Wilson Disease Gene Research Articles

Genotype–phenotype correlations for a wide spectrum of mutations in the Wilson disease gene (ATP7B)

Wilson disease (WND) is caused by mutations in the ATP7B gene and exhibits substantial allelic heterogeneity. In this study we report the results of molecular analyses of 20 WND families not described previously. When combined with our prior results, the cohort includes 93 index patients from 69 unrelated families. Twenty different mutations accounted for 86% of the WND chromosomes. The most frequent were p.H1069Q (35%), p.R969Q (12%), c.2530delA (7%), p.L936X (7%), p.Q289X (7%), and p.I1148T (3%). We also present here a detailed phenotypic assessment for patients whose molecular result was previously reported. Thirty cases were homozygous for 9 different mutations, 13 of which were homozygous for p.H1069Q, and 7 for p.R969Q. Mutations p.H1069Q and p.R969Q appeared to confer a milder disease as patients showed disease onset at a later age, and were associated with milder severity when found in trans with severe mutations. Predicted nonsense and frameshift mutations were associated with severe phenotypic expression with earlier disease onset and lower ceruloplasmin values. WND can be treated by copper-chelation therapy, particularly if the disease is diagnosed before irreversible tissue damage occurs. Our results on the effect of predicted nonsense and frameshift mutations are especially important for early medical intervention in presymptomatic infants and children with these genotypes.

Note: Identification of Three Novel Insertion/Deletion Mutations in Wilson Disease's Gene

Note: Identification of Three Novel Insertion/Deletion Mutations in Wilson Disease's Gene

Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. Since daily copper intake exceeds the body's requirements, effective means of excreting excess copper are essential. These are accomplished by ATP7B, a new member of the cation-transporting p-type ATPase family, which is mainly expressed in the liver and mediates both copper secretion into plasma (coupled with ceruloplasmin synthesis) and its excretion into bile. Thus far, more than 200 mutations of the WD gene have been detected, causing impairment of ATP7B function and, ultimately, copper accumulation. Excess copper, however, induces free-radical reactions and lipid peroxidation. Resultant liver damage leads to steatosis, inflammation, cirrhosis, and, occasionally, fulminant liver failure. The diagnosis of WD is commonly made on the basis of typical clinical and laboratory findings, including low serum ceruloplasmin, increased urinary copper excretion, and increased hepatic copper content. Since liver morphology is non-specific, and copper histochemistry may lead to both false-negative and false-positive results, the pathologist usually only suspects the disease or assists in its confirmation. Although the value of molecular genetic testing is limited due to the high number of possible gene mutations, polymerase chain reaction may be useful for the evaluation of family members of homozygous index patients.

Rapid detection of mutations in Wilson disease gene ATP7B by DNA strip technology.

Wilson disease leads to severe hepatic and neurological pathology resulting from cellular copper overload in the respective tissue. Although the affected gene, ATP7B, has been identified, genetic testing is challenging, time-consuming and expensive. Here we describe the development and use of a novel diagnostic test for four frequent mutations (M769V, W779X, H1069Q and P1134P-fs) found in Germany and many other countries in Europe. The test is based on multiplex polymerase chain reaction and DNA strip technology and was found to be highly sensitive and specific, as well as timely and cost-effective. We conclude that this test is a useful and reliable tool to screen Wilson disease patients and their family members for these mutations and may facilitate diagnosis in this complex disease.

Wilson disease

Wilson disease

Non-radioactive detection of five common microsatellite markers for ATP7B gene in Wilson disease patients

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.

Diagnosis and phenotypic classification of Wilson disease.

Wilson disease is an inherited autosomal recessive disorder of hepatic copper metabolism leading to copper accumulation in hepatocytes and in extrahepatic organs such as the brain and the cornea. Originally Wilson disease was described as a neurodegerative disorder associated with cirrhosis of the liver. Later, Wilson disease was observed in children and adolescents presenting with acute or chronic liver disease without any neurologic symptoms. While diagnosis of neurologic Wilson disease is straightforward, it may be quite difficult in non-neurologic cases. Up to now, no single diagnostic test can exclude or confirm Wilson disease with 100% certainty. In 1993, the gene responsible for Wilson disease was cloned and localized on chromosome 13q14.3 (MIM277900) (1, 2). The Wilson disease gene ATP7B encodes a P-type ATPase. More than 200 disease causing mutations of this gene have been described so far (3). Most of these mutations occur in single families, only a few are more frequent (like H1069Q, 3400delC and 2299insC in Caucasian (4-6) or R778L in Japanese (7), Chinese and Korean patients). Studies of phenotype-genotype relations are hampered by the lack of standard diagnostic criteria and phenotypic classifications. To overcome this problem, a working party discussed these problems in depth at the 8th International Meeting on Wilson disease and Menkes disease in Leipzig/Germany (April 16-18, 2001). After the meeting, a preliminary draft of a consensus report was mailed to all active participants and their comments were incorporated in the final text.

