| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
a author for correspondence: fax 64 3 364-0545, e-mail pgeorge{at}chmeds.ac.nz
Hemochromatosis is a common autosomal recessive disorder of iron metabolism occurring with a prevalence of 0.2–0.5% in Caucasian populations (1)(2)(3)(4)(5)(6). The disease is characterized by the excessive accumulation of dietary iron and a progressive rise in body iron stores, which may lead to serious clinical consequences, including cirrhosis, cardiac failure, diabetes, arthritis, and hepatocellular carcinoma. Treatment involves removal of the iron burden by regular venesection and leads to a normal life expectancy if implemented before the development of cirrhosis (7). Thus early detection and treatment are critically important.
Recent identification of a hemochromatosis gene, (HFE, initially termed HLA-H) by Feder et al.(8) allows for early genetic diagnosis and greatly simplifies the screening of a family once affected individuals have been identified. The HFE gene encodes a protein similar in structure to MHC class I-type molecules (9) that interacts with the transferrin receptor to regulate iron absorption (10). Two mutations have been detected in the HFE gene. Most individuals with hemochromatosis (80–100%) are homozygous for the missense mutation C282Y. In addition, a small number of compound heterozygotes (heterozygous for both C282Y and H63D) may develop clinical iron overload (11)). Homozygosity for H63D is not clearly associated with hemochromatosis. A high prevalence of asymptomatic carriers of C282Y (13.2%) and H63D (24.3%) is found in Caucasian communities (12).
An assay for the identification of these point mutations has been described, but is both expensive and labor-intensive.(8) More recently, Lynas (13)) described a method using two separate PCR reactions and restriction digestion to screen independently for the two mutations. We have used a similar method to study local patients (12)) and found it inconvenient when processing large numbers of samples. We therefore developed a rapid and cost-effective screening procedure that allows both mutations to be detected simultaneously using a multiplex PCR on rapidly extracted patient DNA. The assay is simple, fast, cost-effective, robust, and well-suited for use as a routine diagnostic test.
Blood was obtained in 5-mL EDTA Vacutainer Tubes from which DNA was
extracted by a standard rapid lysis technique (14)) that
requires only 5–7 min of hands-on time for each sample. PCR-mediated
site-directed mutagenesis was used to create unique BbrPI
restriction sites in each product of a duplex PCR that amplifies two
fragments spanning the C282Y and H63D loci. The primers
282mut (GTA CCC CCT GGG GAA GAG CAG AGA TAC A),
282rev (CCA TCC CCT AAC AAA GAG CAG ATC CAC),
63fwd (CAC ACT CTC TGC ACT ACC TCT T), and 63mut
(GGC TCC ACA CGG CGA CTC ACG T) allow simultaneous amplification of the
C282Y locus (188 bp) and the H63D locus (130 bp). As shown in Fig. 1
A, these primers are equally mismatched to both alleles but
uniquely introduce BbrPI sites into the products from both
the wild-type alleles. Digestion using restriction endonuclease
BbrPI (Roche Diagnostics) yields complete digestion of both
wild-type products, generating bands of 162 bp and 110 bp for C282Y and
H63D, respectively. The presence of either mutation destroys the
corresponding restriction site, allowing complete determination of the
patient's genotype in a single reaction.
|
PCR reactions were carried out using a Perkin-Elmer Thermocycler 2400 in 50 µL of total reaction volume. The PCR reaction contains 6.5 pmol of each primer, 1x ThermoPol buffer (New England Biolabs), 200 µmol/L of each dNTP, 5 µL (~50 ng) of DNA, and 0.5 U of Taq DNA Polymerase (Roche Diagnostics). Amplification was performed after a denaturation process of 2 min at 96 °C followed by an initial annealing step of 30 s at 55 °C, with 30 cycles each of 30 s at 72 °C, 30 s at 94 °C, and 15 s at 55 °C. A final extension period of 10 min at 72 °C completed the PCR.
Restriction digestions were carried out for 4 h at 37 °C, using the total PCR product and 5 U of enzyme (prediluted 20-fold with sterile distilled water) added directly to the PCR product. The restriction fragments were separated by gel electrophoresis using 3% Nusieve:Agarose (3:1) gels for 50–60 min at 100V. Products were visualized by staining in 1 mg/L ethidium bromide followed by transillumination at 302 nm.
Using 60 samples, including all possible genotypes, we compared this
method in a blinded experiment with results obtained by use of the
method described previously (13)). In each case,
identical genotypes were obtained. The results from six representative
samples are shown in Fig. 1B
, which illustrates that all combinations
of the two mutations and wild-type alleles can be distinguished
clearly. This method has subsequently been used for >1000 clinical
samples without problems.
Although mutagenic primers are used in this method, it is important to note that they are equally mismatched to both the wild-type and mutant alleles. The specificity is introduced by extension of the primers, which introduces a different base at the first step. This is an important feature of the assay design, removing any possibility of allele-specific amplification, and produces a robust procedure.
This technique provides an improved method of detection for both the C282Y and H63D mutations and diagnosis of hemochromatosis. By testing simultaneously for both mutations, this method decreases both the cost and time involved for each assay. The PCR is rapid and yields a consistently high-quality product. This assay is appropriate as a diagnostic test because results can be obtained quickly and confidently at a low cost, making it suitable for introduction into routine clinical laboratories.
Acknowledgments
This work was supported by the Health Research Council of New Zealand.
Footnotes
Molecular Pathology Laboratory, Canterbury Health Laboratories,
3>P.O. Box 151, Christchurch, New Zealand
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  A. Koeken, E. de Baar, L. Schrauwen, and C. Cobbaert Presence of the Hemochromatosis S65C Mutation Leads to Failure of Amplification in a Multiplex C282Y/H63D PCR Clin. Chem., September 1, 2007; 53(9): 1715 - 1715. [Full Text] [PDF]
|
|
|
|
 |
|
 E. F. Goodall, M. J. Greenway, I. van Marion, C. B. Carroll, O. Hardiman, and K. E. Morrison Association of the H63D polymorphism in the hemochromatosis gene with sporadic ALS Neurology, September 27, 2005; 65(6): 934 - 937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Starczynski, L. Hooper, N. Ali, M. Hill, C. Fegan, and G. Pratt Genetic Screening for Hemochromatosis: A Cautionary Tale Clin. Chem., March 1, 2005; 51(3): 673 - 673. [Full Text] [PDF]
|
|
|
|
|
|
G. G. Donohoe, M. Laaksonen, K. Pulkki, T. Ronnemaa, and V. Kairisto Rapid Single-Tube Screening of the C282Y Hemochromatosis Mutation by Real-Time Multiplex Allele-specific PCR without Fluorescent Probes Clin. Chem., October 1, 2000; 46(10): 1540 - 1547. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bollhalder, C. Mura, O. Landt, and F. E. Maly LightCycler PCR Assay for Simultaneous Detection of the H63D and S65C Mutations in the HFE Hemochromatosis Gene Based on Opposite Melting Temperature Shifts Clin. Chem., December 1, 1999; 45(12): 2275 - 2278. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
X174/HaeIII molecular mass marker;
lane 2, negative control; lane 3,
wild-type for C282Y, homozygous for H63D; lane 4,
wild-type for C282Y, heterozygous for H63D; lane 5,
heterozygous for C282Y, heterozygous for H63D (compound heterozygote);
lane 6, homozygous for C282Y, wild-type for H63D;
lane 7, heterozygous for C282Y, wild-type for H63D;
lane 8, wild-type for both C282Y and H63D.