Genetic And Pathogenic Mechanism Study Of Wilson’s Disease

Objective:1. The ATP7B gene mutation frequency and haplotype analysis were detected in9Chinese Han Wilson’s disease (Wilson’s disease, WD) patients and families diagnosed in the Third Xiangya Hospital from Sep.2010-Dec.2012.2. The clinical features of WD pateints were analyzed, trying to discover the genotype-phenotype relationship.Methods:Polymerse chain reaction (PCR) and gene sequencing were used to detect the ATP7B gene mutations in9WD patients, family members and normal controls.Results:1. One homozygous mutation and8compound heterozygous mutations were detected in9WD families. Eleven mutations were identified, including one nonsense mutation (p.Ser105Stop), one splice mutation (c.1708-1G>C) and9missense mutations (p.Arg778Leu, p.Pro840Leu, p. Ala874Pro, p.Thr888Pro, p.Thr935Met, p.Pro992Leu, p.Asp1047Val, p.Ile1148Thr and p.Glu1173Lys), however, no novel mutation was detected in the9patients. Eight polymorphisms were found, including p.Ile390Val, p.Ser406Ala, p.Val456Leu, p.Leu770Leu, p.Arg832Lys, p.Arg952Lys, p.Val1MOAla and IVS18+6T/C.2. Haplotype analysis were identified in the families with the same mutations, and we found Family M11841and Family M4056had disease haplotype; Family M1841, Family M1856, Family M4061, Family M4062and Family M4174had disease haplotype; and Family M1807and Family M1856had disease haplotype, which suggested founder effect was possibly existed.3. In the11ATP7B gene mutations detected in9families with WD, p. Ala874Pro and p.Thr888Pro were both in exon11, and p.Ile1148Thr and p.Glu1173Lys were in exon16. The gene mutation frequency of p.Arg778Leu (exon8), p.Pro992Leu (exon13) and p.Ile1148Thr (exon16) were11.1%,27.8%and16.7%, respectively. So, we concluded that exons8,11,13, and16of the ATP7B gene may the prior screening exons for the Hunan patients with WD.4. Clinical analysis showed that nine patients were classified phenotypically into hepatic (n=3) and neurological (n=6). The ages of patients with the hepatic form were no older than those of patients with the neurologic form (22.7±15.3vs.20.7±10.9, p>0.05). All patients with neurologic form had hepatic presentations when came to our hospital. The clinical manifestations of the6patients were as follows:extrapyramidal symptoms in6(100%) patients, hepatomegaly in6(100%), splenomegaly in5(83.3%), ascites in4(66.7%), low patelet in4(66.7%), high transaminase in3(50%), jaundice in2(33.3%). Kayser-Fleischer rings and hypoceluroplasminemic (<0.2g/L) were examined in all patients, suggesting the two methods and genetic detection were all effective diagnostic method.Conclusions:1. The ATP7B gene mutations were analyzed in9WD families, including11mutations and8polymorphisms.2. Different families shared the disease haplotypes (Family M1841and Family M4056; Family M1841, Family M1856, Family M4061, Family M4062and Family M4174; Family M1807and Family M1856), suggesting possible founder effect was existed.3. Exons8,11,13, and16on the ATP7B gene might be high mutation frequency regions for WD pateints. Objective:The loss of ATP7B gene function was builded by transfection of HepG2cells with ATP7B siRNA. Furthermore, in vitro effects of ATP7B Silencing on cell viability and apoptosis were deteced by MTT and Hoechst33342stain, and the changes of the genes and proteins of apoptosis-related gene BCL2and BAX, cell cycle-related gene MCM7and lipid metabolism-related gene SREBP1were measured to determine the possible pathogenic mechanism.Methods:1. HepG2cells were transfected with AllStars Negative Control siRNA and ATP7B siRNA, RT-PCR and Western blot analysis were used to detect the expression of ATP7B genes and proteins in order to determinethe the best concentration of ATP7B siRNA and the best