Genomic studies of gene expression: regulation of the Wilson disease gene

Bacterial artificial chromosomes (BACs) have many advantages over other large-insert cloning vectors and have been used for a variety of genetic applications, including the final contigs of the human genome. We describe the utilization of a BAC construct to study gene regulation in a tissue culture-based system, using a 170-kb clone containing the entire Wilson disease (WND) locus as a model. A second BAC construct that lacked a putative negatively regulating promoter sequence was created. A nonviral method of gene delivery was applied to transfect three human cell lines stably with each construct. Our results show correct WND gene expression from the recombinant locus and quantification revealed significantly increased expression from the clone lacking the negative regulator. Comparison with conventional methods confirms the reliability of the genomic approach for thorough examination of gene expression. This experimental system illustrates the potential of BAC clones in genomic gene expression studies, new gene therapy strategies, and validation of potential molecular targets for drug discovery.

Effects of soy protein isolate on LEC rats, a model of Wilson disease: mechanisms underlying enhancement of liver cell damage

Soy-protein isolate (SPI) enhances liver cell damage in Long-Evans rats with a cinnamon-like coat color (LEC rats), which have a defect in Atp7b, the Wilson disease gene. Animals administered an SPI-diet from an age of six weeks died significantly earlier than those administered a control-diet, AIN-93G, from severe liver cell damage associated with jaundice. Since the liver copper level was higher with the SPI-diet than the control-diet, one of the reasons for SPI-toxicity to LEC rats might be due to the higher uptake of copper into liver cells. In the present study, liver levels of glutathione, and liver and intestinal mRNA and protein levels were determined for metallothionein, MT-1 and MT-2. Furthermore, liver and intestinal mRNA expression for the high affinity copper transporter, Ctr1, was determined. None of the parameters showed any significant differences between the SPI-diet and control-diet groups, except for Ctr1 mRNA levels in the liver. It is thus suggested that SPI enhances liver cell copper uptake through induction of Ctr1 expression and this might be the mechanism underlying increased liver damage in LEC rats.

Defective cellular localization of mutant ATP7B in Wilson's disease patients and hepatoma cell lines

Background & Aims: Wilson's disease, a hereditary disorder caused by mutations in the Wilson's disease gene ( ATP7B), leads to hepatic and/or neurological pathology resulting from cellular copper overload. In vitro studies showed that ATP7B, located in the trans-Golgi network, traffics to a cytoplasmic vesicular compartment in response to increased copper concentration. Mislocalization and failed intracellular trafficking of ATP7B mutants are suggested to be among disease-causing mechanisms; however, the effect of mutations on ATP7B localization in human tissues has not been directly shown. Therefore, we characterized the subcellular localization of normal and mutant ATP7B in human livers and in hepatoma cell lines. Methods: Subcellular distribution of ATP7B in liver tissue from 3 control individuals and 3 Wilson's disease patients harboring a homozygous H1069Q-ATP7B mutation was analyzed by using immunogold electron microscopy. In addition, 14 ATP7B mutants tagged to green fluorescent protein were generated and expressed in HuH-7 and HepG2 cells; intracellular localization of these mutants was characterized by confocal microscopy. Results: In hepatocytes, ATP7B was localized in trans-Golgi vesicles, whereas H1069Q-ATP7B was trapped in the endoplasmic reticulum. Similar results were observed for wild-type ATP7B and H1069Q-ATP7B expressed in hepatoma cells. Most ATP7B proteins harboring missense mutations were distributed similarly to wild-type ATP7B. In contrast, truncated ATP7B mutants showed a diffuse, clustered, cytoplasmic pattern, distinct from the trans-Golgi network or endoplasmic reticulum. Conclusions: These results provide a detailed demonstration of the ATP7B distribution in control and diseased human livers and indicate that several Wilson's disease mutations lead to incorrect localization of ATP7B to distinct cell compartments.

