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Detection of prevalent genetic alterations predisposing to hemochromatosis and other common human diseases.

The explosion of recent knowledge in basic genetics has spawned numerous clinical follow-up studies that have confirmed an unequivocal association between the presence of specific prevalent genetic alterations and susceptibility to some very common human diseases. In addition, the imminent completion of the Human Genome Project's sequencing efforts will contribute yet more candidate disease genes that will require both research-based genetic association studies (to confirm suspected disease links) and, if positive, the translation of these disease-genotype associations to routine diagnostic clinical practice. Given this expanding repertoire of confirmed and reputed disease genes (many for common diseases), the demand for rapid, sensitive, specific, inexpensive assays for their clinical- and/or research-based detection is growing quickly. As a consequence, clinical genetic testing laboratories, once accustomed to manual, low-volume, high-labor tests on patients with rare, untreatable classic "genetic" diseases, will soon need to develop more high-throughput, semiautomated methods. In the fast-approaching molecular medicine era, these new genotyping methods will be utilized not only for diagnosing symptomatic patients, but perhaps more importantly, for presymptomatically identifying individuals at risk for common, treatable diseases for whom effective preventative interventions may be available.

A prototypical example of the forthcoming primary public health role of molecular diagnostics is the identification of individuals affected by or at-risk for the iron overload disorder hereditary hemochromatosis. More than 90% of the cases of this most common of all single-gene disorders (present in 0.5% of whites) are caused by the presence of a homozygous well-conserved single nucleotide substitution (nucleotide G845A; amino acid C282Y) in the transferrin receptor binding protein FIFE (1). This loss-of-function mutation abolishes HFE's usual cell surface expression, thus preventing its ability to down-regulate the affinity of transferrin receptor for transferrin-bound iron. The result is a dysregulation of normal cellular iron metabolism and a resulting constitutive intestinal iron absorption. This excess toxic iron deposits in numerous organs and, if not removed, causes progressive chronic damage to the liver, heart, endocrine glands, joints, and skin. Because hemochromatosis is a common, underdiagnosed (but easily diagnosable), progressive chronic disease with late-onset symptomatology for which an effective, safe (preventative) therapy is widely available (phlebotomy), it is perhaps the ideal disease for the implementation of a population-based screening program. Universal population-based hemochromatosis screening (by transferrin saturation) has been recommended by the College of American Pathologists (2), and a more conservative phenotypic screening of "symptomatic" individuals has been recommended by an expert consensus panel of the CDC and NIH (3). More universal recommendations for widespread population screens may result from the recently initiated NIH study (HEIRS) of 100 000 apparently healthy Americans that will evaluate the benefits and risks of iron overload screening by both genotypic and phenotypic determinations.

When population-based screens and overall recognition of iron overload become more widespread, the relatively nonspecific phenotypic diagnostic tests will be increasingly supplemented with the direct C282Y mutation test, particularly for disease confirmation and family member screens. To meet this growing clinical need, accurate, rapid, high-throughput, inexpensive genotyping assays will be required. Toward that goal, Donohoe et al. (4) describe, in this issue of the journal, a novel high-throughput genotyping method for the rapid detection of the FIFE C282Y mutation that utilizes real-time multiplex, allele-specific PCR and melting curves but requires neither fluorescent hybridization probes nor any post-amplification manual processing. In particular, this C282Y genotyping assay requires the design of two unlabeled allele-specific sense-strand PCR primers (and a common antisense primer) to specifically amplify (in the same tube) either the C282 or Y282 allele (or both, in the case of heterozygotes). Nonspecific amplification of the "other" allele is prevented by several deliberate nucleotide mismatches in both allele-specific primers. During and after PCR amplification in a thermal cycler with real-time fluorescent monitoring capabilities, the amplicons are detected fluorescently, not by expensive fluorescently labeled probes, but by a nonspecific double-stranded DNA binding dye (SYBR Green I) included in the reaction. Because one of the allele-specific primers is engineered with a long 5' GC tail to increase the melting temperature of its PCR product, the mutant- and wild-type-specific amplicons are discriminated by a progressive post-PCR temperature surge (with continuous fluorescence monitoring) to generate melting curves with characteristic allele-specific melting temperatures.

