Identification of the Wilson's disease gene: Clues for disease pathogenesis and the potential for molecular diagnosis

HEPATOLOGY EZsewhere T. JAKE LIANG, EDITOR Gastrointestinal Unit

Massachusetts General Hospital 32 Fruit Street Boston. Massachusetts 02114

GRJ-719

Dear readers:

In the past, this section reviewed a potpourri of articles in each issue. We introduce in this issue a new feature of Hepatology Elsewhere. Whenever it is appro- priate, we will showcase articles with a common theme. Given the explosion of new medical information per- taining to hepatobiliary physiology and pathology, we believe that by encompassing a common theme and providing reviews on relevant articles in one issue, you will reap the most benefit from this section. The feature articles and their reviews in this issue provide a comprehensive discussion of a major medical break- through in the field of inherited metabolic liver diseases (i.e., the identification and characterization of the Wilson’s disease gene). We hope you will find this special feature enjoyable.

T. JAKE LUNG and Members of the Advisory Committee

IDENTIFICATION OF THE WILSON’S DISEASE GENE: CLUES FOR DISEASE PATHOGENESIS AND THE POTENTIAL FOR MOLECULAR DIAGNOSIS

Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nature Genet 1993;5:327-337.

ABSTRACT

Wilson disease (WD) is an autosomal recessive disorder of copper transport which maps to chromosome 13q14.3. In pursuit of the WD gene, we developed yeast artificial chromosome and cosmid contigs, and microsatellite markers which span the WD gene region. Linkage disequilibrium and haplotype analysis of 115 WD families confined the disease locus to a single marker interval. A candidate cDNA clone was mapped to this interval which, as shown in the accompanying paper, is very likely the WD gene. Our haplotype and mutation analyses predict that approximately half of all WD mutations will be rare in the American and Russian populations.

Petrukhin K, Fischer SG, Pirastu M, Tanzi RE,

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ADVISORY COMMITTEE M. SAWKAT ANWER, Boston, Massachusetts

BRUCE R. BACON, St. Louis, Missouri Henry C. BODENHEIMER, New York, New York

JAMES M. CRAWFORD, Boston, Massachusetts NORMAN D. GRACE, Boston, Massachusetts

SANJEEV GUPTA, Bronx, New York JOEL LAVINE, Boston, Massachusetts

Chernov I, Devoto M, Brzustowicz LM, Cayanis E, Vitale E, Russo J, Matseoane D, Boukhgalter B, Cao A, Sternlieb I, Evgrafov 0, Parano E, Pavone L, Warburton D, Ott J , Penchaszodeh GK, Scheinberg IH, Gilliam TC. Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nature Genet 1993;5:338-343.

ABSTRACT

Wilson disease (WD) is an autosomal recessive disorder characterized by the toxic accumulation of copper in a number of organs, particularly the liver and brain. As shown in the accompanying paper, linkage disequilibrium & haplotype analysis confirmed the disease locus to a single marker interval at 13q14.3. Here we describe a partial cDNA clone (pWD) which maps to this region and shows a particular 76% amino acid homology to the Menkes disease gene, Mcl. The predicted functional properties of the pWD gene to- gether with its strong homology to Mcl, genetic mapping data and identification of four independent disease-specific mutations, provide convincing evidence that pWD is the Wilson disease gene.

Tanzi RE, Petrukhin K, Chernov I , Pellequer JL, Wasco W, Ross B, Ramano DM, Parano E, Pavone L, Brzustowicz LM, Devoto M, Peppercorn J , Bush AI, Sternlieb I, Pirastu M, Gusella JF, Evgrafov 0, Pen- chaszadeh GK, Honig B, Edelman IS, Soares MB, Scheinberg IH, Gilliam TC. The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nature Genet 1993;5:344-350.

ABSTRACT

Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. The gene (WD) has been mapped to chromosome 13 q14.3. On yeast artificial chromosomes from this region we have identified a sequence, similar to that coding for the proposed copper binding regions of the putative ATPase gene (MNK) defective in Menkes disease. We show that this sequence forms part of a P-type ATPase gene (referred to here as Wcl) that is very similar to MNK, with six putative metal binding regions similar to those found in prokaryotic heavy metal transporters. The gene, expressed in liver and kidney, lies within a 300 kb region likely to include the WD locus. Two WD patients were found to be homozygous for a

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seven base deletion within the coding region of W C l . WC1 is proposed as the gene for WD.