time of treatment.2. Cell viability of AllStars Negative Control siRNA-treated HepG2cells and ATP7B siRNA-treated HepG2cells after exposing at0μM、50μM和200μM copper sulfate were measured by MTT assay.3. Apoptosis analysis of AllStars Negative Control siRNA-treated, ATP7B siRNA-treated,200μM copper sulfate-treated and ATP7B siRNA+200μM copper sulfate-treated HepG2cells were measured by Hoechst33342staining and electron microscopy method.4. The mRNA and protein expression of BCL2, BAX, SREBP1and MCM7of ATP7B siRNA-treated cells were tested by RT-PCR and Western blot analysis.Results:1. Compared to AllStars Negative Control of siRNA treated cells, transfection of HepG2cells with5nM ATP7B siRNA resulted in decreased mRNA expression by86.3%,93.1%and90.8%(All P<0.01) and decreased protein levels by58.5%,85.5%and82.1%at24,48and72hours, respectively (All P<0.01), and transfection with5nM ATP7B siRNA for48hours up to a maximum interference efficiency.2. Cell viability was decreased with the increase of copper sulfate concentration both in AllStars Negative Control siRNA-treated and ATP7B siRNA-treated HepG2cells when incubated at0μM,50μM and200μM copper sulfate for48hours. Exposure to50μM and200μM copper sulfate reduced the cell viability by53.3%and79.8%respectively, as compared with AllStars Negative Control siRNA treated cells alone (All P<0.05). Cell viability decreased by44.0%and77.6%at50μM and200μM copper sulfate as compared with ATP7B siRNA treatment alone (All P<0.05).3. When incubated with5nM ATP7B siRNA for48hours, about10.6%cells underwent apoptosis as compared with AllStars Negative Control siRNA treated cells (P<0.05). Apoptosis increased to18.9%when cells were exposed to200μM copper sulfate as compared with AllStars Negative Control siRNA treated cells (P<0.05). ATP7B siRNA (5nM) treatment also increased copper-induced apoptosis accompanying the higher dose of copper sulfate (200μM) to24.6%as compared with200μM copper sulfate treated cells.4. Real-time RT-PCR analysis showed that the BCL2mRNA expression decreased by90.8%,95.4%and96.1%after HepG2cells were treated with ATP7B siRNA for24h,48h and72h respectively, Whereas BAX expression increased by5.9-fold,6.4-fold and7.2-fold at24,48and72hours, respectively comparing to those of the AllStars Negative Control of siRNA treated cells (All P<0.05). The mRNA levels of SREBP1were significantly decreased by49.7%,59.9%and80.0%and the mRNA levels of the MCM7was significantly decreased by89.2%,95.1%and95.5%in the ATP7B siRNA silenced cells at24,48and72hours, respectively, compared with those of AllStars Negative Control siRNA treated cells (All P<0.05).5. Western blot showed that BCL2decreased to54.6%,19.3%and3.1%, and BAX increased to1.8-fold,1.7-fold and3.2-fold for24h,48h and72hours as compared with AllStars Negative Control siRNA treated cells (All P<0.05). The protein levels of SREBP1were decreased by42.7%,52.2%and94.9%, respectively, and the MCM7protein levels were significant decreased by32.8%,92.4%and93.6%at24,48and72hours, respectively, as compared with AllStars Negative Control siRNA treated cells (P<0.05).Conclusions:1. Our study indicates that ATP7B gene silencing (the target sequence was5’-CCAATTGATATTGAGCGGTTA-3’) was able to effectively inhibit endogenous expression of ATP7B mRNA and protein, which was a good hepatic cell model for the cell study of Wilson’s disease.2. A time-and dose-response relationship was with respect to copper toxicity, ATP7B deficiency may reduce cell viability combined with copper.3. ATP7B siRNA may induce apoptosis combined with high copper in HepG2cells.4. There were significant differences in the expression of BCL2, BAX, SREBP1and MCM7mRNA and protein between ATP7B siRNA treated group and AllStars Negative Control siRNA treated group.