| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1 Laboratory of Biochemistry and Human Molecular Genetics and INSERM U 468, Hôpital Henri-Mondor, AP-HP, 51 Av du Maréchal de Lattre de Tassigny, 94010 Créteil, France
aauthor for correspondence: fax 00-33-1-49-81-28-42, e-mail serge.pissard{at}im3.inserm.fr
Hereditary hemochromatosis [(HC); MIM 235200] is one of the most frequent genetic diseases in Caucasian populations [prevalence up to 1 in 300 in northern Europe, with an estimated carrier frequency of 1 in 10 (1)]. Caused by a progressive iron overload, it is characterized by severe complications and a potentially lethal progression that could be entirely prevented with regular iron removal by means of bleeding (2). The availability of effective preventive treatment highlights the value of early detection. The discovery of the HFE gene by Feder et al. (3) has yielded a genetic test for disease risk. Two mutations, C282Y and H63D, located respectively in exons 4 and 2, impair HFE function (4) and are frequently tested for, along with a third variant located on exon 2 (S65C). Two genotypes (C282Y homozygote and compound heterozygote C282Y/H63D) are associated with risk of HC (5).
Because many could benefit from testing, numerous methods have been described in addition to conventional PCR restriction assays (3). Described methods include real-time PCR with fluorescent probes (6)(7)(8)(9) or SYBR green® melting curve (10), conventional PCR restriction assay with the use of HPLC (11) or capillary electrophoresis to resolve restriction fragments, allele-specific PCR products resolved on slab gels (12)(13) or by capillary electrophoresis (14)(15), and PCR followed by a single-nucleotide extension step (11), electrochemiluminescence detection (16), or reverse hybridization assay (17)(18).
Denaturing HPLC (DHPLC) is a new method for single- nucleotide polymorphism detection, which has been effective in terms of cost per analysis, sensitivity, and specificity (19). However, one limitation of DHPLC is that in most cases it hardly differentiates homozygous mutants from homozygous wild-type genotypes, and therefore, it is not recommended for diseases characterized by a few highly prevalent mutations. DHPLC was used successfully to detect C282Y and H63D heterozygouspatients (20), but detection of homozygous patients required a double analysis: the first using PCR with the sample alone and then a second PCR in which the sample was mixed with a wild-type control to reveal homozygous mutant patients. We have designed a method using an amplification refractory mutation system multiplex PCR assay and DHPLC (ARMS-DHPLC) for HFE genotyping in a two-step procedure.
We used EDTA blood samples obtained from iron-overloaded patients or relatives referred to us for HFE genotyping. DNA was extracted using phenol-chloroform, and the HFE genotypes were determined by PCR restriction assays with Rsa I (C282Y), Hph I (H63D), and HinfI (S65C). All patients gave their informed consent in accordance with regulations.
For each mutation (C282Y and H63D), a set of three primers was designed, two specific for the wild-type and mutant alleles and a third common one. For each set, the primer specific for the mutant allele carried a 12-bp 5' tail (21). To increase the specificity of the r-Y282 primer, we introduced a mismatch close to the 3' end. Primers were selected and computed for compatibility in multiplex PCR using Oligo6® software (22) and purchased from Life Technologies and MWG-Biotech AG. Equalization of amplification efficiency of each set was achieved by adjusting the concentration of each primer. The PCR conditions were as follows: 1x PCR buffer II (PE-Applied Biosystems); 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, pH 8.3), 0.05 mM dNTP, 0.4 µM f-C282Y, 0.36 µM r-C282, 0.04 µM r-Y282, 0.2 µM r-H63D, 0.116 µM f-H63, 0.084 µM f-D63, 1.25 U of Taq polymerase (PE-Applied Biosystems), and 50 ng of genomic DNA in a final volume of 50 µL (primer sequences and expected size for all fragments are given in Table 1
). Cycling conditions were as follows: 94 °C for 5 min, 35 cycles (94 °C for 30 s; 60 °C for 30 s; 72 °C for 45 s), and 72 °C for 7 min. All PCR amplifications were performed on a GeneAmp PCR System 9700 (PE-Applied Biosystems).
|
Multiplex PCR products were visualized on 6% polyacrylamide gels before DHPLC analysis and then analyzed with a Varian ProStar Helix® DHPLC System with the Helix DNA® column set. HPLC buffer compositions were as follows: buffer A, 0.1 mol/L triethylamine acetate (TEAA; pH 7.0) and 0.1 mmol/L EDTA (pH 7.0); and buffer B, 0.1 mol/L TEAA, 250 mol/L acetonitrile, 0.1 mmol/L EDTA, (pH 7.0). Each PCR sample (5 µL) was injected and analyzed by a gradient of buffer B into buffer A as follows: 50% to 52% in 30 s, then a linear gradient from 52% to 60% in 6.5 min. After a wash period (70% buffer B), the column was equilibrated to 50% buffer B for 2.5 min. The detection wavelength was 260 nm.
