Objective: To explore the genotype-phenotype relationship of Wilson\'s disease (WD) and further study the mutation spectrum in the ATP7B gene. Methods: The clinical data and genetic test results of 115 cases with WD diagnosed in the First Affiliated Hospital of Zhengzhou University from 2015 to 2022 were retrospectively analyzed. The rank sum test was used for quantitative data comparison, and χ(2) test was used for count data comparison. Multivariate logistic regression was used to analyze the relationship between patients\' genotype and phenotype. Results: The onset of liver manifestations (hepatic type) accounted for 60.9%, neurological symptoms (cerebral type) for 13.0%, and mixed hepato-cerebral symptoms for 26.1%. Presymptomatic individuals (hepatic types) accounted for 62.9%. Next-generation sequencing- diagnosed WD cases accounted for 87.8%. Combined multiplex ligation-dependent probe amplification assay-diagnosed WD cases accounted for 89.6%. A single case with a detected pathogenic locus accounted for 10.4%. The diagnostic rate of WD by genetic testing combined with clinical data was 100%. A total of 76 ATP7B mutations were detected, and the top three mutation frequencies were c.2333G>T (p.Arg778Leu) (30.7%), c.2975C>T (p.Pro992Leu) (7.3%), and c.2621C>T (p.Ala874Val) (6.4%). The mutations were mainly distributed in exons 8, 11-13, and 15-18, accounting for more than 90% of the total mutations. Eight new mutations were found, including c.3724G>A (p.Glu1242Lys), c.3703G>C (p.Gly1235Arg), c.3593T>C (p.Val1198Ala), c.2494A>C (p.Lys832Gln), c.1517T>A (p.Ile506Lys), c.484G>T (p.Glu162Ter), c.1870-49A>G, and the missing of exons 10-21. Liver histopathology showed cellular edema, degeneration, inflammation, and necrosis, as well as a 42.8% copper staining positive rate. Genotype-phenotype analysis showed that the p.Arg778Leu mutation had higher alanine aminotransferase (ALT) levels than those carrying other mutations (P=0.024), while the homozygous mutation of p.Arg778Leu was associated with cerebral-type patients (P=0.027). Conclusion: Genetic testing plays an important role in the diagnosis of WD. p.Arg778Leu is the first high-frequency mutation in the Chinese population, and patients carrying it have higher ALT levels. The p.Arg778Leu homozygous mutation is prone to causing cerebral-type WD. This study expands the ATP7B gene mutation spectrum.
目的： 探讨肝豆状核变性（WD）的基因型-表型关系，研究ATP7B基因突变谱。 方法： 回顾性分析2015年至2022年在郑州大学第一附属医院确诊的115例WD患者的临床资料和基因检测结果。计量资料比较采用秩和检验，计数资料比较采用χ(2)检验；采用多因素logisitc回归法分析患者基因型和表型的相关性。 结果： 以肝脏表现起病的（肝型）占60.9%，神经系统症状起病的（脑型）占13.0%，肝脑混合型占26.1%。症状前个体占肝型的62.9%。二代测序诊断WD的占87.8%，联合多重连接探针扩增技术诊断WD的占89.6%，仅检测到1个致病位点的占10.4%。基因检测结合临床资料对WD的诊断率为100%。共检出ATP7B变异76种，突变频率最高前3位的为c.2333G>T(p.Arg778Leu，30.7%）、c.2975C>T(p.Pro992Leu，7.3%）和c.2621C>T(p.Ala874Val，6.4%）。变异主要分布在第8、11～13和15～18号外显子，占变异总数的90%以上。发现新变异8个，分别为c.3724G > A（p.Glu1242Lys）、c.3703G > C（p.Gly1235Arg）、c.3593T > C（p.Val1198Ala）、c.2494A > C（p.Lys832Gln）、c.1517T > A（p.Ile506Lys）、c.484G > T（p.Glu162Ter）、c.1870-49A > G和第10～21号外显子缺失。肝组织病理学示细胞水肿变性、炎性和坏死，铜染色阳性率仅为42.8%。基因型-表型分析显示，携带p.Arg778Leu变异者比携带其他变异者的丙氨酸转氨酶（ALT）水平更高（P = 0.024），p.Arg778Leu纯合变异和脑型患者相关（P = 0.027）。 结论： 基因检测在WD确诊中发挥重要作用。p.Arg778Leu为中国人群第1高频变异，携带该变异的患者ALT水平较高；p.Arg778Leu纯合变异易导致脑型WD。该研究扩展了ATP7B基因变异谱。.

