Table of Contents

HK J Paediatr (New Series)
Vol 26. No. 2, 2021

HK J Paediatr (New Series) 2021;26:69-74

Original Article

Genetic Analysis of Wilson Disease in South China: Hotspots and One Novel Mutation in ATP7B

Z Qiao, G Zhou, M Jiang


Objectives: Wilson disease (WD), also known as hepato-lenticular degeneration, is an autosomal recessive inherited disorder of copper metabolism. The gene responsible for WD is located on chromosome 13 at 13q14.3, which encodes copper transporter P-type ATPase, namely ATP7B. Mutation of ATP7B is a genetic signal of highly risk suffering from WD. Here, we aim to analyse the hotspot of ATP7B mutations in WD patients from South China using Sanger sequencing. Methods: In this study, 50 healthy individuals and 32 identified WD patients were enrolled, who manifested with abnormal hepatic function including increased Alanine Transaminase (ALT) or Aspartate Transaminase (AST), and/or dyskinesia. The genomic locus of ATP7B of these patients were amplified by polymerase chain reaction (PCR), then sequenced via Sanger sequencing. Results: Genetic variations were found in all patients, including 17 mutations and nine SNPs, one of which (c.1757T>A) is novel. The most frequent mutations were P.778R>L (22.5%), P.770L>L (17.5%), and P.935T>M (10.0%). These mutations were mainly clustered in the exon 5, 8, 12, and 13 of ATP7B. Conclusions: Based on Sanger sequencing of ATP7B in this study, p.778R>L mutation clustered in the CU channel transmembrane domain consisted over 50% of ATP7B mutations, suggesting hotspots in South China and supplying a suitable strategy focusing on this hotspot for ATP7B screening in South China WD patients. The newly identified mutation, c.1757T>A, is deleterious based on an array of predictions and highly conserved upon comparison among different species. Furthermore, the c.1757T>A is classified as likely pathogenic variant according to American College of Medical Genetics (ACMG) guidelines (2015 Edition).

Keyword : ATP7B; Mutation hotspot; Sanger sequencing; Wilson disease


Wilson disease (WD), also known as hepatolenticular degeneration that was first reported by Kinnear Wilson in 1912,1 is an autosomal recessive inherited disorder of copper metabolism. WD is characterised by impaired synthesis of copper chaperonin leading to lower ceruloplasmin concentration,2 resulting in copper excessive accumulation in organs like liver, brain, cornea, and kidney, thus, leading to a series of complex clinical manifestations, such as acute or chronic hepatitis, liver cirrhosis, hepatic failure, dyskinesia, neuro-psychiatric symptoms, and Kayser-Fleischer corneal ring (K-F ring).3-6

WD is caused by mutations in the ATP7B gene, discovered in 1993, which encodes the copper-specific transporting P-type ATPase and the gene is located on the chromosome 13 at 13q14. The ATP7B is consisted of 21 exons and 20 introns, nearly 7.5 kb in size, encoding a 1465 amino acid-protein that comprises of six copper-binding domains (exons 2-5), eight transmembrane domains of copper channel (exons 6-8, 12-13,19-20), and the ATP-binding domain (exons 10-11,14-18).7,8

The widely accepted incidence of WD is about 1/30 000 worldwide, which is higher in China and Japan (about 1/10 000),9,10 with an estimated carrier frequency of 1/90. This incidence rate was at least partly based on assumption and calculation, and has been questioned.

To date, over 500 mutations have been found in the ATP7B gene, and distribution of the mutations differs among various ethnic groups.11,12 The mutations are scattered throughout within ATP7B gene, however some hotspots were reported to be varied in different populations. For example, the first hotspot in Europeans and North Americans was identified in exon 14, p.His1069Gln.13,14 In some Asian countries, such as Korean and Japan, the first hotspot lies in exon eight, p. Arg778Leu.15,16 More than 100 of these mutations were identified in Chinese populations, characterised by a few hotspot mutations and a variety of rare mutations, with a high genetic heterogeneity and a large variation in prevalence according to the geographic distribution and ethnic background.17 Previous studies had shown that hotspot mutations for some Chinese populations mainly clustered in exons eight with p. Arg778Leu.18,19 In this study, Sanger sequencing was used to identify the mutation spectrum in South Chinese population to investigate hotspot mutation that could facilitate diagnosis confirmation genetically.

Materials and Methods


A total of 32 identified WD patients were analyzed, ranging from 2 to 30 years old, consisted of 71.5% males and 28.5% females. All WD patients were diagnosed and treated at the second affiliated hospital of Wenzhou Medical University among the year of 2010-2017. Diagnosis of WD was based on clinical symptoms, including hepatic dysfunction, and/or typical neurological symptoms, or the presence of Kayser-Fleischer ring, and biochemical parameters, such as low serum ceruloplasmin (<0.2 g/l) and high level of urinary copper (>100 mg/24h).9 Fifty healthy local individuals were enrolled as controls. All patients were provided written informed consent in regarding with the Declaration of Helsinki and ratified by the Ethics Committee of Wenzhou Medical University.