Animal models of copper-associated liver disease

Recent advances in molecular biology have made possible the identification of genetic defects responsible for Wilson's disease, Indian childhood cirrhosis and copper toxicosis in Long Evans Cinnamon rats, toxic milk mice, and Bedlington terriers. The Wilson's disease gene is localized on human chromosome 13 and codes for ATP7B, a copper transporting P-type ATPase. A genetic defect similar to that of Wilson's disease occurs in Long Evans Cinnamon rats and toxic milk mice. Familial copper storage disorders in Bedlington and West Highland white terriers are associated with early subclinical disease, and copper accumulation with subsequent liver injury culminating in cirrhosis. The canine copper toxicosis locus in Bedlington terriers has been mapped to canine chromosome region CFA 10q26. Recently, a mutated MURR1 gene was discovered in Bedlington terriers affected with the disease. Idiopathic childhood cirrhosis is biochemically similar to copper toxicosis in Bedlington terriers, but clinically much more severe. Both conditions are characterized by the absence of neurologic damage and Kayser-Fleisher rings, and normal ceruloplasmin levels. A recent study added North Ronaldsay sheep to the list of promising animal models to study Indian childhood cirrhosis. Morphologic similarities between the two conditions include periportal to panlobular copper retention and liver changes varying from active hepatitis to panlobular pericellular fibrosis, and cirrhosis. Certain copper-associated disorders, such as chronic active hepatitis in Doberman pinschers and Skye terrier hepatitis are characterized by copper retention secondary to the underlying disease, thus resembling primary biliary cirrhosis in humans. Copper-associated liver disease has increasingly being recognized in Dalmatians. Copper-associated liver diseases in Dalmatians and Long Evans Cinnamom rats share many morphologic features. Fulminant hepatic failure in Dalmatians is characterized by high serum activities of alanine aminotransferase and aspartate aminotransferase, and severe necrosis of centrilobular areas (periacinar, zone 3) hepatocytes. Macrophages and surviving hepatocytes contain copper-positive material. Liver disease associated with periacinar copper accumulation has also been described in Siamese cats. Many questions regarding copper metabolism in mammals, genetic background, pathogenesis and treatment of copper-associated liver diseases remain to be answered. This review describes the similarities between the clinico-pathological features of spontaneous copper-associated diseases in humans and domestic animals.

Identification of novel mutations and the three most common mutations in the human ATP7B gene of Korean patients with Wilson disease

Purpose: Wilson disease, an autosomal recessive disorder of copper transport, is probably the most common inherited metabolic disorder in Korea. In Wilson disease, synthesis of a defective copper transporting enzyme leads to the accumulation of copper in the liver, brain, and kidney. The product of the Wilson disease gene is a copper transporting P-type ATPase (ATP7B). In this study, efforts were made to identify novel mutations and investigate the frequency of the common mutations in Korean patients with Wilson disease.Methods: This study includes 37 patients from 33 unrelated Korean families with Wilson disease. Genomic DNA from peripheral leukocytes or skin fibroblasts and cDNA from liver tissue were polymerase chain reaction—amplified exon by exon and subsequently analyzed using heteroduplex or single-strand conformation polymorphism analysis. Specimens showing mobility shift on those studies were directly sequenced.Results: Twelve different mutations in 33 Korean families with Wilson disease were identified: Arg778Leu (R778L), Asn1270Ser (N1270S), Ala874Val (A874V), 2303–2305delC, 2630–2656del, 2460–2462insC, Cys656Stop (C656X), Pro768His (P768H), Leu1083Phe (L1083F), Ala1168Ser (A1168S), Leu1255Ile (L1255I), and Asp1267Ala (D1267A). Among these, six mutations (2630–2656del, 2460–2462insC, C656X, P768H, A1168S, and L1255I) are novel. The R778L mutation is known to be highly prevalent in Asian patients. The allele frequency of R778L in Korean patients with Wilson disease was 37.9%, which was significantly higher than those of Japanese and Taiwanese patients. The N1270S mutation, originally described in an Italian patient, was the next most common mutation in Korean patients, with an allele frequency of 12.1%, which was presumed to disrupt the ATP hinge domain of the ATP7B protein. The A874V mutation was the third most common mutation with an allele frequency of 9.4%, which was presumed to disrupt the Td domain of the ATP7B protein.Conclusion: The R778L, N1270S, and A874V mutations are three major mutations representing approximately 60% of mutated alleles, although Korean patients with Wilson disease are genetically heterogeneous.

