Wilson disease novel mutations in the ATP7B gene and clinical correlation in Brazilian patients.

MUTATION IN BRIEFHUMAN MUTATION Mutation in Brief ##698 (2004) Online© 2004 WILEY-LISS, INC. DOI: 10.1002/humu.9227 Received 30 October 2003; accepted revised manuscript 28 January 2003.Wilson Disease: Novel Mutations in the ATP7B Gene and Clinical Correlation in Brazilian PatientsMarta M. Deguti 1, Janine Genschel 4, Eduardo L.R. Cancado 1, Egberto R. Barbosa 2, Bettina Bochow 4, Marcos Mucenic 1, Gilda Porta 3, Herbert Lochs 4, Flair J. Carrilho 1, and Hartmut H.-J. Schmidt 4*Departments of 1Gastroenterology, 2Neurology, and 3Pediatrics Hepatology of Sao Paulo University School of Medicine, Sao Paulo, Brazil; 4Med. Klinik Gastroenterologie, Hepatologie und Endokrinologie, Charité, Berlin, Germany*Correspondence to: PD Dr. H. Schmidt, Medizinische Klinik mit Schwerpunkt Gastroenterologie, Hepatologie und Endokrinologie, Campus Charité Mitte, Schumannstr. 20–21, 10117 Berlin, Germany; Tel.: +49 30 450 514 102; Fax: +49 30 450 514 916; E-mail: hartmut.schmidt@charite.deGrant sponsor: German Academic Exchange Service; Grant number: DAAD #A/01/17629Communicated by Peter ByersWilson disease (WD) is a rare inherited autosomal recessive disorder caused by a defect in a metal transporting P-type ATPase, resulting in copper overload in various tissues and cells. The aim was to assess both the phenotype in Brazilian WD patients and the corresponding ATP7B genotype. Sixty subjects belonging to 46 pedigrees diagnosed as WD were included in this study. Direct sequencing of all 21 exons within ATP7B and their flanking introns was performed. Demographic, clinical, laboratory and histopathological data at the time of diagnosis were obtained. We identified twenty-five mutations, twelve of them reported for the first time. The c.3402delC mutation had the highest allelic frequency (30.8%), followed by the c.2123T>C (p.L708P) (16.7%). Exons 8 and 15 were the site of 62.5% of the mutations. The common European mutation c.3207C>A (p.H1069Q) was not present at all. Phenotype varied greatly among individuals with the same ATP7B genotype. Our data confirm the heterogeneity of ATP7B genotype in Brazilian WD patients. The mutational spectrum is compatible with the Brazilian history of Mediterranean immigration; however, new mutations, and different frequencies and phenotype associated with the previously known mutations characterize this population. Exons 8 and 15 should be preferentially screened in WD cases from Brazil. Phenotype variation among subjects with the same ATP7B genotype suggests that modifying factors play an additional role in the pathogenesis of WD. © 2004 Wiley-Liss, Inc.KEY WORDS: ATP7B gene; Wilson disease; mutation; sequencing; cirrhosis; neurology; psychiatryINTRODUCTIONWilson disease (WD, MIM# 277900) is an autosomal recessive disorder of biliary excretion of copper, first described as hepatolenticular degeneration (Wilson, 1912). The excess copper accumulates in various tissues, mainly in the liver and in the brain, leading to clinical manifestations such as liver disease, hemolysis, renal alterations, and neuropsychiatric disease. Kayser-Fleischer (KF) rings in the periphery of the cornea and copper2 Deguti et al.abnormalities in serum and urinary tests are frequent. In addition, serum ceruloplasmin levels are usually decreased. Hepatic intracellular accumulation of copper may be identified by rhodanine staining (Scheinberg and Sternlieb, 1984), but any available staining procedures may not be necessarily positive in the presence of excess copper. Therefore, copper quantitation is more suitable. Prevalence worldwide at birth is estimated to be about 30 million, with demographic differences related to the consanguinity rate (Hoogenraad, 1996).The underlying genetic defect of WD has been identified within the ATP7B gene,mapped to chromosome 13q14.1 (Bull et al., 1993; Tanzi et al., 1993). This gene encodes a copper transporting ATPase of 1465 amino acids, with six copper-binding domains, eight transmembrane domains (Tm), a transduction domain