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Improved molecular diagnosis of hereditary hemochromatosis using a DNA enzyme immunoassay.

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism characterized by excessive absorption of dietary iron from the small intestine, leading to gradual accumulation in several organ systems (1).

In the Caucasian population, HH affects ~3-8 in 1000 individuals, with an estimated prevalence of heterozygous carriers of 1 in 10 (2). The molecular basis for HH was completely unknown until the identification by Feder et al. (3) in 1996 of a gene on chromosome 6p, designated HLA-A. The gene, subsequently renamed HFE by the Nomenclature Committee of the Genome Database, is a MHC class Ib gene.

A single missense mutation, a G-to-A transition at nucleotide 845 that produces a cysteine-to-tyrosine substitution at position 282 (C282Y) in the HFE protein, has been observed in the majority of HH patients, with frequencies ranging from 64% to 100% in different geographic areas. The role of a second mutation, which produces a histidine-to-aspartic acid substitution at position 63 (H63D), remains controversial, although it is clearly associated with HH.

The strategies developed to screen HH chromosomes for the C282Y (845A) mutation include allele-specific oligonucleotide hybridization (4), restriction enzyme analysis (5), and oligonucleotide ligation assays (3). However, the use of all of these techniques is still confined to research laboratories because they are relatively expensive, time-consuming, and complicated for routine screening. In this report, we describe a PCR-based method that uses a DNA enzyme immunoassay (DEIA) for the specific detection of C282Y mutations.

After receiving informed consent, we analyzed 75 subjects. Of these, 46 were unrelated cases with a suspicion of hemochromatosis, whereas the remaining 29 subjects were members of five families with HH, comprising five probands and their relatives. Patients with HH had been diagnosed previously according to clinical criteria (signs and symptoms, increased transferrin saturation and/or serum ferritin, and presence of parenchymal iron overload on liver biopsy specimen).

After genomic DNA was extracted from peripheral blood leukocytes, 200 ng of template was amplified by PCR using 50 pmol of forward (5'-TGG CAA GGG TAA ACA GAT CC-3') and reverse (5'-CTC AGG CAC TCC TCT CAA CC-3') primers according to Feder et al. (3) in 100 [micro]L of reaction mixture containing 2.5 U of Taq DNA Polymerase (Life Technologies), 1 X PCR buffer (20 mmol/L Tris-HCl pH 8.4, 50 mmol/L KCl), 200 [micro]mol/L deoxynucleotides (Amersham Pharmacia Biotech), and 3 mmol/L [MgCl.sub.2]. Amplification was as follows: initial denaturation of 94[degrees]C for 2 min, followed by 30 cycles of 94[degrees]C for 40 s, 55[degrees]C for 40 s, and 72[degrees]C for 40 s (additional 5 min in the last cycle). All samples were tested for the HFE genotype by restriction enzyme analysis and further analyzed by DEIA as follows. Briefly, streptavidin-coated microtiter plates (ETI-IEMA; DiaSorin) were incubated overnight at 4[degrees]C with 40 ng/well of biotinylated oligonucleotide probes in 100 [micro]L of Tris-EDTA (10 mmol/L Tris, pH 8.0, 1 mmol/L EDTA). Each amplified specimen was added to a well coated with the 845G capture probe (5'-GAT ATA CGT GCC AGG TGG-3'), specific for the wild-type allele, and a second well coated with the 845A oligonucleotide (5'-GAT ATA CGT ACC AGG TGG-3'), specific for the mutant allele. Each test routinely included control samples for C282Y homozygotes, heterozygotes, and wild types. Two additional blank wells containing only chromogen/substrate were also included to check for cross-contamination. The solid phase was washed five times with 300 [micro]L of washing solution containing 6.7 mmol/L phosphate buffer, pH 6.4, 0.13 mol/L sodium chloride, 0.04 g/L Cialit, and 1 mL/L Tween 20. The crude thermally denatured PCR products (15 [micro]L) were added to the coated wells and incubated at 50[degrees]C for 1 h in 100 [micro]L of hybridization buffer (1 X standard saline citrate, 2X Denhardt's solution, 10 mmol/L Tris-HCI, pH 7.5, and 1 mmol/L EDTA). After five washes with washing solution, hybrids between the allele-specific probes and the HH gene sequences were detected by the addition of 100 [micro]L of a 1:100 dilution of a standard preparation of anti-DNA monoclonal antibody (MAb 27-14-D9; DiaSorin) in phosphate-buffered saline containing 100 mL/L fetal calf serum; this monoclonal antibody recognizes only double-stranded DNA. At the end of a 30-min incubation at room temperature, the wells were washed five times, and 100 /,L of an enzyme tracer [horseradish peroxidase conjugated to protein A (DiaSorin) diluted 1:20 000 in phosphate-buffered saline containing 100 mL/L fetal calf serum] was added to each well to reveal the bound antibody; the plates were then incubated another 30 min at room temperature. After the wells were washed five times to remove the samples, positive reactions were detected by a colorimetric reaction, which involved addition of 100 [micro]L of ETI-IEMA chromogen/substrate mixture (27 g/L tetramethylbenzidine, 0.1 mmol/L hydrogen peroxide) and incubation in the dark for 30 min at room temperature. The reaction was stopped with 200 [micro]L of 0.5 mol/L sulfuric acid, and the genotype of each amplified sample was detected by measuring the absorbance at 450 nm with a spectrophotometer.