Yamaguchi Y, Heiny ME, Gitlin JD. Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease. Biochem Biophys Res Commun 1993;197:271-277.

ABSTRACT

The putative copper and ATP-binding domains of the human Menkes disease gene were used as probes to screen a human liver cDNA library at reduced strin- gency. Sixty-five clones which remained positive after tertiary screening were subcloned and sequenced. One of these cDNA clones contains an open reading frame with 65% amino acid homology to the Menkes protein. Southern blot analysis localizes this cDNA to the region of the Wilson disease locus on chromosome 13. This cDNA detects a 7.5 kB transcipt which is present in human liver and cell lines devoid of the Menkes transcript and which is absent in liver from a patient with Wilson disease. These data suggest that this cDNA is a candidate gene for Wilson disease and that the protein encoded at this locus is a member of the P-type ATPase family.

COMMENTS

The study of inherited metabolic disorders has fre- quently led to better understanding of normal physiological function. New information about copper metabolism in human beings has emerged from recent molecular genetic studies of two unique inherited disorders of copper metabolism, Wilson’s and Menkes’ diseases.

The identification of the Wilson’s disease gene- recently accomplished almost simultaneously by labora- tories in New York, Toronto and St. Louis-follows by less than a decade the localization of this disorder to chromosome 13 by classic genetic linkage studies (1). Few would have predicted that the isolation of the gene for Menkes’ disease (2-41, a disorder manifesting signs and symptoms of copper deficiency, would play a significant role in the discovery of the pathogenesis of a disorder of copper toxicosis, Wilson’s disease. The information gained from these discoveries is reshaping our conception of the pathobiology of copper metabolism.

Led by Conrad Gilliam at Columbia University, the New York group utilized positional cloning and classic genetic linkage analysis to identify the location of the Wilson’s disease gene with microsatellites within a region of chromosome 13. The serendipitous isolation of a novel cDNA clone with metal binding regions from a brain cDNA library by Tanzi and his colleagues from Harvard led Gilliam’s group to directly determine that this cDNA was encoded from the region of chromosome 13 encompassed by the Wilson’s disease gene.

An entirely different approach toward the isolation of the Wilson’s disease gene was taken by the groups from Toronto and St. Louis. These groups began searching for

a gene with homology to the newly isolated Menkes’ disease gene, which encodes a putative copper-transport protein. The Toronto group screened both cosmid and cDNA libraries and reported on the complete cDNA sequence encoded by a gene homologous to that for Menkes’ disease. Confirmation that this was the Wil- son’s disease gene was made by means of identification of a specific deletion of a portion of the gene in two patients with Wilson’s disease. Also utilizing the Menkes’ disease gene product as a probe, the St. Louis group screened a liver cDNA library and obtained a homologous partial cDNA clone. They postulated that this clone encoded part of the Wilson’s disease gene, on the basis of Northern-blot analysis indicating reduced expression of the gene product in liver samples from two patients with Wilson’s disease.

These genetic analyses indicate that the approximately 7.5-kb Wilson’s disease gene transcript encodes a transmembrane protein with a high degree of DNA and amino acid homology to the Menkes’ disease gene product (2-4). Both of these newly discovered genes encode membrane-spanning proteins that contain copper binding regions and P-type ATPase motifs bearing remarkable similarity to prokaryotic heavy metal transport proteins (5) and other calcium-transporting ATPases (6), respectively.

That the gene for Wilson’s disease encodes a copper- transport protein fits well with earlier physiological studies of copper metabolism in patients with Wilson’s disease (7) and in animal models with inherited hepatic copper toxicosis (8)’ suggesting reduced hepatic excretion of copper into bile as the basis for hepatic copper accumulation. These studies concluded that a mutation of a transport protein inhibits the entry of copper into biliary excretory pathways, perhaps by acting to reduce copper flux across lysosomal membranes or directly at the bile canaliculus. Consequently, genetic studies and physiological modeling of inherited disorders of copper metabolism converge on the concept of mutations of metal ion transporters as the basis for disease pathogenesis.