Abnormal mRNA splicing resulting from consensus sequence splicing mutations of ATP7B

More than 200 Wilson disease (WD) disease-causing mutations have been defined to date. Missense mutations are largely prevalent while splice-site mutations are limited in number. Most reside in the splice donor or acceptor sites and only a minority are detected in splicing consensus sequences. Furthermore, only a few splicing mutations have been studied at the RNA level to date. In this study, using the RT-PCR method we performed the molecular characterization of four consensus splice-site mutations identified by DNA analysis in patients with WD. One of them, previously described 1707+3insT, occurred at position 3 in the donor splice site of intron 4, while the other three, 2122-8T>G, 2866-6T>G, and 3061-12T>A, are novel and occurred in the acceptor splice sites of introns 7, 12, and 13, respectively. Analysis revealed a prevalently abnormal splicing in the samples carrying the mutations compared to the normal controls. Comparison of RNA splicing with normal controls in liver and lymphocytes further suggests that abnormal splicing of the WD gene is also present and differentially regulated in normal tissues. The results produced in this study strongly suggest that DNA mutations residing in the consensus sequence of WD gene splice sites result in the WD phenotype by interfering with the production of the normal WD protein. Further studies are necessary to better quantify the amount of different transcripts produced by these mutations, and establish their correlation with the disease phenotype.

Wilson's disease.

Wilson's disease (WD), an inborn error of copper (Cu) metabolism, is now one of the leading liver diseases in children in India. The clinical presentation can be extremely varied viz.,--all forms of acute and chronic liver disease, minimal to severe neurological disease, psychiatric problems, bony deformities, hemolytic anemia and endocrine manifestations. A high index of suspicion is necessary along with a judicious battery of investigations for diagnosis. Hepatic copper estimation is the most reliable test but is not easily available in India. Liver biopsy may not be possible because of bleeding problems and histological features are often not diagnostic of WD. In the absence of hepatic Cu, a low ceruloplasmin, high 24 hour urinary copper and presence of KF rings aid in making the diagnosis. The mainstay of initial therapy is Cu-chelators like D-Penicillamine, and Trientine for reduction in body copper to sub-toxic levels. Subsequent maintenance therapy is necessarily lifelong with D-Penicillamine, Trientine or Zinc. Children on therapy must be monitored regularly for response, side-effects, compliance and rehabilitation. Response to therapy may be unpredictable, but acute and early presentations like fulminant hepatic failures have a poor outcome. All siblings must be screened for WD as early diagnosis and treatment result in a good outcome. The identification of the WD gene on chromosome 13 has led to the possible use of molecular genetics (haplotype and mutational analyses) in the diagnosis of WD. Parent groups/associations must take active part in holistic management of WD.

Nuclear proteins that bind to metal response element a (MREa) in the Wilson disease gene promoter are Ku autoantigens and the Ku-80 subunit is necessary for basal transcription of the WD gene.

Wilson disease (WD), an inherited disorder affecting copper metabolism, is characterized by hepatic cirrhosis and neuronal degeneration, which result from toxic levels of copper that accumulate in the liver and brain, respectively. We reported previously that the approximately 1.3-kb promoter of the WD gene contains four metal response elements (MREs). Among the four MREs, MREa plays the most important role in the transcriptional activation of the WD promoter. Electrophoretic mobility shift assays (EMSAs) using synthetic MREa and an oligonucleotide containing the binding site for transcription factor Sp1 revealed the presence of nuclear factors that bind specifically to MREa. Two MREa-binding proteins of 70 and 82 kDa were purified using avidin-biotin affinity chromatography. Amino acid sequences of peptides from each protein were found to be highly homologous to the Ku proteins. Immunoblot analysis and EMSAs showed that the MREa-binding proteins are immunologically related to the Ku proteins. To study further the functional significance of these Ku-related proteins in transcriptional regulation of the WD gene, we performed RNA interference (RNAi) assays using a Ku-80 inverted-repeat gene to inhibit expression of the Ku-80 gene in vivo. Results of the RNAi assays showed that expression of the Ku-80 protein was suppressed in transfected cells, which in turn led to the suppression of the WD gene. In addition, a truncated Ku-80 (DeltaKu-80) mutant inhibited WD promoter activity in HepG2 cells in a dominant-negative manner. We also found that WD promoter activity was decreased in Xrs5 cells, which, unlike the CHO-K1 cells, are defective in the Ku-80 protein. When Ku-80 cDNA was transfected into Xrs5 and CHO cells, WD promoter activity was recovered only in Xrs5 cells. Taken together, our findings suggest that the Ku-80 subunit is required for constitutive expression of the WD gene.

