Novel ATPase Cu2+ Transporting Beta Polypeptide Mutations in Chinese Families with Wilson's Disease

Wilson's disease (WD) is an autosomal recessive inherited disorder caused by mutations in the ATPase Cu2+ transporting beta polypeptide gene (ATP7B). The detailed metabolism of copper-induced pathology in WD is still unknown. Gene mutations as well as the possible pathways involved in the ATP7B deficiency were documented. The ATP7B gene was analyzed for mutations in 18 Chinese Han families with WD by direct sequencing. Cell viability and apoptosis analysis of ATP7B small interfering RNA (siRNA)-treated human liver carcinoma (HepG2) cells were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst 33342 staining. Finally, the expression of B-cell CLL/lymphoma 2 (BCL2), BCL2-associated X protein (BAX), sterol regulatory element binding protein 1 (SREBP1), and minichromosome maintenance protein 7 (MCM7) of ATP7B siRNA-treated cells were tested by real-time polymerase chain reaction (real-time PCR) and Western blot analysis. Twenty different mutations including four novel mutations (p.Val145Phe, p.Glu388X, p.Thr498Ser and p.Gly837X) in the ATP7B gene were identified in our families. Haplotype analysis revealed that founder effects for four mutations (p.Arg778Leu, p.Pro992Leu, p.Ile1148Thr and p.Ala1295Val) existed in these families. Transfection of HepG2 cells with ATP7B siRNA resulted in decreased mRNA expression by 86.3%, 93.1% and 90.8%, and decreased protein levels by 58.5%, 85.5% and 82.1% at 24, 48 and 72 hours, respectively (All P<0.01). In vitro study revealed that the apoptotic, cell cycle and lipid metabolism pathway may be involved in the mechanism of WD. Our results revealed that the genetic cause of 18 Chinese families with WD and ATP7B deficiency-induce apoptosis may result from imbalance in cell cycle and lipid metabolism pathway.


Introduction
Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism. The disease is caused by mutations in the ATPase Cu 2+ transporting beta polypeptide gene (ATP7B), a cellular copper transporting ATPase. The incidence of WD among different populations varies from 1/30,000 to 1/100,000 [1]. The hallmarks of the disease are the presence of liver disease, neurologic symptoms and Kayser-Fleischer (K-F) rings. The deficiency of ATP7B disrupts copper homeostasis, particularly in the liver, by greatly decreasing the ability of exporting excess copper from hepatocytes to bile. Copper accumulation causes severe morphological and functional changes, including cirrhosis, hepatitis and liver failure. There is wide variability in clinical manifestation and age at the onset (from 3 to 70 years) of this disease, and typical biochemical features may not always be present. Therefore, genetic analysis provides the potential for more reliable early diagnosis, and prompt treatment [2]. Genetic analysis reveals at least 506 distinct mutations, including missense and nonsense mutations, deletions and insertions (http://www. wilsondisease.med.ualberta.ca/database.asp), but a detailed mechanistic understanding of copper-induced pathology in WD is still lacking. Knowledge of the distribution of particular mutations may help to design shortcuts for genetic screening strategies of WD. To evaluate the frequency of the ATP7B mutations in Chinese Han patients with WD, to explore genotype-phenotype correlations and to possibly unveil the pathways involved in the ATP7B deficiency, we screened 18 families with WD and inhibited the ATP7B gene expression in human liver carcinoma (HepG2) cells.

Subjects
Eighteen Chinese Han WD families (Figure 1), consisting of 38 family members, and 100 normal age-and ethnic-matched unrelated controls (50 males and 50 females) were enrolled in this study. The mean age of disease onset of patients was 17610 years (range 2-41 years) ( Table 1). All patients were examined and diagnosed at the Third Xiangya Hospital. Their evaluations consisted of medical history, physical examination, ophthalmologic slit-lamp examination, abdominal ultrasound, live function tests, serum copper and ceruloplasmin, and 24-hour urinary copper levels. The Third Xiangya Hospital Institutional Review Board approved this proposal: Identification of the Gene Mutation of Wilson Disease, following the Declaration of Helsinki. Informed consents were written by all participating individuals or guardians on the behalf of the minors/children participants involved in the study.