That the Wilson’s disease gene encodes a protein with 76% amino acid homology to that encoded by the Menkes’ disease gene appears surprising gven the distinctly disparate clinical manifestations of these two disorders. However, careful inspection of studies on copper metabolism in tissues and cells from patients with Menkes’ disease suggest this to be a disorder of ineffective intracellular transfer of copper. The more global effects of the Menkes’ disease gene on copper metabolism in almost all tissues of the body and the confinement of the effects of Wilson’s disease mostly to the liver and brain is consistent with evidence that the mRNA for the Menkes’ disease gene is poorly expressed in liver but widely expressed in other tissues (2, 3), whereas the mRNA for the Wilson’s disease gene is highly expressed in liver but exhibits only limited expression in other tissues. Indeed, liver transplantation cures Wilson’s disease (9) but not Menkes’ disease.

Important information with respect to the future of

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molecular diagnosis of this disorder was provided by the study by Petrukhin et al. In their analysis of 115 patients, the two most frequent mutations (a point mutation resulting in a C- to A-transversion and a frame-shift mutation) were present in 50% of patients from Russia and in 30% of American patients. The remaining patients contained different mutations scat- tered across the gene, reminiscent of the reports on the numerous mutations of the gene for cystic fibrosis (10). Because Wilson’s disease is inherited in a recessive fashion, the predicted frequency of patients homozygous for the single most common mutation (those possessing two alleles with the same mutation) would be in most cases far less than the 25% predicted for the Russian group. Most patients would therefore have alleles with two different mutations of the Wilson’s disease gene.

The presence of multiple mutations of the Wilson’s disease gene and the necessity of searching for two mutant alleles to establish the diagnosis make DNA analysis for the individual patient difficult at best, given our limited knowledge of disease-specific mutations and present technological limitations. Despite these ob- stacles, these studies do demonstrate that accurate molecular diagnosis for family members of an affected patient can be accomplished by means of haplotype analysis. Using physically mapped microsatellite markers, Petrukhin et al. produced a set of high- resolution haplotypes across the Wilson’s disease gene locus. Mutation analysis revealed that disease specific mutations were correlated closely with distinct haplotypes; therefore haplotype analysis could predict the presence of the disease in family members of affected individuals with nearly 100% accuracy, the only caveat being the potential for the rare occurrence of more than one mutation bearing identical haplotypes. The near- perfect accuracy of molecular diagnosis on haplotype analysis represents a significant improvement on previous restriction polymorphism analysis. The only limi- tation of this testing is the availability of DNA from the affected patient, which can be obtained from WBCs or extracted from stored tissues or biopsy samples if the patient is no longer living or available for testing.

Despite these advances in molecular genetics, the initial diagnosis of Wilson’s disease still depends on careful slit-lamp examination for Kayser-Fleischer rings and measurement of serum ceruloplasmin and hepatic copper determinations. It should be noted that if family members of a patient have been screened over time for these clinical and biochemical parameters and found to lack Kayser-Fleischer rings and have normal levels of serum ceruloplasmin and parameters of liver function, it is extremely unlikely that they have Wilson’s disease.

Because therapy for Wilson’s disease is lifelong, and the consequences of its disruption potentially fatal, it is important to resolve the diagnostic problem that occa- sionally arises in family members of patients with Wilson’s disease who have decreased levels of serum ceruloplasmin. Genetic testing represents an alternAve means to liver biopsy and radiocopper anuysis for determining whether these individuals are hG.llozygous

for the Wilson’s disease gene and require treatment or are merely asymptomatic carriers. Certainly this diagnostic mode should now be utilized in patients when the disease diagnosis is in doubt and, arguably, for all siblings and offspring of newly identified patients.

Because genetic testing can identify individuals het- erozygous for the Wilson’s disease gene, it is important that these individuals be made aware that they will not suffer any of the pathological effects of the disease. Their identification becomes a matter of interest for genetic counseling when the other partner is known to be a carrier or homozygous for the Wilson’s disease gene. In this instance, partners could be made aware of the odds of producing a normal or affected child before conception takes place.

The ability to perform genetic testing for Wilson’s disease raises other important medical and ethical questions. Although the limitations described above preclude the widespread use of genetic testing as a cost-effective means of screening for Wilson’s disease, there remains the question of how early in life genetic testing should be performed for family members of affected patients and for pregnant patients with Wil- son’s disease. Biochemical parameters have allowed detection of the disease in patients as young as 6 mo, and genetic testing could theoretically be performed from intrauterine samples obtained by chorionic villus sampling or from amniocentesis. Because current treatment for Wilson’s disease is simple and effective, it is this author’s view that the presence of this gene in utero should not be utilized as the sole criterion for termi- nation of pregnancy. Furthermore, the slim possibility of obtaining maternal DNA by means of these methods of intrauterine sampling suggests that DNA testing from samples obtained after birth should be preferred.

