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Yang W, Yang Y, Wang H, Wang J, Zhang S. Clinical and genetic characterization of patients with late onset Wilson's disease. NPJ Genom Med 2024; 9:71. [PMID: 39719440 DOI: 10.1038/s41525-024-00459-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2024] [Accepted: 12/17/2024] [Indexed: 12/26/2024] Open
Abstract
Wilson's disease (WD) typically manifests in children and young adults, with little knowledge of its late-onset forms. In this study, we performed a retrospective cohort study of 105 WD patients (99 index cases, 6 siblings) with an onset age ≥35 years. We compared 99 index late-onset patients with 1237 early-onset patients and analyzed the ATP7B variant penetrance referring to the Genome Aggregation Database (gnomAD). Sixty-two ATP7B variants were identified in the late-onset patients, among which A874V, V1106I, R919G, and T935M were correlated with late presentation of WD. Regarding gnomAD, V1106I and T935M exhibited significantly low penetrance, and there is a lack of patients carrying a genotype of V1106I/V1106I, R919G/R919G, T935M/T935M, V1106I/T935M, V1106I/R919G, or T935M/R919G. Our data revealed that patients carrying a combination of two late-onset variants may be overlooked due to atypical or lack of WD symptoms, which may provide valuable insights into the genetic basis and diagnosis of WD.
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Affiliation(s)
- Wenming Yang
- Department of Neurology, the First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, China
| | - Yue Yang
- Department of Neurology, the First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, China
| | - Han Wang
- Department of Neurology, the First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, China
| | - Jiuxiang Wang
- Experimental Center of Clinical Research, the First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, China
| | - Shijie Zhang
- Experimental Center of Clinical Research, the First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, China.
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Roy S, Ghosh S, Ray J, Ray K, Sengupta M. Missing heritability of Wilson disease: a search for the uncharacterized mutations. Mamm Genome 2023; 34:1-11. [PMID: 36462057 DOI: 10.1007/s00335-022-09971-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2022] [Accepted: 11/24/2022] [Indexed: 12/05/2022]
Abstract
Wilson disease (WD), a copper metabolism disorder caused by mutations in ATP7B, manifests heterogeneous clinical features. Interestingly, in a fraction of clinically diagnosed WD patients, mutations in ATP7B appears to be missing. In this review we discuss the plausible explanations of this missing heritability and propose a workflow that can identify the hidden mutations. Mutation analyses of WD generally includes targeted sequencing of ATP7B exons, exon-intron boundaries, and rarely, the proximal promoter region. We propose that variants in the distal cis-regulatory elements and/or deep intronic variants that impact splicing might well represent the hidden mutations. Heterozygous del/ins that remain refractory to conventional PCR-sequencing method may also represent such mutations. In this review, we also hypothesize that mutations in the key copper metabolism genes, like, ATOX1, COMMD1, and SLC31A1, could possibly lead to a WD-like phenotype. In fact, WD does present overlapping symptoms with other rare genetic disorders; hence, the possibility of a misdiagnosis and thus adding to missing heritability cannot be excluded. In this regard, it seems that whole-genome analysis will provide a comprehensive and rapid molecular diagnosis of WD. However, considering the associated cost for such a strategy, we propose an alternative customized screening schema of WD which include targeted sequencing of ATP7B locus as well as other key copper metabolism genes. Success of such a schema has been tested in a pilot study.
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Affiliation(s)
- Shubhrajit Roy
- S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India
- Post-doctoral Fellow, Physiology Department, Johns Hopkins University, Baltimore, USA
| | - Sampurna Ghosh
- Department of Genetics, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada
| | - Jharna Ray
- S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India
| | - Kunal Ray
- Ramakrishna Mission Vivekananda Educational and Research Institute, Narendrapur, Kolkata, 700 103, India.
| | - Mainak Sengupta
- Department of Genetics, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India.
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3
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Jia S, Li X, Zhang W, Zhang B, Wu Z, Duan W, Ou X, Zhou D, Huang J. Laboratory and clinical evaluation of a microarray for the detection of ATP7B mutations in Wilson disease in China. J Clin Lab Anal 2022; 36:e24735. [PMID: 36253962 DOI: 10.1002/jcla.24735] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2022] [Revised: 10/02/2022] [Accepted: 10/05/2022] [Indexed: 11/05/2022] Open
Abstract
BACKGROUND AND OBJECTIVE Wilson disease (WD) is an autosomal recessive copper metabolic disorder caused by mutations in ATP7B. Sanger sequencing is currently used for ATP7B variant identification. However, the ATP7B gene contains 21 exons, which makes sequencing of the entire gene both complex and time-consuming. Therefore, a simpler assay is urgently needed. METHODS We performed a laboratory and clinical evaluation of an oligonucleotide microarray for the detection of 24 ATP7B recurrent mutations (except p.P992L) in Chinese patients with WD. RESULTS The accuracy of the microarray was evaluated by screening for ATP7B mutations in 126 patients including 106 suspected WD samples and 20 patients with other liver diseases as negative control. Results were confirmed by Sanger sequencing. We established a reliable microarray system for the rapid detection of the 24 ATP7B mutations, with a sensitivity of 30 ng/test genomic DNA and specificity of 100% for all loci; the coefficient of variation in repeatability tests was <10%. Clinical evaluation showed an overall concordance between the microarray detection and sequencing of 100%, and 81.13% (86/106) of suspected WD cases showed ATP7B mutations by microarray detection. Microarray and Sanger sequencing identified p.R778L (50.94%), p.A874V (17.92%), p.P992L (11.32%), p.V1106I (11.32%), and p.I1148T (6.60%) as the most common mutations in WD patients. CONCLUSIONS Our microarray system is customizable and easily used for high-throughput detection of certain recurrent ATP7B mutations, providing a simpler method suitable for WD genetic diagnosis in China.