Patients homozygous for the C282Y mutation were unambiguously identified by development of color only in the wells containing the 845A-specific probe. Samples from homozygous wild-type individuals gave a positive reaction only with the 845G-specific probe, whereas heterozygous carriers hybridized with both the wild-type probe and the oligonucleotide specific for the mutated gene.

Among the 75 subjects enrolled in this study, we detected 43 wild types, 22 heterozygotes, and 10 C282Y homozygotes. All of the latter group belonged to HH families (five probands and four relatives) other than one with a histological diagnosis related to hemochromatosis.

Deviations of 2[degrees]C from the selected hybridization temperature produced cross-hybridization between mismatched probes and target DNAs. The cutoff value, calculated in each experiment as the mean absorbance of two negative controls plus 3 SD, was 0.110 [+ or -] 0.04 (mean [+ or -] SD; range, 0.097-0.124). Under these conditions, we could always discriminate between positive and negative reactions. In all experiments at the selected cutoffs, we found no false-positive results, and the absorbance at 450 nm was 1.290-2.560 after specific hybridization with both probes. The signal generated by the heterozygous DNA with the two specific probes was roughly one-half the signal generated by the homozygous DNA hybridized with the complementary probe (Table 1). We assessed the specificity of the method by adding unrelated PCR products of noncomplementary sequences (HLA and ([beta]-globin) to microtiter plates containing HH probes; the signals obtained were indistinguishable from background. Moreover, the results obtained with DEIA completely matched the results obtained with restriction enzyme analysis genotyping both in terms of specificity and sensitivity.

Recently, Jeffrey et al. (6) reported that use of the original primers described by Feder et al. (3) in samples with the 5569G/A polymorphism may lead to misidentification of C282Y heterozygotes as homozygotes. To avoid erroneous genotyping and overestimation of homozygotes, Jeffrey et al. (6) developed a new antisense primer that excluded the site of the new polymorphism. These alternative primers can be substituted directly into our method because they give a PCR product almost identical to the one obtained with the primers used by Feder et al. (3), other than the 5569G/A site, and do not influence the binding regions of the specific probes that we used in the DEIA.

In conclusion, we have developed a nonisotopic hybridization system for the detection of the C282Y mutation in HFE gene sequences amplified by PCR. The method provides definitive data and is highly specific and sensitive. In addition, the procedure is simpler, faster, and less expensive than electrophoresis-based mutation analyses as well as other ELISA-coupled assays, such as oligonucleotide ligation assays, that require the use of fluorochrome-labeled probes. In the DEIA assay, the use of an anti-double-stranded DNA monoclonal antibody directly identifies the hybridization event itself and therefore bypasses the difficulty of DNA modification. The reagents and general assay scheme are well suited for routine analysis. Moreover, the use of 96-well microtiter plates allows simultaneous testing of several samples, greatly facilitating the large-scale screening of inherited diseases.

References

(1.) Adams PC, Valberg LS. Evolving expression of hereditary haemochromatosis. Semin Liver Dis 1996;16:47-54.

(2.) Gerhard GS, Ten Elshof AE, Chorney MJ. Hereditary haemochromatosis as an immunological disease. Br J Haematol 1998;100:247-55.

(3.) Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399-408.

(4.) Beutler E, Gelbart T, West C, Lee P, Adams M, Blackstone R, et al. Mutation analysis in hereditary haemochromatosis. Blood Cells Mol Dis 1996;22:187-94.

(5.) Adams PC, Chakrabarti S. Genotypic/phenotypic correlation in genetic haemochromatosis: evolution of diagnostic criteria. Gastroenterology 1998;114: 319-23.

(6.) Jeffrey GP, Chakrabarti S, Hegele RA, Adams PC. Polymorphism in intron 4 of HFE may cause overestimation of C282Y homozygote prevalence in haemochromatosis. Nat Genet 1999;22:325-6.

Raffaella Biasin, [1] Tosca Bertin, [2] Giuseppe Sardeo, [3] Paolo Fabris, [4] Enzo Venza, [l] and Domenico Infantolino [1] * ([1] U.O. Istologia ed Anatomia Patologica, Ospedale Civile, 31033 Castelfranco Veneto, Italy; [2] Medicina III, Ospedale "S Bortolo", 36100 Vicenza, Italy; [3] Medicina I, Ospedale Civile, 31044 Montebelluna, Italy; [4] U.O. Malattie Infettive, Ospedale "S Bortolo", 36100 Vicenza, Italy; * address correspondence to this author at: Modulo di Diagnostica Molecolare, U.O. Anatomia Patologica, Ospedale Civile, 31033 Castelfranco Veneto, Italy; fax 39-423-732280, e-mail infantolino@ulssasolo.ven.it)
Table 1. Characterization of amplified DNA by DEIA
hybridization.

Genotype at [A.sub.450], mean [+ or -] SD
 nucleotide Subjects
position 845 analyzed 845A capture probe 845G capture probe

 A/A 10 1.840 [+ or -] 0.476 0.182 [+ or -] 0.045
 A/G 22 0.737 [+ or -] 0.180 1.319 [+ or -] 0.533
 G/G 43 0.114 [+ or -] 0.034 2.314 [+ or -] 0.401
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Title Annotation:Technical Briefs
Author:Biasin, Raffaella; Bertin, Tosca; Sardeo, Giuseppe; Fabris, Paolo; Venza, Enzo; Infantolino, Domenic
Publication:Clinical Chemistry
Date:May 1, 2000
Words:1679
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