Genetic analysis
Polymerase chain reaction (PCR) amplified all coding regions and intron/exon boundaries of the ATP7B gene. The primers sequences are available on demand (RefSeq NG_008806). PCR products were directly sequenced on 3130 Genetic Analyzer. Given that WD is an autosomal recessive disorder with an estimated carrier frequency of 1/90 [1], and the variant found in a patient was considered as a polymorphism other than as a mutation if it exists as a homozygous statue in normal controls.
Cell culture, small interfering RNA (siRNA) transfection and RNA extraction HepG2 cells (ATCC HB-8065, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum, 1% L-glutamine and 1% penicillin/streptomycin. All experiments were repeated at least three times. In vitro transient transfection was done as described previously [3]. ATP7B siRNA were purchased from Qiagen. The target sequence was 59-CCAATTGATATTGAGCGGTTA-39. Briefly, Cells from passages 10 to 20 were used and they were seeded at 60% confluency into six-well or 96-well dish. The ATP7B siRNA and 5.0 ml HiPerFect Reagent (Qiagen, Melbourne, Australia) were diluted into a final volume of 100 ml in Opti-MEM (Gibco, Grand Island, NY), respectively, and gently mixed and incubated at room temperature for 10 min, then 800 ml Opti-MEM was added to the mixture. The above transfection solution was overlaid onto cells at a final concentration of 5 nM siRNA. Transfection of HepG2 cells with AllStars Negative Control siRNA (Qiagen, Melbourne, Australia) served as a negative control. After 12-hour incubation at 37uC in the presence of 5% CO 2 , 2 ml of complete medium with 10% FBS was added to each well of transfected cells to replace transfection solution.

Real-time polymerase chain reaction (Real-time PCR)
Total RNAs were prepared and cDNA was synthesized by using the SuperScript First-Strand kit (Invitrogen, Carlsbad, CA, USA) as per the manufacturer's instructions. The cDNA samples were amplified by the following primers (Table S1). PCR was done with 1 mg of cDNA and 100 ng/mL of sense and antisense primers in a total volume of 20 mL.

Analysis of cell viability
HepG2 cells were seeded per well in a 96-well plate and incubated (37uC, 5% CO 2 ) for 12 h, then transfected with 5 nM ATP7B siRNA for 24 h and recovery for 3 h with cell culture medium. Copper sulfate (0, 50 and 200 mM; Sigma, St Louis, MO, USA) at various concentration was added for 24 hours. The supernatant was aspirated and 200 ml medium mixed with 3 ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reagent (5 mg/ml) was added to each well. After a 3-h incubation at 37uC, 200 ml of DMSO was added to dissolve the formazan crystals, and the absorbance was then measured at 490 nm using a Digiscan Microplate Reader [4]. Wells without cells were used as blanks and were subtracted as background from each sample. Results were expressed as a percentage of control.

Identification and quantification of apoptotic cells
Treated cells were stained for 10 min with 1 mM Hoechst 33342 to assess nuclear morphology, and with 1 mM calcein AM and 2 mM propidium iodide (PI) to evaluate membrane integrity. About 100 cells were examined in each field at 2006 magnification, and at least five random fields were chosen [4].

Statistical analysis
MTT and cell apoptosis assays were performed in triplicate in three separate experiments. Statistical analysis was performed using SPSS17.0. Results are presented as means 6 SD. Data analysis was performed using one-way ANOVA. The level of significant difference was set at P,0.05 and P,0.01, as indicated.  Table 1. Clinical data from the 20 patients with the ATP7B gene mutations.