Common mutations of ATP7B in Wilson disease patients from Hungary.

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. The H1069Q mutation in exon 14 of ATP7B is far the most frequent in Wilson patients of European origin. Mutations in exon 8 and 15 are also common among the over 150 described mutations in the WD gene. The aim was to investigate the frequency of these common WD gene mutations in Hungarian patients. A total of 42 patients with WD from 39 Hungarian families were examined. The H1069Q mutation was assessed by a seminested polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assay, while mutations in exons 8, 13, 15, and 18 of WD gene were identified by sequencing. In addition, haplotype analysis was performed using three common microsatellite markers (D13S314, D13S301, D13S316). The H1069Q mutation was found in 27 patients (64.3%). Nine patients were H1069Q homozygous. Eighteen patients were H1069Q compound heterozygous, two of them had H1069Q/P969Q and one patient H1069Q/3400delC genotype. In two of the 15 H1069Q-negative patients a novel mutation in exon 13 (T977M) was detected. One H1069Q-negative patient had a mutation in exon 8 (G710S). None of the studied mutations was detected in 12 WD patients. H1069Q-positive patients from various European countries had the same haplotype pattern. The H1069Q point mutation is frequent in Hungarian patients with WD and appears to have originated from a single founder in Eastern Europe. In contrast, mutations in exons 8, 13, 15, and 18 are uncommon in Hungarian WD patients.

Estimate of the frequency of Wilson's disease in the US Caucasian population: a mutation analysis approach.

The frequency of Wilson's disease in many populations is thought to be about one in 40000 persons, based on case and autopsy studies. Although the Wilson's disease gene has been identified, there is such a large number of mutations already known that it is not currently feasible to determine disease gene frequency by mutation analysis of a population. We have used a novel approach to obtain an estimate of the number of cases of Wilson's disease expected at birth in the US Caucasian population. We used data from four studies to determine that approximately one-third of Wilson's disease mutations in US Caucasian Wilson's disease patients are due to His-->Gln at the 1069 position. We then determined the frequency of this mutation in random DNA samples from 2601 US Caucasian newborns to be 0.285%. Multiplying by three gives an expected Wilson's disease heterozygote frequency of 0.855% and an allele frequency of 0.428%, or 0.00428. These data translate into a Wilson's disease frequency of about one in 55000 births. The 95% confidence interval is rather broad, ranging from about one in 18000 to one in 700000 births, but will be reduced as more data are added.

Identification of a high frequency of mutation at exon 8 of the ATP7B gene in a Chinese population with Wilson disease by fluorescent PCR.

Wilson disease (WD) is an autosomal recessive disorder of copper transport. Mutation analysis has led to the discovery of more than 100 mutations at ATP7B, and most of them are population specific. To verify the high frequency of mutation at exon 8 of ATP7B in Chinese patients with WD and to establish a DNA diagnostic method for WD. University medical centers. Screening for mutations at exon 8 of ATP7B by fluorescent polymerase chain reaction analysis and restriction analysis was conducted in 106 unrelated Chinese patients with WD and in 55 individuals from 10 Chinese families with WD. Five homozygotes and 32 heterozygotes were identified. Sequence analysis showed a missense mutation (2273G-->T) and a nonsense mutation (2250C-->G) together at exon 8. The rate of gene mutation in 106 patients was 35% (5% homozygous and 30% heterozygous). Samples of DNA from 55 individuals from 10 Chinese families with WD were examined by fluorescent polymerase chain reaction. We found that 13 siblings were carriers (24%). A high frequency of mutation at exon 8 of the ATP7B gene exists in the Chinese population, and fluorescent polymerase chain reaction analysis may be an effective and accurate assay in detection of the WD gene.

