Elsevier

Molecular Genetics and Metabolism

Volume 86, Issues 1–2, September–October 2005, Pages 277-285
Molecular Genetics and Metabolism

Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease

https://doi.org/10.1016/j.ymgme.2005.05.004Get rights and content

Abstract

Wilson disease (WD) is an autosomal recessive disorder of copper transport. WD patients are presenting with a wide range of heterogeneous clinical syndromes including hepatic, neurological, or psychiatric presentations. The disease is caused by mutations in the ATP7B gene. This study presents the results of comprehensive mutation analysis in 227 WD patients from 200 unrelated families (173 from Czech Republic and 27 from Slovakia). More than 80% of all mutant alleles were identified, using a combination of PCR/RFLP, DGGE, TTGE, DHPLC, and sequencing. A total of 40 different mutations and 18 polymorphisms were detected on 400 independent mutant chromosomes. The most common molecular defect was H1069Q (57% of all 400 studied alleles). Each of the other 39 mutations was present in no more than 4% of WD alleles and 23 mutations were found in only one WD allele each (0.25%). Thirteen novel mutations were identified, including seven missense mutations (L641S, T737R, D918E, T1033S, G1111D, D1271N, and G1355C), four small deletions (19_20delCA, 1518_1522del5, 3140delA, and 3794_3803del10), and two splice-site mutations (2446-2A>G, 2865+1G>A). We did not find a significant correlation between H1069Q homozygosity and age of onset, and clinical and biochemical manifestation. Our data provide evidence that the H1069Q mutation—the most common molecular defect of the ATP7B gene in the Caucasian population—originates from Central/Eastern Europe. Screening of five prevalent mutations is predicted to reveal 70% of all mutant alleles presented in WD patients. This will provide a good starting point for early clinical classification of WD in our population.

Introduction

Wilson disease (WD, MIM 277900) is an autosomal recessive disorder of copper excretion caused by deficiency of a P-type ATPase, encoded by the ATP7B gene. The principle of this disease is failure of copper secretion from the liver to the bile and a defective incorporation of copper into ceruloplasmin. The disorder usually manifests early as chronic liver disease and/or later as neurological impairment due to the toxic accumulation of copper in several tissues, principally the liver and the brain. Wilson disease is lethal if left untreated. Incidence is estimated to be 1:30,000 in most populations [1]. Biochemical diagnosis is based on raised urinary Cu after penicillamine, low plasma ceruloplasmin, and elevated Cu concentrations in the liver biopsy [2]. However, some patients may be biochemically indistinguishable from healthy carriers [3] and WD can sometimes be difficult to diagnose. Among diagnostic tests concerning WD, DNA analysis now represents the most sensitive and specific approach.

The gene defective in Wilson disease has been identified and found to encode a copper-transporting P-type ATPase (ATP7B) [4], [5], [6]. The ATP7B gene has 21 exons, encodes a protein containing 1465 amino acids, and is predominantly expressed in the liver with reduced expression in the brain, kidney, and placenta [4], [5], [7]. Large numbers of mutations and polymorphisms were identified in the ATP7B gene, some of which are found in probands from a variety of different ethnic backgrounds [8], [9], [10]. Others appear to be population specific [11]. The most common mutation found in a number of Caucasian populations seems to be H1069Q, which disrupts the unique amino acid SEHPL motif and leads to a deficiency of ATP binding [9], [12], [13], [14], [15], [16], [17], [18], [19], [20].

Conflicting results on genotype–phenotype correlations of the most common mutations have been reported. A correlation between the prevalent H1069Q mutation in the homozygote state and the late onset form of WD frequently associated with neurological symptoms was described [12], [14], [19], while no correlations were found in other studies [9], [21].

The aims of this study were to identify the spectrum of mutations in a large group of Czech and Slovak WD patients and to provide insight into the possible relationship between genotype and phenotype. Analyzed patients represent the homogeneous middle European population, thus this study contributes to an enrichment of knowledge about the population genetics of WD in Europe.

Section snippets

Patients

Two hundred and twenty-seven WD patients from 200 unrelated families (173 from Czech Republic and 27 from Slovakia) and their relatives were examined. One hundred and twenty-six male and 101 female patients were observed, with mean age at onset of the disease 15.2 ± 5.3 years. In the absence of the Kayser–Fleischer ring and neurological symptoms, diagnosis of WD was based on the presence of at least two abnormal biochemical tests: raised urinary copper excretion (basal >1.5 μmol/24 h or >5 μmol/24 h

Mutation analysis

A multiple step approach using PCR/RFLP, DGGE, TTGE, DHPLC, and sequencing was applied for the detection of mutations in the ATP7B gene. The promoter region and the whole coding region with flanking intronic sequences of the ATP7B gene were analyzed. Examples of newly identified mutations in the ATP7B gene are shown in Fig. 1.

Results are summarised in Table 4. With the strategy used, we were able to identify disease causing mutations on 321 (80.3%) of 400 mutant alleles. A total of 40 different

Discussion

By detailed scanning of the ATP7B gene, a mutation detection rate of 80.3% was achieved in our study. As can be seen in Table 4, 40 different mutations were identified on 400 mutant alleles. We found that the spectrum of WD mutations in the Czech and Slovak population consists of a small number of relatively frequent mutations, and a large number of rarely occurring mutations. The H1069Q missense mutation in exon 14 of the ATP7B gene is the major molecular defect in our Slavic population with a

Acknowledgments

We are thankful to all physicians who referred patients: Drs. Brotanek, Bzduch, Cermakova, Durovic, Fiedler, Gaillyova, Grochova, Kofer, Kovacova, Kupcova, Kvasnicova, Malikova, Nebesnakova, Prasilova, Prochazkova, Siruckova, Smolka, Starha, Sulakova, Trunecka, Uteseny, and Vejvalkova. This work was supported by Grants IGA MZ CR No. NR/8451-3 and AV CR S4031209.

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