Novel ATP7B gene mutations in Chinese Han patients with hepatolenticular degeneration

BACKGROUND： ATP7B gene exon 8 Arg778Leu and exon 12 Arg952Lys are gene mutation hot spots in Chinese Han patients with hepatolenticular degeneration, or Wilson＇s disease （WD）. However, the gene fragments are too short for detection and the mutation detection rate remains low. OBJECTIVE： To analyze DNA sequences of ATP7B gene exon 8-exon 9 and exon 10-exon 12 sections. DESIGN, TIME AND SE＇I-rlNG： A concurrent, non-randomized, controlled, genetic polymorphism study was performed at the Anhui Medical Genetics Center, Anhui, China from March to July in 2009. PARTICIPANTS： Fifty patients, who were admitted to the Department of Neurology at the First Affiliated Hospital of Anhui Traditional Chinese Medical College between March and July in 2009, were diagnosed with WD. The WD group comprised 32 males and 18 females, with an average age of （18.8 ± 8.3） years. WD was confirmed by clinical observation, as well as physical, imaging, and biochemical examinations, including testing for serum copper, ceruloplasmin, and copper oxidase. The control group comprised 20 normal subjects, who underwent physical examination at the First Affiliated Hospital of Anhui Traditional Chinese Medical College, and included 13 males and 7 females, with an average age of （27.9 ± 2.4） years. All subjects were Chinese Han population. METHODS： Genomic DNA was extracted from 50 WD patients and 20 normal controls. Polymerase chain reaction amplification of ATP7B gene exon 8-exon 9 （about 1 100 bp） and exon 10-exon 12 （about 850 bp） segments was performed. DNA exon-intron amplification products from all subjects were processed through direct bidirectional sequencing, and sequencing results were analyzed. MAIN OUTCOME MEASURES： Sequence changes of ATPTB gene exon 8-exon 9 and exon 10-exon 12 segments. RESULTS： In the 50 included WD patients, ATP7B gene intron 8 nt53592A → G with nt53671G→ A homozygous mutation was detected between exon 8-exon 9 in seven cases; exon 8 Arg778Leu mutations with Leu770Leu synonymous mutation was detected in four cases; exert 11 Gly790Arg heterozygous missense mutation between exon 10-exon 12 was found in four cases; exon 12 Arg952Lys heterozygous missense mutation was seen in 11 cases; and two additional cases were associated with exon 1211e929Val polymorphism. CONCLUSION： ATP7B gene intron 8 mutation is a possible pathogenic mutation that is associated with WD pathogenesis. The exon 11 mutation rate accounts for 8% of all WD patients, and the very few previously reported cases deserve further study.

Detection of distribution of copper inside and outside of lysosomes in cultured hepatolenticular degeneration fibroblasts by electron probe X-ray microanalysis

OBJECTIVE: To observe the distribution of copper in the subcellular structure for the understanding of primary pathogenesis of hepatolenticular degeneration (HLD). METHODS: Skin fibroblasts taken from HLD patients were cultured as an in vitro model of HLD, and the control cells taken from healthy volunteers were clutured in the same way. The distribution of copper inside and outside of lysosomes in fibroblasts was detected by quantitative electron probe X-ray microanalysis. The relationship between the subcellular location of copper and the genotype of the patients, and relationship between the distribution of copper and the course of the disease were analyzed. RESULTS: The content of Cu^(2+) inside lysosomes of HLD cells (14.6±2.1 mmol/kg) and of heterozygote cells (11.6±0.6 mmol/kg) was higher than that of normal cells (4.5±1.2 mmol/kg) (P<0.01). The content of Cu^(2+) outside lysosomes of HLD cells (17.5±4.2 mmol/kg) and of heterozygote cells (12.0±0.9 mmol/kg) was higher than that of normal cells (4.7±1.2 mmol/kg) (P<0.01). The distribution of copper in the subcellular structure was correlated with disease courses of HLD patients. With the progression of the disease, more copper was deposited in lysosomes (r=0.85, P<0.01). The content of copper in the diffused cytoplasmic compartment in HLD cells was correlated with that of sulfur (r=0.86, P<0.05), but not in heterozygote and normal cells. CONCLUSIONS: In the early stage of HLD, copper is accumulated outside lysosome, which is paralleled with increase of metallothionein-like proteins (copper and sulfur-binding proteins). With the development of the disease, more copper is deposited inside lysosome than outside lysosome. We conclude that the up-regulation expression of copper and sulfur-binding proteins and copper accumulation in lysosomes may play an important role in lowering the ATP7B gene mutation-induced toxic effects of free copper on the cell.