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Affiliation(s)
- Siyu Jia
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.,Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China.,Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Xiaojin Li
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.,Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China.,Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Wei Zhang
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China.,Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China.,Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
| | - Bei Zhang
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.,Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China.,Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Zhen Wu
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China.,Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China.,Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
| | - Weijia Duan
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China.,Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China.,Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
| | - Xiaojuan Ou
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China.,Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China.,Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
| | - Donghu Zhou
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.,Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China.,Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Jian Huang
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.,Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China.,Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China.,Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
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4
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Panzer M, Viveiros A, Schaefer B, Baumgartner N, Seppi K, Djamshidian A, Todorov T, Griffiths WJH, Schott E, Schuelke M, Eurich D, Stättermayer AF, Bomford A, Foskett P, Vodopiutz J, Stauber R, Pertler E, Morell B, Tilg H, Müller T, Kiechl S, Jimenez‐Heredia R, Weiss KH, Hahn SH, Janecke A, Ferenci P, Zoller H. Synonymous mutation in adenosine triphosphatase copper-transporting beta causes enhanced exon skipping in Wilson disease. Hepatol Commun 2022; 6:1611-1619. [PMID: 35271763 PMCID: PMC9234614 DOI: 10.1002/hep4.1922] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/10/2021] [Revised: 11/25/2021] [Accepted: 12/22/2021] [Indexed: 12/21/2022] Open
Abstract
Wilson disease (WD) is caused by biallelic pathogenic variants in adenosine triphosphatase copper-transporting beta (ATP7B); however, genetic testing identifies only one or no pathogenic ATP7B variant in a number of patients with WD. Synonymous single-nucleotide sequence variants have been recognized as pathogenic in individual families. The aim of the present study was to evaluate the prevalence and disease mechanism of the synonymous variant c.2292C>T (p.Phe764=) in WD. A cohort of 280 patients with WD heterozygous for a single ATP7B variant was investigated for the presence of c.2292C>T (p.Phe764=). In this cohort of otherwise genetically unexplained WD, the allele frequency of c.2292C>T (p.Phe764=) was 2.5% (14 of 560) compared to 7.1 × 10-6 in the general population (2 of 280,964 in the Genome Aggregation Database; p < 10-5 ; Fisher exact test). In an independent United Kingdom (UK) cohort, 2 patients with WD homozygous for p.Phe764= were identified. RNA analysis of ATP7B transcripts from patients homozygous or heterozygous for c.2292C>T and control fibroblasts showed that this variant caused high expression of an ATP7B transcript variant lacking exon 8. Conclusion: The synonymous ATP7B variant c.2292C>T (p.Phe764=) causes abnormal messenger RNA processing of ATP7B transcripts and is associated with WD in compound heterozygotes and homozygotes.
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Affiliation(s)
- Marlene Panzer
- Department of Medicine IMedical University of InnsbruckInnsbruckAustria
- VASCage Research Center on Vascular Ageing and StrokeInnsbruckAustria
| | - André Viveiros
- Department of Medicine IMedical University of InnsbruckInnsbruckAustria
| | - Benedikt Schaefer
- Department of Medicine IMedical University of InnsbruckInnsbruckAustria
| | - Nadja Baumgartner
- Department of Medicine IMedical University of InnsbruckInnsbruckAustria
| | - Klaus Seppi
- Department of NeurologyMedical University of InnsbruckInnsbruckAustria
| | - Atbin Djamshidian
- Department of NeurologyMedical University of InnsbruckInnsbruckAustria
| | - Theodor Todorov
- Department of Medical Genetics and Molecular BiologyUniversity Hospital LozenetzSofiaBulgaria
| | - William J. H. Griffiths
- Cambridge Liver UnitCambridge University Hospitals National Health Service (NHS) Foundation TrustCambridgeUK
| | - Eckart Schott
- Helios Klinikum Emil von Behring GmbHKlinik für Innere Medizin IIBerlinGermany
| | - Markus Schuelke
- Department of NeuropediatricsCharité University Medical Center BerlinBerlinGermany
| | - Dennis Eurich
- Department of SurgeryCharité University Medical Center BerlinBerlinGermany
| | - Albert Friedrich Stättermayer
- Division of Gastroenterology and HepatologyDepartment of Internal Medicine IIIMedical University ViennaViennaAustria
| | - Adrian Bomford
- Institute of Liver StudiesKing's College Hospital NHS Foundation TrustLondonUK
| | - Pierre Foskett
- Institute of Liver StudiesKing's College Hospital NHS Foundation TrustLondonUK
| | - Julia Vodopiutz
- Division of Pediatric Pulmology, Allergology, and EndocrinologyDepartment of Pediatrics and Adolescent MedicineComprehensive Center for PediatricsMedical University of ViennaViennaAustria
| | - Rudolf Stauber
- Division of Gastroenterology and HepatologyDepartment of Internal MedicineMedical University of GrazGrazAustria
| | - Elke Pertler
- Department of Medicine IMedical University of InnsbruckInnsbruckAustria
- Christian Doppler Laboratory on Iron and Phosphate BiologyInnsbruckAustria
| | - Bernhard Morell
- Department of Gastroenterology and HepatologyUniversity Hospital ZurichZurichSwitzerland
| | - Herbert Tilg
- Department of Medicine IMedical University of InnsbruckInnsbruckAustria
| | - Thomas Müller
- Department of Pediatrics IMedical University of InnsbruckInnsbruckAustria
| | - Stefan Kiechl
- Department of NeurologyMedical University of InnsbruckInnsbruckAustria
| | - Raul Jimenez‐Heredia
- Ludwig Boltzmann Institute for Rare and Undiagnosed DiseasesViennaAustria
- Department of Pediatrics and Adolescent MedicineMedical University of ViennaViennaAustria
- St. Anna Children's Cancer Research InstituteViennaAustria
| | - Karl Heinz Weiss
- Internal MedicineKrankenhaus Salem der Evangelischen StadtmissionHeidelbergGermany
| | - Si Houn Hahn
- University of Washington School of MedicineSeattle Children’s HospitalSeattleWashingtonUSA
