- Email: [email protected]
Non-radioactive detection of five common microsatellite markers for ATP7B gene in Wilson disease patients
Molecular and Cellular Probes 17 (2003) 271–274 www.elsevier.com/locate/ymcpr
Non-radioactive detection of five common microsatellite markers for ATP7B gene in Wilson disease patientsq A. Bobbaa,*, E. Marraa, D.M. Fathallahb, S. Giannattasioa a
Istituto di Biomembrane e Bioenergetica, CNR, Via Amendola 165/A, 70126 Bari, Italy b Molecular Biotechnology Group, Institut Pasteur de Tunis, Tunis, Tunisia Received 2 April 2003; accepted for publication 11 July 2003
Abstract Haplotype analysis using microsatellite markers is a useful indicator of specific mutations and is often exploited as the first large-scale screening technique to carry out the molecular characterization of the disease gene in probands from a specific population. However, the methodologies available are still cumbersome and require the use of either radioactive compounds or specialized equipment suitable to follow fluorescent dyes. Both these techniques may not be available for newly developing clinical laboratories. We have set up a sensitive and easy-to-use protocol to characterize five closely spaced, highly polymorphic microsatellite polymorphisms (CA repeats) that span the Wilson disease (WD) region, i.e. D13S316, D13S133, D13S301, D13S314, D13S315. The technique described here for the analysis of the WD gene microsatellite system relies on the quick detection method of silver staining, avoiding the use of toxic or sophisticated equipment. This approach could be the method of choice to implement molecular genetic testing in clinical laboratories, even those not especially equipped for DNA analysis and in particular in newly developed molecular genetics centers in countries whose population has not yet been characterized for WD-causing ATP7B gene mutations. q 2003 Elsevier Ltd. All rights reserved. Keywords: Microsatellite; Wilson disease; ATP7B gene
Wilson disease (WD) is an autosomal recessive disorder of copper transport, characterized by impaired biliary excretion and failure to incorporate copper into ceruloplasmin. Toxic accumulation of copper causes tissue damage primarily in the liver, brain and kidneys. The disease frequency is estimated to be between 1 in 35,000 and 1 in 100,000 live births, with a carrier frequency of 1 in 90 [1 – 3]. The gene responsible for WD is ATP7B. It has been mapped to chromosome 13q and is split into 22 exons spanning a DNA region of about 100 kb [4]. ATP7B protein product is a copper-transporting P-type ATPase with a high degree of amino acid sequence identity with that of ATP7A, the gene responsible for Menkes disease, an X-linked disorder of copper transport [5]. So far more than 200 mutations have been identified in the ATP7B gene of WD patients of different ethnic origin, q The work has been carried out at the Istituto di Biomembrane e Bioenergetica, CNR, Bari, Italy. * Corresponding author. Tel.: þ 39-080-5442412; fax: þ 39-0805443317. E-mail address: [email protected] (A. Bobba).