converting the energy of ATP hydrolysis to cation transport, a Tm cation channel and a phosphorylation domain (Petrukhin et al., 1994). Up to the present time, more than 250 mutations have been identified within the ATP7B gene (/wilson/index.html). Disease rarity and the high number of mutations, often in the compound heterozygous state, make the correlation between genotype and phenotype difficult. Some mutations have been reported at higher frequencies: in Eastern Europe up to 38% of WD patients carry the substitution p.H1069Q in the homozygous state, which is much less frequent in the Mediterranean region and absent in Asia (Figus et al., 1995; Loudianos et al., 1996; Caca et al., 2001). In the island of Gran Canary, the very rare mutation p.L708P (c.2123T>C) is highly prevalent, while the p.R778L (c.2333G>T) is frequent in Asia but not found among Caucasians (Thomas et al., 1996; Garcia-Villarreal et al., 2000).So far, there are no data on WD genotype in South America. We genetically analyzed the ATP7B gene in 60 WD subjects derived from 46 unrelated Brazilian families and correlated the results with detailed clinical, laboratory and histopathological data. Our findings extend the current clinical and genetic information on WD, which add to the present understanding on its genotype/phenotype relationship.METHODSCohort of PatientsThe 46 index cases consisted of 30 males (65.2%) and 16 females (34.8%). Thirty-three were white (71.7%), 10 mixed black and white (21.7%), and 3 black (6.5%) subjects. Patients were admitted at the clinics of Gastroenterology, Neurology and Pediatric Hepatology of the Hospital das Clínicas, a reference center in Sao Paulo, Brazil. This research was approved by the local Ethics Committee and all participants gave informed written consent.Clinical dataA diagnosis of WD was established by a combination of clinical, biochemical, imaging and histochemical evaluations as described (Scheinberg and Sternlieb, 1984). Neurological, psychiatric and hepatic presentations were described.Mutational analysis of the ATP7B geneGenomic DNA was extracted from whole venous blood collected in EDTA using a salt precipitation method (Gustincich et al., 1991). The 21 exons of the WD gene were amplified in a thermal cycler (Biometra T3 Thermocycler, Göttingen, Germany) as described elsewhere (Waldenström et al, 1996). Direct sequencing of the amplified PCR products was performed by cycle sequencing using fluorescent dye terminators in an automatic sequencer (Applied Biosystems, Darmstadt, Germany).Mutations were quoted according to the guidelines from /mutnomen/, using the reference sequence with the GenBank accession number NM_00053.1. The nucleotide +1 ist the A of the ATG-translation initiation codon. The ATG-translation initiation codon is also the first codon.RFLP analysis and Allele-specific PCRThe novel mutations modifying endonuclease restriction sites were studied by specific RFLP analysis. The novel mutations that did not modify any known restriction sites were analyzed by allele-specific PCR (Table 1B and C). To assess the possibility that the mutations p.N41S (c.122A>G), p.V949G (c.2846T>G), p.F1094L(c.3282C>G), p.T1232P (c.3694A>C) and p.L1373R (c.4118T>G) were polymorphisms, they were also tested in60 healthy control individuals of the Brazilian population.Wilson Disease in Brazil 3RT-PCR analysisWhole blood from two siblings homozygous for c.51+4A>T (IVS1+4A>T) and two patients homozygous for c.1543+1G>C (IVS3+1G>C) was collected in PaxGene Blood RNA tubes (Qiagen, Hilden, Germany), in order to assess if the splice sites found altered the predicted splicing. mRNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and the corresponding cDNA was obtained using the Omniscript Reverse Transcriptase Kit (Qiagen, Hilden, Germany). PCRs of the regions spanning exon 1 to exon 2 and exon 3 to exon 5 were performed using the primers forward 5´gat gcc tga gca gga gag ac3´ and reverse 5´ccc aag atc ctg act gtg ct3´, and forward 5´tgt gca tcc tgt gtg tct aa3´ and reverse 5´cat ttg tcc tcg tga gtt tg3´, respectively. The PCR products