Identifying regulatory variants in Indian Wilson’s disease patients with missing heritability

Abstract

Wilson’s disease (WD) is a rare autosomal recessive copper metabolism disorder that primarily affects hepatic and neuronal tissues. The condition is caused by mutations in the ATP7B gene. Our group conducted extensive molecular genetic studies, identifying 13 clinically diagnosed Indian WD patients lacking the coding variant of ATP7B and 17 patients with a single mutated allele. We hypothesize that in these patients, unidentified mutations may reside in cis-regulatory elements of ATP7B or that a WD-like phenotype results from the cumulative effect of hypofunctional alleles of other key genes in the copper metabolism pathway. In this study, we employed an established bioinformatic pipeline to identify and screen cis-regulatory elements of ATP7B in WD patients with missing heritability through polymerase chain reaction sequencing. Although no pathogenic variants were identified, our analysis revealed two heterozygous single nucleotide polymorphisms (rs2181891 and rs747781) in two patients. Notably, rs2181891 showed strong regulatory potential with a RegulomeDB score of 1d. The genotype-specific expression profile for rs2181891 revealed it to be an expression quantitative trait locus for ATP7B in the cerebellum. In addition, the Genotype-Tissue Expression portal data suggest that the T allele of rs2181891 is associated with higher expression of ATP7B, making it unlikely to contribute to the WD phenotype. This novel study is the first to identify and screen ATP7B cis-regulatory elements in Indian WD patients with missing heritability.

Keywords

Wilson’s disease / ATP7B / Cis-regulatory elements / Missing heritability

Funding

Shubhrajit Roy was supported by the University Grants Commission Junior Research fellowship (UGC-JRF) from UGC, Govt. of India.

Conflict of interest

The authors declare that they have no conflicts of interest.

References

[1] Członkowska A, Litwin T, Dusek P, et al. Wilson disease. Nat Rev Dis Primers. 2018; 4(1):21. doi: 10.1038/s41572-018-0018-3

[2] Lutsenko S, Roy S, Tsvetkov P. Mammalian copper homeostasis: Physiological roles and molecular mechanisms. Physiol Rev. 2025; 105(1):441-491. doi: 10.1152/physrev.00011.2024

[3] Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW.The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. [Published correction appears in Nat Genet. 1994; 6( 2):214]. Nat Genet. 1993; 5(4):327-337. doi: 10.1038/ng1293-327

[4] Thomas GR, Forbes JR, Roberts EA, Walshe JM, Cox DW.The Wilson disease gene: Spectrum of mutations and their consequences. [Published correction appears in Nat Genet. 1995; 9( 4):451]. Nat Genet. 1995; 9(2):210-217. doi: 10.1038/ng0295-210

[5] Kumar M, Gaharwar U, Paul S, et al. WilsonGen a comprehensive clinically annotated genomic variant resource for Wilson’s disease. Sci Rep. 2020; 10(1):9037. doi: 10.1038/s41598-020-66099-2

[6] Wang J, Tang L, Xu A, Zhang S, Jiang H, Pei P, et al. Identification of mutations in the ATP7B gene in 14 Wilson disease children: Case series. Medicine (Baltimore). 2021; 100(16):e25463. doi: 10.1097/MD.0000000000025463

[7] Beyzaei Z, Mehrzadeh A, Hashemi N, Geramizadeh B. The mutation spectrum and ethnic distribution of Wilson disease, a review. Mol Genet Metab Rep. 2023;38:101034. doi: 10.1016/j.ymgmr.2023.101034

[8] Gomes A, Dedoussis GV. Geographic distribution of ATP7B mutations in Wilson disease. Ann Hum Biol. 2016; 43(1):1-8. doi: 10.3109/03014460.2015.1051492

[9] Das SK, Ray K. Wilson’s disease: An update. Nat Clin Pract Neurol. 2006; 2(9):482-493. doi: 10.1038/ncpneuro0291

