Novel mutations found in the ATP7B gene in Chinese patients with Wilson's disease

Abstract Background Wilson's disease (WD) is an autosomal recessive genetic disease caused by mutations in ATP7B and characterized by copper metabolism disorders. Methods Direct sequencing of the ATP7B gene is the most sensitive and widely used confirmatory testing method. Fourteen probands with WD and 12 family members participated in this study. The ATP7B gene was analyzed by direct sequencing. Results Twenty‐nine different variants (27 substitutions, 1 duplication, 1 deletion) were found. Of the 23 reported variants, nine nondisease variants, 11 disease variants, one silent variant, and two variants with uncertain functions were identified. The six novel variants included c.1875T>A, c.2306T>C, c.3028A>G, c.3243G>A, c.3437_3438 delTG, and c.3903+5G>A. Conclusion These findings will assist in the diagnosis of WD. The novel variants have enriched the WD database.


| BACKGROUND
Wilson's disease (WD, OMIM#277900) is an autosomal recessive genetic disease caused by mutations in ATP7B (HGNC: 870, VERSION NG_008806.1). ATP7B is located on 13q14.3 and contains 20 introns and 21 exons, for a total genomic length of 80 kb . ATP7B encodes copper-transporting P-type ATPase, which is a group of transmembrane copper transport proteins . This protein is composed of 1,465 amino acids that contain a phosphatase domain (A-domain), a phosphorylation domain (P-domain), a nucleotide-binding domain (Ndomain), and eight transmembrane ion channels (M-domain) (Cater, Fontaine, & Mercer, 2007).
Mutation of the ATP7B gene is closely linked to the impairment of copper excretion, leading to abnormal deposition of copper in the target organs (Dong & Wu, 2012). Variants in the ATP7B gene have been reported in almost all exons. More than 700 variants in ATP7B have been identified, of which single-nucleotide missense and nonsense mutations is the most common, followed by insertions/deletions and splice site mutations. Most patients are compound heterozygotes, carrying different mutations on each copy of the chromosome. Due to the diverse clinical manifestations of WD, it can sometimes be difficult to diagnose.
We conducted a molecular analysis of 14 probands and 12 family members and identified six novel variants in the ATP7B gene.

Novel mutations found in the ATP7B gene in Chinese patients with Wilson's disease 2 | PATIENTS AND METHODS
Fourteen probands (three males and 11 females, age from 4 to 43 years old), who presented with hepatic symptoms and decreased ceruloplasmin (<200 mg/L, normal 200-400 mg/L), were diagnosed with WD from 2012 to 2015 in the YouAn Hospital of Capital Medical University. All probands had at least four points according to the WD scoring system (European Association for the Study of the Liver, 2012). Additionally, 11 parents and one sibling of the 14 probands were recruited for the study. They were of the Han ethnicity from North China. Written informed consent was obtained from the participants or their guardians before the genetic investigation was conducted. The Ethics Committee of the Beijing YouAn Hospital of Capital Medical University approved the present work. This study protocol conformed to the ethical guidelines of the Declaration of Helsinki.
The ATP7B gene was analyzed by direct sequencing using genomic DNA extracted from leukocytes in peripheral blood (QIAGEN, Germany). The Primers used for PCR assay were showed in Table 1. The amplified products were detected by agarose gel electrophoresis and sequenced using an ABI3730 DNA Analyzer (Applied Biosystems, USA). The pathogenicity of the genetic variants was ascertained using the WD allelic variant database (http://www.wilsondisease.med.ualberta.ca/database.asp).

| RESULTS
By direct sequence analysis of the entire ATP7B gene coding and promoter regions, we identified 29 different variants.
In our study, we found six novel variants, of which two were synonymous mutations (c.1875T>A and c.3243G>A) and four were possible DVs (c.2306T>C, c.3028A>G, c.3437_3438 delTG, and c.3903+5G>A).
The c.2306T>C (ATG-ACT, p.Met769Thr) mutation was newly found. At the same amino acid position, two mutations (c.2305A>G, ATG-GTG, p.Met769Val and c.2306T>G, ATG-AGG, p.Met769Arg) have been reported as DVs. The novel c.2306T>C heterozygous mutation was found in a child proband and his father. This mutation affects Cu transport by creating a conservative amino acid change in Tm4. The c.3028A>G(AAG-GAG, p.Lys1010Glu)mutation is regarded as a new DV. At the same amino acid position, three DVs have been verified previously (Santhosh et al., 2008). It is found a compound heterozygote patient carrying c.3028A>G mutation and the known pathogenic variant c.3053C>T. We found a novel variant in exon 16, c.3437_3438 delTG (p.Val1146Ala fs*6). In a previous study, it was found that the c.3436G>A (p.Val1146Met) missense mutation at amino acid position 1146 is a DV (Antonietta et al., 2008). Generally, frameshift and missense mutations are associated with more severe phenotypes of WD. Furthermore, the compound heterozygote proband with the novel variant (c.3437_3438 delTG) and another DV (c.4114C>T, p.Gln1372Ter) was diagnosed with WD. This proband's father had a novel variant, and the proband's mother had another DV (c.4114C>T, p.Gln1372Ter); both were diagnosed as WD carriers because they were simple heterozygotes. Therefore, the novel variant (c.3437_3438 delTG) is likely to be a DV. A novel variant (c.3903+5G>A) was found in exon 18. Similarly, the c.3903+6T>C splice variant is a nondisease variant (NDV) (Gu et al., 2003) and the novel c.3903+5G>A splice variant was speculated to be a NDV. However, in our study, a patient carried the novel variant (c.3903+5G>A), a DV (c.2621C>T), and three NDVs (c.2855A>, c.3419C>T, and c.3903+6C>T). The pathological significance of the novel variant (c.3903+5G>A) requires more study in future.
Summary, genetic testing is a valuable tool to detect WD. The results add data to the spectrum of known mutations in the ATP7B gene in Chinese Han population.