Whom and how to screen for Wilson disease

Wilson disease is a genetic disorder of hepatic copper excretion leading to copper accumulation in various tissues. The disease expression is highly variable, ranging from totally asymptomatic subjects to patients with severe liver disease or movement disorders. Thus, it is difficult to define in which patient Wilson disease has to be considered as diagnosis. The suspicion should be high in patients presenting with extrapyramidal disorders or with liver diseases or of unknown origin. For diagnosis, in many patients a combination of tests reflecting disturbed copper metabolism may be needed. Not a single test is per se specific and, thus, a range of tests has to be applied (presence or absence of Kayser–Fleischer rings or neurologic symptoms, serum ceruloplasmin, liver copper content, urinary copper excretion, mutation analysis; rated –1 to 4 depending on the test) and clinical symptoms. A diagnostic sum score of ≥4 confirms the diagnosis.     

The Overuse of Serum Ceruloplasmin Measurement

Background

Wilson disease is rare, found in 3 of 100,000 people (0.03%). Ceruloplasmin is often ordered to evaluate liver enzyme elevations. Because Wilson disease often presents before middle-age, the American Association for the Study of Liver Disease recommends screening patients between the ages of 3 and 55 years with liver abnormalities of uncertain cause. We evaluate guideline adherence and the clinical and economic impact of current clinical use of ceruloplasmin.

Methods

We reviewed all ceruloplasmin measurements at a clinical laboratory that serves a large primary care network, specialty clinics, and a 600-bed tertiary referral center between January 1, 2003, and December 12, 2011.

Results

Ceruloplasmin was measured 5325 times in 5023 unique patients, resulting in 8 (0.16%) new Wilson disease diagnoses. Ceruloplasmin's positive predictive value was 8.4% (95% confidence interval, 7.7-9.3) and false-positive rate was 98.1% (95% confidence interval, 96.2-99.1). A total of 1109 ceruloplasmin levels (20.8%) were ordered in the 1066 patients aged more than 55 years (none with Wilson disease). A “shotgun” approach to liver disease diagnosis was found: Ceruloplasmin was ordered on the same day as hepatitis B (81.0%), hepatitis C (76.0%), autoimmune hepatitis (75.1%), and hemochromatosis (73.1%). Of 424 positive ceruloplasmin results, 91% were not pursued further.

Conclusions

Guideline adherence restricts ceruloplasmin use to a population with a higher pre-test probability of Wilson disease: patients with chronic liver disease aged 3 to 55 years who have been tested for common causes of liver disease. The majority of the serum ceruloplasmin was measured in patients not indicated by the guidelines, resulting in poor test performance and wasted healthcare resources. Our data on ceruloplasmin use could serve as a touchstone for broader discussions on rational clinical decision making.     

Pathogenesis and management of Wilson disease

Hepatolenticular degeneration, commonly known as Wilson disease, is an autosomal recessive inherited disease of abnormal copper metabolism, characterized by the accumulation of copper in the body due to decreased biliary excretion of copper from hepatocytes. Wilson disease protein, ATP7B, functions in copper excretion into bile and in copper secretion to the bloodstream coupled with ceruloplasmin synthesis. Various kinds of mutations of ATP7B cause Wilson disease. Wilson disease is a rare genetic disease that can be treated pharmacologically. Recognition and prompt diagnosis are very important, because Wilson disease is fatal if left untreated. In this review, I summarize the pathogenesis and management of Wilson disease.     

COMPARATIVE SENSITIVITY OF SERUM CHOLYLGLYCINE CONCENTRATION AND BROMSULPHALEIN RETENTION IN PATIENTS WITH EARLY AND LATE ALCOHOLIC LIVER DISEASE

Measurement of serum bile acids has been claimed to be a sensitive and specific biochemical test of hepatic function. We have prospectively measured post-prandial serum glycocholate (cholylglycine) concentrations in 31 patients with alcoholic liver disease and compared these measurements with those of bromsulphalein (BSP) retention, prothrombin time, and serum albumin. In the patients with early (non-cirrhotic) alcoholic liver disease (N = 14) BSP retention was abnormal significantly more frequently than was serum cholylglycine concentration (100% vs 29%, p < 0.001). In contrast, amongst patients with late (cirrhotic) alcoholic liver disease, BSP retention and serum cholylglycine were abnormal with equal frequency (94%). In both groups of patients BSP retention and serum cholylglycine were abnormal significantly more often than were prothrombin time and serum albumin concentrations. We conclude that moderately severe hepatocellular dysfunction is required before serum cholylglycine can become a reliable biochemical indicator of liver disease.     