| | - Andreas Janecke
- Department of Pediatrics IMedical University of InnsbruckInnsbruckAustria
| | - Peter Ferenci
- Division of Gastroenterology and HepatologyDepartment of Internal Medicine IIIMedical University ViennaViennaAustria
| | - Heinz Zoller
- Department of Medicine IMedical University of InnsbruckInnsbruckAustria
- Christian Doppler Laboratory on Iron and Phosphate BiologyInnsbruckAustria
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Huang C, Fang M, Xiao X, Gao Z, Wang Y, Gao C. Genetic studies discover novel coding and non-coding mutations in patients with Wilson's disease in China. J Clin Lab Anal 2022; 36:e24459. [PMID: 35470480 PMCID: PMC9169201 DOI: 10.1002/jcla.24459] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2022] [Revised: 04/02/2022] [Accepted: 04/16/2022] [Indexed: 11/13/2022] Open
Abstract
OBJECTIVES Wilson disease (WD) is a rare autosomal recessive genetic disorder associated with various mutations in the ATP7B gene and leads to significant disability or death if untreated. Early diagnosis and proper therapy usually predict a good prognosis, especially in pre-symptomatic WD. Genetic testing provides an accurate and effective diagnostic method for the early diagnosis of WD. METHODS We recruited 18 clinically diagnosed WD patients from 16 unrelated families and two independent individuals. The next-generation sequencing of the ATP7B gene was performed. The 293T cell lines were divided into wild-type (WT) ATP7B and mutated ATP7B groups. Cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay and apoptosis was detected by Annexin V/propidium iodide (PI) assays. RESULTS Pedigree analysis showed that compound heterozygous variants (17/18, 94.44%) were present in the majority of WD patients. A total of 33 ATP7B gene variants were identified, including three variants with uncertain significance (VUS) [two splice mutations (c.51+2T>G, c.1543+40G>A) and one frameshift mutation (c.3532_3535del)]. The CCK-8 and apoptosis assays demonstrated that the VUS of ATP7B could significantly affect the transportation of copper. CONCLUSIONS The study revealed genetic defects of 16 Chinese families and two independent individuals with WD, which enriched the mutation spectrum of the ATP7B gene worldwide and provided valuable information for studying the mutation types of ATP7B in the Chinese populations. Genetic testing in WD patients is necessary to shorten the time to initiate therapy, reduce damage to the liver and improve the prognosis.
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Affiliation(s)
- Chenjun Huang
- Department of Clinical Laboratory Medicine CenterYueyang Hospital of Integrated Traditional Chinese and Western MedicineShanghai University of Traditional Chinese MedicineShanghaiChina
| | - Meng Fang
- Department of Laboratory MedicineShanghai Eastern Hepatobiliary Surgery HospitalShanghaiChina
| | - Xiao Xiao
- Department of Clinical Laboratory Medicine CenterYueyang Hospital of Integrated Traditional Chinese and Western MedicineShanghai University of Traditional Chinese MedicineShanghaiChina
| | - Zhiyuan Gao
- Department of Clinical Laboratory Medicine CenterYueyang Hospital of Integrated Traditional Chinese and Western MedicineShanghai University of Traditional Chinese MedicineShanghaiChina
| | - Ying Wang
- Department of Laboratory MedicineShanghai Eastern Hepatobiliary Surgery HospitalShanghaiChina
| | - Chunfang Gao
- Department of Clinical Laboratory Medicine CenterYueyang Hospital of Integrated Traditional Chinese and Western MedicineShanghai University of Traditional Chinese MedicineShanghaiChina
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6
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Li M, Ma J, Wang W, Yang X, Luo K. Mutation analysis of the ATP7B gene and genotype-phenotype correlation in Chinese patients with Wilson disease. BMC Gastroenterol 2021; 21:339. [PMID: 34470610 PMCID: PMC8411542 DOI: 10.1186/s12876-021-01911-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/24/2020] [Accepted: 08/23/2021] [Indexed: 11/10/2022] Open
Abstract
AIM To discover the novel ATP7B mutations in 103 southern Chinese patients with Wilson disease (WD), and to determine the spectrum and frequency of mutations in the ATP7B gene and genotype-phenotype correlation in a large-scale sample of Chinese WD patients. METHODS One hundred three WD patients from 101 unrelated families in southern China were enrolled in this study. Genomic DNA was extracted from the peripheral blood. Direct sequencing of all 21 exons within ATP7B was performed. Subsequently, an extensive study of the overall spectrum and frequency of ATP7B mutations and genotype-phenotype correlation was performed in all Chinese patients eligible from the literature, combined with the current southern group. RESULTS In 103 patients with WD, we identified 48 different mutations (42 missense mutations, 4 nonsense mutations and 2 frameshifts). Of these, 3 mutations had not been previously reported: c.1510_1511insA, c.2233C>A (p.Leu745Met) and c.3824T>C (p.Leu1275Ser). The c.2333G>T (p.Arg778 Leu) at exon 8, was the most common mutation with an allelic frequency of 18.8%, followed by c.2975C>T (p.Pro992Leu) at exon 13, with an allelic frequency of 13.4%. In the comprehensive study, 233 distinct mutations were identified, including 154 missense mutations, 23 nonsense mutations and 56 frameshifts. Eighty-five variants were identified as novel mutations. The c.2333G>T (p.Arg778 Leu) and c.2975C>T (p.Pro992Leu) were the most common mutations, with allelic frequencies of 28.6% and 13.0%, respectively. Exons 8, 12, 13, 16 and 18 were recognised as hotspot exons. Phenotype-genotype correlation analysis suggested that c.2333G>T (p.Arg778 Leu) was significantly associated with lower levels of serum ceruloplasmin (P = 0.034). c.2975C>T (p.Pro992Leu) was correlated with earlier age of disease onset (P = 0.002). Additionally, we found that the c.3809A>G (p.Asn1270Ser) mutation significantly indicated younger onset age (P = 0.012), and the c.3884C>T (p.Ala1295Val) mutation at exon 18 was significantly associated with hepatic presentation (P = 0.048). Moreover, the patients with mixed presentation displayed the initial WD features at an older onset age than the groups with either liver disease or neurological presentation (P = 0.039, P = 0.015, respectively). No significant difference was observed in the presence of KF rings among the three groups with different clinical manifestations. CONCLUSION In this study, we identified three novel mutations in 103 WD patients from the southern part of China, which could enrich the previously established mutational spectrum of the ATP7B gene. Moreover, we tapped into a large-scale study of a Chinese WD cohort to characterise the overall phenotypic and genotypic spectra and assess the association between genotype and phenotype, which enhances the current knowledge about the population genetics of WD in China.