BACKGROUND: Wilson disease (WD) is a rare autosomal recessive disorder of copper metabolism caused due to mutations in the copper transporter ATP7B. There is often a striking variability of clinical manifestations among patients with ATP7B mutations, including in siblings. This phenomenon may be caused by individual differences in copper accumulation in hepatocytes and intolerance to copper toxicity as governed by genetic variations in copper metabolism genes acting as modifier loci to the disease.
OBJECTIVE: To elucidate the genetic basis of striking clinical heterogeneity among two siblings of two families with WD.
METHODS: The disease diagnosis and subsequent clinical examinations were performed by expert clinicians. The younger siblings in both families presented with early neurological manifestations at a younger age than their older siblings. Interestingly, only the younger siblings were reported to have had hepatic manifestations. Exome sequencing of all the four individuals was performed to understand their heterogeneous phenotypic outcomes.
RESULTS: Genetic screening revealed no difference in the ATP7B variant spectrum between the siblings of each family. However, the siblings of both the families were found to harbor mutually exclusive pathogenic variants in suspected modifier genes implicated in copper metabolism and/or other neurological and hepatic disorders having overlapping symptoms with WD, viz., CFTR, PPARG, ABCB11, ATP7A, CYP2D6, mTOR, TOR1A, and CP, which can potentially explain their differential clinical phenotypes.
CONCLUSIONS: Clinical heterogeneity between siblings with WD with the same ATP7B mutation profile may be attributed to the presence of different pathogenic variants in potential modifier genes.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.

Evidence indicates that patients with Alzheimer\'s dementia (AD) show signs of copper (Cu) dyshomeostasis. This study aimed at evaluating the potential of Cu dysregulation as an AD susceptibility factor. We performed a meta-analysis of 56 studies investigating Cu biomarkers in brain specimens (pooled total of 182 AD and 166 healthy controls, HC) and in serum/plasma (pooled total of 2929 AD and 3547 HC). We also completed a replication study of serum Cu biomarkers in 97 AD patients and 70 HC screened for rs732774 and rs1061472 ATP7B, the gene encoding for the Cu transporter ATPase7B. Our meta-analysis showed decreased Cu in AD brain specimens, increased Cu and nonbound ceruloplasmin (Non-Cp) Cu in serum/plasma samples, and unchanged ceruloplasmin. Serum/plasma Cu excess was associated with a three to fourfold increase in the risk of having AD. Our replication study confirmed meta-analysis results and showed that carriers of the ATP7B AG haplotype were significantly more frequent in the AD group. Overall, our study shows that AD patients fail to maintain a Cu metabolic balance and reveals the presence of a percentage of AD patients carrying ATP7B AG haplotype and presenting Non-Cp Cu excess, which suggest that a subset of AD subjects is prone to Cu imbalance. This AD subtype can be the target of precision medicine-based strategies tackling Cu dysregulation.

OBJECTIVE: To explore the genetic basis for three children with Menkes disease.
METHODS: The patients were subjected to next-generation sequencing (NGS) to detect potential variants of the ATP7A gene. Suspected variants were verified by Sanger sequencing of their family members and 200 healthy individuals. Multiplex ligation-dependent probe amplification (MLPA) was also carried out to detect potential deletions in their family members and 20 healthy individuals.
RESULTS: Variants of the ATP7A gene were detected in all of the three families, including a novel c.1465A>T nonsense variant in family 1, a novel c.3039_3043del frame-shifting variant in family 2, and deletion of exons 3 to 23 in family 3, which was reported previously. Based on the standards and guidelines of American College of Medical Genetics and Genomics, the c.1465A>T and c.3039_3043del variants of ATP7A gene were predicted to be likely pathogenic (PVS1+PM2).
CONCLUSIONS: Variants of the ATP7A gene may underlay the Menkes disease in the three children. Above findings have facilitated clinical diagnosis and enriched the spectrum of genetic variants of Menkes disease.

Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR.