DNA Extraction

We used a salt precipitation and extraction method to extract genomic DNA from peripheral blood samples. In brief, two ml venous blood was extracted into ethylenediaminetetraacetic acid (EDTA) sample tube and the genomic DNA was extracted from peripheral blood leukocytes using the standard phenol/chloroform extraction protocol.

PCR and Sanger Sequencing

All the exons and intron/exon boundaries of ATP7B genes were amplified by polymerase chain reaction (PCR), using a set of twenty-five primer pairs designed before.20 PCR was performed using GoTaq Green Master Mix (Promega) with 100 ng of genomic DNA in a mix containing 10 pmol of each primer, 12.5 μl of 2xGoTaq Green Master mix in a total volume of 25 μl. The thermocycle programme consisted of an initial denaturation at 94&deg;C for five min, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s and 72°C for 30 s, with a final extension at 72°C for five min. The size and quantity of PCR products were verified by electrophoresis in 2% (w/v) agarose gel. Then the PCR products were sequenced on an ABI PRISM 3730 DNA Sequencer.

Prediction of the Virulence of Gene Mutation

Three bioinformatics software that contains polymorphism phenotyping (PolyPhen)-2 (, sorting intolerant from tolerant (SIFT) ( and Taster Mutation ( were used to predict the virulence of mutations in ATP7B.


Identification of Genetic Variants

All of the exons and intron/exon boundaries of ATP7B genes were amplified by PCR and sequenced for mutation. A total of 26 genetic variations, including 17 mutations (12 missense mutations, two synonymous mutations, two splicing mutations and one deletion) and nine SNPs, were identified (Tables 1 and 2). All the mutations were heterozygous. Among those mutations, we found one missense mutation (c.1757T>A) was novel (Figure 1) after a search in a series of databases, including the 1000 Genome, dbSNPs (v130), Wilson disease Mutation Database, and HapMap. No mutations were found in the healthy control group.

Table 1 Analysis of ATP7B mutations and prediction of the functional effects of the mutation
NT change AA change Location Mutation type Functional region PROVEAN PolyPhen-2 Allele count Allele
Freq %
Prediction score
c.1708-1G>C   IVS5-1 Splicing MBD6 NA NA NA 1 1.56
c.1708-5G>C   IVS5-5 Splicing MBD6 NA NA NA 1 1.56
c.3799delG   Exon18 Deletion ATP hinge NA NA NA 1 1.56
c.1351G>A p.451G>S Exon3 Missense MBD5 Neutral Benign 0.004 1 1.56
c.1757T>A* p.586L>H Exon5 Missense MBD6 Deleterious Probably damaging 1.000 1 1.56
c.1857C>A p.619I>I Exon5 Synonymous MBD6 MBD6 NA NA 2 3.12
c.2294A>G p.765D>G Exon8 Missense Tm4 Deleterious Probably damaging 1.000 1 1.56
c.2306T>C p.769M>T Exon8 Missense Tm4 Deleterious Probably damaging 1.000 1 1.56
c.2310C>G p.770L>L Exon8 Synonymous NA NA NA NA 7 10.92
c.2333G>T P.778R>L Exon8 Missense Tm4 Deleterious Probably damaging 1.000 9 14.04
c.2621C>T p.874A>V Exon11 Missense ATPase Deleterious Possible damaging 0.832 1 1.56
c.2755C>G p.919R>G Exon12 Missense Tm5 Deleterious Probably damaging 1.000 1 1.56
c.2804C>T p.935T>M Exon12 Missense Tm5 Deleterious Possible damaging 0.832 4 6.24
c.2828G>A p.943G>D Exon12 Missense Tm5 Deleterious Possible damaging 0.832 1 1.56
c.2930C>T p.977T>M Exon13 Missense Ch/Tm6 Deleterious Probably damaging 1.000 2 3.12
c.2975C>T p.992P>L Exon13 Missense Ch/Tm6 Deleterious Probably damaging 1.000 4 6.24
c.3960G>C p.1320R>S Exon19 Missense Tm7 Deleterious Probably damaging 1.000 1 1.56
Abbreviations: AA, amino acid; ATPase, copper-(or silver)-translocating P-type ATPase segment; MBD, mental-binding domain; Ch, ion channel region;Tm, transmembrane segment.
*Represent newly discovered mutations.