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Affiliation(s)
- Mingming Li
- Department of Infectious Diseases, Institute of Hepatology, The Second Xiangya Hospital, Central South University, Changsha, 410011, Hunan Province, China
| | - Jing Ma
- Department of Infectious Diseases, Institute of Hepatology, The Second Xiangya Hospital, Central South University, Changsha, 410011, Hunan Province, China
| | - Wenlong Wang
- Department of Infectious Diseases, Institute of Hepatology, The Second Xiangya Hospital, Central South University, Changsha, 410011, Hunan Province, China
| | - Xu Yang
- Department of Infectious Diseases, Institute of Hepatology, The Second Xiangya Hospital, Central South University, Changsha, 410011, Hunan Province, China
| | - Kaizhong Luo
- Department of Infectious Diseases, Institute of Hepatology, The Second Xiangya Hospital, Central South University, Changsha, 410011, Hunan Province, China.
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7
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Investigation of the Wilson gene ATP7B transcriptional start site and the effect of core promoter alterations. Sci Rep 2021; 11:7674. [PMID: 33828154 PMCID: PMC8027023 DOI: 10.1038/s41598-021-87000-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Accepted: 03/22/2021] [Indexed: 02/01/2023] Open
Abstract
Pathogenic genetic variants in the ATP7B gene cause Wilson disease, a recessive disorder of copper metabolism showing a significant variability in clinical phenotype. Promoter mutations have been rarely reported, and controversial data exist on the site of transcription initiation (the core promoter). We quantitatively investigated transcription initiation and found it to be located in immediate proximity of the translational start. The effects human single-nucleotide alterations of conserved bases in the core promoter on transcriptional activity were moderate, explaining why clearly pathogenic mutations within the core promoter have not been reported. Furthermore, the core promoter contains two frequent polymorphisms (rs148013251 and rs2277448) that could contribute to phenotypical variability in Wilson disease patients with incompletely inactivating mutations. However, neither polymorphism significantly modulated ATP7B expression in vitro, nor were copper household parameters in healthy probands affected. In summary, the investigations allowed to determine the biologically relevant site of ATP7B transcription initiation and demonstrated that genetic variations in this site, although being the focus of transcriptional activity, do not contribute significantly to Wilson disease pathogenesis.
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Zhou X, Zhou W, Wang C, Wang L, Jin Y, Jia Z, Liu Z, Zheng B. A Comprehensive Analysis and Splicing Characterization of Naturally Occurring Synonymous Variants in the ATP7B Gene. Front Genet 2021; 11:592611. [PMID: 33719328 PMCID: PMC7947925 DOI: 10.3389/fgene.2020.592611] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Accepted: 12/16/2020] [Indexed: 12/18/2022] Open
Abstract
Next-generation sequencing is effective for the molecular diagnosis of genetic diseases. However, the identification of the clinical significance of synonymous variants remains a challenge. Our previous study showed that some synonymous variants in ATP7B gene produced splicing disruptions, leading to Wilson disease (WD). To test the hypothesis that synonymous variants of ATP7B cause abnormal splicing by disrupting authentic splice sites or splicing regulatory elements, we used computational tools and minigene assays to characterize 253 naturally occurring ATP7B gene synonymous variants in this study. Human Splicing Finder (HSF) and ESE Finder 3.0 were used to predict the impact of these rare synonymous variants on pre-mRNA splicing. Then, we cloned 14 different wild-type Minigene_ATP7B_ex constructs for in vitro minigene assay, including 16 exons of ATP7B gene. After computational prediction, 85 candidate variants were selected to be introduced into the corresponding Minigene_ATP7B_ex constructs for splicing assays. Using this two-step procedure, we demonstrated that 11 synonymous variants in ExAc database (c.1620C>T, c.3888C>T, c.1554C>T, c.1677C>T, c.1830G>A, c.1875T>A, c.2826C>A, c.4098G>A, c.2994C>T, c.3243G>A, and c.3747G>A) disrupted RNA splicing in vitro, and two (c.1620C>T and c.3243G>A) of these caused a complete exon skipping. The results not only provided a reliable experimental basis for the genetic diagnosis of WD patients but also offered some new insights into the pathogenicity of synonymous variants in genetic diseases.
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Affiliation(s)
- Xiaoying Zhou
- Department of Gastroenterology, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Wei Zhou
- Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Chunli Wang
- Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Lan Wang
- Department of Gastroenterology, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Yu Jin
- Department of Gastroenterology, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Zhanjun Jia
- Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Zhifeng Liu
- Department of Gastroenterology, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Bixia Zheng
- Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China
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Poon KS, Teo ZH, Yap JH, Koay ES, Tan K. Challenges in molecular diagnosis of Wilson disease: viewpoint from the clinical laboratory. J Clin Pathol 2019; 73:231-234. [PMID: 31796634 DOI: 10.1136/jclinpath-2019-206054] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2019] [Revised: 09/01/2019] [Accepted: 09/13/2019] [Indexed: 01/11/2023]
Affiliation(s)
- Kok-Siong Poon
- Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital, Singapore
| | | | | | - Evelyn Sc Koay
- Department of Pathology, National University of Singapore, Singapore
| | - Karen Tan
- Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospitals, Singapore
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10
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Li X, Zhang W, Zhou D, Lv T, Xu A, Wang H, Zhao X, Zhang B, Li Y, Jia S, Wang Y, Wang X, Wu Z, Duan W, Wang Q, Nan Y, Shang J, Jiang W, Chen Y, Zheng S, Liu M, Sun L, You H, Jia J, Ou X, Huang J. Complex ATP7B mutation patterns in Wilson disease and evaluation of a yeast model for functional analysis of variants. Hum Mutat 2019; 40:552-565. [PMID: 30702195 DOI: 10.1002/humu.23714] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2018] [Revised: 01/21/2019] [Accepted: 01/22/2019] [Indexed: 12/16/2022]
Abstract
Wilson disease (WD) is a rare autosomal recessive genetic disorder that is associated with various mutations in the ATP7B gene. Although ATP7B variants are frequently identified, the exact mutation patterns remain unknown because of the absence of pedigree studies, and the functional consequences of individual ATP7B variants remain to be clarified. In this study, we recruited 65 clinically diagnosed WD patients from 60 unrelated families. Pedigree analysis showed that besides several ATP7B homozygous variants (8/65, 12.3%), compound heterozygous variants (43/65, 66.2%) were present in the majority of WD patients. There were 20% of the patients had one (12/65, 18.5%) or multiple (1/65, 1.5%) variants in only a single allele, characterized by a high ratio of splicing or frameshift variants. Nine ATP7B variants were cloned into the pAG426GPD yeast expression vector to evaluate their functional consequences, and the results suggested different degrees of functional disruption from mild or uncertain to severe, consistent with the corresponding phenotypes. Our study revealed the complex ATP7B mutation patterns in WD patients and the applicability of a yeast model system to the evaluation of the functional consequences of ATP7B variants, which is essential for WD cases that are difficult to interpret.