0890-8508/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0890-8508(03)00065-3
with the H1069Q mutation being the most common (ranging from 10 to 70%) in the majority of the populations studied [6]. Notwithstanding this, the frequency and distribution of WD mutations and their linked haplotypes vary worldwide. Thus, the definition of the mutation spectrum in a definite population is a prerequisite for genotype/phenotype correlation studies as well as for the development of carrier detection and genetic counselling protocols in that population. Due to the length and molecular organization of the ATP7B gene, together with the large number of WD mutations so far identified in the 4.1 kbp coding region of the WD gene, complete molecular characterization of WD chromosomes is still mainly performed through DNA single strand conformation polymorphism analysis of each of the 22 ATP7B gene exons [4,7]. Thus, alternative approaches seem worth developing to implement molecular genetic testing in new molecular genetics centers in countries whose population has not yet been characterized for WD-causing ATP7B gene mutations. This is especially true in populations of the Mediterranean area, in which high
272
A. Bobba et al. / Molecular and Cellular Probes 17 (2003) 271–274
heterogeneity has been found so far in the molecular basis of WD [7,8]. Haplotype data are important as a guide for mutation detection. Several highly polymorphic microsatellite (CA repeats) systems in linkage disequilibrium with the WD locus have been identified allowing haplotype analysis of WD chromosomes from different populations [7,9 – 14]. The frequency and distribution of haplotypes associated with WD chromosomes is a useful tool to carry out indirect molecular diagnosis through linkage analysis in families with affected siblings. Most available up-dated methodologies for haplotype analysis require the use of either radioactive compounds or dedicated equipments suitable to trace fluorescent dyes. Yet, these facilities are not always available in newly developing clinical laboratories. We have developed a sensitive and easy-to-use protocol to characterize ATP7B gene haplotypes through analysis of five highly informative microsatellites in linkage disequilibrium with the WD locus, i.e. D13S316, D13S133, D13S301, D13S314, D13S315. In particular, the D13S301 marker is proximal and , 40 kbp from the ATP7B gene [4, 9]. The remaining markers are distal to the ATP7B gene, with D13S314 being the closest and D13S316 the farthest, within a region of , 1 Mbp [9,10]. The use of several markers for haplotype definition may limit the occurrence of misdiagnosis resulting from recombination or instability of repeats. The typical experimental procedure was as follows. 200 –300 ng of genomic DNA, extracted from whole-blood samples of the members of four WD families of Tunisian origin with at least one sibling affected by WD and one single patient, was used as a template in PCR reactions in the presence of 50 mM KCl, 10 mM Tris –HCl, pH 9.0, 0.1% Triton X-100, 1.5 mM MgCl2, 200 mM of each deoxynucleotide, 25 pmol of the two amplification primers [4,10] and 1.5U Amplitaq DNA Polymerase (Perkin Elmer, Norwalk, CT) in 100 ml reaction volume. The length of the expected PCR amplification products were in the 118– 194 bp range for D13S316, 134 –187 bp for D13S133, 135– 150 bp for D13S301 and D13S314, and 150– 180 bp for D13S315. Aliquots (10 ml) of the solution containing the amplification products, ranging in size from 118 to 194 bp, were loaded on a 12% polyacrylamide gel (20 £ 40 £ 0.8 cm3) and electrophoretic run was performed at 450 V for about 22 h at room temperature. The gel was then transferred to a plastic sheet to facilitate handling. The gel portion containing the relevant electrophoretic bands was cut and immediately stained by silver staining protocol with all steps performed under gentle shaking. The gel was fixed for 5 min in 10% ethanol and then incubated in 1% nitric acid for 4 min, washed with deionized water for 20 s and incubated for 20 min in 0.2% silver nitrate. The gel was then washed with water and incubated in the developing solution (0.28 M sodium carbonate, 0.05% formaldehyde).
The developing reaction was stopped with 5% acetic acid. Alternatively the gel was stored in a fixing solution containing acetic acid/methanol/H2O ¼ 1/4/5 (v/v/v) before performing silver staining. After staining the gel was dried and the electrophoretic pattern was analysed by using a GS-700 Imaging Densitometer (Bio Rad Laboratories). The dimension of each allele was determined by analysing both the dried gel and the densitometric image, with the same result obtained. To permit consistency in the naming of the alleles each of the following criteria were taken into consideration: (a) comparison with the 50 bp-DNA sequencing laddering (Invitrogen, Carlsbad, CA, USA), used as molecular size marker, (b) comparison with control DNA samples for which the sizes of alleles were already known, (c) separate reading of each electrophoretic pattern by two investigators. The results of a typical analysis of D13S315 