[10] Taly AB, Meenakshi-Sundaram S, Sinha S, Swamy HS, Arunodaya GR. Wilson disease: Description of 282 patients evaluated over 3 decades. Medicine (Baltimore). 2007; 86(2):112-121. doi: 10.1097/MD.0b013e318045a00e

[11] Saha A, Das S, De S, et al. An effort to identify genetic determinants in siblings with wilson disease manifesting striking clinical heterogeneity: An exome profiling study of two Indian families. Pediatr Neurol. 2024; 155:1-7. doi: 10.1016/j.pediatrneurol.2024.03.005

[12] Martinelli D, Travaglini L, Drouin CA, et al.MEDNIK syndrome: A novel defect of copper metabolism treatable by zinc acetate therapy. [Published correction appears in Brain. 2013; 136( Pt 10):e256]. Brain. 2013;136(Pt 3): 872-881. doi: 10.1093/brain/awt012

[13] Chiplunkar S, Bindu PS, Nagappa M, et al.Huppke-brendel syndrome in a seven months old boy with a novel 2-bp deletion in SLC33A1. Metab Brain Dis. 2016; 31(5):1195-1198. doi: 10.1007/s11011-016-9854-6

[14] Gonzales E, Davit-Spraul A, Baussan C, Buffet C, Maurice M, Jacquemin E. Liver diseases related to MDR3 (ABCB4) gene deficiency. Front Biosci (Landmark Ed). 2009; 14(11):4242-4256. doi: 10.2741/3526

[15] Marchi G, Busti F, Lira Zidanes A, Castagna A, Girelli D. Aceruloplasminemia: A severe neurodegenerative disorder deserving an early diagnosis. Front Neurosci. 2019;13:325. doi: 10.3389/fnins.2019.00325

[16] Jaeken J, Matthijs G. Congenital disorders of glycosylation. Annu Rev Genomics Hum Genet. 2001; 2:129-151. doi: 10.1146/annurev.genom.2.1.129

[17] Ranucci G, Iorio R. Disorders that mimic Wilson disease. In: Clinical and Translational Perspectives on Wilson Disease. Amsterdam, Netherlands: Elsevier. 2019;41:419-426. doi: 10.1016/B978-0-12-810532-0.00041-0

[18] Mukherjee S, Dutta S, Majumdar S, et al. Genetic defects in Indian Wilson disease patients and genotype-phenotype correlation. Parkinsonism Relat Disord. 2014; 20(1):75-81. doi: 10.1016/j.parkreldis.2013.09.021

[19] Todorov T, Balakrishnan P, Savov A, Socha P, Schmidt HH. Intragenic deletions in ATP7B as an unusual molecular genetics mechanism of Wilson’s disease pathogenesis. PLoS One. 2016; 11(12):e0168372. doi: 10.1371/journal.pone.0168372

[20] 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):1-11. doi: 10.1007/s00335-022-09971-y

[21] Teschke R, Eickhoff A. Wilson disease: Copper-mediated cuproptosis, iron-related ferroptosis, and clinical highlights, with comprehensive and critical analysis update. Int J Mol Sci. 2024; 25(9):4753. doi: 10.3390/ijms25094753

[22] Liu Y, Li C, Shen S, et al. Discovery of regulatory noncoding variants in individual cancer genomes by using cis-X. Nat Genet. 2020; 52(8):811-818. doi: 10.1038/s41588-020-0659-5

[23] Subramanian A, Su S, Moding EJ, Binkley MS. Investigating the tissue specificity and prognostic impact of cis-regulatory cancer risk variants. Hum Genet. 2023; 142(9):1395-1405. doi: 10.1007/s00439-023-02586-6

[24] Ichiyama-Kobayashi S, Hata K, Wakamori K, et al. Chromatin profiling identifies chondrocyte-specific Sox9 enhancers important for skeletal development. JCI Insight. 2024; 9(11):e175486. doi: 10.1172/jci.insight.175486