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Affiliation(s)
- Xiaojin Li
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Wei Zhang
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Donghu Zhou
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Tingxia Lv
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Anjian Xu
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Hejing Wang
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Xinyan Zhao
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Bei Zhang
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Yanmeng Li
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Siyu Jia
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Yu Wang
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Xiaoming Wang
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Zhen Wu
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Weijia Duan
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Qianyi Wang
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Yuemin Nan
- Department of Traditional and Western Medical Hepatology, Third Hospital of Hebei Medical University, Shijiazhuang, China
| | - Jia Shang
- Department of Infectious Diseases, Henan Provincial People's Hospital, Zhengzhou, Henan, China
| | - Wei Jiang
- Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Yongpeng Chen
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
| | - Sujun Zheng
- Artificial Liver Center, Beijing You-An Hospital, Capital Medical University, Beijing, China
| | - Mei Liu
- Artificial Liver Center, Beijing You-An Hospital, Capital Medical University, Beijing, China
| | - Liying Sun
- Liver Transplant Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
| | - Hong You
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Jidong Jia
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Xiaojuan Ou
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
| | - Jian Huang
- Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Beijing Key Laboratory of Translational Medicine on Liver Cirrhosis, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
- Clinical Research Center for Rare Liver Diseases, Capital Medical University, Beijing, China
- Liver Research Center, National Clinical Research Center for Digestive Diseases, Beijing, China
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Chen HI, Jagadeesh KA, Birgmeier J, Wenger AM, Guturu H, Schelley S, Bernstein JA, Bejerano G. An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease. Eur J Hum Genet 2018; 26:1810-1818. [PMID: 30087448 PMCID: PMC6244090 DOI: 10.1038/s41431-018-0221-4] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2017] [Revised: 05/09/2018] [Accepted: 06/26/2018] [Indexed: 12/16/2022] Open
Abstract
Approximately 2% of the human genome accounts for protein-coding genes, yet most known Mendelian disease-causing variants lie in exons or splice sites. Individuals who symptomatically present with monogenic disorders but do not possess function-altering variants in the protein-coding regions of causative genes may harbor variants in the surrounding gene regulatory domains. We present such a case: a male of Afghani descent was clinically diagnosed with Wilson Disease-a disorder of systemic copper buildup-but was found to have no function-altering coding variants in ATP7B (ENST00000242839.4), the typically causative gene. Our analysis revealed the homozygous variant chr13:g.52,586,149T>C (NC_000013.10, hg19) 676 bp into the ATP7B promoter, which disrupts a metal regulatory transcription factor 1 (MTF1) binding site and diminishes expression of ATP7B in response to copper intake, likely resulting in Wilson Disease. Our approach to identify the causative variant can be generalized to systematically discover function-altering non-coding variants underlying disease and motivates evaluation of gene regulatory variants.
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Affiliation(s)
- Heidi I Chen
- Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA
| | - Karthik A Jagadeesh
- Department of Computer Science, Stanford University School of Engineering, Stanford, CA, USA
| | - Johannes Birgmeier
- Department of Computer Science, Stanford University School of Engineering, Stanford, CA, USA
| | - Aaron M Wenger
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA
| | - Harendra Guturu
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA
| | - Susan Schelley
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA
| | - Jonathan A Bernstein
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
| | - Gill Bejerano
- Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA.
- Department of Computer Science, Stanford University School of Engineering, Stanford, CA, USA.
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
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Abstract
Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism. Its incidence is higher in China than in western countries. ATP7B is the causative gene and encodes a P-type ATPase, which participates in the synthesis of holoceruloplasmin and copper excretion. Disease-causing variants of ATP7B disrupt the normal structure or function of the enzyme and cause copper deposition in multiple organs, leading to diverse clinical manifestations. Given the variety of presentations, misdiagnosis is not rare. Genetic diagnosis plays an important role and has gradually become a routine test in China. The first Chinese spectrum of disease-causing mutations of ATP7B has been established. As a remediable hereditary disorder, most WD patients have a good prognosis with an early diagnosis and chelation treatment. However, clinical trials are relatively few in China, and most treatments are based on the experience of experts and evidences from other countries. It is necessary to study and develop appropriate regimens specific for Chinese WD patients.
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Affiliation(s)
- Juan-Juan Xie
- Department of Neurology and Research Center of Neurology in the Second Affiliated Hospital, and the Collaborative Innovation Center for Brain Science, Zhejiang University School of Medicine, Hangzhou, 310009, China
| | - Zhi-Ying Wu
- Department of Neurology and Research Center of Neurology in the Second Affiliated Hospital, and the Collaborative Innovation Center for Brain Science, Zhejiang University School of Medicine, Hangzhou, 310009, China.