microsatellite are shown in Fig. 1. The presence of multiple, non-specific electrophoretic bands within a molecular weight range greater than that expected (150 –180 bp) might be due to Taq DNA polymerase splippage events which are likely to occur during PCR amplification of CA repeats. Control samples, whose haplotype is known, were loaded in lane i (178, 178 bp) and lane j (178, 180 bp) and considered as internal standards. Subjects carrying heterozygote alleles were identified by the presence of two more intense bands (as an example see lane f). Bands that differ by only two bases are clearly detectable as shown in lane o in which 172 and 174 bp DS13315 alleles have been identified. Homozygote individuals were clearly identified by the presence of one electrophoretic band with an intensity double that in the case of heterozygotes as for the proband in lane b in which 180 bp DS13315 alleles have been identified. Six D13S315 alleles were identified among WD and normal chromosomes (Table 1) and can be used to trace WD chromosomes in the four families analysed. In three families the probands were found to be homozygous for different DS13315 alleles: 178 bp for both probands of Family 1 (lanes c, d); 176 bp for proband of Family 2 (lane k) and 174 bp for proband of Family 4 (lane q). Only in the case of Family 3 the proband (lane n) was found to be heterozygous for 172, 176 bp alleles. In all cases the exact segregation of ATP7B alleles was observed in each family analysed (Fig. 1). The same experimental setting was used to genotype the other four microsatellites, D13S314, D13S301, D13S133 and D13S316 thus allowing, in each case, the identification of eight, six, eight and two alleles, respectively (Table 1). For each microsatellite marker, the size of the identified alleles did not affect the resolution of the electrophoretic analysis through which it was possible to discriminate between two alleles differing by only one CA repeat. The informativity of haplotype analysis to genotype WD family members for genetic counselling and prenatal diagnosis is greatly increased by the analysis of all five microsatellites. Early molecular genetic diagnosis is important in WD, since it is a treatable inherited disease [14,15]. Molecular
A. Bobba et al. / Molecular and Cellular Probes 17 (2003) 271–274
273
Fig. 1. Polyacrylamide gel electrophoresis analysis of the D13S315 microsatellite in the WD locus in four WD families and one WD patient. The relevant STR alleles are indicated. Lane b is a single WD patient. Lanes i and j: control samples used as internal standards. Lanes a and t: 50 bp-DNA size marker. Family 1: (c) proband; (d) proband, brother of c; (e) maternal grandmother; (f) maternal grandfather; (g) the same as in c; (h) father. Family 2: (k) proband; (l) proband, cousin of k; (m) father of l. Family 3: (n) proband; (o) father; (p) mother; Family 4: (q) proband; (r) father; (s) mother.
genetic testing of inherited diseases with high allelic heterogeneity, such as WD, is often performed by cumbersome mutation detection techniques. On the other hand, microsatellite analysis is usually exploited as the first largescale screening technique to carry out the molecular characterization of the disease gene in probands from a specific population. Thus, in a population in which WDcausing ATP7B gene mutations have not been identified, it is extremely useful to carry out haplotype analysis. To this aim, microsatellite marker analysis is usually performed by PCR amplification in the presence of radioactive or fluorescent nucleotide precursors followed by autoradiography (1–5 days) or detection through fluorescence analysers, respectively. The technique described here for the analysis of the WD gene microsatellite systems, on the other hand, relies on a quick detection method such as silver staining, which does not Table 1 ATP7B-linked microsatellite alleles identified in this study D13S315
D13S314
D13S301
D13S133
D13S316
182 180 178 176 174 172
147 145 143 141 139 137 135 133
148 146 144 142 140 126
181 173 171 169 161 149 131 129
142 140
Note: numbers indicate allele size in base pairs.
make use of hazardous compounds such as radioactive nucleotide precursors, making it the method of choice in clinical laboratories even not especially equipped for DNA analysis. Indeed, in developing countries in which implementation of innovative health care programme is in progress, the availability of such a simple and reproducible method to perform haplotype analysis without the need to buy dedicated instruments or to use hazardous material, could be the starting point to implement molecular genetic testing and to develop new clinical genetics laboratories.
Acknowledgements This work was partially financed by MURST Piani di Potenziamento della Rete Scientifica e Tecnologica-Cluster 03 Project ‘Analisi molecolare di patologie umane e marcatori di variabilita genetica’. The authors thank Dr Loudianos who kindly provided us with three control DNA samples for which the sizes of D13S301 and D13S316 alleles were already known, and Richard Lusardi for linguistic consultation.