obtained were sequenced (Applied Biosystems, Darmstadt, Germany).Haplotype analysisHaplotype analysis was performed in six cases found to be homozygous for p.L708P and in three subjects with the same genotype from the Canarian series to determine a potential founder effect (Garcia-Villarreal et al., 2000). We chose the markers spanning a region of approximately 2 cM around the ATP7B locus, which were reported to have the most significant linkage disequilibrium with the p.L708P: D13S270, D13S284 and D13S153 (Garcia-Villarreal et al., 2000). According to the Ensemble database (), these markers are mapped to 47.3 Mb, 45.7 Mb and 42.8 Mb, respectively, flanking the ATP7B gene, which is at 46.5 Mb. Allele size was determined semi-automatically using the ABI Prism 310 automatic sequencer and the GeneScan Software (Applied Biosystems, Darmstadt, Germany).RESULTSTwenty-five distinct mutations were detected in this cohort of 60 WD patients derived from 46 unrelated Brazilian families. According to the literature and the "Wilson disease mutation database" (/wilson/index.html), the mutations p.N41S (c.122A>G), c.1436delC, c.2296dupA, c.2438_2440delTAAinsAT, p.S932X (c.2795C>A), p.V949G (c.2846T>G), p.L1088X (c.3263T>A), p.F1094L (c.3282C>G), p.T1232P (c.3694A>C), p.K1258X (c.3772A>T), p.L1373R (c.4118T>G) and c.1543+1G>C (IVS3+1G>C) are being reported for the first time affecting various protein domains of ATPase 7B (Table 1A). These novel mutations were confirmed by RFLP analysis and allele-specific PCR, respectively (Table 1B and 1C). None of the missense mutations were detected among 120 alleles of a Brazilian control population. The alteration c.1543+1G>C (IVS3+1G>C) was confirmed to be a mutation, as the RT-PCR resulted in an alternatively spliced product (Figure 1). In contrast, the potential splice mutation c.51+4A>T (IVS1+4A>T) did not result in any aberrant product (data not shown).Table 1A. Characteristics of Novel ATP7B MutationsExon Nucleotide change Predictedchange Type Theoretically affectedProtein Domain1 c.122A>G p.N41S Substitution Cu13 c.1436delC p.P478fs* Deletion Cu4-5*8 c.2296dupA p.T766fs* Insertion Tm3*9 c.2438_2440delTAAinsAT p.L813fs* Deletion and Insertion Tm4*12 c.2795C>A p.S932X* Stop codon Tm5*12 c.2846T>G p.V949G Substitution Tm515 c.3263T>A p.L1088X* Stop codon ATP loop*15 c.3282C>G p.F1094L Substitution ATP loop17 c.3694A>C p.T1232P Substitution ATP binding domain18 c.3772A>T p.K1258X* Stop codon ATP hinge*20 c.4118T>G p.L1373R Substitution Tm8c.1543+1G>C (IVS3+1G/C) p.E515fs* Splice Cu5-6**mutation may result in rapid mRNA degradation, therefore the corresponding predicted amino acid change and affected protein domain is theoretically presented.4 Deguti et al.Table 1B: RFLP Analysis to Confirm Novel ATP7B MutationsMutationRestriction Endonuclease Endonuclease Specificity Size of Fragments (bp) c.122A>GTspI mutation 139, 168, 95 c.2438_2440delTAAinsAT MseI wild type 177, 88 c.2795C>A HindII wild type 223, 135c.3282C>G Cac8I mutation 108, 106, 64, 50, 30 c.3263T>A BfaI mutation 208, 150 c.3772A>T MseI mutation 220, 146 c.4118T>GHhaImutation129, 124Table 1C: Allele-specific PCR to Confirm Novel ATP7B MutationsMutation Primer Specificity Primersc.2296dupA mutation 5‘-ctg tga cat tct tcg aca acg c-3‘ 5‘-tgg gat ttc aga agt agt gac caa-3‘wild type 5‘-ctg tga cat tct tcg aca cgc-3‘ c.2846T>G mutation 5‘-cgg ttt tat cga ttt ttg gtg gt-3’ 5‘-cct gca gaa gga gag tga ctg-3'wild type 5‘-cgg ttt tat cga ttt ttg gtg tt-3‘ c.3694A>Cmutation5‘-ggg gag cca ctg cga aga tt-3‘ 5‘-aag ggc tgt acc tgg ggg g-3’ wild type5‘-aag ggc tgt acc tgg gtg gc-3‘GenBank accession number: NM_00053.1.