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Lv T, Li X, Zhang W, Zhao X, Ou X, Huang J. Recent advance in the molecular genetics of Wilson disease and hereditary hemochromatosis. Eur J Med Genet 2016; 59:532-539. [PMID: 27592149 DOI: 10.1016/j.ejmg.2016.08.011] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2016] [Revised: 07/12/2016] [Accepted: 08/31/2016] [Indexed: 02/07/2023]
Abstract
Metabolic liver diseases such as Wilson disease (WD) and hereditary hemochromatosis (HH) possess complicated pathogenesis and typical hereditary characteristics with the hallmarks of a deficiency in metal metabolism. Mutations in genes encoding ATPase, Cu + transporting, beta polypeptide (ATP7B) and hemochromatosis (HFE) or several non-HFE genes are considered to be causative for WD and HH, respectively. Although the identification of novel mutations in ATP7B for WD and HFE or the non-HFE genes for HH has increased, especially with the application of whole genome sequencing technology in recent years, the biological function of the identified mutations, as well as genotype-phenotype correlations remain to be explored. Further analysis of the causative gene mutation would be critical to clarify the mechanisms underlying specific disease phenotypes. In this review, we therefore summarize the recent advances in the molecular genetics of WD and HH including the updated mutation spectrums and the correlation between genotype and phenotype, with an emphasis on biological functional studies of the individual mutations identified in WD and HH. The weakness of the current functional studies and analysis for the clinical association of the individual mutation was also discussed. These works are essential for the understanding of the association between genotypes and phenotypes of these inherited metabolic liver diseases.
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Affiliation(s)
- Tingxia Lv
- Liver Research Center, Experimental Center, Beijing Friendship Hospital, Capital Medical University, 95 Yong-an Road, Xuan-wu District, Beijing, 100050, China.
| | - Xiaojin Li
- Liver Research Center, Experimental Center, Beijing Friendship Hospital, Capital Medical University, 95 Yong-an Road, Xuan-wu District, Beijing, 100050, China.
| | - Wei Zhang
- Liver Research Center, Beijing Friendship Hospital, Capital Medical University, 95 Yong-an Road, Xuan-wu District, Beijing, 100050, China.
| | - Xinyan Zhao
- Liver Research Center, Beijing Friendship Hospital, Capital Medical University, 95 Yong-an Road, Xuan-wu District, Beijing, 100050, China.
| | - Xiaojuan Ou
- Liver Research Center, Beijing Friendship Hospital, Capital Medical University, 95 Yong-an Road, Xuan-wu District, Beijing, 100050, China.
| | - Jian Huang
- Liver Research Center, Experimental Center, Beijing Friendship Hospital, Capital Medical University, 95 Yong-an Road, Xuan-wu District, Beijing, 100050, China.
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Chandhok G, Horvath J, Aggarwal A, Bhatt M, Zibert A, Schmidt HHJ. Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines. World J Gastroenterol 2016; 22:4109-4119. [PMID: 27122662 PMCID: PMC4837429 DOI: 10.3748/wjg.v22.i16.4109] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2015] [Revised: 01/24/2016] [Accepted: 02/22/2016] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture.
METHODS: The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined.
RESULTS: Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability.
CONCLUSION: The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines.
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Abstract
CONTEXT Geographic distribution of ATP7B mutations in different populations. OBJECTIVE To summarise common mutations in the ATP7B gene and graphically illustrate their prevalence in different populations. METHODS A literature search was done using PubMed and the Wilson Disease Mutation Database (http://www.wilsondisease.med.ualberta.ca/database). RESULTS p.His1069Gln is the most prevalent mutation seen in Europe. In the Mediterranean countries, the array of prevalent mutations is different from the rest of Europe. In Far East Asian countries, the mutation p.Arg778Leu is the most common. In India, no single mutation seems to be dominant, owing to the vast ethnic diversity of the country. The p.Cys271* mutation is dominant in the east, west and south, but not reported in the north. In the Middle East, data from Saudi Arabia shows the p.Gln1399Arg mutation as the most prevalent. In the US, the p.His1069Gln is dominant, whereas in Brazil the mutation c.3402delC dominates. CONCLUSION Clinical features in WD patients can be misleading and often absent. Genetic testing is used to confirm the diagnosis. However, owing to the large gene size and vast diversity in the mutations, genetic testing can be time-consuming and tedious. This study reviews ATP7B mutations seen in different populations and can help develop time-saving methods and expediate the process of genetic analysis of WD.
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Affiliation(s)
- Amanda Gomes
- a Department of Dietetics and Nutrition , Harokopio University of Athens , Athens , Greece and.,b Wilson Disease Clinic, Kokilaben Dhirubhai Ambani Hospital & Medical Research Institute , Mumbai , India
| | - George V Dedoussis
- a Department of Dietetics and Nutrition , Harokopio University of Athens , Athens , Greece and
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Lin YJ, Ho TJ, Lin TH, Hsu WY, Huang SM, Liao CC, Lai CH, Liu X, Tsang H, Lai CC, Tsai FJ. P-coumaric acid regulates exon 12 splicing of the ATP7B gene by modulating hnRNP A1 protein expressions. Biomedicine (Taipei) 2015; 5:10. [PMID: 26048696 PMCID: PMC4502042 DOI: 10.7603/s40681-015-0010-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2015] [Accepted: 03/20/2015] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND Wilson's disease (WD) is a genetic disorder involving the metabolism of copper. WD patients exhibit a wide range of disease phenotypes, including Kayser-Fleischer rings in the cornea, predominant progressive hepatic disease, neurological diseases, and/or psychiatric illnesses, among others. Patients with exon12 mutations of the ATP7B gene have progressive hepatic disease. An ATP7B gene that lacks exon12 retains 80% of its copper transport activities, suggesting that alternative splicing of ATP7B gene may provide alternative therapeutic ways for patients with inherited sequence variants and mutations of this gene. PURPOSE We aimed to search for possible Chinese herbs and related compounds for modulating ATP7B premRNA splicing. METHODS We used an ATP7B exon11-12-13 mini-gene vector as a model and screened 18 Chinese herbal extracts and four compounds from Schizonepeta to determine their effects on ATP7B pre-mRNA splicing in vitro. RESULTS We found that Schizonepeta demonstrated the greatest potential for alternative splicing activity. Specifically, we found that p-coumaric acid from this herb enhanced ATP7B exon12 exclusion through the down-regulation of heterogeneous ribonucleoprotein (hnRNP) A1 protein expressions. CONCLUSION These results suggest that there are herbs or herb-related compounds that could modify the alternative splicing of the ATP7B gene via a mechanism that regulates pre-mRNA splicing.