Mutation analysis
Thirty-six sequence variants, including 16 polymorphisms  Figure 1A). Twenty mutations, including 3 nonsense mutations (p.Ser105X, p.Gln388X and p.Gly837X), one deletion mutation (c.2659delG), one splice mutation (c.1708-1G.C) and 15 missense mutations (p.Val145Phe, p.Thr498Ser, p.Asp765Gly, p.Arg778Leu, p.Pro840Leu, p.Gly869Arg, p.Ala874Pro, p.Thr888Pro, p.Thr935Met, p.Pro992Leu, p.Asp1047Val, p.Ile1148Thr, p.Glu1173Lys, p.Ala1295Val and p.Arg1320Ser) were identified by further analysis of these variants in 100 normal controls (All novel mutations and variants included in the article had been submitted to NCBI, Table S2-4). Among these mutations, Four (p.Val145Phe, p.Gln388X, p.Thr498Ser and p.Gly837X) of them are novel ATP7B gene mutations [6][7][8] ( Figure 1B). Three homozygous mutations and 15 compound heterozygous mutations were found in probands from 2 consanguinity and 16 non-consanguinity families, respectively. Further analysis of family members showed that homozygous or compound heterozygous mutations in the ATP7B gene were the cause of WD in our families. Single heterozygous mutation in one of the two copies of the ATP7B gene was insufficient to cause this disorder, consistent with a loss-of-function mechanism of the ATP7B mutations.

Haplotype analysis
Haplotype analysis of different families with the same mutation revealed that clear founder effects for p.Arg778Leu

In vitro effects of ATP7B silencing
Compared to AllStars Negative Control of siRNA treated cells, transfection of HepG2 cells with 5 nM ATP7B siRNA resulted in decreased mRNA expression by 86.3%, 93.1% and 90.8% (All P,0.01; Figure 2) and decreased protein levels by 58.5%, 85.5% and 82.1% at 24, 48 and 72 hours, respectively (All P,0.01; Figure 3A; B). There was no significant difference for mRNA or protein expression between AllStars Negative control siRNA treated cells and untreated cells at 24, 48 and 72 hours (All P .0.05). When incubated with 5 nM ATP7B siRNA for 48 hours, the cell viability decreased by 17.2% and 16.6% (as compared with untreated cells and AllStars Negative Control siRNA treated cells, respectively) by MTT analysis (P,0.05; Figure 4). Using the same incubation method, about 5.6% cells underwent apoptosis ( Figure 5).
To examine the combined effects of ATP7B deficiency and copper, HepG2 cells were treated with 5 nM ATP7B siRNA for 48 hours and then exposed to 50 and 200 mM of copper sulfate for 24 hours. Exposure to 50 mM copper sulfate further reduced the cell viability by 53.3%, as compared with AllStars Negative Control siRNA treated cells alone (P,0.05), and by 44.0% as compared with ATP7B siRNA treatment alone (P,0.05). Cell viability decreased more obviously when ATP7B-deficient cells were exposed to higher concentration of copper sulfate (decreased by 79.8% for 200 mM) as compared with AllStars Negative Control siRNA treated cells alone (P,0.05), and by 77.6% as compared with ATP7B siRNA treatment alone (P,0.05; Figure 4). Apoptosis increased to 17.3% and 24.6%, respectively, when cells were exposed to 50 mM and 200 mM copper sulfate (P,0.05; Figure 5). ATP7B siRNA (5 nM) treatment also increased copperinduced apoptosis accompanying the higher dose of copper sulfate ( Figure 5).