The acetonitrile gradient (slope and range) giving the best resolution in the range 100–200 bp was determined using a 25-bp DNA ladder.
With the oven temperature set at 45 °C, the four peaks corresponding to the four alleles (i.e., H63, 122 bp; D63, 134 bp; C282, 155 bp; and Y282, 167 bp) were accurately separated (Fig. 1A
), allowing a clear determination of the HFE genotype. We analyzed 96 patients, including controls, to assess the accuracy and reproducibility of the chromatograms. The accuracy of ARMS-DHPLC is very good because we observed no discrepancy with previous genotypes obtained with PCR restriction assays, with the exception of one patient who was incorrectly genotyped by the PCR restriction assay. Calculated from 20 chromatograms, the CV of the retention time (SE/mean) was good for each expected peak ( H63, 0.0121; D63, 0.0097; C282, 0.009; and Y282, 0.009), thus providing evidence for the good reproducibility of the chromatograms. When the patient was heterozygous for C282Y, there was minor interference in the migration pattern of the peaks corresponding to C282 and Y282, leading to a "two-phase" appearance of the last peak. We do not completely understand this phenomenon, which is presumably attributable to heteroduplex tail formation. However, this does not modify the peaks for H63D mutation and does not generate ambiguities in the interpretation of the chromatograms.
|
DHPLC methodology is well known for its ability to reveal most of the sequence variation within a PCR product. We tried to use this property in the same analytical run to search for the S65C variant. The exon 2 primer set was designed to bracket the nt 193 A
T (S65C) site, and the optimal denaturing condition (58 °C) was chosen with in silico modeling of the melting behavior of both fragments, making use of the software of Hansen and Oefner (23). The same batch of PCR products used for accuracy and reproducibility testing was run under denaturing conditions, and the same good size discrimination observed under nondenaturing conditions (45 °C) was reproduced (data not shown). In a patient heterozygous for the S65C mutation located in exon 2 and in a patient heterozygous for the rare E277K mutation located in exon 4, analysis under denaturing conditions revealed an extra peak in the H63 and C282 peaks, respectively (Fig. 1, B and C
), providing evidence of the ability of ARMS-DHPLC to detect mutations within PCR products in addition to the two main HFE mutations.
The ARMS-DHPLC method combines the sizing and DNA conformation detection properties of DHPLC, thereby allowing fast and reliable HFE genotyping. The key features of this method are that it makes use of only one basic PCR reaction using standard buffer and DNA polymerase, regular primers (i.e., no fluorescent labeling or terminal modification), standard PCR tubes without further reaction steps or handling, and 260 nm DNA absorbance measurement. This should be compared with the technical constraints of real-time fluorescent PCR methods and other methods (i.e., double-dye labeling of primers or probes, special reaction buffers and reaction vessels, several PCR reactions, and several PCR manipulations). The purchase of relatively expensive DHPLC instrumentation is made acceptable by the low cost of each analysis and the savings in work time.
The ARMS-DHPLC assay is a novel, highly automated, two-step genotyping method. Applied to HFE genotyping, it gives unambiguous results for the two most frequent mutations associated with HC (C282Y and H63D), and it is able to detect other mutations in the amplified PCR products without loss of accuracy for the two main mutations, as shown for the less frequent mutation S65C and the rare E277K mutant.
One feature of HC is that in a substantial number of patients displaying the typical HC phenotype, conventional HFE genotyping (i.e., the detection of the three most frequent mutations) detects only one mutation or no mutations. This peculiarity prompted extensive screening of the HFE gene sequence to identify rare or private mutations, and several investigators have characterized eight private or rare mutations (24)(25)(26). Interestingly, a recent report describes a full HFE screening procedure using DHPLC methodology (27). Building on the same technology, these two approaches could be complementary in a full evaluation of the HFE involvement in HC. This would allow identification of iron-overloaded patients in whom HFE is not involved in the disease and who are therefore candidates for the exploration of other genes involved in iron-metabolism disorders.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  S. Fruchon, M. Bensaid, N. Borot, M.-P. Roth, and H. Coppin Use of Denaturing HPLC and a Heteroduplex Generator to Detect the HFE C282Y Mutation Associated with Genetic Hemochromatosis Clin. Chem., May 1, 2003; 49(5): 822 - 824. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
X174-HaeIII); lane 2, control; lane 3, test sample.