Wilson Disease Mutations in the American Population: Identification of Five Novel Mutations in ATP7B

Wilson disease is an autosomal recessive disorder characterized by toxic accumulation of copper in a number of organs such as liver and brain, which results in significant disability or death if left untreated. Wilson disease is caused by mutations in ATP7B, a copper transporter. We analyzed 108 American Wilson disease patients, who are predominantly White, for mutations in ATP7B. Consistent with studies from other populations, H1069Q was the most common mutation in this group of patients, accounting for 40.3% of the sequenced alleles; 26 of the 108 patients were homozygous, and 35 patients were heterozygous for this mutation. We also identified 24 additional ATP7B mutations, of which five were novel. The five new mutations consist of two insertion mutations (2302insT and 3843insT), one splice site mutation (IVS11+2:T>A), one combination of deletion (2bp) and insertion (19bp) (3693-3697delins19bp), and one missense mutation (G1213S). All variants are predicted to be disease-causing mutations. Ninety six percent of all mutations we identified were clustered in regions encoding the C-terminal half (catalytic domain) of the ATP7B protein. Furthermore, we found that 84% of the mutant alleles identified in the American population are located in exons 14 and 18. INTRODUCTION Wilson disease (WD) is an autosomal recessive disorder of copper transport with a world-wide prevalence of 30 per million [1-3]. WD is associated with decreased incorporation of copper into ceruloplasmin and a buildup of copper in target tissues, which result in hepatocellular failure and/or basal ganglia degeneration [2, 4]. This disorder is one of a few hepatic or neurological disorders that is treatable [1, 2, 4]. Thus, identification of genetic mutations for suspected WD patients may play a critical role in successful treatment. The WD gene ATP7B, comprised of 21 exons spanning 80 kb of genomic DNA, encodes a copper-transporting Ptype ATPase [5]. Over 380 mutations in the WD gene have now been identified from a multitude of populations [6] (http://www.medicalgenetics.med.ualberta.ca/wilson/index.p hp). Screening for mutations can thus be laborious, time consuming, and expensive. Since selected mutations appear to be restricted to specific populations [7], establishing the most common mutations in a given population may simplify initial efforts to identify WD gene mutations in specific families. To date, two large studies have analyzed WD patients from North America. Tanzi et al. [8] studied 50 unrelated families and identified four mutations, though they only screened part of the WD gene. Shah et al. [9] studied 128 North American patients and found that the H1069Q was the most common mutations in the WD gene. In this study, we analyzed 108 unrelated American patients; we find that most *Address correspondence to this author at the Departments of Molecular and Integrative Physiology, 7629 Medical Sciences II Box 5622, 1301 East Catherine Street, Ann Arbor, MI 48109-5622, USA; E-mail: [email protected] mutations are located within exons 14 and 18 and describe five new mutations in the WD gene. SUBJECTS AND METHODS This study included 108 unrelated subjects from the United States with confirmed WD. The diagnosis of Wilson disease was confirmed by a combination of a variety of clinical symptoms, biochemical tests, and detection of gene mutations, which are examined by a group of doctors headed by Dr. Brewer at University of Michigan. Mutation analysis was performed on DNA samples isolated from patient blood samples by PCR amplification, purification, and sequencing of all 21 exons from each of the patients. The primer design and use were based on previously published data [10]. When two different mutations were found in the same exon from the same patient, the PCR products were subcloned and individual clones were sequenced to confirm that they occurred on different chromosomes (demonstrating that the patients were compound heterozygotes). In addition, 98 normal genomic DNA samples from White Americans were analyzed as controls to confirm the absence of the novel ATP7B mutations we found in this study. RESULTS AND DISCUSSION We sequenced ATP7B gene on 216 chromosomes from 108 American WD patients and found mutations in 128 of these. There was a remarkable clustering of mutations in exons 14 and 18 (93 and 14 of 128 mutations, respectively); these two exons account for 84% of the total number of hits in the ATP7B gene. We were able to identify both ATP7B mutations in 47 (44%) of the American patients (Table 1). H1069Q repre2 The Open Hepatology Journal, 2009, Volume 1 Kuppala et al. sented the most common mutation found in this cohort. Twenty-six (24%) patients were homozygous for H1069Q, and two patients were confirmed compound heterozygous for E1064A/H1069Q (by subcloning and sequencing exon 14). The frequent occurrence of H1069Q in Caucasian patients with WD, including those in the United States [9], is well documented [7]. N1270S was the second most commonly identified mutation in our study, occurring in four of 108 patients (Tables 1 and 2). Most populations have distinct mutations that are relatively specific for the group [7, 11]. H1069Q, for instance, is found exclusively in patients of European origin [7]. Conversely, R778G mutation is extremely common for Asians [12]. In addition to these two common mutations, many other rare mutations tend to have regional clustering [7]. N1270S, however, is distinguished by its common occurrence in a large number of groups, including our American cohort, Chinese [12, 13], Korean [14, 15], Japanese [16], Indian [17], Sicilian [8], Bulgarian [18], Table 1. Mutations Identified in American WD Patients Mutations N a Exon (Intron) N/total patients