Late diagnosed Wilson disease with hepatic and neurological manifestations

A 50‐year‐old woman was referred to our hospital due to liver dysfunction and progressive neurological symptoms. She had previously been diagnosed with nonalcoholic steatohepatitis (NASH). Ursodeoxycholic acid (UDCA) had effectively normalized her serum aminotransferase levels, however, she presented with loss of balance, dysarthria and difficulty in handwriting. Autoantibodies and hepatitis virus markers were negative. Serum ceruloplasmin and copper levels were noted to be 9 mg/dL and 32 µg/dL, respectively. The 24‐h urinary copper excretion was 331.8 µg/day. Kayser‐Fleischer ring was demonstrated. Histological examination of the liver revealed inflammatory infiltrate and fibrosis, and the hepatic copper concentration was 444.4 µg/g dry weight. We diagnosed her as having Wilson disease and started treatment with trientine. Immuohistochemistry for keratin 8 and p62 demonstrated Mallory‐Denk bodies. Many of the p62‐expressing cells were positive for 4‐Hydroxy‐2‐nonenal (HNE). Few Ki‐67‐positive hepatocytes were present in the liver. Wilson disease is one of the causes of NASH and UDCA may be a supportive therapeutic agent for Wilson disease. Cell proliferation is suppressed under copper‐loaded conditions and this phenomenon may be associated with the clinical course of Wilson disease.     

Hemolytic anemia in wilson disease: Clinical findings and biochemical mechanisms

Two patients with Wilson disease who presented with severe hemolytic anemia are described. One was noted to have unusually high serum copper levels (369 μg/100 ml). A review of similar such patients in the literature suggests that, rather than having a low serum copper, patients with hemolysis accompanying Wilson disease have very high serum copper levels. For this reason, in vitro studies of the toxic effects of copper on erythrocytes were undertaken. It was found that, although copper does not have a major direct inhibitory effect on glycolytic enzymes such as hexokinase, the metal does inhibit hexokinase as a consequence of its interaction with oxyhemoglobin. However, such inhibition does not appear to be a major factor in copper-induced hemolysis. On the other hand, the addition of the lipid antioxidant butylated hydroxyanisole (BHA) suppresses hemolysis in copper-treated cells. These experiments suggest that the primary toxic effect of copper is mediated through its oxidant actions on membrane phospholipids rather than through its potential inhibitory effects on intracellular enzymes.     

不同临床分型的Wilson’s病临床特点分析

探讨不同亚型Wilson’s病（WD）的临床特点及肝型患者预后转归。方法收集256例WD患者的临床资料并随访，根据临床表现将患者分为不同的临床亚型，进一步分析比较肝型、脑型和混合型WD患者的临床特点差异和随访追踪肝型WD患者的预后。结果在256例WD患者中，以混合型（152例，59.4%）和肝型（74例，28.9%）患者常见，而脑型（27例，10.5%）和其他亚型（3例，1.2%）较少；肝型WD患者失代偿期肝硬化比例（78.4%）高于混合型患者（22.0%，P〈0.001）；肝型WD患者肝脏血清生化学指标（转氨酶、ALP、GGT、胆红素以及球蛋白水平）高于混合型WD患者（P〈0.05）；肝型WD患者血清铜[（1.04±1.50） mg/L]水平明显低于脑型WD患者[（2.96±2.88） mg/L]和混合型WD患者[（2.34±2.68） mg/L，P〈0.001]，但两者铜蓝蛋白和尿铜水平无统计学差异（P〉0.05）；肝型WD患者K-F环检出率（64.9%）低于脑型WD患者（92.6%）和混合型WD患者（90.1%，P〈0.05）；经Logistic回归分析显示角膜K-F的有无与年龄（OR=0.922，P=0.014）、血清铜蓝蛋白（OR=35902.1，P=0.015）相关；平均随访31例肝型WD患者（8.3±5.8）年，3例（9.7%）进展为混合型WD患者。结论 WD以混合型和肝型最多见，肝型患者肝脏损害比混合型更为严重，提示肝脏是WD最主要的靶器官。     