The timing of the initiation of treatment, whether with penicillamine or with other agents, may be moved to very early ages with the advent of genetic testing. A recent retrospective survey of the outcome of treatment for Wilson’s disease in 32 asymptomatic children suggests that safe and effective long-term treatment was achieved by penicillamine prophylaxis beginning as early as 1.5 yr of age (11). These results suggest that treatment, whether with penicillamine or with alter- native agents, should probably begin by at least 3 yr of age. There are no existing data for the efficacy and safety of treatment begun at earlier ages.

The discovery that Wilson’s disease is the result of a mutation of a copper-transport protein helps to explain the characteristic reduction of serum ceruloplasmin activity observed in more than 90% of patients with Wilson’s disease. Recent physiological studies from our laboratory utilizing an animal model of Wilson’s disease, the Long-Evans Cinnamon rat, suggest that the effect of the single gene mutation leading to hepatic copper accumulation may be pleomorphic- that is, it may alter copper transport at cellular sites other than those involved in the lysosomal-biliary excretory pathway (12). From these studies we postulated that the transport of copper into the pathway for ceruloplasmin biosynthesis

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was reduced by the LEC mutation and that the same alteration occurs in Wilson’s disease. Recent studies in our laboratory indicating the presence of a rat homolog to the human Wilson’s disease gene product (Schilsky ML, Unpublished observation) provide further support for extending our observations on this animal model to human beings.

How, then, do we account for the normal levels of ceruloplasmin in a minority of patients with Wilson’s disease? Akin to the varied functional effects on chloride ion transport of some of the differing mutations of the cystic fibrosis gene (lo), it is possible that the variety of mutations of the Wilson’s disease gene alter copper transport to different degrees and, possibly, at different cellular sites. Hence we speculate that certain mutations of the Wilson’s disease gene could allow normal transport of copper for ceruloplasmin synthesis but reduce transport into excretory pathways. The varied effects of other mutations could account for the observed heterogeneity of clinical and biochemical manifestations among patients with this disorder.

We offer a similar hypothesis for the reduced levels of circulating ceruioplasmin manifested by patients with hereditary hypoceruloplasminemia (13). Here the mutation in these patients could inhibit normal copper transfer into the biosynthetic pathways for ceruloplasmin, resulting in reduced copper incorporatior_ and decreased circulating levels of this protein. The normal hepatic copper contents found in liver biopsy samples from these patients suggest adequate hepatic copper transport into excretory pathways. Similarly, the reduced serum ceruloplasmin levels in newborns could also be the consequence of delayed expression or function of the Wilson’s disease gene product. The proof of these postulates awaits studies of the expression and subcellular localization of the Wilson’s disease protein, characterization of its copper transport function, and further genetic and biochemical analyses of patients with hereditary hypoceruloplasminemia.

It is generally accepted that the incidence of HCC in patients with Wilson’s disease is not higher than that for the general population. Studies on the LEC rat indicate that long-term exposure to elevated hepatic copper contents leads to preneoplastic liver lesions and ultimately to HCC in aged animals (8). The initial hepatocellular injury and the late occurrence of cellular transformation in LEC rats have been reported to be halted by the administration of penicillamine (14). Because it is the rare patient with Wilson’s disease who survives to old age without treatment, it is likely that the rarity of HCC among patients with Wilson’s disease is the result of treatment. A recent occurrence of HCC in a patient poorly compliant with pharmacotherapy (Sternlieb I, Personal communication) supports this contention.

The discovery of the gene for Wilson’s disease is the first requisite for the future treatment of this disorder by gene therapy. Gene therapy for Wilson’s disease will likely need to overcome problems not faced by other

diseases in which the replacement of enzymatic function or production of secretory factors by only a fraction of cells may be adequate to ameliorate the disease process. Because all hepatocytes in a liver are affected by mutation of the Wilson’s disease gene, each will accumulate copper and ultimately suffer toxic injury, death or possible cellular transformation. It is therefore unlikely that the replacement of the normal gene in only a fraction of the liver cells will effect a cure of the disease. It is, however, possible that the restoration of this gene’s function in some of the cells could alter the pathogenesis of the disease by halting the cycle of hepatic injury and the extrahepatic de- position of copper. This may occur because the copper that reenters the bloodstream from metal-laden hepatocytes after their injury after a critical phase of copper accumulation could be efficiently transferred into bile by the genetically altered cells. Unfortunately, this would not alleviate the potential carcinogenic action of copper in other untreated cells or a possible fibrogenic response after hepatocellular injury. The development of techniques for the efficient transfer of genes into 100% of hepatocytes in a liver are probably necessary for effecting a molecular cure for this disorder.