In comparison with the predominantly manual single nucleotide mutation assays used in most clinical molecular pathology laboratories--typically with a time-consuming hands-on post-PCR analysis step (5)--the real-time PCR melting curve method of Donohoe et al. (4) could benefit laboratories interested in its faster turnaround time, single-tube format (without any post-PCR manipulation), and resulting minimization of potential amplicon carryover and sample-tracking errors. In addition, this particular real-time PCR melting curve genotyping method detects amplified products with the inexpensive, universally applicable DNA binding dye SYBR Green I rather than the expensive target-specific fluorescently labeled hybridization probes used by other investigators (6-12). Like other melting curve-based genotyping methods, the presence of an unexpected nucleotide alteration adjacent to the expected nucleotide substitution would likely manifest as a small but detectable shift in the predicted melting temperature. As shown by Lyon et al. (12), who used melting curve reagents specific for factor V Leiden (G1691A) to detect an adjacent nonpathogenic, silent A1692C factor V polymorphism, an unexpected nucleotide alteration would likely not be detectable with a comparable PCR restriction fragment length polymorphism (RFLP) genotyping method. Finally, although Donohoe et al. (4) do not specifically address the reagent and labor costs associated with their assay, the avoidance of both fluorescent probes and manual post-PCR processing would likely reduce the total direct assay cost and thus further promote its real-world applicability, perhaps despite the increased costs associated with the acquisition of the obligate real-time PCR hardware and software.

As addressed by Donohoe et al. (4), a potential drawback of the SYBR Green I melting curve genotyping method is its reliance on the careful design and optimization of allele-specific primers that are truly allele specific. When applying this method to new genetic targets without preoptimized primers, the effort spent in optimizing primer design could then obviate the labor savings associated with its eventual application. Additional disadvantages of the SYBR Green I melting curve genotyping method include the possible nonspecific amplification of the unwanted other allele (likely primer and temperature dependent) and the limitations imposed by the use of a single-color fluorescent detection signal (SYBR Green I), which will likely prevent single-tube, multiplex genotype analysis. In comparison, real-time PCR melting curve-based genotyping assays using allele-specific fluorescent probes (and unlabeled non-allele-specific PCR primers) (6-11) avoid the problem of nonspecific amplicon generation in exchange for higher reagent costs. These hybridization probes offer the additional benefit of being available in a variety of different fluorescent colors (9-11), thus extending their potential utility to the multiplex genotyping needs of the future.

Real-time PCR melting curve-based genotype determinations are but one of an increasing number of tools that have recently become available for the accurate high-throughput determination of disease-associated single nucleotide alterations. Present-day applications of these methods in the clinical diagnostic laboratory include direct DNA tests to detect such common clinical syndromes as hemochromatosis (caused by FIFE gene alterations) (9-11) and thrombotic predisposition [caused by mutations in factor V (R506Q and A4070G) (7,12), factor II (prothrombin G20210A) (7), methylenetetrahydrofolate reductase (MTHFR C677T) (7), factor XIII (Va134Leu) (13), plasminogen activator inhibitor-1 (4G/5G) (14), and angiotensin 1-converting enzyme (insertion/ deletion) (15)]. Additional clinical and/or research applications include prediction of cardiovascular disease risk [apolipoprotein E genotyping (8), platelet glycoprotein la (C807T) (16), factor VII (R353Q) (17), platelet glycoprotein IIIa (Leu33Pro) (18)], posttransfusion purpura (platelet antigen 1 mutations) (6), neurodegenerative disease risk (apolipoprotein E genotyping) (8), and numerous other commonly encountered clinical scenarios. Given the overlapping phenotypes associated with these and other prevalent genetic alterations, the demand for symptom- or risk-based multiplex genotyping assays will almost certainly substantially broaden in the coming molecular medicine era.

Even among the genotyping methods that utilize hybridization melting curves to discriminate variant alleles, the configuration of primers, probes, and fluorophores varies considerably. Widely available methods include those utilizing (a) adjacent fluorophore-labeled hybridization probes requiring fluorescent resonance energy transfer for signaling (6-12); (b) hairpin probes with doubly labeled quencher and fluorophore moieties (19); (c) 5' exonuclease activity with doubly end-labeled quencher and fluorophore moieties (20); (d) and now allele-specific PCR with nonspecific amplicon detection (4). Alternative single nucleotide detection technologies that are now (or soon will be) clinically available (and do not require expensive real-time PCR instrumentation) include multichannel solid-phase hybridization arrays (21), enzymatic recognition and cleavage of nucleotide mismatches (22), heteroduplex discrimination by HPLC (23), direct DNA sequencing (24), and many others. Each of these methods offers some advantages over traditional laborious PCR-RFLP approaches for the detection of disease-associated DNA alterations, particularly in the routine clinical laboratory having to do progressively more work with fixed or shrinking laboratory staffs.


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(2.) Witte DL, Crosby WH, Edwards CQ, Fairbanks VF, Mitros FA. Practice guideline development task force of the College of American Pathologists. Hereditary hemochromatosis. Clin Chim Acta 1996;245:139-200.

(3.) Cogswell ME, McDonnell SM, Khoury MJ, Franks AL, Burke W, Brittenham G. Iron overload, public health, and genetics: evaluating the evidence for hemochromatosis screening. Ann Intern Med 1998;129:971-9.