Berberine alleviates ovarian tissue damage in mice with hepatolenticular degeneration by suppressing ferroptosis and endoplasmic reticulum stress

Objective:Hepatolenticular degeneration (HLD) is an autosomal recessive disorder that manifests as multiorgan damage due to impaired copper (Cu) metabolism. Female patients with HLD often experience reproductive impairments. This study investigated the protective effect of berberine against ovarian damage in toxic-milk (TX) mice, a murine model for HLD.Methods:Mice were categorized into control group, HLD TX group (HLD group), penicillamine (Cu chelator)-treated TX group and berberine-treated TX group. Body weight, ovary weight and the number of ovulated eggs were recorded. Follicular morphology and cellular ultrastructure were examined. Total iron, ferrous iron (Fe2+) and trivalent iron (Fe3+) levels, as well as malondialdehyde (MDA), glutathione(GSH) and oxidized glutathione (GSSG), were measured in the ovaries. Western blot analysis was used to analyze the expression of proteins related to ferroptosis and endoplasmic reticulum (ER) stress.Results:Ovarian tissue damage was evident in the HLD group, with a significant increase in ferroptosis and ER stress compared to the control group. This damage was inhibited by treatment with penicillamine,a Cu chelator. Compared with the HLD group, berberine increased the number of ovulations, and improved ovarian morphology and ultrastructure. Further, we found that berberine reduced total iron,Fe2+, MDA and GSSG levels, elevated GSH levels, decreased the expression of the ferroptosis marker protein prostaglandin-endoperoxide synthase 2 (PTGS2), and increased glutathione peroxidase 4 (GPX4)expression. Furthermore, berberine inhibited the expression of ER stress-associated proteins mediated by the protein kinase RNA-like ER kinase (PERK) pathway.Conclusion:Ferroptosis and ER stress are involved in Cu-induced ovarian damage in TX mice. Berberine ameliorates ovarian damage in HLD TX mice by inhibiting ferroptosis and ER stress.

Observation of the Clinical Therapeutic Effect of Gandou Tablet Ⅰ(肝豆片Ⅰ号) on Hepatolenticular Degeneration

Objective: To investigate the therapeutic efficacy of Gandou Tablet Ⅰ (肝豆片Ⅰ号) in treating hepatolenticular degeneration (HLD) and its effect on urinary trace element in 24 hours. Methods: Thirty four patients with HLD took Gandou Tablet Ⅰ for 4 weeks, their changes of symptoms, signs and daily life pattern were observed closely, and the levels of urinary trace element in 24 hours before the therapy and every week after initiation of treatment were measured. Results: The total effective rate reached 70.59%, with markedly effective rate being 8.82%.The therapeutic effect was much better in the child and mild patients. After treatment, the daily urinary copper output was obviously increased every week compared with that before ( P < 0.01 ). Although it was highly negative linear correlated with the duration of treatment (γ=-0.96, P < 0.05 ), it has insignificant difference between any two weekly excretion of urinary copper per 24 hours after treatment ( P >0.05). So its effect on urinary copper excretion was not decreased. Conclusion: Gandou Tablet Ⅰ could increase the output of urinary copper, hence it was effective in treating HLD without apparent toxic or side reaction.

Application of ARFI technology to explore the clinical study of Gandou Tang in treating liver fibrosis of wilson's disease with damp-heat accumulation

Objective:Acoustic radiation force impulse(ARFI)was applied to measure Shear wave velocity(SWV)of liver in patients with Wilson's disease(WD).To investigate the relationship between SWV and the serological indexes of liver fibrosis,such as type Ⅳ collagen(CⅣ),hyaluronic acid(HA),type Ⅲ procollagen peptide(PⅢNP),laminin(LN),APRI score(Asparate Aminotransfer to Platelet Ratio Index),and FIB-4 index(FIB-4 index).The clinical efficacy of GandouTang(GDT)in the treatment of liver fibrosis in WD with damp-heat internalization was also observed.Methods:80 cases of WD patients who met the inclusion criteria were randomly divided into the treatment group and the control group,with 40 cases in each group.The control group was treated with Sodium Dimercaptopropylsulfonate(DMPS)and the treatment group was additionally treated with the traditional Chinese medicine GDT.One course for 8 days,a total of 6 courses.The levels of SwV and four serological indicators of liver fibrosis(PⅢNP,HA,CⅣ,LN),APRI score and FIB-4 index were compared before and after treatment.Pearson correlation test was used to analyze the correlation between SWV and HA,CⅣ,LN,PⅢNP,APRI score and FIB-4 index.The effects of GDT on SWV,liver fiber,APRI and FIB-4 were evaluated according to the treatment plan.Results:①The SWV was positively correlated with FIB-4(r=0.83),APRI(r=0.82),HA(r=0.87),CⅣ(r=0.71),LN(r=0.85)and PINP(r=0.77).②Before treatment,there were no significant differences in SWV level,PⅢNP,HA,CⅣ,LN,APRI and FIB-4 levels between two groups(P>0.05).After treatment,the levels of PⅢNP,HA,LN,SWV,APRI and FIB-4 in both groups were significantly decreased(P<0.05,P<0.01),and the levels in the treatment group were lower than those in the control group.There were no significant specific changes in CⅣ level(P>0.05).Conclusion:SWV value can reflect the degree of WD liver fibrosis,and is positively correlated with HA,PⅢNP,CⅣ,LN,FIB-4 index and APRI score.On the basis of the treatment of protecting liver and expelling copper with western medicine,plus the treatment of traditional Chinese medicine GDT can effectively improve the degree of liver fibrosis in WD patients with damp-heat accumulation.