References [1] Scheinberg IH, Sternlieb I. Wilson’s disease. In: Smith LH, editor. Major problems in internal medicine, vol. 23. Philadelphia: W.B. Saunders; 1984.
274
A. Bobba et al. / Molecular and Cellular Probes 17 (2003) 271–274
[2] Danks DM. Disorders of copper transport. In: Beauder AL, Sly WS, Valle D, editors. Metabolic basis of inherited disease. New York: McGraw-Hill; 1989. p. 1411–31. [3] Subramanian I, Vanek ZF, Bronstein JM. Diagnosis and treatment of Wilson’s disease. Curr Neurol Neurosci Rep 2002;2:317–23. Review. [4] Thomas GR, Forbes JR, Roberts EA, Walshe JM, Cox DW. The Wilson disease gene: spectrum of mutations and their consequences. Nat Genet 1995;9:210–7. [5] Tanzi RE, Petrukhin K, Chernov I, Pellequer JL, Wasco W, Ross B, Romano DM, Parano E, Pavone L, Brzustowicz LM, Devoto M, Peppercorn J, Bush AI, Sternlieb I, Pirastu M, Gusella JF, Evgrafov O, Penchaszadeh GK, Honig B, Edelman IS, Soares MB, Scheinberg IH, Gilliam TC. The Wilson diseases gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat Genet 1993;5:344–50. [6] Riordan SM, Williams R. The Wilson’s disease gene and phenotypic diversity. J Hepatol 2001;34:165 –71. Review. [7] Figus A, Angius A, Loudianos G, Bestini C, Dessi V, Loi A, Deiana M, Lovicu M, Olla N, Sole G, De Virgiliis S, Lilliu F, Farci AMG, Nurchi A, Giacchino R, Barbino A, Marazzi M, Cancan L, Greggio NA, Marcellini M, Solinas A, Depilano A, Barbera C, Devoto M, Ozsoylu S, Kocak N, Akar N, Karayalcin S, Molini V, Cullufi P, Balestrieri A, Cao A, Pirastu M. Molecular pathology and haplotype analysis of Wilson disease in Mediterranean populations. Am J Hum Genet 1995;57:1318–24. [8] Kalinsky H, Funes A, Zeldin A, et al. Novel ATP7B mutations causing Wilson disease in several Israeli ethnic groups. Hum Mutat 1998;11:145 –51.
[9] Petrukhin K, Fischer SG, Pirastu M, Tanzi RE, Chernov I, Devoto M, Brzustowicz LM, Cayanis E, Vitale E, Russo JJ, Matseoane D, Boukhgalter, Wasco W, Figus AL, Loudianos J, Cao A, Sternlieb I, Evgrafov O, Parano E, Pavone L, Warburton D, Ott J, Penchaszadeh GK, Scheinberg JH, Gilliam TC. Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nat Genet 1993;5:338–43. [10] Thomas GR, Bull PC, Roberts EA, Walshe JM, Cox DW. Haplotype studies in Wilson disease. Am J Hum Genet 1994;54:71–8. [11] Loudianos G, Dessi V, Lovicu M, Angius A, Kanavakis E, Tzetis M, Kattamis C, Manolaki N, Vassiliki G, Karpathios T, Cao A, Pirastu M. Haplotype and mutation analysis in Greek patients with Wilson disease. Eur J Hum Genet 1998;6:487–91. [12] Nanji MS, Nguyen VTT, Kawasoe JH, Inui K, Endo F, Nakajima T, Anezaki T, Cox DW. Haplotype and mutation analysis in Japanese patients with Wilson disease. Am J Hum Genet 1997;60: 1424–9. [13] Firneisz G, Lakatos PL, Szalay F, Polli C, Glant TT, Ferenci P. Common mutations of ATP7B in Wilson disease patients from Hungary. Am J Hum Genet 2002;108:23–8. [14] Lovicu M, Dessi V, Zappu A, De Virgiliis S, Cao A, Loudianos G. Efficient strategy for molecular diagnosis of Wilson disease in the Sardinian population. Clin Chem 2003;49:496–8. [15] Loudianos G, Gitlin JD. Wilson’s diseases. Semin Liver Dis 2000;20: 353 –64.