The discovery of the Wilson’s disease gene represents a significant step toward better understanding of copper metabolism in human beings. The immediate applied benefit of the gene identification is the ability to perform accurate genetic diagnosis for family members of identified patients. The long-term goal of genetic therapy for Wilson’s disease awaits the improvement of technology for this mode of treatment.

MICHAEL L. SCHILSKY, M.D. Marion Bessin Liver Research Center Albert Einstein College of Medicine Bronx, New York 10461

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REFERENCES Frydman M, Bonne-Tammir B, Farrer LA, Conneally PM, Maga- zanik A, Ashbel S , Goldwitch Z. Assignment of the gene for Wilson disease to chromosome 13: linkage to the esterase D locus. Proc Natl Acad Sci U S A 1985;82:1819-1821. Vulpe C, Levinson B, Whitney S, Packman S, Gitschier J. Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nature Genet 1993;3:7-13. Chelly J, Tumer A, Tonnensen T, Petterson A, Ishikawa-Brush Y, Tommerup N, Horn N, et al. Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nat Genet 1993;3:14-19. Mercer JFB, Livingston J, Hall B, Paynter JA, Begy C, Chan- drasekharappa S, Lockart P, et al. Isolation of a partial candidate gene for Menkes disease by positional cloning. Nat Genet 1993;3:

Silver S, Nucifora G, Chu L, Misra TK. Bacterial resistance ATPases: primary pumps for exporting toxic cations and anions. Trends Biol Sci 1989;14:76-80. Jencks WP. On the mechanism of ATP-driven CA” transport by the calcium ATPase of sarcoplasmic reticulum. Ann N Y Acad Sci 1992;671:49-57. Sternlieb I, van den Hamer CJA, Morel1 AG, Alpert S, Gregoriadis G, Scheinberg IH. Lysosomal defect of hepatic copper excretion in Wilson’s disease (hepatolenticular degeneration). Gastroenter-

20-25.

010a 1973;64:99-105.

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8. Schilsky ML, Sternlieb I. Animal models of copper toxicosis. In: Cornelius C, ed. Advances in veterinary science and comparative medicine. Vol 37. San Diego: Academic Press, 1993:357-377.

9. Schilsky ML, Scheinberg IH, Sternlieb I. Liver transplantation for Wilson’s disease: indications and outcome. HEPATOLOGY 1994; 19:

10. Tizzano EF, Buchwald M. Recent advances in cystic fibrosis research. J Pediatr 1993;122:985-988.

11. Collins JC, Scheinberg IH, Sternlieb I. Penicillamine prophylaxis for asymptomatic children with Wilson’s disease [Abstract], HEPATOLOGY 1993; 18: 128A.

12. Schilsky ML, Stockert RJ, Sternlieb I. Pleiotropic effect of the LEC mutation: A rodent model of Wilson’s disease. Am J Physiol 1994;266:G907-G913.

13. Edwards CQ, Williams DM, Cartwright GE. Hereditary hypoceruloplasminemia. Clin Gen 1979;15:311-316,

14. Jong-Hon K, Togashi Y, Kasai €I, Hosokawa M, Takeichi N. Prevention of spontaneous hepatocellular carcinoma in Long- Evans Cinnamon rats with hereditary hepatitis by the administration of D-PeniCillamine. HEPATOLOGY 1993;18:614-620.

AT LONG LAST: AN ANIMAL MODEL OF WILSON’S DISEASE

Mori M, Hattori A, Sawaki M, Tsuzuki N, Sawada N, Oyamada M, Sugawara N, Enomoto K. The LEC rat: a model for human hepatitis, liver cancer and much more. Am J Pathol 1994;144:200-204.