血浆铜蓝蛋白在乙型肝炎不同肝功能状态的水平及意义

目的探讨肝豆状核变性、乙型肝炎不同肝功能状态时血浆铜蓝蛋白水平的差异及其临床意义。方法用终末期肝病模型(model for end-stage liver disease,MELD)评分衡量肝功能损害的严重性,采用散射比浊法检测并比较分析肝豆状核变性、慢加急性肝衰竭极期以及恢复期、慢性乙型肝炎患者的血浆铜蓝蛋白水平。结果肝豆状核变性(n=50)、慢加急性肝衰竭极期和恢复期(n=30)、慢性乙型病毒性肝炎患者(n=50)的MELD评分分别为11.1±6.5、20.4±4.2、10.9±4.9、9.6±9.8,血浆铜蓝蛋白分别为(0.065±0.036)g/L、(0.176±0.037)g/L、(0.210±0.056)g/L、(0.197±0.038)g/L;血浆铜蓝蛋白水平4组之间的两两比较存在差异(F=111.4,P<0.001),肝豆状核变性患者低于其他3组(P<0.001)。结论肝豆状核变性血浆铜蓝蛋白水平显著降低。相对于肝豆状核变性,没有肝衰竭的慢性乙型肝炎血浆铜蓝蛋白水平几乎正常。慢加急性肝衰竭极期血浆铜蓝蛋白水平仅轻度下降,随着肝功能衰竭的恢复,血浆铜蓝蛋白恢复正常。     

Special stain and X‐ray probe microanalysis of livers with Wilson disease

Aim: Primary copper toxicosis due to Wilson disease is clinically complex, often leading to delayed diagnosis. Because the metabolic disorder is frequently complicated by iron overload due to hypoceruloplasminemia, either a special stain or microanalysis has been recommended for liver biopsy specimens. Methods: Liver biopsy was performed in three patients in whom Wilson disease was highly suspected. Light microscopic study included rubeanic acid stain for copper and Berlin blue stain for iron. To improve the resolution of ultra‐structures and preservation of toxic metals, short‐term fixation with a 0.1% osmic acid solution was applied for X‐ray probe microanalysis. Their diagnosis was confirmed by genetic study and copper chelation therapy. Results: Two patients at the age of 17 and 23 years, respectively, demonstrated cirrhotic livers surrounded by fibrous septa, while a 7‐year‐old patient demonstrated fatty liver with mildly expanded portal tracts. Both copper grains stained with rubeanic acid and cuprothionein by microanalysis were found in the cirrhotic livers of aged patients. However, either morphological method failed to detect copper deposition in fatty liver tissues from the young patient. Iron deposits were also found in the cirrhotic livers of aged patients. The molecular basis of Wilson disease was confirmed by gene analysis. All patients responded to copper chelation therapy. Conclusion: A morphological method of special staining or microanalysis improved with a new fixative may be unreliable for detecting diffusely distributed copper in the early stage of Wilson liver disease.     

Lentiviral gene transfer ameliorates disease progression in Long-Evans cinnamon rats: An animal model for Wilson disease

Objective. Wilson disease is a copper storage disorder caused by mutations in the ATP7B gene leading to liver cirrhosis. It has previously been shown that lentiviral vectors can govern an efficient delivery and stable expression of a transgene. The aim of this pilot study was to prove the principle of a lentiviral gene transfer in the Long-Evans cinnamon (LEC) rat, an animal model of Wilson disease. Material and methods. LEC rats were treated either by systemic application of lentiviral vectors or by intrasplenic transplantation of LEC-rat hepatocytes lentivirally transduced with ATP7B. The ATP7B gene expression was analyzed by RT-PCR and immunofluorescence analysis. The therapeutic effect was assessed by analysis of liver histology, serum ceruloplasmin oxidase activity, and liver copper content. Results, Hepatic expression of the transgene was detected at different time-points post-treatment and lasted for up to 24 weeks (end of experiment). Liver copper levels were lowered in all treatment groups compared to untreated LEC rats. Twenty-four weeks after treatment, the area of the examined liver-tissue sections occupied by fibrosis was 48.3–57.9% in untreated LEC rats and 10.7–19.8% in rats treated with cell therapy. In systemically treated rats, only small fibrous septa could be observed. Conclusions. These data prove for the first time that lentiviral ATP7B gene transfer is feasible in Wilson disease. In our pilot study the systemic approach was more promising in ameliorating disease progression than the transplantation of lentivirally transduced hepatocytes.     

中国肝病患者血清铜蓝蛋白水平的研究

目的　探讨我国不同肝病患者血清铜蓝蛋白 (CP)水平交叉程度 ,为肝豆状核变性(WD)的诊断和鉴别诊断提供科学的依据。方法　测定 90 5例正常人、WD及其他各种肝病患者血清CP水平 ,采用SPSS12统计软件进行统计分析。结果　WD患者血清CP平均为 (93 9± 98 1)mg/L ,与其他各组相比差异有非常显著性 ,72 7%的患者低于 10 0mg/L ,其中 4 2 9%的患者低于 5 0mg/L ,但是也有 9 1%的患者其血清CP正常 ,其中 3例高于 4 0 0mg/L ,最高达 5 0 1mg/L。 6 8%的非WD患者血清CP低于正常 ,最低为 2 8mg/L。急性肝炎患者血清CP平均为 (398 4± 15 1 3)mg/L ,显著高于其他各组。重型肝炎患者血清CP平均为 (2 96 5± 10 6 5 )mg/L ,显著低于其他各组 ,其中18 8%的患者低于正常。结论　WD患者CP水平显著低于正常人和其他肝病患者 ,但是与其他肝病有一定程度的交叉 ,单凭CP水平不足以确诊或排除WD。     