Mutation analysis and the correlation between genotype and phenotype of Arg778Leu mutation in chinese patients with Wilson disease.

The defective gene (ATP7B) that causes Wilson disease (WD) codes for a putative copper-transporting P-type adenosine triphosphatase. After cloning of ATP7B, the spectrum of mutations and their clinical consequences have been investigated in patients with WD in different ethnic populations. However, the spectrum of mutations and the correlation of genotype-phenotype in the Chinese population have not been extensively studied. To investigate the characterization of mutations of ATP7B and the correlation between genotype and phenotype in the Chinese population. We studied 60 unrelated healthy Chinese and 65 unrelated Chinese families, including 84 patients with WD and 126 parents. Genomic DNA was prepared from peripheral blood leukocytes using a salt-precipitation method. Polymerase chain reaction single-strand conformation polymorphism and subsequent direct sequencing were used to identify the mutations and polymorphisms of ATP7B. Statistical analysis was performed using t test or chi(2) test. We identified 18 mutations (7 novel) and 11 polymorphisms (3 novel). The novel mutations are -36C-->T, Trp650ter, Gln914ter, 2810delT, Thr935Met, Arg1041Pro, and Glu1173Lys. The novel polymorphisms are 1168A-->G (Ile390Val), 2785A-->G (Ile929Val), and 3316G-->A (Val1106Ile). Two mutations, Arg778Leu and Thr935Met, are relatively frequent, representing 37.7% and 10.0% of patients, respectively. To our knowledge, we are the first to report the correlation between the genotype and phenotype of Arg778Leu. The result shows that Arg778Leu homozygotes are associated with the early onset of WD with hepatic presentation. The Arg778Leu and Thr935Met mutations are hot spots in the Chinese population. The features of mutations of ATP7B differ between the Chinese and Western ethnic populations. The Arg778Leu mutation has severe effects on the function of ATP7B. These findings are valuable for developing a fast and effective method to diagnose the presence of the WD gene.

Morbus Wilson: Identification of three additional novel mutations in the ATPase7B gene

Background: Mutations in the copper transporting P-type ATPase gene have been reported to result in the clinical phenotype Wilson disease. The disease is a rare inherited autosomal recessive disorder, characterized by impaired incorporation of copper into ceruloplasmin and by impaired excretion via the bile. The gene consists of 21 exons and encodes a protein of 1,465 amino acids. About 190 mutations have been detected throughout the whole gene sequence with certain accumulation in hot spots. The underlying mutations are very heterogeneous, thus the fast and reliable genetic screening for mutations as the underlying cause of the disease is hampered. The aim of this investigation was to assess novel mutations in the recently discovered Wilson disease gene. Methods: We screened 57 individuals of european origin for mutations in the 21 exons and their flanking intronic sequences using direct sequence analysis. All exons and exon/intron boundaries of patients suffering from Wilson disease were amplified by PCR. Subsequently, cycle sequencing of the purified PCR products using the fluorescent dye terminating system (PE Applied Biosystems) was performed. Detected mutations were confirmed using single strand conformation analysis of the PCR products. Results: So far we detected in 114 analyzed alleles 22 disease causing mutations affecting the ATPase 7B gene. Ten of these mutations were novel mutations (4 missense, 2 nonsense, 1 insertion, 2 deletions, t splice site) (Hum Murat 14 (1):88; 18(3):278). The mutations Pro76OLeu, Leu1305Pro und GIn1351Stop in the exons 8, 19, and 20 have not yet been reported. All patients were heterozygous for the mutation. The most common HislO69GIn mutation was found in 30 of 57 (53%) patients. 14 patients were homozygous carriers (47%), 16 patients were heterozygous carriers. The His1069GIn occurred with an allele frequency of 38.5% in this cohort. Conclusions: These data confirm, that Wilson disease in our patients results from a frequent mutation His1069GIn and/or from a large number of rare mutations. The mutation heterogeneity complicates genetic testing to confirm the diagnosis of WD, although DNAbased genetic analysis is still an important supplement to clinical diagnostics. The most recommendable procedure for genetic analysis is the analysis of the common mutation His1069GIn using PCR and subsequent restriction endonuclease digestion. In the absence of homozygosity of this mutation direct analysis of all exons including the promoter is required.