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Gene & Protein in Disease Regulatory elements of ATP7B in WD

presenting with either one mutated allele or no mutation high DNase I hypersensitivity, H3K4me3 and H3K27ac
in the coding region of ATP7B. signals, and low CTCF Z-scores. Two of these regulatory
elements exhibit distal regulation, while one shows proximal
2. Materials and methods regulation of ATP7B gene expression (Table 1). The DNase I

2.1. Study subjects hypersensitivity indicates the open chromatin conformation,
whereas H3K4Me3 and H3K27Ac denote the enhancer
A total of 30 Indian WD patients, comprising 12 females region. CTCF is a transcription factor generally present in a
and 18 males, were included in this study, selected from a DNA element that potentially acts as a repressor.
larger cohort of Indian WD patients who have undergone
mutation screening in the coding region of ATP7B. The 2.3. Polymerase chain reaction (PCR) and Sanger
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patients were diagnosed by clinicians primarily at the sequencing
Bangur Institute of Neurosciences in Kolkata, National PCR was performed to amplify three regulatory elements
Medical College in Kolkata, and King Edward Memorial
Hospital in Pune, Maharashtra, following Sternleib’s of the ATP7B gene. Each PCR reaction was conducted in
criteria. Key diagnostic features included the presence of a 20 µL volume, using 80 ng of genomic DNA and GoTaq
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Kayser-Fleischer rings, elevated 24-h urinary copper levels, Green PCR Master Mix (Promega, India) using specific
low plasma ceruloplasmin levels, and abnormal hepatic primers. The details of the primers are provided in Table S1.
and neurological features. Among these 30 clinically Following PCR amplification, bi-directional sequencing of
diagnosed WD patients, 13 lacked any coding mutations the products was performed using the same set of primers
in ATP7B, while the remainder presented with only one on an ABI 3100 sequencer (Applied Biosystem, California,
mutated allele. Genomic DNA isolated from the peripheral USA). Nucleotide changes were detected by comparing the
blood of the patients was used for all genetic screenings. sequences obtained in the chromatogram with the normal
gene sequence using pairwise BLAST.
2.2. Prioritization of cis-regulatory elements for
ATP7B screening 2.4. In-silico analysis of the variants
The ENCODE portal provides experimentally validated The regulatory potential of the identified variants was
information about regulatory elements (enhancers, silencers, validated using RegulomeDB, a variant annotation software
repressors, and promoter regions) present in the genome. It that identifies DNA features and regulatory elements in non-
integrates DNase I hypersensitive sites sequencing, chromatin coding regions of the human genome. Users can input variant
immunoprecipitation sequencing, H3K4me3, H3K27ac, IDs, Browser Extensible Data files, Variant Call Format files,
and CCCTC-binding factor (CTCF) data generated by or General Feature Format3 files, and RegulomeDB returns
ENCODE and Roadmap Epigenomics consortia. Using a score assessing the regulatory potential of each variant. The
DNase 1 hypersensitivity, H3K4me3, H3K27ac, and CTCF data supporting the inference by data type and cell type can
signals, candidate regulatory elements were classified into also be obtained. Accordingly, each variant we identified
enhancer-like sequences, promoters, and CTCF-bound was assigned a score based on its expression quantitative
repressors, validated across several cell types and tissues. trait locus (eQTL), transcription factor binding, DNase I
This classification is based on data from the ENCODE hypersensitivity, and DNase I footprinting data. RegulomeDB
database, which utilized the human genome assembly hg19. assigns scores ranging from 1 to 6 to variants based on their
In a recent review, a total of eight candidate regulatory regulatory potential. In addition, the variants were tested
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elements for the ATP7B gene were identified in the HepG2 for genotype-specific expression patterns and eQTL scores
cell line, with proximal or distal modes of regulation. in tissues from the Genotype-Tissue Expression (GTex)
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In this study, we selected three regulatory elements with portal. The GTEx portal is a comprehensive public resource
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Table 1. Regulatory elements of ATP7B prioritized from the Encyclopedia of DNA Elements database

Genomic coordinates Cell Distance from ATP7B Length of Type of
type transcription start site (bp) cis‑regulatory element regulation
(bp)
chr13: 52,572,128 – 52,572,425 HepG2 13,122 297 Distal
chr13: 52,552,999 – 52,553,472 HepG2 3,695 473 Distal
chr13: 52,585,439 – 52,585,709 HepG2 0 a 270 Proximal (5’UTR)
Note: Partially overlapping with the first exon and 5’UTR of ATP7B.
a
Abbreviations: bp: Base pair; HepG2: Human liver cancer cell line; UTR: Untranslated region.

Volume 4 Issue 2 (2025) 3 doi: 10.36922/gpd.7503