肝豆状核变性与慢性乙型肝炎重度患者临床特征分析*

目的 观察肝豆状核变性（WD）伴黄疸患者的临床特征,探讨肝豆状核变性黄疸患者临床鉴别诊断的侧重点。方法 回顾性分析52例以“黄疸待查”初次入院的WD患者的临床资料,包括临床表现和常规实验室指标的变化。采用序列分析法检测全血基因组8、12、13、16外显子的突变情况。结果 本研究纳入WD患者24例和慢性乙型肝炎重度患者28例。WD患者发病中位年龄（33.2岁）显著低于乙型肝炎（41.8岁,P=0.049）,WD患者自起病至明确诊断的中位时间（8.2月）显著长于乙型肝炎（1月,P<0.001）;WD患者24 h尿铜水平（919.83±1017.15 μg）显著高于乙型肝炎（204.79±191.85 μg,P<0.001）;WD患者血清γ-谷氨酰转肽酶水平（175.74±245.99 U/L）显著高于乙型肝炎（133.44±115.95 U/L,P=0.004）,且与24 h尿铜水平呈正相关（r=0.552,P=0.012）;24例WD患者全部,而仅4例乙型肝炎患者可检出ATP7B突变。结论 肝豆状核变性伴黄疸患者除可利用血清铜、铜蓝蛋白检测进行筛选性诊断外,24 h尿铜检测为重要的鉴别诊断指标,而血清γ-谷氨酰转肽酶可能是该病潜在的诊断指标。     

Copper toxicosis gene MURR1 is not changed in Wilson disease patients with normal blood ceruloplasmin levels

AIM: To analyze our Wilson disease patient cohort (n=106) for alterations in the gene coding for MURR1. METHODS: Patients with an established diagnosis of Wilson disease but normal ceruloplasmin blood levels were chosen for our study (n = 14). Patients with two known disease-causing mutations in the ATP7B gene were not included. The three exons of the human MURR1 gene were sequenced after amplification of the genomic DNA by polymerase chain reaction. RESULTS: Our study did not reveal any mutations leading to an amino acid change in the MURR1 sequence of Wilson disease patients. A polymorphism at 472 bp of the coding sequence could be confirmed. CONCLUSION: The MURR1 gene plays no role in the pathogenesis of Wilson disease patients with normal serum ceruloplasmin levels.     

Copper toxicosis gene MURRl is not changed in Wilson disease patients with normal blood ceruloplasmin levels

AIM: To analyze our Wilson disease patient cohort (n = 106) for alterations in the gene coding for MURR1. METHODS: Patients with an established diagnosis of Wilson disease but normal ceruloplasmin blood levels were chosen for our study (n = 14). Patients with two known disease-causing mutations in the ATP7B gene were not included. The three exons of the human MURR1 gene were sequenced after amplification of the genomic DNA by polymerase chain reaction. RESULTS: Our study did not reveal any mutations leading to an amino acid change in the MURR1 sequence of Wilson disease patients. A polymorphism at 472 bp of the coding sequence could be confirmed. CONCLUSION: The MURR1 gene plays no role in the pathogenesis of Wilson disease patients with normal serum ceruloplasmin levels.     

以非结合胆红素增高为主要表现的Wilson病（附1例报告）

Wilson病是一种罕见的常染色体隐性遗传性铜代谢障碍性疾病。临床症状复杂多样,常见的有肝损害及神经系统病变,而以非结合胆红素增高为主要表现的罕见。诊断上需综合考虑临床表现、角膜Kayser—Fleischer环、铜蓝蛋白检测及肝脏组织学。治疗上常用药物驱铜和肝移植。     

Diminution of toxic copper accumulation in toxic milk mice modeling Wilson disease by embryonic hepatocyte intrasplenic transplantation