Effect of ATP7B silencing on apoptosis, cell cycle and lipid metabolism
To determine if ATP7B siRNA treatment may alter translation of apoptosis-related proteins, the expression of the BCL2 and BAX were measured. Real-time RT-PCR analysis showed that the BCL2 mRNA expression decreased by 90.8%, 95.4% and 96.1% after HepG2 cells were treated with ATP7B siRNA for 24 h, 48 h and 72 h respectively, as compared with AllStars Negative Control siRNA treated cells (All P,0.05). Whereas BAX expression increased by 5.9-fold, 6.4-fold and 7.2-fold at 24, 48 and 72 hours, respectively comparing to those of the AllStars Negative Control of siRNA treated cells (All P,0.05). Consistently, Western blot showed that BCL2 decreased to 54.6%, 19.3% and 3.1%, and BAX increased to1.8-fold, 1.7-fold and 3.2-fold for 24 h, 48 h and 72 h ( Figure 3A, C, D). These results indicate that ATP7B siRNA silencing induced a decrease in BCL2 expression, and an increase in BAX expression, indicative of apoptosis.
Previous in vivo data suggested cell cycle machinery and lipid metabolism were selectively affected by copper overload in Atp7b 2/2 mice [9][10][11]. Therefore, to explore possible mechanisms in human, we examined the effects of ATP7B siRNA silencing on the expression of cell cycle-related gene(s) and lipid metabolismrelated gene(s). The mRNA levels of SREBP1, a lipid metabolismrelated gene, were significantly decreased by 49.7%, 59.9% and 80.0% in the ATP7B siRNA silenced cells at 24, 48 and 72 hours, respectively, compared with those of AllStars Negative Control siRNA treated cells (P,0.05). Consistently, the protein levels were decreased by 42.7%, 52.2% and 94.9%, respectively. The mRNA levels of the MCM7, a cell cycle-related gene, was significantly decreased by 89.2%, 95.1% and 95.5%, and the MCM7 protein levels were significant decreased by 32.8%, 92.4% and 93.6% at 24, 48 and 72 hours, respectively (P,0.05; Figure 3A, E, F).