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Affiliation(s)
- Ying-Ju Lin
- Genetic Center, Department of Medical Research, China Medical University Hospital, No. 2, Yuh-Der Road, 404, Taichung, Taiwan,
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Usta J, Wehbeh A, Rida K, El-Rifai O, Estiphan TA, Majarian T, Barada K. Phenotype-genotype correlation in Wilson disease in a large Lebanese family: association of c.2299insC with hepatic and of p. Ala1003Thr with neurologic phenotype. PLoS One 2014; 9:e109727. [PMID: 25390358 PMCID: PMC4229086 DOI: 10.1371/journal.pone.0109727] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2014] [Accepted: 09/04/2014] [Indexed: 12/15/2022] Open
Abstract
Genotype phenotype correlations in Wilson disease (WD) are best established in homozygous patients or in compound heterozygous patients carrying the same set of mutations. We determined the clinical phenotype of patients with WD carrying the c.2298_2299insC in Exon 8 (c.2299insC) or the p. Ala1003Thr missense substitution in Exon 13 mutations in the homozygous or compound heterozygous state. We investigated 76 members of a single large Lebanese family. Their genotypes were determined, and clinical assessments were carried out for affected subjects. We also performed a literature search retrieving the phenotypes of patients carrying the same mutations of our patients in the homozygous or compound heterozygous state. There were 7 consanguineous marriages in this family and the prevalence of WD was 8.9% and of carriers of ATP7B mutation 44.7%. WD was confirmed in 9 out of 76 subjects. All 9 had the c.2299insC mutation, 5 homozygous and 4-compound heterozygous with p. Ala1003Thr. Six of our patients had hepatic, 2 had neurologic and 1 had asymptomatic phenotype. Based on our data and a literature review, clear phenotypes were reported for 38 patients worldwide carrying the c.2299insC mutation. About 53% of those have hepatic and 29% have neurologic phenotype. Furthermore, there were 10 compound heterozygous patients carrying the p. Ala1003Thr mutation. Among those, 80% having c.2299insC as the second mutation had hepatic phenotype, and all others had neurologic phenotype. We hereby report an association between the c.2299insC mutation and hepatic phenotype and between the p. Ala1003Thr mutation and neurologic phenotype.
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Affiliation(s)
- Julnar Usta
- Department of Biochemistry and Molecular Genetics; Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Antonios Wehbeh
- Faculty of Medicine, American University of Beirut Medical Center, Beirut, Lebanon
| | - Khaled Rida
- Department of Biochemistry and Molecular Genetics; Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Omar El-Rifai
- Department of Biochemistry and Molecular Genetics; Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | | | - Tamar Majarian
- Department of Biochemistry and Molecular Genetics; Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Kassem Barada
- Division of Gastroenterology, Department of Internal Medicine, American University of Beirut Medical Center, Faculty of Medicine, Beirut, Lebanon
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Mukherjee S, Dutta S, Majumdar S, Biswas T, Jaiswal P, Sengupta M, Bhattacharya A, Gangopadhyay PK, Bavdekar A, Das SK, Ray K. Genetic defects in Indian Wilson disease patients and genotype-phenotype correlation. Parkinsonism Relat Disord 2013; 20:75-81. [PMID: 24094725 DOI: 10.1016/j.parkreldis.2013.09.021] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2013] [Revised: 08/15/2013] [Accepted: 09/17/2013] [Indexed: 01/20/2023]
Abstract
Wilson disease (WD) is caused by defects in ATP7B gene due to impairment of normal function of the copper transporting P-type ATPase. This study describes a comprehensive genetic analysis of 199 Indian WD patients including mutations detected in our previous studies, undertakes functional assessment of the nucleotide variants in ATP7B promoter and correlates genotype with disease phenotype. The patient cohort harbors a total of 10 common and 48 rare mutations in the coding region of ATP7B including 21 novel changes. The common mutations represent 74% of characterized coding mutant alleles with p.C271X (63/260) and p.G1101R (7/31) being the most prevalent in eastern and western Indian patients, respectively. The mutation spectrum between east and west is mostly different with only three mutations (p.G1061E, p.N1270S and p.A1049A-fs) being shared between both the groups. Eight novel and 10 reported variants have been detected in the promoter and non-coding regions (5' and 3'UTRs) of ATP7B. Promoter reporter assay demonstrated that 3 novel variants and 5 reported polymorphisms alter the gene expression to a considerable extent; hence might play important role in ATP7B gene regulation. We devised the neurological involvement score to capture the spectrum of neurological involvement in WD patients. By utilizing the age at onset, neurological involvement score and ATP7B mutation background, we generated a genotype-phenotype matrix that could be effectively used to depict the phenotypic spectra of WD affected individuals and serve as a platform to identify prospective "outliers" to be investigated for their remarkable phenotypic divergence.
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Affiliation(s)
| | - Shruti Dutta
- CSIR-Indian Institute of Chemical Biology, Kolkata, India
| | | | | | - Preeti Jaiswal
- CSIR-Indian Institute of Chemical Biology, Kolkata, India
| | | | | | | | | | | | - Kunal Ray
- CSIR-Indian Institute of Chemical Biology, Kolkata, India.
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Medici V, Shibata NM, Kharbanda KK, LaSalle JM, Woods R, Liu S, Engelberg JA, Devaraj S, Török NJ, Jiang JX, Havel PJ, Lönnerdal B, Kim K, Halsted CH. Wilson's disease: changes in methionine metabolism and inflammation affect global DNA methylation in early liver disease. Hepatology 2013; 57:555-65. [PMID: 22945834 PMCID: PMC3566330 DOI: 10.1002/hep.26047] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/20/2012] [Accepted: 08/14/2012] [Indexed: 12/18/2022]
Abstract
UNLABELLED Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilson's disease (WD) through the inhibition of S-adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S-adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx-j mouse (tx-j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down-regulated at baseline in tx-j mice, further down-regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx-j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf-α) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-α and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels. CONCLUSION Reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx-j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD.