Non-radioactive detection of five common microsatellite markers for ATP7B gene in Wilson disease patientsq A. Bobbaa,*, E. Marraa, D.M. Fathallahb, S. Giannattasioa a
Istituto di Biomembrane e Bioenergetica, CNR, Via Amendola 165/A, 70126 Bari, Italy b Molecular Biotechnology Group, Institut Pasteur de Tunis, Tunis, Tunisia Received 2 April 2003; accepted for publication 11 July 2003
Abstract Haplotype analysis using microsatellite markers is a useful indicator of specific mutations and is often exploited as the first large-scale screening technique to carry out the molecular characterization of the disease gene in probands from a specific population. However, the methodologies available are still cumbersome and require the use of either radioactive compounds or specialized equipment suitable to follow fluorescent dyes. Both these techniques may not be available for newly developing clinical laboratories. We have set up a sensitive and easy-to-use protocol to characterize five closely spaced, highly polymorphic microsatellite polymorphisms (CA repeats) that span the Wilson disease (WD) region, i.e. D13S316, D13S133, D13S301, D13S314, D13S315. The technique described here for the analysis of the WD gene microsatellite system relies on the quick detection method of silver staining, avoiding the use of toxic or sophisticated equipment. This approach could be the method of choice to implement molecular genetic testing in clinical laboratories, even those not especially equipped for DNA analysis and in particular in newly developed molecular genetics centers in countries whose population has not yet been characterized for WD-causing ATP7B gene mutations. q 2003 Elsevier Ltd. All rights reserved. Keywords: Microsatellite; Wilson disease; ATP7B gene
Wilson disease (WD) is an autosomal recessive disorder of copper transport, characterized by impaired biliary excretion and failure to incorporate copper into ceruloplasmin. Toxic accumulation of copper causes tissue damage primarily in the liver, brain and kidneys. The disease frequency is estimated to be between 1 in 35,000 and 1 in 100,000 live births, with a carrier frequency of 1 in 90 [1 – 3]. The gene responsible for WD is ATP7B. It has been mapped to chromosome 13q and is split into 22 exons spanning a DNA region of about 100 kb [4]. ATP7B protein product is a copper-transporting P-type ATPase with a high degree of amino acid sequence identity with that of ATP7A, the gene responsible for Menkes disease, an X-linked disorder of copper transport [5]. So far more than 200 mutations have been identified in the ATP7B gene of WD patients of different ethnic origin, q The work has been carried out at the Istituto di Biomembrane e Bioenergetica, CNR, Bari, Italy. * Corresponding author. Tel.: þ 39-080-5442412; fax: þ 39-0805443317. E-mail address: [email protected] (A. Bobba).
0890-8508/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0890-8508(03)00065-3
with the H1069Q mutation being the most common (ranging from 10 to 70%) in the majority of the populations studied [6]. Notwithstanding this, the frequency and distribution of WD mutations and their linked haplotypes vary worldwide. Thus, the definition of the mutation spectrum in a definite population is a prerequisite for genotype/phenotype correlation studies as well as for the development of carrier detection and genetic counselling protocols in that population. Due to the length and molecular organization of the ATP7B gene, together with the large number of WD mutations so far identified in the 4.1 kbp coding region of the WD gene, complete molecular characterization of WD chromosomes is still mainly performed through DNA single strand conformation polymorphism analysis of each of the 22 ATP7B gene exons [4,7]. Thus, alternative approaches seem worth developing to implement molecular genetic testing in new molecular genetics centers in countries whose population has not yet been characterized for WD-causing ATP7B gene mutations. This is especially true in populations of the Mediterranean area, in which high
272
A. Bobba et al. / Molecular and Cellular Probes 17 (2003) 271–274