Copper transportion of WD protein in hepatocytes from Wilson disease patients in vitro

AIM: To study the effect of copper transporting P-type ATPase in copper metabolism of hepatocyte and pathogenesis of Wilson disease (WD). METHODS: WD copper transporting properties in some organelles of the cultured hepatocytes were studied from WD patients and normal controls.These cultured hepatocytes were incubated in the media of copper 15 mg x L(-1) only, copper 15 mg x L(-1) with vincristine (agonist of P-type ATPase) 0.5mg x L(-1), or copper 15 mg x L(-1) with vanadate (antagonist of P-type ATPase) 18.39 mg x L(-1) separately. Microsome (endoplasmic reticulum and Golgi apparatus), lysosome, mitochondria, and cytosol were isolated by differential centrifugation. Copper contents in these organelles were measured with atomic absorption spectrophotometer, and the influence in copper transportion of these organelles by vanadate and vincristine were comparatively analyzed between WD patients and controls. WD copper transporting P-type ATPase was detected by SDS-PAGE in conjunction with Western blot in liver samples of WD patients and controls. RESULTS: The specific WD proteins (M(r)155,000 lanes) were expressed in human hepatocytes, including the control and WD patients. After incubation with medium containing copper for 2 h or 24 h, the microsome copper concentration in WD patients was obviously lower than that of controls, and the addition of vanadate or vincristine would change the copper transporting of microsomes obviously. When incubated with vincristine, levels of copper in microsome were significantly increased, while incubated with vanadate, the copper concentrations in microsome were obviously decreased. The results indicated that there were WD proteins, the copper transportion P-type ATPase in the microsome of hepatocytes. WD patients possessed abnormal copper transporting function of WD protein in the microsome, and the agonist might correct the defect of copper transportion by promoting the activity of copper transportion P-type ATPase. CONCLUSION: Copper transportion P-type ATPase plays an important role in hepatocytic copper metabolism. Dysfunction of hepatocytic WD protein copper transportion might be one of the most important factors for WD.

Currently Clinical Views on Genetics of Wilson＇s Disease

Objective： Tile objective of this study was to review the research on clinical genetics of Wilson＇s disease （WD）. Data Sources： We searched docunlents from PubMed and Wanfang databases both in English and Chinese up to 2014 using the keywords WD in combination with genetic,ATP7B gene, gene mntation, genotype, phenotype. Study Selection： Publications about the ATP7B gene and protein timction associated with clinical features were selected. Results： Wilson＇s disease, also named hepatolenticular degeneration, is an autosomal recessive genetic disorder characterized by abnormal copper metabolism caused by mutations to the copper-transporting gene A TP7B. Decreased biliary copper excretion and redtlced incorporation of copper into apoeeruloplasmin caused by defunctionalization of ATP7B protein lead to acct, mulation of copper in many tissues and organs, including liver; brain, and cornea, finally restllting in liver disease and extrapyramidal symptoms. It is the most common genetic neurological disorder in the onset of adolescents, second to muscular dystrophy in China. Early diagnosis and medical therapy are of great significance tbr improving the prognosis of WD patients. However, diagnosis of this disease is usually diffict.lt because of its complicated phenotypes. In the last l0 years, an increasing number of clinical studies have used molecular genetics techniques. Improved diagnosis and prediction ol＇the progression of this disease at the molecular level will aid in the development of more individualized and effective intervcntions, which is a key to transition from molecular genetic research to the clinical study. Conclusions： Clinical genetics studies are necessary to understand the mechanism underlying WD at the molecular level from the genotype to the phenotype. Clinical genetics research benefits newly emerging medical treatments including stem cell transplantation and gone therapy for WD patients.