(4.) Donohoe GG, Laaksonen M, Pulkki K, Rbnnemaa T, Kairisto V. Rapid single-tube screening of the C282Y hemochromatosis mutation by real-time multiplex allele-specific PCR without fluorescent probes. Clin Chem 2000; 46:1540-7.

(5.) Lutz CT, Foster PA, Noll WW, Voelkerding KV, Press RD, McGlennen RC, Kirschbaum NE. Multicenter evaluation of PCR methods for the detection of factor V Leiden (R506Q) genotypes. Clin Chem 1998;44:1356-8.

(6.) Nauck MS, Gierens H, Nauck MA, Marz W, Wieland H. Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the LightCycler. Br J Haematol 1999;105:803-10.

(7.) von Ahsen N, Schutz E, Armstrong VW, Oellerich M. Rapid detection of prothrombotic mutations of prothrombin (G20210A), factor V (G1691A), and methylenetetrahydrofolate reductase (C677T) by real-time fluorescence PCR with the LightCycler. Clin Chem 1999;45:694-6.

(8.) Aslanidis C, Schmitz G. High-speed apolipoprotein E genotyping and apolipoprotein B3500 mutation detection using real-time fluorescence PCR and melting curves. Clin Chem 1999;45:1094-7.

(9.) Mangasser-Stephan K, Tag C, Reiser A, Gressner AM. Rapid genotyping of hemochromatosis gene mutations on the LightCycler with fluorescent hybridization probes. Clin Chem 1999;45:1875-8.

(10.) Bernard PS, Ajioka RS, Kushner JP, Wittwer CT. Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes. Am J Pathol 1998;153:1055-61.

(11.) Phillips M, Meadows CA, Huang MY, Millson A, Lyon E. Simultaneous detection of C282Y and H63D hemochromatosis mutations by dual-color probes. Mol Diagn 2000;5:107-16.

(12.) Lyon E, Millson A, Phan T, Wittwer CT. Detection and identification of base alterations within the region of factor V Leiden by fluorescent melting curves. Mol Diagn 1998;3:203-9.

(13.) Catto AJ, Kohler HP, Coore J, Mansfield MW, Stickland MH, Grant PJ. Association of a common polymorphism in the factor XIII gene with venous thrombosis. Blood 1999;93:906-8.

(14.) Nauck M, Wieland H, Marz W. Rapid, homogeneous genotyping of the 4G/5G polymorphism in the promoter region of the PAII gene by fluorescence resonance energy transfer and probe melting curves. Clin Chem 1999;45:1141-7.

(15.) Philipp CS, Dilley A, Saidi P, Evatt B, Austin H, Zawadsky J, et al. Deletion polymorphism in the angiotensin-converting enzyme gene as a thrombophilic risk factor after hip arthroplasty. Thromb Haemost 1998;80:869-73.

(16.) Santoso S, Kunicki TJ, Kroll H, Haberbosch W, Gardemann A. Association of the platelet glycoprotein la C807T gene polymorphism with nonfatal myocardial infarction in younger patients. Blood 1999;93:2449-53.

(17.) Iacoviello L, Di Castelnuovo A, De Knijff P, D'Orazio A, Amore C, Arboretti R, et al. Polymorphisms in the coagulation factor VII gene and the risk of myocardial infarction. N Engl J Med 1998;338:79-85.

(18.) Walter DH, Schachinger V, Elsner M, Dimmeler S, Zeiher AM. Platelet glycoprotein Illa polymorphisms and risk of coronary stent thrombosis. Lancet 1997;350:1217-9.

(19.) Tyagi S, Bratu DP, Kramer FR. Multicolor molecular beacons for allele discrimination. Nat Biotechnol 1998;16:49-53.

(20.) Happich D, Schwaab R, Hanfland P, Hoernschemeyer D. Allelic discrimination of factor V Leiden using a 5' nuclease assay. Thromb Haemost 1999;82:1294-6.

(21.) Hacia JG, Fan JB, Ryder O, Jin L, Edgemon K, Ghandour G, et al. Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonuclectide arrays. Nat Genet 1999;22:164-7.

(22.) Ryan D, Nuccie B, Arvan D. Non-PCR-dependent detection of the factor V Leiden mutation from genomic DNA using a homogeneous invader microtiter plate assay. Mol Diagn 1999;4:135-44.

(23.) O'Donovan MC, Oefner PJ, Roberts SC, Austin J, Hoogendoom B, Guy C, et al. Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection. Genomics 1998;52:44-9.

(24.) Frank TS. Laboratory determination of hereditary susceptibility to breast and ovarian cancer. Arch Pathol Lab Med 1999;123:1023-6.

Richard D. Press

Departments of Pathology, and Molecular & Medical Genetics

Oregon Health Sciences University

3181 SW Sam Jackson Park Rd.

Portland, OR 97201

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Title Annotation:Editorial
Author:Press, Richard D.
Publication:Clinical Chemistry
Date:Oct 1, 2000
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