heterogeneity has been found so far in the molecular basis of WD [7,8]. Haplotype data are important as a guide for mutation detection. Several highly polymorphic microsatellite (CA repeats) systems in linkage disequilibrium with the WD locus have been identified allowing haplotype analysis of WD chromosomes from different populations [7,9 – 14]. The frequency and distribution of haplotypes associated with WD chromosomes is a useful tool to carry out indirect molecular diagnosis through linkage analysis in families with affected siblings. Most available up-dated methodologies for haplotype analysis require the use of either radioactive compounds or dedicated equipments suitable to trace fluorescent dyes. Yet, these facilities are not always available in newly developing clinical laboratories. We have developed a sensitive and easy-to-use protocol to characterize ATP7B gene haplotypes through analysis of five highly informative microsatellites in linkage disequilibrium with the WD locus, i.e. D13S316, D13S133, D13S301, D13S314, D13S315. In particular, the D13S301 marker is proximal and , 40 kbp from the ATP7B gene [4, 9]. The remaining markers are distal to the ATP7B gene, with D13S314 being the closest and D13S316 the farthest, within a region of , 1 Mbp [9,10]. The use of several markers for haplotype definition may limit the occurrence of misdiagnosis resulting from recombination or instability of repeats. The typical experimental procedure was as follows. 200 –300 ng of genomic DNA, extracted from whole-blood samples of the members of four WD families of Tunisian origin with at least one sibling affected by WD and one single patient, was used as a template in PCR reactions in the presence of 50 mM KCl, 10 mM Tris –HCl, pH 9.0, 0.1% Triton X-100, 1.5 mM MgCl2, 200 mM of each deoxynucleotide, 25 pmol of the two amplification primers [4,10] and 1.5U Amplitaq DNA Polymerase (Perkin Elmer, Norwalk, CT) in 100 ml reaction volume. The length of the expected PCR amplification products were in the 118– 194 bp range for D13S316, 134 –187 bp for D13S133, 135– 150 bp for D13S301 and D13S314, and 150– 180 bp for D13S315. Aliquots (10 ml) of the solution containing the amplification products, ranging in size from 118 to 194 bp, were loaded on a 12% polyacrylamide gel (20 £ 40 £ 0.8 cm3) and electrophoretic run was performed at 450 V for about 22 h at room temperature. The gel was then transferred to a plastic sheet to facilitate handling. The gel portion containing the relevant electrophoretic bands was cut and immediately stained by silver staining protocol with all steps performed under gentle shaking. The gel was fixed for 5 min in 10% ethanol and then incubated in 1% nitric acid for 4 min, washed with deionized water for 20 s and incubated for 20 min in 0.2% silver nitrate. The gel was then washed with water and incubated in the developing solution (0.28 M sodium carbonate, 0.05% formaldehyde).
The developing reaction was stopped with 5% acetic acid. Alternatively the gel was stored in a fixing solution containing acetic acid/methanol/H2O ¼ 1/4/5 (v/v/v) before performing silver staining. After staining the gel was dried and the electrophoretic pattern was analysed by using a GS-700 Imaging Densitometer (Bio Rad Laboratories). The dimension of each allele was determined by analysing both the dried gel and the densitometric image, with the same result obtained. To permit consistency in the naming of the alleles each of the following criteria were taken into consideration: (a) comparison with the 50 bp-DNA sequencing laddering (Invitrogen, Carlsbad, CA, USA), used as molecular size marker, (b) comparison with control DNA samples for which the sizes of alleles were already known, (c) separate reading of each electrophoretic pattern by two investigators. The results of a typical analysis of D13S315 microsatellite are shown in Fig. 1. The presence of multiple, non-specific electrophoretic bands within a molecular weight range greater than that expected (150 –180 bp) might be due to Taq DNA polymerase splippage events which are likely to occur during PCR amplification of CA repeats. Control samples, whose haplotype is known, were loaded in lane i (178, 178 bp) and lane j (178, 180 bp) and considered as internal standards. Subjects carrying heterozygote alleles were identified by the presence of two more intense bands (as an example see lane f). Bands that differ by only two bases are clearly detectable as shown in lane o in which 172 and 174 bp DS13315 alleles have been identified. Homozygote individuals were clearly identified by the presence of one electrophoretic band with an intensity double that in the case of heterozygotes as for the proband