-500-404-331-242-190-147-111-67277 bp-383 bp-WTc.1543+1G>C (IVS3+1G>C)c.1543+1G>C (IVS3+1G>C)--- AAA GAA GCT G c t aag aga tga --- Lys Glu Ala Ala Lys Arg Stop--- AAA GAA GCT GAA AGC TGT ------ Lys Glu Ala Glu Arg Cys ---Ex33 new amino acids, stopWTc.1543+1G>C (IVS3+1G>C)DNA mRNA ProteinEx3Ex4ABFigure 1: A: Schematic representation of the altered splicing caused by the c.1543+1G>C (IVS3+1G>C) mutation. The mutation results in an aberrant spliced mRNA with additional 106 bases derived from the intronic sequence. The predicted protein sequence contains the first 514 amino acids coded by exon 1-3, followed by three additional amino acids and a stop codon. B: RT-PCR analysis confirming the splice mutation c.1543+1G>C (IVS3+1 G/C). For the mutation a splice product of about 380 bp was identified in the region spanning exon 3 to exon 5 (normal 277 bp). Sequence analysis revealed a fragment of 383 bp including an additional 106 bp fragment derived from intron 3.Wilson Disease in Brazil 5Among the 60 patients studied, age at diagnosis ranged from 9 to 35 years old, with an average of 17 years of age. Table 2 summarizes the various identified genotypes and the corresponding mean age at disease onset and at diagnosis plus the involved organ liver, brain and kidney, respectively. Due to the mutational heterogeneity a genotype/phenotype correlation in this cohort is very difficult to obtain. Among the 46 index cases, 22 (47.8%) had at least one relative also affected. A disease-causative mutation in all studied subjects was detected in 81.7% (49/60) on both alleles; in 16.7% (10/60) of the patients we identified a mutation only on one allele. In one patient we were not able to detect any mutation at all. The promotor region was not examined.The frameshift c.3402delC (exon 15), identified in 24 distinct pedigrees, was the most common mutation, with an allelic frequency of 34.8%. It has been previously reported as a common mutation in patients from Russia, North America, Sweden and East Germany (Waldenström et al., 1996; Shah et al., 1997; Caca et a., 2001). Patients with genotype homozygous for c.3402delC had disease onset varying between 13 and 20 years of age, except for a non-index that was asymptomatic at the age of 31 years. In this group, six index cases had severe neurological disease and hepatic involvement, and two had decompensated liver disease without neurological symptoms.Table 2. ATP7B Genotype and Clinical Presentation of WD Patients (n=60)Main affectedorganGenotype index / non-index (n) Mean age at disease onset / at diagnosis (years)a Liver (n) Brain(n)Organs involved*[c.3402delC]+[c.3402delC] 8 , 1 16 , 18 2 6 L, B, A [c.3402delC]+[c.2304dupC] 1 , 0 9 , 10 1 0 L [c.3402delC]+[-]3 , 0 12 , 14 1 2 L, B [c.3402delC]+[p.P1273L] 1 , 2 34 , 35 0 1 L, B [c.3402delC]+[p.N41S] 1 , 0 24 , 24 1 0 L [c.3402delC]+[c.1436delC] 1 , 0 24 , 24 1 0 L, B [c.3402delC]+[p.G1061E] 1 , 0 9 , 10 1 0 L, B [c.3402delC]+[p.L1088X] 1 , 0 16 , 16 0 1 L, B [c.3402delC]+[p.R1319X] 1 , 0 9 , 16 1 0 L, B [c.3402delC]+[p.R1041W]1 , 0 30 , 31 0 1 L, B [c.3402delC]+[c.3713_14delAA]1 , 1 19 , 19 0 1 L, B [c.3402delC]+[c.2438_2440delTAAinsAT] 1 , 0 9 , 9 0 1 L, B [c.3402Del C]+[p.L708P] 3 , 0 10 , 13 12 L, B [p.L708P]+[p.L708P] 4 ,3 15 , 16 2 2 L, B, K [p.L708P]+[p.M645R] 1 , 0 23 , 23 0 1 L, B [p.L708P]+[p.S932X] 1 , 1 18 , 22 0 1 L, B [p.S932X]+[p.S932X] 1 , 0 24 , 24 1 0 L [p.S932X]+[p.P1273L] 1 , 0 20 , 21 1 0 L, B [c.2304dupC]+[p.V949G] 1 , 0 26 , 29 0 1 L, B [c.2304dupC]+[-]1 , 0 10 , 10 1 0 L, B [p.T1232P]+[p.G1061E] 1 , 0 14 , 16 0 1 L, B [p.T1232P]+[c.3449delA] 1 , 0 15 , 15 0 1 L, B [c.2296dupA]+[c.2296dupA] 1 , 3 A , 12 1 0 L [p.F1094L]+[p.F1094L] 1 , 0 25 , 27 0 1 L, B [p.F1094L]+[-]0 , 2 A , 11 0 0 L, K [p.D765N]+[p.D765N]1 , 0 25 , 26 0 1 L, B [c.3713_14delAA]+[p.N1270S] 1 , 0 19 , 19 1 0 L [p.R1319X]+[-] 1 , 0 16 , 17 0 1 L, B [p.K1258X]+[-] 1 , 0 A , 14 1 0 L [p.L1373 R]+[-] 1 , 0 19 , 21 0 1 L, B [p.S921N]+[-]1 , 0 A , 11 1 0 L [c.1543+1G>C]+[c.1543+1G>C] 1 , 1 14 , 14 0 1 L, B [-]+[-]1 , 0 15 , 15 1 0 Laconsidering only symptomatic index case; *A: asymptomatic; L: liver, B: brain, K: kidney. GenBank accession number: NM_00053.1.6 Deguti et al.The second most frequent mutation was p.L708P (exon 8) (14.1%), affecting 9 distinct pedigrees. It had been considered almost exclusively to Gran Canary, which might be the place of its origin, from one unique mutant ancestor (Garcia-Villarreal et al., 2000). All index cases homozygous for p.L708P presented with liver disease of varying severity; two of them had neurological