Discussion
Wilson's disease, a genetic disorder of copper metabolism, is caused by mutations in the ATP7B gene. The disease has significant phenotypic diversity, and occurs widely in all ethnic populations [12]. The phenotype of WD usually manifests as a range or a spectrum, and clinical presentations include symptomatic live disease, neuropsychiatric disorders, and other features (hematologic, renal, etc). Affected patients in our families began to have classical symptoms of WD with hepatic or neurological features, and K-F ring, from 2-41 years of age. Our cases also shared clinical features similar to other ATP7B cases, including beneficial response to D-penicilliamine [13]. The age of onset is similar within sibships in our study (Family M41 and Family M1279), which may be attributed to the similar genetic background [14]. All of patients had hepatic diseases, whereas only 60% (12/20) of patients had neurological presentations as the early manifestations. Although the hepatic presentation was assumed to mean more severely deranged ATP7B function, modifier genes, such as the 5,10-methylenetetrahydrofolate reductase gene, environmental factors, such as nutritional copper intake, infectious disease, drug and toxin, and other epigenetic factors must be at play in the overall phenotypic expression of WD in individuals [15,16]. We found three homozygous ATP7B mutations and 15 compound heterozygous mutations in 2 consanguinity pedigrees and 16 non-consanguinity pedigrees, respectively. Four of these 20 mutations are novel. Among the novel mutations identified in this study, p.Val145Phe was present in two compound heterozygous patients (sib) and their unaffected mother was in heterozygous status (M1279). p.Thr498Ser was found in a 20-year female WD patient (M1038, II:1) with compound heterozygous mutations (p.Thr498Ser and p.Pro992-Leu), and the age at the onset was 12 years with neurological presentation. Compound heterozygous ATP7B mutations  (p.Gly837X/p.Pro992Leu) were found in an 18-year WD female (M1031, II:1) presented with liver disease at the onset age of 2 years old, the earliest reported onset age. All these four novel mutations were not detectable in 100 normal controls. Some reports suggested that the early onset of severe liver disease occurs with frameshift or nonsense mutations [17]. However, our study does not confirm that, consistent with other reports [18,19]. Homozygous Pro992Leu mutation, probably involved in poor phosphorylation, was found in a 36-year old female patient, she developed abdominal pain and vomiting at age of 18 years, rapidly progressed to acute liver failure and received urgent liver transplantation. On neurological examination, abnormal involuntary movement, festinating gait, dysmetria and bilateral intention tremor on knee-to-heel and finger-to-nose testing, dysdiadochokinesis, and scanning dysarthria were found. Homozygous Ile1148Thr mutation, expected to impair the function of the WD protein by altering the secondary structure of the ATP loop, was found in a 43-year old female with both hepatic and neurologic manifestations. She developed abdominal pain and jaundice at 41 years of age, and abnormal rigidity, bilateral static tremor and dysarthria were found on neurological examination. Although consanguineous marriage were denied, parents from the same village and the identical haplotype between the parents who are from the same village indicates that the mutation may be inherited from a same ancestor. Homozygous p.Ala1295Val mutation, located in the conserved adenosine triphosphate (ATP) hinge region, was found in a female with hepatic manifestation as early symptoms, confirming the association between homozygous mutation in the ATP hinge region of ATP7B and hepatic phenotype [20]. p. Arg778Leu and p.Pro992Leu mutations, was found with high allele frequencies in WD form Asia [1], and a similar high allele frequencies (15% for p. Arg778Leu and 22.5% for p.Pro992Leu) were found in our study. Interestingly, haplotype analysis suggested that the different pedigrees existed founder effects for p.Arg778Leu, p.Pro992Leu, p.Ile1148Thr and p.Ala1295Val mutations, suggesting these mutations may be not located on a hot mutation site but due to founder effects.
Several studies suggest that a single ATP7B mutation may cause WD [1]. In the few patients in whom only a heterozygous ATP7B mutation was detected. It is possible that a second mutation escaped identification by the methods employed or that some mutations in heterozygous forms are sufficient to cause disease [1,4]. However, in our study, none of the heterozygous carriers were affected, consistent with loss-of-function as the underlying molecular mechanism of ATP7B mutations in WD. Therefore, caution should be exercised in the interpretation of heterozygous forms in WD individuals.
Our study indicates that ATP7B gene silencing is able to effectively inhibit endogenous expression of ATP7B mRNA and protein. ATP7B deficiency may reduce cell viability by inducing apoptosis in HepG2 cells. The ATP7B deficiency, coupled with exposure to copper, may further aggravate apoptosis in a dosedependent manner for copper. The increased BAX expression and decreased BCL2 expression in HepG2 cells treated with ATP7B siRNA correlates with reduced cell viability and apoptosis.
Our discovery of the reduced expression of SREBP1 and MCM7 mRNA and protein by ATP7B silencing in HepG2 cells provides evidence that the deficiency of the ATP7B gene is involved in the initiation of the lipid metabolism and cell cycle pathway, although the precise mechanism is unknown. SREBP1 is a fundamental regulator of fatty acid desaturation, elongation and phospholipid biosynthesis. Our results of decreased expression of SREBP1 mRNA and protein by the ATP7B inhibition is consistent with the findings in a recent publication of 45 WD patients with the lower levels of total cholesterol and LDL cholesterol compared with the control group [21], and reduced SREBP1 expression in liver of Atp7b knockout mice [22].
MCM7 is involved in replicative licensing and synthesis of DNA [23]. In contrast to our result, MCM7 was previously identified as an overexpressed gene in the liver of Atp7b knockout mouse in cDNA microarray studies [22]. However, our findings of deceased MCM7 expression in ATP7B deficiency cells is consistent with previous observation of overexpression of MCM7 in cancers [24][25][26], which is the opposite extreme for apoptosis.
The detrimental effects of ATP7B silencing on hepatocytes may be attributed to its role in promoting reactive oxygen species generation via Cu 2+ accumulation induced oxidation of biomolecules such as lipids, proteins, and nucleic acids [27,28].
In summary, 4 novel mutations were identified in our study and apoptosis may result from ATP7B deficiency-induced imbalance in cell cycle and lipid metabolism pathway. The results add data to the spectrum of the mutations in the ATP7B gene in Chinese Han population.