Identification and analysis of mutations of the Wilson disease gene in Chinese population

OBJECTIVE: To investigate the characteristics of mutations in exon 3-20 of Wilson disease (WD) gene and their consequences in Chinese population. METHODS: Sixty unrelated normal Chinese and forty-four unrelated WD patients were studied. Genomic DNA was prepared from peripheral blood leukocytes by a salt-out method. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and subsequently direct sequencing were used to identify the mutations and polymorphisms of WD gene. RESULTS: Ten different mutations have been found, accounting for 52% of the mutant genes. Five of them are identified as novel missense mutations. Mutations Arg778Leu, Thr935Met and Ala874Val were represented respectively in 28.4%, 6.8% and 3.4% of WD chromosomes. The remaining mutations were found rare and limited to one or two patients. A total of 11 patients were homozygous for a single mutation, and 17 patients were in a compound heterozygous state with or without a known mutation. CONCLUSION: In Chinese, WD seems to result from two or three relatively common mutations and a large number of rare mutations. Arg778Leu and Thr935Met might be hotspots of mutation in Chinese population. The results indicated that the feature of mutations of WD gene is different between Chinese and the Western. Instead of exon 14 and exon 18, we had to select exon 8 and exon 12 first to detect mutations of WD gene in Chinese. It is of great importance to establish a direct diagnostic method for WD. This study improves our knowledge on functional domains of the WD gene, and helps elucidate the wide spectrum of manifestations of the disease as well.

Clinical efficacy and safety of Gandouling plus low-dose D-penicillamine for treatment of Wilson's disease with neurological symptoms

OBJECTIVE： To investigate the effect and safety of Gandouling plus low-dose D-penicillamine for treating patients with Wilson＇s disease （WD） who have neurological symptoms. METHODS： WD patients with neurological symptoms were divided into two groups： a treatment group （n = 53） and a control group （n = 50）. The treatment group received anti-copper therapy with a combination of Gandouling and low-dose D-peni- cillamine （10 mglkg）, whereas the control group was with conventional dose D-penicillamine （20 rag/ kg） monotherapy. The clinical efficacies, adverse re- actions, and results of the various hematological and biochemical investigations were recorded and analyzed statistically. RESULTS： Overall, 98.11% of the WD patients treated with the combined therapy experienced alleviation of their neurological condition （paralleled by a significantly improved Global Assessment Scale score or remained stable）. Their white blood cell and platelet counts stabilized, and their liver function was improved or remained stable. The combined therapy also obviously promoted improved 24-h urinary copper excretion. Only 15.09% of the WD patients with the combined therapy experienced adverse reactions, including neurological deterioration in one case （1.89%） and hepatic worsening in one case （1.89%）, which was less frequent than that in the control group given conventional-dose D-penicillamine monotherapy. CONCLUSION： Treating WD patients with neurological symptoms using Gandouling plus low-close D-penicillamine is effective and safe.

A case on acupuncture for Wilson disease

Wilson disease(WD),an autosomal recessive disease with abnormal copper metabolism,is caused by mutations in the ATP7 B gene.The symptoms are characterized by liver,nervous system,and ocular manifestations related to the deposition of copper in the liver,lens nucleus,and cornea.The case reported in this article was a 30-year-old woman with high muscle tension,unable to stand or walk,low voice,and short-term memory loss.Brain MRI showed that patchy Tl,long T2 signals,and high FLAIR presented signals in the basal ganglia thalamus area,subcortex,and brain stem.Low signal shadows were presented in magnetically sensitive bilateral basal ganglia region,callus,and cerebellar dentate nucleus.Ceruloplasmin was significantly reduced.With conventional treatments by western medicine,the symptoms were not alleviated.However,after the application of acupuncture-assisted treatment,the patient is capable with self-care ability,thus a desired effect was achieved.It is therefore suggested that acupuncture shall be applied to assist the treatment of WD to relieve symptoms and help patients regain self-care ability.

Subnormal Serum Liver Enzyme Levels:A Review of Pathophysiology and Clinical Significance

Subnormal levels of liver enzymes,below the lower limit of normal on local laboratory reports,can be useful diagnostically.For instance,subnormal levels of aminotransferases can be observed in vitamin B6 deficiency and chronic kidney disease.Subnormal alkaline phosphatase levels may indicate the presence of hypophosphatasia,Wilson's disease,deficiencies of divalent ions,or malnutrition.Subnormal levels of gamma glutamyl transferase may be seen in cases of acute intrahepatic cholestasis,the use of certain medications,and in bone disease.Finally,subnormal levels of 5'-nucleotidase have been reported in lead poisoning and nonspherocytic hemolytic anemia.The aim of this review is to bring attention to the fact that subnormal levels of these enzymes should not be ignored as they may indicate pathological conditions and provide a means of early diagnosis.