involvement and nephrocalcinosis. All patients with nephrocalcinosis (n=7) at the time of diagnosis had the mutation p.L708P in at least one of the alleles; however, the sample size does no allow us to speculate on a cause-effect relationship between these findings (Hoppe et al., 1993; Sözeri et al., 1993; Garcia-Villarreal et al., 2000). The results of the haplotype analysis in patients homozygous for p.L708P are depicted in Table 3. For D13S153, the allele sizes of haplotypes B, C and D corresponded to 216, 220 and 222 bp, respectively; for D13S284, B and G were 204 and 216 bp; and for D13S270, B and D corresponded to 85 and 91 bp. As shown the siblings (BR1.1-1.3) had the same haplotype as the Canarian cases (GC1.1-3.1) for D13S153, D13S284 and D13S270. Patients 2.1 and 4.1 had each only one of the three markers in common with the Canarian cases, whereas patient 4.1 had two markers in common. All Brazilian cases carrying p.L708P were from cities located in Minas Gerais or Bahia State.Table 3. Haplotype Analysis of Patients p.L708P +/+ from Brazil and Gran Canary, Spain Patient D13S270 D13S284 D13S153GC1.1 D D B B D DGC2.1 D D B B D DGC3.1 D D B B D DBR1.1 D D B B D DBR1.2 D D B B D DBR1.3 D D B B D DBR2.1 B B B B B BBR3.1 D D B B B BBR4.1 D D G G B CNotes: patients are identified by origin - Gran Canary (GC) or Brazil (BR), number of pedigree and number of family member.The mutation c.2304dupC was identified in three heterozygous white subjects of our cohort. This mutation is not uncommon in various different populations (Figus et al., 1995; Shah et al.,1997; Riordan and Williams, 2001). Interestingly, the patient with the genotype [c.3402delC]+[c.2304dupC] had moderately strong Perls' staining within the liver before initiating therapy and without any additional evidence of hemochromatosis; he responded well to penicillamine, but developed elastosis perforans serpiginosa eight years later (Deguti et al., 2002).The splicing defect c.1543+1G>C (IVS3+1 G>C) was identified in two homozygous first cousins with history of consanguinity. Both manifested severe neurological disease with dysarthria, dysphagia and tremors followed by acute hepatitis between the ages of 13 and 15 years.DISCUSSIONThis study extends the mutational analysis of ATP7B to South America. The direct sequencing of the gene yielded a detection rate of 90.0% (108/120) of disease-causative mutations. Both c.3402delC and p.L708P together represented 47.5%, about half of the mutations identified in this cohort. The c.3402delC was shown to be the most common mutation among Brazilian patients, with an allelic frequency of 30.8%. The highest frequency reported for this mutation so far was 19% among a Russian studied cohort (Riordan and Williams, 2001). Interestingly, this mutation was not described in the Mediterranean region, which is the main origin of the immigrants that colonized Brazil (Figus et al., 1995; Loudianos et al., 1996). On the other hand, the absence of the mutation p.H1069Q, frequent (10-63%) in populations of U.S. and Central Europe, is consistent with the fact that immigrations from these areas were low (Shah et al., 1997; Caca et al., 2001). The ethnic background of patients could not be investigated in detail, because the Brazilian population is highly multiracial mixed.Wilson Disease in Brazil 7 The results of haplotype analysis of the homozygous p.L708P subjects do not convincingly support the hypothesis of the same founder for both Brazilian and Canarian cases. The Canary islands were on the route of the European going to South America during the 16th to 19th centuries.WD presentation varied greatly among patients carrying the same homozygous mutation. The pedigree with the frameshift c.2296dupA illustrates this point, as one individual presented with acute liver failure at childhood while the other with decompensated liver disease of late onset. Furthermore, the predicted function of a mutant protein did not often correspond to the expected clinical phenotype. The substitution mutation p.D765N was demonstrated not to interfere with the copper-transport