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Technical Briefs |
1 Instituto di Neurogenetica e Neurofarmacologia, Consiglio Nazionale Richerche, Cagliari, via Boccaccio 8, 0947 Selagius, Italy
2 Dipartmento di Scienze Biomediche e Biotecnologie, Università di Cagliari, via Jenner s/n, 09121 Cagliari, Italy
3 Ospedale Regionale per Le Microcitemie, Azienda Sanitaria Locale 8, Cagliari, Via Jenner s/n, 09131 Cagliari-Sardegna, Italy
aauthor for correspondence: fax 39-070-503696, e-mail gloudian{at}mcweb.unica.it
Wilson disease (WD) is an autosomal recessive disorder of copper transport resulting from the defective function of a copper-transporting ATPase (ATP7B) (1)(2)(3). More than 200 disease-causing mutations have been identified (4). In the Sardinian population, WD has an incidence of 
1 in 7000 live births (5). Using single-strand conformation polymorphism (SSCP) and sequencing methods for mutation analysis, we have characterized 92% of the chromosomes analyzed and identified 16 different WD-causing mutations, 6 of which (-441/-427del, 213–214delAT, 1512–1513insT, R778W, 2463delC, and V1146M) are relatively common and account for 85% of chromosomes (6). On the basis of these data, we developed a reverse dot-blot (RDB) method as a practical solution to mutation screening in this population.
DNA samples from Sardinian WD patients carrying different combinations of the most common mutations (-441/-427del, 2463delC, V1146M, 213–214delAT, 1512–1513insT, and R778W) were used as controls. Our aim was to obtain the same PCR conditions for all six pairs of primers that were used to amplify the regions containing the six most common mutations. We therefore designed primers with an identical melting temperatures and tested their specificity first in single and then in multiplex PCRs. We also wanted to obtain relatively equal yields for all PCR products to obtain comparable signals using the RDB method. We therefore tested different concentrations for each pair of primers and finally established primer concentrations that allowed us to obtain approximately equal yields for the six PCR products. After repeated experiments and adjustment of the primer lengths and concentrations, we finally obtained six pairs of primers that permitted specific amplification using an annealing temperature of 60 °C (Fig. 1A
and Table 1
). The PCR products ranged from 93 to 430 bp in size (Fig. 1A
and Table 1
). The experiments were performed in 25 µL of 1x buffer containing 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 0.05 mM biotin 16-dUTP, and 1 U of Taq polymerase. Primers were used at the concentrations described in Table 1
. The reactions were performed on 100 ng of genomic DNA in a Gene-Amp 2400 (Perkin-Elmer) with an initial denaturation of 5 min at 94 °C, followed by 30 cycles of denaturation for 30 s at 94 °C and annealing for 30 s at 60 °C, with extension for 1 min at 72 °C for each cycle and a final extension of 10 min at 72 °C. After amplification, 5 µL of the PCR mixture was analyzed on a 2% agarose gel.
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The criteria used to set up the RDB assay were (a) a stringent hybridization to differentiate between oligonucleotides differing by only one base pair and (b) the same hybridization conditions for all wild-type/mutant (WT/MT) oligonucleotide pairs. Different oligonucleotides were synthesized and covalently attached to membranes via an amino linker. By varying the length and by experimentally testing the oligonucleotides, we obtained uniform hybridization conditions for all WT and MT probes at 53 °C. The oligonucleotide concentrations were the second variable investigated. They were tested in the same set of experiments using different concentrations for each probe. The WT oligonucleotide probes, which have a strong signal when hybridized to the WT PCR products and no signal when hybridized to the MT PCR products, were chosen as the WT probe; those that gave no signal when hybridized to WT products and a strong signal when hybridized to MT PCR products were chosen as the MT probes (Fig. 1B
). This was repeated for all six mutations. The sequences and the concentrations of the oligonucleotides used in the final assay are given in Table 1
.
Preparation of the Biodyne C membrane (Pall BioSupport) was essentially as described by Zang et al. (7). The membrane strips were prehybridized in 3 mL of 2x standard saline citrate, pH 7, containing 1 g/L sodium dodecyl sulfate in a plastic bag at 53 °C for 15 min. We denatured 20 µL of each multiplex PCR product at 95 °C for 10 min, put the denatured products on ice for 5 min, added the hybridization solution, and incubated the products individually with the membrane strips at 53 °C for 1 h. After hybridization, the strips were washed with 5 mL of fresh hybridization solution at 53 °C for 10 min. After washing, strips were incubated with 5 mL of fresh hybridization solution containing 3 units of streptavidin-alkaline phosphatase conjugate at room temperature for 30 min. Excess conjugate was removed with two 5-min washes in 0.1 mol/L diethanolamine, pH 10, containing 1 mmol/L MgCl2 and 0.1 g/L sodium azide. The strips were then incubated in the dark with 0.1 mol/L Tris, pH 9.5, containing 0.1 mol/L NaCl, 50 mmol/L MgCl2, 0.6 mg of nitroblue tetrazolium, and 0.3 mg of 5-bromo-4-chloro-3-indolyl phosphate for 30 min, after which the strips were blocked with a 5 mL of 20 mmol/L Tris, pH 7.5, containing 5 mmol/L EDTA.
In this study, exploiting the allelic homogeneity of WD in this population, we developed a RDB system that simplifies mutation screening because by screening for only the six most common mutations, we cover 85% of the total number of WD chromosomes.
Using the RDB method and SSCP analysis, we analyzed 63 individuals referred to our center for molecular analysis of WD. The obtained results agreed with both the RDB and the SSCP methods of analysis (our unpublished data). In our hands, the RDB assay is a rapid, sensitive, and easy system for screening multiple mutations in a single assay and allows testing for the majority of WD defects (85%) in this population. Another advantage of the RDB assay is that one patient can be screened with only one strip. This minimizes sample confusion in all of the steps. On the basis of these data, we can conclude that the most efficient strategy in the molecular diagnosis of WD in the Sardinian population is a first-step mutation screening for the six most common mutations with multiplex PCR followed by RDB analysis. Samples not characterized with this first step will be subsequently characterized by SSCP analysis of all of the exons and the promoter region of the ATP7B gene.
Acknowledgments
This work was supported by grants from Assessorato Igiene e Sanità Regione Sardegna-L.R (11.30.04.1990) and Programa di Educazione Sanitaria (DGR 1380/98-60% and 40%-97) to A. Cao.
References
The following articles in journals at HighWire Press have cited this article:
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  C. M. Mak, C.-W. Lam, and S. Tam Diagnostic Accuracy of Serum Ceruloplasmin in Wilson Disease: Determination of Sensitivity and Specificity by ROC Curve Analysis among ATP7B-Genotyped Subjects Clin. Chem., August 1, 2008; 54(8): 1356 - 1362. [Abstract] [Full Text] [PDF]
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X174 marker; lanes 2–6, multiplex PCR products of different WD patients. The amplified regions and the mutations contained in each region are indicated on the right. UTR, untranslated region. (B), examples of DNA analysis of WD patients carrying different combinations for the six most common Sardinian mutations. Samples homozygous for a MT (M) or WT (N) allele hybridize only with the oligonucleotide that detects the respective allele. Heterozygous samples hybridize with both the MT and the WT oligonucleotides. Normal, WT control.