AIM: To observe the therapeutic effect of intrasplenic transplantation with embryonic hepatocytes on amelioration of hereditary copper accumulation in toxic milk (TX) mouse modeling Wilson disease. METHODS: Donor hepatocytes were harvested from 14-d fetal liver of a pregnant homogeneous DL mouse. These cells were successively cultured, labeled with fluorescein dye Hoechst 33342 for 24 h, and sequentially infused into the spleen parenchyma of the recipient TX mice. No host immunosuppression measures were taken. Two and four weeks after transplantation, the recipients were killed for routine histologic investigation and immunohistochemistry study up to 4 wk after transplantation. The serum copper and ceruloplasmin concentrations of the recipient mice were determined by graphite furnace atomic absorption spectroscopy. RESULTS: In the following 2nd and 4th wk after transplantation, the donor hepatocytes could be visualized in the livers of 47.3% recipients. The serum ceruloplasmin and copper concentrations increased by 1.6-fold after 2 wk and 2.0-fold times after 4 wk respectively, which ultimately rose from about 30% of the normal level to nearly 60% (P<0.01). The hepatic copper concentration decreased 7.2%, 4 wk after transplantation. Pathologic examination showed that there were many actively proliferative hepatocyte precursor cells with specific embryonic hepatocyte marker AFP migrated into hepatic sinusoids of the recipients. A large number of cells carrying hepatocytes marker and albumin were observed in the recipient spleen tissues. CONCLUSION: Embryonic hepatocytes are capable of differentiating into mature hepatocytes in vivo. After transplantation, the hereditary abnormalities of copper metabolism in TX mice could be corrected partially by intrasplenic transplantation of homogeneous embryonic hepatocytes.     

Copper incorporation into ceruloplasmin is regulated by Niemann–Pick C1 protein

Aim: Wilson disease is a genetic disorder of copper metabolism characterized by impaired biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. Our previous study showed the late endosome localization of ATP7B and described the copper transport pathway from the late endosome to trans‐Golgi network (TGN). However, the cellular localization of ATP7B and copper metabolism in hepatocytes remains controversial. The present study was performed to evaluate the role of Niemann–Pick type C (NPC) gene product NPC1 on intracellular copper transport in hepatocytes. Methods: We induced the NPC phenotype using U18666A to modulate the vesicle traffic from the late endosome to TGN. Then, we examined the effect of NPC1 overexpression on the localization of ATP7B and secretion of holo‐Cp, a copper‐binding mature form of Cp. Results: Overexpression of NPC1 increased holo‐Cp secretion to culture medium of U18666A‐treated cells, but did not affect the secretion of albumin. Manipulation of NPC1 function affected the localization of ATP7B and late endosome markers, but did not change the localization of a TGN marker. ATP7B co‐localized with the late endosome markers, but not with the TGN marker. Conclusion: These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via an NPC1‐dependent pathway and incorporated into Cp.     

Anomalies du métabolisme du cuivre chez l’adulte

Copper is essential for many enzymatic reactions and in neurotransmitter biosynthesis. Its deficiency or its excess has consequences on many organs, especially the liver and the brain. The biochemical tests performed in case of suspicion of copper metabolism disorder are difficult to analyse. They include the measurement of serum ceruloplasmin, serum copper and 24 h urinary copper excretion. The interpretation must take into account the clinical features. We distinguish mainly: (1) copper deficiency, acquired in malabsorption or in copper diet deficiency, or related to a genetic disease (Menkes disease); (2) copper overload, acquired or from a genetic origin (Wilson disease); (3) aceruloplasminemia, reducing ferroxidase activity leading to iron overload. It is important to diagnose these diseases as some of them have an effective treatment if it is started early enough.     

Thyroid dysfunction in primary biliary cirrhosis,primary sclerosing cholangitis and non‐alcoholic fatty liver disease

Background/Aims: Primary biliary cirrhosis (PBC) is frequently associated with autoimmune diseases, including thyroid disease, although it is uncertain that this association is higher than in other liver diseases. Methods: We compared the prevalence and incidence of thyroid dysfunction (TD) in a series of patients with PBC (n=67) with patients with primary sclerosing cholangitis (PSC) (n=79) and non‐alcoholic fatty liver disease (NAFLD) (n=97) seen in a tertiary referral centre who had previously participated in clinical trials. Results: At initial evaluation, prevalence of TD in PBC was 13% compared with 11% in PSC (P=0.71) and 25% in NAFLD (P=0.08). Incidence of TD was 2.9 patients per 100 person‐years in PBC compared with 2.1 patients per 100 person‐years in PSC (P=0.57) and 1.8 patients per 100 person‐years in non‐alcoholic liver disease (P=0.45). Older age, female gender, biochemical abnormalities and concurrent autoimmune disorders were not predictive of the development of TD. Conclusions: TD was unexpectedly as common in patients with PBC as in patients with PSC and NAFLD, yet significantly more common than expected in the general population. Further investigation of thyroid disease in PSC and NAFLD is warranted.