function in vitro; nevertheless, our patient with genotype [p.D765N]+[p.D765N] had severe neurological and hepatic symptoms (Forbes and Cox, 1998). In contrast, the case [p.S932X]+[p.S932X] had late disease onset, liver cirrhosis, severe coagulopathy, without brain involvement and was highly responsive to penicillamine. Other authors have reported another nonsense mutation in the same exon, p.W779X; and their patient had also moderate neurological disease of late onset, but displayed rapid deterioration after penicillamine treatment (Waldenström et al., 1996). It would be expected that stop codon mutations in exon 12 could disrupt the ATPase domain, leading to complete protein dysfunction, resulting in an extremely severe disease. The most likely explanation for these relatively benign clinical presentations is the alternative splicing involving exons 6-8 and 12-13 (Petrukhin et al., 1996).Figure 2: The ATP7B gene is schematically shown consisting of 21 exons. The six copper binding domains (Cu), transmembrane domains (Tm), a transduction domain converting the energy of ATP hydrolysis to cation transport (Td), the transmembrane cation channel/phosporylation domain (Ch Ph), and the ATP binding domain (ATP hinge) are illustrated. All here reported mutations in Brazilian WD subjects are visualized. Newly described mutations are indicated in red.Similarly, the substitution of leucine by proline in codon 708 would be expected to result in a misfolded ATPase, because proline is a helix breaker for the secondary structure of the protein. Nevertheless, the clinical picture of the homozygous patients varied greatly, and, despite two siblings with neurological sequelae, all of them responded well to penicillamine treatment. .As the phenotype varies considerably for the same genotype, environmental factors and/or additional genetic and epigenetic aspects might be involved in the pathogenesis of WD. A putative modifying factor is the high oral intake of copper in Brazil. Bean (1304.6 µg/ 100 mg) and coffee (1433.2 µg/ 100 mg) consumption is very popular. Nevertheless, it is not possible to establish a relationship between disease severity and daily copper8 Deguti et al.ingestion, since detailed data are not available. Age of disease onset or disease severity seems also not to be significantly different from WD cohorts, originating from other countries.Mutations in the region of the six copper-binding domains (exons 1-6) represented < 3.0% (Figure 2). Scarcity of missense mutations in this region has been observed in the literature and the hypothesis of functional redundancy among the repeated motifs has been proposed to explain this phenomenon (Forbes and Cox, 1998). On the other hand, mutations within exons 8 and 15, corresponding to Tm 2-4 and ATP loop domains, harbored together 62.5% of all studied alleles.In conclusion, WD genotyping in Brazil revealed novel mutations and some peculiarities related to the allelic frequency and the associated phenotype. Almost two thirds of the mutations are located in exons 8 and 15. The ATP7B genotype alone does not determine WD manifestations. Therefore, modifying factors, which have to be determined, play an additional role in the timing of disease onset, clinical phenotype and maybe even in the response to treatment in Wilson disease.ACKNOWLEDGMENTSWe appreciate the participation of the investigated subjects and control group volunteers. We are thankful to Dr. RM Martin for expertise assistance and all physicians who referred patients: AQ Farias, M Dias, B Della-Guardia, JE Medeiros-Filho, MSC Santos, CM Tani, JM Oliveira and M Kallas. Special acknowledgements are given to Dr.A Tugores for sending DNA samples of the Canarian patients. Dr. MM Deguti was supported by a grant from the Deutsche Akademische Austauschdienst (DAAD# A/01/17629) and from Capes. This work was supported by a grant from the Humboldt University and the BMBF.REFERENCESBull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. 1993. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. 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