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ABSTRACT

The LEC rat is an inbred mutant strain with spontaneous hepatitis isolated from Long-Evans rats. Since approximately 40% of LEC rats die of fulminant hepatitis, the rat serves an animal model for studying the pathogenesis and treatment of human fulminant hepatitis. The remaining 60% of LEC rats survive and develop chronic (prolonged) hepatitis and subse- quently develop liver cancer. Therefore, the LEC rat serves an important animal model for studying the significance of chronic hepatitis in the development of human liver cancer, which often develops in asso- ciation with chronic hepatitis. The LEC rat can also be used as an animal model of Wilson’s disease, since recent studies have disclosed high copper accumulation in the liver and low ceruloplasmin concen- tration in the serum of this mutant rat.

COMMENTS Wilson’s disease is a relatively uncommon (1 : 30,000)

but important autosomal recessive copper-storage disorder that is uniformly fatal without treatment. The Wilson’s disease gene was recently cloned and localized to chromosome 13q14.3 (1, 2). Because of defective biliary secretion of copper and hepatic synthesis of ceruloplasmin, hepatic accumulation of copper occurs during the first two decades of life in Wilson’s disease, inevitably progressing to acute or chronic hepatitis, cirrhosis or fulminant liver failure. After the hepatic storage capacity for copper is exceeded, copper is redistributed to other tissues, leading to central nervous system, renal and corneal involvement. Thus, before age 10 yr, 83% of patients appear with hepatic symptoms and only 17% have neuropsychiatric manifestations, between 10 and 20 yr of age approximately 50% of patients have hepatic symptoms and thereafter 75% have neuropsychiatric symptoms (3-5). Many patients

are completely asymptomatic when abnormal liver function test results lead to an investigation that yields the diagnosis, or if siblings of known patients are screened. Without therapy, progressive liver failure or severe neuropsychiatric problems eventually cause death (3). Therapy has been directed at reducing the burden of copper by chelating with D-penicillamine or trientine (31, possibly impairing copper absorption with oral zinc supplements (6) or inducing the synthesis of metallothionein to “detoxify” copper by these same agents (7). The critical role of the disturbed hepatic copper metabolism in producing the multisystem clinical abnormalities of Wilson’s disease is supported by the improved neurological function and prevention of neu- rologic disease in those who have undergone liver transplantation (8). Most of the therapeutic advances in Wilson’s disease are the result of carefully conducted clinical trials in afflicted patients with agents that would theoretically be of benefit. It has not been possible to test these agents adequately in an animal model for Wilson’s disease because none existed. In addition, the absence of such a model precluded the identification of the under- lying molecular and biochemical basis for Wilson’s disease, as well as the pathological processes that lead to cellular injury and fibrosis of the liver and other tissues.

The identification of the Long-Evans Cinnamon (LEO rat may have helped open the door to these types of investigations. The article by Mori et al. summarizes many of the recent advances in this unique inbred strain of rats. In the LEC rat (a mutant strain of Long-Evans rat), discovered in 1983 at the Center for Experimental Plants and Animals of Hokkaido University in Sapporo, Japan, hepatitis develops spontaneously at 3 to 4 mo of age, with an initial high mortality rate (40%) (9). Chronic hepatitis and cholangioproliferative fibrosis develop in survivors. Hyperplastic nodules with pleomorphic, polypoid nuclei are common by 1 yr of life, with a high incidence of HCC after 1 yr. It was not until 1991 that increased hepatic and brain copper concentrations were noted in the LEC rat, with concomitant low serum ceruloplasmin concentrations (10,111. Thus the LEC rat was characterized by marked hepatic copper accumulation in the first few months of life associated with very low circulating levels of ceruloplasmin, followed by acute (sometimes fulminant) hepatitis and the development of chronic liver disease and HCC in survivors. Other than the rarity of HCC in affected human patients, the LEC rat shares many important clinical, biochemical and histological features with human Wilson’s disease.

Previous putative models of Wilson’s disease have not satisfied all the basic features of the human disorder, although hepatic copper overload and liver injury were observed. For example, the Bedlington terrier may ex- hibit an autosomal recessive copper toxicosis causing cirrhosis and liver failure that responds to D-penicillamine and other copper chelators (12). Enormous hepatic copper concentrations cause liver damage that re- sembles that seen in Wilson’s disease. Biliary excretion of copper is impaired; however, plasma ceruloplasmin concentrations are normal. Therefore, compared with

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Identification of the Wilson's disease gene: Clues for disease pathogenesis and the potential for molecular diagnosis