHh43.41017.9HHhh10.911.8Total11756Allele frequencies for the C282Y genotype were 204/234 (87.1%) for patients with iron overload and 7/112 (6.3%) for controls. For the H63D genotype, allele frequencies were 10/234 (4.3%) for patients with iron overload and 12/112 (10.7%) for controls.* hh designates homozygosity for the mutation; Hh designates heterozygosity; HH designates homozygous wild type. Open table in a new tab Allele frequencies for the C282Y genotype were 204/234 (87.1%) for patients with iron overload and 7/112 (6.3%) for controls. For the H63D genotype, allele frequencies were 10/234 (4.3%) for patients with iron overload and 12/112 (10.7%) for controls. Ninety-eight (83.8%) of the patients and none of the controls were homozygous for the C282Y mutation. The C282Y mutation was found in 87% of patient chromosomes and 6.3% of chromosomes from normal controls. The H63D mutation was found in 11% of control chromosomes and only 4.3% of patient chromosomes. There were eight patients and seven normal controls heterozygous for the C282Y mutation. One-half of the C282Y heterozygous patients carried the H63D mutation, whereas there were no compound heterozygous genotypes among the controls. 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. Multiplex technology continues to advance both research and routine diagnostics. Sensitive methods of multiplex analysis combined with improved methods of DNA preparation increase the density of information obtained from small amounts of whole blood.23Ricciardone MD Lins AM Schumm JW Holland MM Multiplex systems for the amplification of short tandem repeat loci: evaluation of laser fluorescence detection.BioTechniques. 1997; 23: 742-747PubMed Google Scholar, 24Vandenberg N van Oorschot RAH Mitchell RJ An evaluation of selected DNA extraction strategies for short tandem repeat typing.Electrophoresis. 1997; 18: 1624-1626Crossref PubMed Scopus (24) Google Scholar This reduces the cost and invasiveness of sample collection and is useful in the analysis of rare samples. Population screening for presymptomatic diagnosis of hereditary hemochromatosis by DNA analysis has been proposed since the first report of the hemochromatrosis gene.8Feder JN Gnirke A Thomas W Tsuchihashi Z Ruddy DA Basava A Dormishian F Domingo Jr, R Ellis MC Fullan A Hinton LM Jones NL Kimmel BE Kronmal GS Lauer P Lee VK Loeb DB Mapa FA McClelland E Meyer NC Mintier GA Moeller N Moore T Morikang E Prass CE Quintana L Starnes SM Schatzman RC Brunke KJ Drayna DT Risch NJ Bacon BR Wolff RK A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nature Genet. 1996; 13: 399-408Crossref PubMed Scopus (3402) Google Scholar, 9Beutler E Gelbart T West C Lee P Adams M Blackstone R Pockros P Kosty M Venditti CP Phatak PD Seese NK Chorney KA Ten Elshof AE Gerhard GS Chorney M Mutation analysis in hereditary hemochromatosis.Blood Cells Mol Dis. 1996; 22: 187-194Crossref PubMed Scopus (387) Google Scholar, 25Edwards CQ Griffen LM A
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