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Affiliation(s)
- Valentina Medici
- Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of California Davis
| | - Noreene M. Shibata
- Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of California Davis
| | - Kusum K. Kharbanda
- Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska
| | - Janine M. LaSalle
- Department of Medical Microbiology and Immunology, University of California Davis
| | - Rima Woods
- Department of Medical Microbiology and Immunology, University of California Davis
| | - Sarah Liu
- Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of California Davis
| | | | | | - Natalie J. Török
- Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of California Davis
| | - Joy X. Jiang
- Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of California Davis
| | - Peter J. Havel
- Department of Molecular Biosciences, University of California Davis
- Department of Nutrition, University of California Davis
| | - Bo Lönnerdal
- Department of Nutrition, University of California Davis
| | - Kyoungmi Kim
- Department of Public Health Sciences, Division of Biostatistics, University of California Davis
| | - Charles H. Halsted
- Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of California Davis
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Abstract
Wilson disease (WD) is an autosomal recessive inherited disease caused by abnormalities of the copper-transporting protein encoding gene ATP7B. In this study, we examined ATP7B for mutations in 114 individuals of Chinese Han population living in north China who were diagnosed as WD. Totally, we identified 36 mutations and 11 single-nucleotide polymorphisms (SNPs), of which 14 mutations have never been reported previously and 5 were firstly described in Chinese. Among these, p.R778L (21.5%), p.A874V (7.5%) and p.P992L (6.1%) were the most frequent mutations. A genotype of p.L770L+p.R778L+p.P992L was the most frequent triple mutations and two pairs of mutations, p.L770L/p.R778L and p.A874V/p.I929V, were closely related. In addition, a database was established to summarize all ATP7B mutations, including those reported previously and those identified in this study. Popular algorithms were used to predict the functional effects of these mutations, and finally, by comparative genomics approaches, we predicted a group of mutation hot spots for ATP7B. Our study will broaden our knowledge about ATP7B mutations in WD patients in north China, and be helpful for clinical genetic testing.
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Weiss KH, Stremmel W. Evolving perspectives in Wilson disease: diagnosis, treatment and monitoring. Curr Gastroenterol Rep 2012; 14:1-7. [PMID: 22083169 DOI: 10.1007/s11894-011-0227-3] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
Wilson disease (WD), the autosomal recessively inherited copper overload disorder, remains a diagnostic and therapeutic challenge. In the last decade, direct sequencing of the affected gene ATP7B became commercially available, but interpretation of the results still requires careful attention. Thus, a combination of tests reflecting the disturbed copper metabolism is needed to make the final diagnosis. Because of the low disease frequency, the existing treatment concepts are not based on controlled trails. Here, recent outcome reports of larger cohort studies challenge the recommended therapies and call for individualized treatment strategies. The notion, that certain medical regimens may either be insufficient to upkeep copper homeostasis or may lead to a clinically relevant overtreatment, demand a continuous monitoring of patients even after decades of therapy. In this article, we review current diagnostic and therapeutic approaches in WD.
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Affiliation(s)
- Karl Heinz Weiss
- University Hospital Heidelberg, Internal Medicine IV, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany.
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Wei X, Ju X, Yi X, Zhu Q, Qu N, Liu T, Chen Y, Jiang H, Yang G, Zhen R, Lan Z, Qi M, Wang J, Yang Y, Chu Y, Li X, Guang Y, Huang J. Identification of sequence variants in genetic disease-causing genes using targeted next-generation sequencing. PLoS One 2011; 6:e29500. [PMID: 22216297 PMCID: PMC3244462 DOI: 10.1371/journal.pone.0029500] [Citation(s) in RCA: 128] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2011] [Accepted: 11/29/2011] [Indexed: 01/17/2023] Open
Abstract
Background Identification of gene variants plays an important role in research on and diagnosis of genetic diseases. A combination of enrichment of targeted genes and next-generation sequencing (targeted DNA-HiSeq) results in both high efficiency and low cost for targeted sequencing of genes of interest. Methodology/Principal Findings To identify mutations associated with genetic diseases, we designed an array-based gene chip to capture all of the exons of 193 genes involved in 103 genetic diseases. To evaluate this technology, we selected 7 samples from seven patients with six different genetic diseases resulting from six disease-causing genes and 100 samples from normal human adults as controls. The data obtained showed that on average, 99.14% of 3,382 exons with more than 30-fold coverage were successfully detected using Targeted DNA-HiSeq technology, and we found six known variants in four disease-causing genes and two novel mutations in two other disease-causing genes (the STS gene for XLI and the FBN1 gene for MFS) as well as one exon deletion mutation in the DMD gene. These results were confirmed in their entirety using either the Sanger sequencing method or real-time PCR. Conclusions/Significance Targeted DNA-HiSeq combines next-generation sequencing with the capture of sequences from a relevant subset of high-interest genes. This method was tested by capturing sequences from a DNA library through hybridization to oligonucleotide probes specific for genetic disorder-related genes and was found to show high selectivity, improve the detection of mutations, enabling the discovery of novel variants, and provide additional indel data. Thus, targeted DNA-HiSeq can be used to analyze the gene variant profiles of monogenic diseases with high sensitivity, fidelity, throughput and speed.
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Affiliation(s)
- Xiaoming Wei
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Xiangchun Ju
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Xin Yi
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Qian Zhu
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Ning Qu
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Tengfei Liu
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Yang Chen
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Hui Jiang
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Guanghui Yang
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Ruan Zhen
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | | | - Ming Qi
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Jinming Wang
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Yi Yang
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Yuxing Chu
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Xiaoyan Li
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Yanfang Guang
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
| | - Jian Huang
- Beijing Genomics Institute at Shenzhen, Shenzhen, China
- Shanghai-Ministry Key Laboratory of Disease and Health Genomics, National Engineering Center for Biochip at Shanghai, Shanghai, China
- * E-mail:
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Avances en el diagnóstico molecular de la enfermedad de Wilson. GASTROENTEROLOGIA Y HEPATOLOGIA 2011; 34:428-33. [DOI: 10.1016/j.gastrohep.2011.01.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/30/2010] [Accepted: 01/15/2011] [Indexed: 11/23/2022]
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