Broadening the implications of gene discovery

Potential conflict of interest: Nothing to report. See Articles on Pages 1828 And 1842 In the mid‐1980s the enthusiastic rationale for identifying the gene somehow responsible for any given monogenic disease was, first, to facilitate accurate diagnosis and, second, to cure the disease. When gene analysis was extremely labor‐intensive and gene replacement therapy proved difficult or even perilous to patients, such motivation seemed like pure hype. Yet unquestionably, for clinically pleomorphic diseases like cystic fibrosis (CF) and Wilson disease (WD) genotype determination promoted accurate diagnosis. Fast‐forward to 2012: inclusive analysis of the large ATP7B gene associated with WD is realistic and almost routine. Some CF patients can effectively be cured with a drug, ivacaftor, which permits the modestly abnormal CF transmembrane conductance regulator protein of G551D homozygotes to function adequately within the cell membrane. This treatment would not have been possible without identifying the gene first, yet it is very different from gene replacement or gene editing. However, there was another rationale for this gene discovery research—though much less glamorous. Identifying the gene was expected to energize research into relevant physiology and help elucidate the pathophysiology of the disease. For WD, this was certainly the case. Complementing research already under way with various bacteria and yeasts, within a few years of identifying the human gene ATP7B, key features of the structure of its gene product, ATP7B, the Wilson adenosine triphosphatase (ATPase), were worked out1 and copper‐responsive intracellular movement of this protein was reported.2 Understanding the role of metals such as copper and zinc, and more recently platinum and manganese, in hepatocellular function acquired new interest. The Wilson ATPase is a metal‐transporting P1‐type ATPase. The paradoxical clinical features of WD (hepatic copper overload but low serum copper) became explicable when it was apparent that the Wilson ATPase has two functions within the hepatocyte. When ambient hepatocellular copper concentrations are relatively low, it participates in the incorporation of copper into apoceruloplasmin. When the Wilson ATPase is dysfunctional, copper is not made available for incorporation and apoceruloplasmin devoid of copper is produced. Serum copper is consequently measured as subnormal. When bioavailable hepatocellular copper concentrations are high, the Wilson ATPase expedites biliary excretion of copper. Failure at this function leads to toxic accumulation of copper in the hepatocyte. Evidently the Wilson ATPase has at least one other function. It serves as a copper sensor within the hepatocyte. Persisting questions include the following: What regulates Wilson ATPase function? Given multiple mechanisms, how does copper actually get excreted into bile? Answers to such questions may reveal new opportunities for therapeutic intervention, if a therapy could compensate for, or potentiate, an imperfect Wilson ATPase. Much is known about selected disease‐causing mutations among the hundreds of gene alterations thus far identified in ATP7B.3 Some result in such severe disruption of Wilson ATPase structure that effectively no protein is produced, a situation resembling the knockout mouse. Other mutations result in misfolded proteins that get hung up in the endoplasmic reticulum. Still others may produce variably dysfunctional Wilson ATPase whose functional compromise depends on bioavailable cellular copper concentration. Some Wilson ATPase mutants may retain the copper‐transporting function for holoceruloplasmin production but lack the biliary excretion action. Thus, gene discovery shifts the focus from gene to gene product. Two articles in this issue of hepatology4 have opened the research aperture relating to WD even wider. Each seeks to understand Wilson ATPase function in terms of the hepatocellular machinery within which it operates. This could be called an “ecological” approach, but the usual description is a “systems” approach, although it is not, strictly speaking, “systems biology.” Its hallmark is that it attaches importance to the context—or system—within which the entity operates. It begins by interrogating that system as a whole to see what composes that system and then proceeds by zooming in on features of interest. This experimental strategy invites the application of omics methodology. An early example of a “systems” approach to WD and the physiology of hepatocellular copper disposition involves metalloproteomics.6 In these pioneering experiments, the distal objective of determining the Cu metalloproteome in hepatocytes was to see how it changed if the Wilson ATPase was defective; the immediate objective was to investigate the Cu metalloproteome in the healthy liver. Numerous proteins not expected to have anything to do with copper handling were found in this hepatic Cu metalloproteome, including protein disulfide isomerase, an endoplasmic reticulum chaperone, later demonstrated to be capable of binding Cu(I), the form of copper found in the liver cell. Similarly, both of these articles begin with omics and move in new and exciting directions. In the first, Hamilton et al.4 used messenger RNA profiling and subsequent pathway analysis to recognize that abnormalities in the Atp7b−/− mouse reflected down‐regulation of cholesterol biosynthesis due to inhibition of nuclear receptor signaling, notably of liver X receptor/retinoid X receptor (LXR/RXR). Similar results were obtained in livers of patients with WD. Thus, they investigated the role of LXR/RXR in WD. In the second article, Chesi et al.5 began by comparing the transcriptomes of hepatocytes expressing wild‐type or H1069Q‐Wilson