Nanobodies are genetically engineered single domain antibodies derived from the unusual heavy-chain only antibodies found in llamas and camels. The small size of the nanobodies and flexible selection schemes make them uniquely versatile tools for protein biochemistry and cell biology. We have developed a panel of nanobodies against the metal binding domains of the human copper transporter ATP7B, a multidomain membrane protein with a complex regulation of enzymatic activity and intracellular localization. To enable the use of the nanobodies as tools to investigate copper transport in the cell, we characterized their binding sites and affinity by isothermal titration calorimetry and NMR. We have identified nanobodies against each of the first four metal binding domains of ATP7B, with a wide affinity range, as evidenced by dissociation constants from below 10-9 to 10-6 M. We found both the inhibitory and activating nanobodies among those tested. The diverse properties of the nanobodies make the panel useful for the structural studies of ATP7B, immunoaffinity purification of the protein, modulation of its activity in the cell, protein dynamics studies, and as mimics of copper chaperone ATOX1, the natural interaction partner of ATP7B.

The PharmacoScan pharmacogenomics platform screens for variation in genes that affect drug absorption, distribution, metabolism, elimination, immune adverse reactions and targets. Among the 1,191 genes tested on the platform, 12 genes are expressed in the red cell membrane: ABCC1, ABCC4, ABCC5, ABCG2, CFTR, SLC16A1, SLC19A1, SLC29A1, ATP7A, CYP4F3, EPHX1 and FLOT1. These genes represent 5 ATP-binding cassette proteins, 3 solute carrier proteins, 1 ATP transport protein and 3 genes associated with drug metabolism and adverse drug reactions. Only ABCG2 and SLC29A1 encode blood group systems, JR and AUG, respectively. We propose red cells as an ex vivo model system to study the effect of heritable variants in genes encoding the transport proteins on the pharmacokinetics of drugs. Altered pharmacodynamics in red cells could also cause adverse reactions, such as haemolysis, hitherto unexplained by other mechanisms.

Copper\'s essentiality and toxicity mean it requires a sophisticated regulation system for its acquisition, cellular distribution and excretion, which until now has remained elusive. Herein, we applied continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy in solution to resolve the copper trafficking mechanism in humans, by considering the route travelled by Cu(I) from the metallochaperone Atox1 to the metal binding domains of ATP7B. Our study revealed that Cu(I) is most likely mediated by the binding of the Atox1 monomer to metal binding domain 1 (MBD1) and MBD4 of ATP7B in the final part of its extraction pathway, while the other MBDs mediate this interaction and participate in copper transfer between the various MBDs to the ATP7B membrane domain. This research also proposes that MBD1-3 and MBD4-6 act as two independent units.

Hepatocyte polarity is essential for the development of bile canaliculi and for safely transporting bile and waste products from the liver. Functional studies of autologous mutated proteins in the context of the polarized hepatocyte have been challenging because of the lack of appropriate cell models. The aims of this study were to obtain a patient-specific hepatocyte model that recapitulated hepatocyte polarity and to employ this model to study endogenous mutant proteins in liver diseases that involve hepatocyte polarity.
Urine cell-derived pluripotent stem cells, taken from a patient with a homozygous mutation in ATP7B and a patient with a heterozygous mutation, were differentiated towards hepatocyte-like cells (hiHeps). HiHeps were also derived from a patient with MEDNIK syndrome.
Polarized hiHeps that formed in vivo-like bile canaliculi could be generated from embryonic and patient urine cell-derived pluripotent stem cells. HiHeps recapitulated polarized protein trafficking processes, exemplified by the Cu2+-induced redistribution of the copper transporter protein ATP7B to the bile canalicular domain. We demonstrated that, in contrast to the current dogma, the most frequent yet enigmatic Wilson disease-causing ATP7B-H1069Q mutation per se did not preclude trafficking of ATP7B to the trans-Golgi Network. Instead, it prevented its Cu2+-induced polarized redistribution to the bile canalicular domain, which could not be reversed by pharmacological folding chaperones. Finally, we demonstrate that hiHeps from a patient with MEDNIK syndrome, suffering from liver copper overload of unclear etiology, showed no defect in the Cu2+-induced redistribution of ATP7B to the bile canaliculi.
Functional cell polarity can be achieved in patient pluripotent stem cell-derived hiHeps, enabling, for the first time, the study of the endogenous mutant proteins, patient-specific pathogenesis and drug responses for diseases where hepatocyte polarity is a key factor.
This study demonstrates that cells that are isolated from urine can be reprogrammed in a dish towards hepatocytes that display architectural characteristics similar to those seen in the intact liver. The application of this methodology to cells from patients diagnosed with inherited copper metabolism-related liver diseases (that is, Wilson disease and MEDNIK syndrome) revealed unexpected and novel insights into patient mutation-specific disease mechanisms and drug responses.