[25] Topfer SK, Feng R, Huang P, et al. Disrupting the adult globin promoter alleviates promoter competition and reactivates fetal globin gene expression. Blood. 2022; 139(14):2107-2118. doi: 10.1182/blood.2021014205

[26] Kleinjan DA, Seawright A, Schedl A, Quinlan RA, Danes S, van Heyningen V. Aniridia-associated translocations, DNase hypersensitivity, sequence comparison and transgenic analysis redefine the functional domain of PAX6. Hum Mol Genet. 2001; 10(19):2049-2059. doi: 10.1093/hmg/10.19.2049

[27] Pradhan S, Sengupta M, Dutta A, et al. Indian genetic disease database. Nucleic Acids Res. 2011;39:D933-D938. doi: 10.1093/nar/gkq1025

[28] Gupta A, Chattopadhyay I, Dey S, et al. Molecular pathogenesis of Wilson disease among Indians: A perspective on mutation spectrum in ATP7B gene, prevalent defects, clinical heterogeneity and implication towards diagnosis. Cell Mol Neurobiol. 2007; 27(8):1023-1033. doi: 10.1007/s10571-007-9192-7

[29] Gupta A, Aikath D, Neogi R, et al. Molecular pathogenesis of Wilson disease: Haplotype analysis, detection of prevalent mutations and genotype-phenotype correlation in Indian patients. Hum Genet. 2005; 118(1):49-57. doi: 10.1007/s00439-005-0007-y

[30] Aggarwal A, Bhatt M. Update on Wilson disease. Int Rev Neurobiol. 2013;110:313-348. doi: 10.1016/B978-0-12-410502-7.00014-4

[31] Cullen LM, Prat L, Cox DW. Genetic variation in the promoter and 5’ UTR of the copper transporter, ATP7B, in patients with Wilson disease. Clin Genet. 2003; 64(5):429-432. doi: 10.1034/j.1399-0004.2003.00160.x

[32] Loudianos G, Dessi V, Lovicu M, et al. Molecular characterization of Wilson disease in the Sardinian population- -evidence of a founder effect. Hum Mutat. 1999; 14(4):294-303. doi: 10.1002/(SICI)1098-1004(199910)14:4<294:AID-HUMU4>3.0.CO;2-9

[33] Yang CS, Liang XL, Li JY, Yan ZW, Huang F. Effect of the mutation of promoter region in Wilson disease ATP7B gene on the expression of reporter gene. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005; 22(5):566-568.

[34] Wan L, Tsai CH, Hsu CM, et al. Mutation analysis and characterization of alternative splice variants of the Wilson disease gene ATP7B. Hepatology. 2010; 52(5):1662-1670. doi: 10.1002/hep.23865

[35] Chen HI, Jagadeesh KA, Birgmeier J, et al. An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease. Eur J Hum Genet. 2018; 26(12):1810-1818. doi: 10.1038/s41431-018-0221-4

[36] Sternlieb I. Perspectives on Wilson’s disease. Hepatology. 1990; 12(5):1234-1239. doi: 10.1002/hep.1840120526

[37] Boyle AP, Hong EL, Hariharan M, et al. Annotation of functional variation in personal genomes using RegulomeDB. Genome Res. 2012; 22(9):1790-1797. doi: 10.1101/gr.137323.112

[38] GTEx Consortium.The genotype-tissue expression (GTEx) project. Nat Genet. 2013; 45(6):580-585. doi: 10.1038/ng.2653

[39] Ward LD, Kellis M. HaploReg: A resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants. Nucleic Acids Res. 2012;40:D930-D934. doi: 10.1093/nar/gkr917

[40] Hintze JL, Nelson RD. Violin plots: A box plot-density trace synergism. Am Stat. 1998; 52(2):181-184. doi: 10.1080/00031305.1998.10480559