Table 2 Information of ATP7B SNPs
SNP RS-ID Location 1000G Freq
c.-123_-119dupCGCCG rs148013251 5'UTR C=0.3704
c.1543-93A>C rs3742288 Intron2 G=0.3776
c.1707-53A>C rs2147363 Intron3 T=0.3882
c.2448-25G>A rs9526811 Intron9 T=0.3069
c.3903+6C>T rs2282057 Intron18 G=0.4982
c.1216T>G rs1801243 Exon2 C=0.3762
c.1366G>C rs1801244 Exon3 G=0.3770
c.2495A>G rs1061472 Exon10 C=0.4976
c.2855G>A rs732774 Exon12 T=0.4690

Prediction of Functional Virulence Caused by ATP7B Mutations

We combined PolyPhen-2, SIFT, and Taster Mutation to predict the effects of the missense mutations on ATP7B function. Results showed that all of the missense mutations were deleterious except c.1351G>A, which was calculated as a benign one. Two synonymous mutations (c.1857C>A and c.2310C>G) were considered as polymorphic variations. The deletion mutation, c.3799delG, would lead to frame shift or PTC-further downstream change, which was predicted to be deleterious according the Taster Mutation prediction. The splicing mutations, c.1708-1G>C, first reported by Thomas,13 was predicted as a 'pathogenic variant'. The splicing mutation, c.1708-5T>G, first reported by Norikazu Shimizu,20 was predicted as 'variants with uncertain significance'. Particularly, the novel missense mutation, c.1757T>A, a heterozygous mutation in exon five, was identified in this study as a disease-causing mutation predicted by all three software. This mutation would lead to the replacement of a highly conserved Leucine with a Histidine at the 586 amino acid position (p. L586H) (Figure 2), which is deleterious for the last copper-binding domain.


WD is a life threatening autosomal recessive disorder, caused by abnormal copper metabolism. However, early diagnosis and effective therapy can prevent disease progression and minimise injuries to the organs, including liver, brain, cornea.21,22 To date, the diagnosis of WD is mainly based on typical clinical manifestations and laboratory findings, including decreasing concentration of ceruloplasmin. Nevertheless, the clinical manifestations of WD are extraordinarily diverse and atypical, so that establishing timely diagnosis based on clinical manifestations is not easy, especially in adolescent patients.21,23 As to the ceruloplasmin, which is commonly used as a serum marker for WD, is a kind of positive acute phase reactant in nature. In the case of acute inflammation, the ceruloplasmin concentration would increase, which might sometime be mislead when the WD patients undergoing infection. On the other hand, the concentration of ceruloplasmin will be declined in chronic liver disease, which will also be troublesome in diagnosing WD patients along with chronic liver disease. Therefore, genetic confirmation of WD diagnosis is necessary, which would be facilitated with hotspot in the ATP7B.

In this study, 16 known mutations and one novel ATP7B mutation (p. L586H) were found in those patients from South China. The mutations were mainly clustered in exons 8 (25%), 12 (18.7%), 4 (12.5%), 5 (12.5%), and 13 (12.5%), the same as previous results from North China.19,24 However, P.778A>L was not detected in this study, though this mutation was recognised as the most frequent mutation in Chinese population.24 Meanwhile, 50% (8/16) of the mutations happened to CU channel transmembrane domain, indicating a hotspot region for genetic screening of WD diagnosis in South China.

Interestingly, the mutation c.2310C>G and c.2333G>T as a unit, comprised 18.7% of the mutations in these WD patients, indicating these two mutations might act on ATP7B in the manner of cis-action (Table 3). In this study, p.778R>L showed the highest allele frequency (Table 1), indicating a hotspot for genetic confirmation of WD.

The newly identified mutation p.586L>H, not found in the 50 healthy individuals meaning <1% allele frequency, is a missense mutation existing in exon five, affecting the CU binding domain, which was predicted as deleterious. The 586 amino acid is a highly conserved region when compared between human and other species, indicating an indispensable impact on ATP7B function (Figure 3).

In summary, we recommend that sequencing of the CU channel binding domain of ATP7B in genetic screening for WD patients, which cover over 50% in south China. And the mutation p.778R>L might also serve as a hotspot for genetic confirmation of WD. The newly identified mutation, p.586L>H, is an addition for the genetic mutation base of WD.

Note: The mutations detected in this study. The novel one identified in this study was indicated in red letters.
Figure 1 Mutations in the different domains of the ATP7B gene.

Figure 2 Sequencing diagram of the new mutation.

Figure 3 Multiple-sequence alignment in ATP7B from different species revealed that codon 586 was located within a highly conserved region.