in lane b in which 180 bp DS13315 alleles have been identified. Six D13S315 alleles were identified among WD and normal chromosomes (Table 1) and can be used to trace WD chromosomes in the four families analysed. In three families the probands were found to be homozygous for different DS13315 alleles: 178 bp for both probands of Family 1 (lanes c, d); 176 bp for proband of Family 2 (lane k) and 174 bp for proband of Family 4 (lane q). Only in the case of Family 3 the proband (lane n) was found to be heterozygous for 172, 176 bp alleles. In all cases the exact segregation of ATP7B alleles was observed in each family analysed (Fig. 1). The same experimental setting was used to genotype the other four microsatellites, D13S314, D13S301, D13S133 and D13S316 thus allowing, in each case, the identification of eight, six, eight and two alleles, respectively (Table 1). For each microsatellite marker, the size of the identified alleles did not affect the resolution of the electrophoretic analysis through which it was possible to discriminate between two alleles differing by only one CA repeat. The informativity of haplotype analysis to genotype WD family members for genetic counselling and prenatal diagnosis is greatly increased by the analysis of all five microsatellites. Early molecular genetic diagnosis is important in WD, since it is a treatable inherited disease [14,15]. Molecular
A. Bobba et al. / Molecular and Cellular Probes 17 (2003) 271–274
273
Fig. 1. Polyacrylamide gel electrophoresis analysis of the D13S315 microsatellite in the WD locus in four WD families and one WD patient. The relevant STR alleles are indicated. Lane b is a single WD patient. Lanes i and j: control samples used as internal standards. Lanes a and t: 50 bp-DNA size marker. Family 1: (c) proband; (d) proband, brother of c; (e) maternal grandmother; (f) maternal grandfather; (g) the same as in c; (h) father. Family 2: (k) proband; (l) proband, cousin of k; (m) father of l. Family 3: (n) proband; (o) father; (p) mother; Family 4: (q) proband; (r) father; (s) mother.
genetic testing of inherited diseases with high allelic heterogeneity, such as WD, is often performed by cumbersome mutation detection techniques. On the other hand, microsatellite analysis is usually exploited as the first largescale screening technique to carry out the molecular characterization of the disease gene in probands from a specific population. Thus, in a population in which WDcausing ATP7B gene mutations have not been identified, it is extremely useful to carry out haplotype analysis. To this aim, microsatellite marker analysis is usually performed by PCR amplification in the presence of radioactive or fluorescent nucleotide precursors followed by autoradiography (1–5 days) or detection through fluorescence analysers, respectively. The technique described here for the analysis of the WD gene microsatellite systems, on the other hand, relies on a quick detection method such as silver staining, which does not Table 1 ATP7B-linked microsatellite alleles identified in this study D13S315
D13S314
D13S301
D13S133
D13S316
182 180 178 176 174 172
147 145 143 141 139 137 135 133
148 146 144 142 140 126
181 173 171 169 161 149 131 129
142 140
Note: numbers indicate allele size in base pairs.
make use of hazardous compounds such as radioactive nucleotide precursors, making it the method of choice in clinical laboratories even not especially equipped for DNA analysis. Indeed, in developing countries in which implementation of innovative health care programme is in progress, the availability of such a simple and reproducible method to perform haplotype analysis without the need to buy dedicated instruments or to use hazardous material, could be the starting point to implement molecular genetic testing and to develop new clinical genetics laboratories.
Acknowledgements This work was partially financed by MURST Piani di Potenziamento della Rete Scientifica e Tecnologica-Cluster 03 Project ‘Analisi molecolare di patologie umane e marcatori di variabilita genetica’. The authors thank Dr Loudianos who kindly provided us with three control DNA samples for which the sizes of D13S301 and D13S316 alleles were already known, and Richard Lusardi for linguistic consultation.
References [1] Scheinberg IH, Sternlieb I. Wilson’s disease. In: Smith LH, editor. Major problems in internal medicine, vol. 23. Philadelphia: W.B. Saunders; 1984.