ATPase: gene ontology analysis showed increased expression of genes related to regulating cell death and stress responses. The gene products of several of the implicated genes participate in the mitogen‐activated protein kinase signaling pathway mediated by p38 and c‐Jun N‐terminal kinase (JNK), possibly reflecting the role of reactive oxygen species in the pathogenesis of hepatocellular damage in WD. Further they compared interactomes of wild‐type and H1069Q‐Wilson ATPase. Whereas wild‐type Wilson ATPase interacted prominently with intracellular trafficking proteins, H1069Q‐Wilson ATPase interacted with proteins of the endoplasmic reticulum‐associated quality control machinery. They therefore focused on whether activation of p38 and JNK might enhance degradation of the abnormal H1069Q‐Wilson ATPase. The point here is not that the omics methodology was convenient for finding focus and generating hypotheses. Rather, it was the appropriate experimental design for interrogating the system within which the Wilson ATPase function was to be studied: this is a key aspect of system‐driven research. For Hamilton et al. interest in the relationship between the Wilson ATPase and lipid metabolism was not accidental: besides previous reports from this research group,7 the inverse relation between copper sufficiency and cholesterol level has been known for years, notably in chickens. In contradistinction to the claim by Chesi et al., omics methodology only aspires to being unbiased. It trades the constraints of preconceived hypothesis for mainly specifiable bias. Both articles illustrate how a system‐driven approach can generate possibly surprising results. Importantly, the findings in both studies generate potential therapeutic innovations. In the Hamilton et al. article, treatment with the LXR agonist TO901317 improved lipid metabolism with increases in plasma cholesterol, low‐density lipoprotein, high‐density lipoprotein, and hepatic triglyceride concentrations. Parameters associated with fibrosis and inflammation were diminished in the treated Atp7b−/− mice, without removal of copper from liver parenchyma. A similar point was made in a recent study of farnesoid X receptor (FXR) and RXR in the Atp7b−/− mouse.8 Elevated copper concentrations were shown to interfere with binding of FXR and RXR to appropriate response elements. Provision of excess zinc restored binding in a dose‐dependent fashion. The 3‐month‐old Atp7b−/− mouse had decreased expression of FXR target genes, and its response to the potent FXR agonist GW4064 was slender compared to wild type. A high‐zinc diet was associated with improved nuclear receptor function with increased Bsep, SHP, and Rxr messenger RNA levels, though not Fxr. Thus, zinc pharmacodynamics may be more complicated than just interfering with intestinal copper uptake, and zinc may be synergistic with specific enhancers of nuclear receptor function. In the Chesi et al. study, inhibition of specific mitogen‐activated protein kinase pathways, namely p38 and JNK (known to be involved in proteostasis of the CF transmembrane conductance regulator), “rescued” the H1069Q‐Wilson ATPase and enhanced its delivery to the Golgi; in the presence of elevated intracellular copper concentrations, this mutant Wilson ATPase then trafficked appropriately. This recovered trafficking was associated with copper excretion. Various p38 and JNK inhibitors are currently being developed for clinical therapeutics. Similar enhancement of function for the mutant R778L‐Wilson ATPase was described in hepatocyte‐like induced pluripotent stem cells treated with curcumin.9 The contrarian may ask whether we really need new drugs for WD. Available therapy works well: early institution of effective treatment restores most patients to normal health and longevity. No established treatment for WD is problem‐free. Notably, d‐penicillamine has numerous nontrivial adverse effects, and zinc poses problems for compliance. Some patients never normalize the serum aminotransferases: 36% in the study of 109 children with WD cited by Chesi et al., although their clinical course was otherwise favorable.10 New drug treatments allowing fine‐tuned individualized treatment and perhaps once‐daily dosing to enhance compliance would be advantageous. If your health/well‐being depends on availability of effective drug treatment, as it does in WD, you want as many options as possible. You also want drugs at a price point whereby you can afford to take them on a daily basis for your entire life. These complex studies require the spectrum of experimental models now available. Cell culture models and induced pluripotent stem cells play an important role, despite their obvious limitations. The Atp7b−/− mouse represents the very severe version of WD. The tx‐j mouse, homozygous for a point mutation (exon 8, G712D), has early accumulation of hepatic copper but milder liver disease. It resembles many humans with WD. The role of epigenetic factors in its liver disorder is being shown.11 Examining these mechanistic issues and proposed interventions in the tx‐j mouse would be of interest. Moreover, with the currently available models and technology, we have yet another excellent reason to study WD. This disease may provide the best model for investigating the mechanisms of liver disease which involve oxidative stress and it may be relevant to disordered hepatic lipid metabolism. None of this is realistically possible without a clear and detailed description of copper handling in hepatocytes and how it changes when the Wilson ATPase is abnormal. Thus, the implications of the ATP7B gene discovery have become very extensive indeed, potentially beyond WD itself. Author names in bold designate shared co‐first authorship.

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