Table 3 Informations for patients with WD
No Gender Age
K-Fring Manifestation Mutations classification ACMG of the variants Literatures
1 Male 5 8.8 6.8 None Liver ribs 3.0 cm c.1757T>A PM1 This study
              c.2310C>G BA1 Ref.8
              c.2333G>T PM1 Ref.7
2 Female 13 9.7 8.9 Yes Liver ribs 2.0 cm c.1857C>A BP7 Ref.11
3 Female 3 8.0 7.6 Yes Liver ribs 2.0 cm c.2310C>G BA1 Ref.8
              c.2333G>T PM1 Ref.7
              c.3799delG PM4 Ref.8
4 Male 2 2.6 8.9 None Liver ribs 1.0 cm c.1857C>A BP7 Ref.11
5 Female 7 6.8 8.4 Yes Liver ribs 4.0 cm c.2306T>C PM1 Ref.7
6 Male 12 8.8 8.9 None Liver ribs 2.0 cm c.2306T>C PM1 Ref.7
              c.2310C>G BA1 Ref.8
              c.2333G>T PM1 Ref.7
7 Male 3 4.8 8.0 None Liver Not touched c.2755C>G PM1, PM4 Ref.8
8 Female 16 4.5 10.0 None Liver Not touched c.2828G>A PM1, PM4 Ref.7
9 Male 8 7.2 11.3 Yes Liver cirrhosis c.2294A>G PM1, PM4 Ref.8
10 Male 11 <2.0 6.1 Yes Liver ribs 1.0 cm c.2333G>T PM1 Ref.7
11 Male 4 3.3 14.9 None Liver ribs 1.0 cm c.2975C>T PM4 Ref.8
12 Male 16 2.4 11.0 None Liver ribs 2.0 cm c.1857C>A BP7 Ref.11
13 Female 15 <2.0 8.8 None Liver ribs 1.5 cm c.2804C>T PM1 Ref.8
              c.2310C>G BA1 Ref.8
              c.2333G>T PM1 Ref.7
14 Male 5 4.0 10.0 None Liver ribs 2.0 cm c.3960G>C PM4 Ref.11
              c.2294A>G PM1, PM4 Ref.8
15 Male 19 <2.0 10.1 Yes Liver Not touched c.2310C>G BA1 Ref.8
            Limb tremor Erectile dysfunction c.2333G>T PM1 Ref.7
16 Male 28 4.1 14.3 None Liver cirrhosis c.1351G>A BP4 Ref.11
              c.2930C>T PM4 Ref.8
              c.2975C>T PM4 Ref.8
17 Male 26 2.2 11.0 None Liver Not touched c.2310C>G BA1 Ref.8
              c.2333G>T PM1 Ref.7
18 Male 2 2.6 8.9 None Liver ribs 1.0 cm c.2828G>A PM1, PM4 Ref.7
19 Female 3 4.1 11.6 None Liver Not touched c.2804C>T PM1 Ref.8
              c.2975C>T PM4 Ref.8
20 Female 4 9.7 15.0 None Liver ribs 1.0 cm c.2310C>G BA1 Ref.8
              c.2333G>T PM1 Ref.7
21 Male 21 3.6 6.8 None Liver ribs 1.0 cm c.2930C>T PM4 Ref.11
              c.2975C>T PM4 Ref.8
22 Male 10 8.0 10.1 None Liver ribs 1.0 cm c.3960G>C PM4 Ref.11
23 Male 6 5.8 9.7 None Liver Not touched c.1708-3G>C BP4 Ref.8
24 Male 30 4.0 11.5 Yes Liver ribs 1.5 cm c.2621C>T PM4 Ref.8
              c.2333G>T PM1 Ref.7
25 Male 9 <2.0 10.1 None Liver Not touched c.2804C>T PM1 Ref.8
              c.-123_-119dupCGCCG PM4 Ref.7
26 Male 2 4.0 8.1 None Liver Not touched c.-123_-119dupCGCCG PM4 Ref.7
27 Female 3 7.6 10.1 None Liver Not touched c.2755C>G PM1, PM4 Ref.11
28 Male 5 14.0 6.8 None Liver Not touched c.1751G>A PM1 Ref.7
29 Female 8 13.5 8.8 None Liver Not touched c.2621C>T PM4 Ref.8
30 Male 9 4.1 9.0 Yes Liver ribs 1.0 cm c.1708-5G>C BP4 Ref.8
            Liver cirrhosis Haematuria c.-123_-119dupCGCCG PM4 Ref.7
31 Male 14 3.0 7.9 Yes Liver ribs 1.5 cm
c.2294A>G PM1, PM4 Ref.8
32 Male 11 3.8 8.9 None Liver ribs 2.0 cm c.2306T>C PM1 Ref.7
Reference range: CER, 19-67mg/dl; CU level, 11.8-39.3 umol/l.
According to ACMG, PM1-PM6 refers to moderate evidence for pathogenicity; BA1 refers to alone evidence for benign impact; BP1-BP7 refers to supporting evidence of benign impact.
Ref. refers to Reference attached at the end of the paper.

Declaration of Interests

The authors declare no competing financial interests.


This study was supported by the Bureau of Science and Technology of Wenzhou city (NO: Y20160515).


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