274
A. Bobba et al. / Molecular and Cellular Probes 17 (2003) 271–274
[2] Danks DM. Disorders of copper transport. In: Beauder AL, Sly WS, Valle D, editors. Metabolic basis of inherited disease. New York: McGraw-Hill; 1989. p. 1411–31. [3] Subramanian I, Vanek ZF, Bronstein JM. Diagnosis and treatment of Wilson’s disease. Curr Neurol Neurosci Rep 2002;2:317–23. Review. [4] Thomas GR, Forbes JR, Roberts EA, Walshe JM, Cox DW. The Wilson disease gene: spectrum of mutations and their consequences. Nat Genet 1995;9:210–7. [5] Tanzi RE, Petrukhin K, Chernov I, Pellequer JL, Wasco W, Ross B, Romano DM, Parano E, Pavone L, Brzustowicz LM, Devoto M, Peppercorn J, Bush AI, Sternlieb I, Pirastu M, Gusella JF, Evgrafov O, Penchaszadeh GK, Honig B, Edelman IS, Soares MB, Scheinberg IH, Gilliam TC. The Wilson diseases gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat Genet 1993;5:344–50. [6] Riordan SM, Williams R. The Wilson’s disease gene and phenotypic diversity. J Hepatol 2001;34:165 –71. Review. [7] Figus A, Angius A, Loudianos G, Bestini C, Dessi V, Loi A, Deiana M, Lovicu M, Olla N, Sole G, De Virgiliis S, Lilliu F, Farci AMG, Nurchi A, Giacchino R, Barbino A, Marazzi M, Cancan L, Greggio NA, Marcellini M, Solinas A, Depilano A, Barbera C, Devoto M, Ozsoylu S, Kocak N, Akar N, Karayalcin S, Molini V, Cullufi P, Balestrieri A, Cao A, Pirastu M. Molecular pathology and haplotype analysis of Wilson disease in Mediterranean populations. Am J Hum Genet 1995;57:1318–24. [8] Kalinsky H, Funes A, Zeldin A, et al. Novel ATP7B mutations causing Wilson disease in several Israeli ethnic groups. Hum Mutat 1998;11:145 –51.
[9] Petrukhin K, Fischer SG, Pirastu M, Tanzi RE, Chernov I, Devoto M, Brzustowicz LM, Cayanis E, Vitale E, Russo JJ, Matseoane D, Boukhgalter, Wasco W, Figus AL, Loudianos J, Cao A, Sternlieb I, Evgrafov O, Parano E, Pavone L, Warburton D, Ott J, Penchaszadeh GK, Scheinberg JH, Gilliam TC. Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nat Genet 1993;5:338–43. [10] Thomas GR, Bull PC, Roberts EA, Walshe JM, Cox DW. Haplotype studies in Wilson disease. Am J Hum Genet 1994;54:71–8. [11] Loudianos G, Dessi V, Lovicu M, Angius A, Kanavakis E, Tzetis M, Kattamis C, Manolaki N, Vassiliki G, Karpathios T, Cao A, Pirastu M. Haplotype and mutation analysis in Greek patients with Wilson disease. Eur J Hum Genet 1998;6:487–91. [12] Nanji MS, Nguyen VTT, Kawasoe JH, Inui K, Endo F, Nakajima T, Anezaki T, Cox DW. Haplotype and mutation analysis in Japanese patients with Wilson disease. Am J Hum Genet 1997;60: 1424–9. [13] Firneisz G, Lakatos PL, Szalay F, Polli C, Glant TT, Ferenci P. Common mutations of ATP7B in Wilson disease patients from Hungary. Am J Hum Genet 2002;108:23–8. [14] Lovicu M, Dessi V, Zappu A, De Virgiliis S, Cao A, Loudianos G. Efficient strategy for molecular diagnosis of Wilson disease in the Sardinian population. Clin Chem 2003;49:496–8. [15] Loudianos G, Gitlin JD. Wilson’s diseases. Semin Liver Dis 2000;20: 353 –64.