Association Of ATP7B Mutation Genotype And Clinical Phenotype In Patients With Wilson 's Disease In Chinese Han Nationality

Hepatolenticuler degeneration (HLD) is an autosomal recessive disorder of copper transport diseases, since Wilson described this disease in detail earliest in 1912, it also known as Wilson disease (WD). The disease caused by mutations in the copper-transporting P-type ATPase gene (ATP7B), that result in reduction or loss of protein function, as well as disorder of copper transport. Excessive copper build-up in the liver, brain, kidneys and other important organs leads to corresponding tissue degeneration. The main clinical symptoms of WD are liver damage, extrapyramidal symptoms, kidney damage, mental disorders, skeletal malformations, etc. WD is also one of a few genetic diseases which could be treated effectively. However, it relies on early diagnosis and treatment. Due to its complex and diverse clinical symptom, and biochemical testing can’t report a clear distinction between disease gene carriers and presymptomatic patients, early diagnosis is difficult. Identifying characteristics of ATP7B mutations in Chinese population and establishing effective treatment, revealing the genotype and phenotype correlation, will provide guidance for more efficient early diagnosis and prognosis and further support the use of copper chelating agents to prevent the onset of WD in asymptomatic individuals with ATP7B mutations.Clinical and genetic analyses were performed on 361 unrelated WD patients. ATP7B mutations were screened by direct sequencing in all exons and promoter region.27 novel and 45 known mutations were identified. Over 91.13%(329/361) of patients with a clinical diagnosis of WD were found to have two mutations (homozygous or compound heterozygous) in the ATP7B gene. Among the remaining 32 patients,13 were heterozygous for an ATP7B mutation carrying only a single identified mutant ATP7B allele and another 19 patients contained no identified mutation in the ATP7B gene. R778L, P992L, T935M, N1270S, Q511X, R919G and G943D were seven most common mutations, cover 301 patients (91.13% of the total cases). Make use of R778L, P992L, T935M, N1270S, Q511X and R919G mutations, multiple allele specific PCR method has been established for rapid screening A TP7B mutants.Through correlation statistical analysis between ATP7B common mutations and age of onset, concentration of serum ceruloplasmin and classification of clinical subtypes, we identified the onset age and concentration of serum ceruloplasmin of R778L homozygote and P992L homozygote are comparable to that of N-terminal mutant homozygote or heterozygote (P>0.1). It indicates that R778L, P992L, Q511X or other N-terminal truncated mutation are serious mutant forms. The age of onset and concentration of serum ceruloplasmin of 94 patients with T935M or R919G mutation are significantly higher than those not carry these mutations. It indicates that T935M or R919G are modest mutant forms.Further analysis of protein structure modeling and copper damage tolerance comparison experimental in stable expression of mutant ATP7B protein in CHO cell line model reveals R778L or P992L mutations are serious than T935M. R778L and P992L can alter positioning of the TM segments in the membrane and influence the conformation of the ion channel, and leading to lower copper damage tolerance capability than T935M mutation. Addition of zinc to culture systems that contained cells with WD causing mutations protected these cells against copper damage. Of notable interest, administration of zinc resulted in the reduction of the cell death rate and improvement of cell survival in the cell line bearing R778L. The functional analysis data further support the idea that zinc agents can be used in the treatment of WD and the prevention of the onset of the disease in individuals who have WD causing mutations of ATP7B.In this study, through direct DNA sequencing performed on 361 unrelated WD patients, we summarize the prevalence of ATP7B mutations in the Chinese WD population; establish a simple and effective method of ATP7B mutation detection (cover 88.45% caces). Through correlation analysis between mutant genotype and clinical phenotype, protein structure and cell function experiments, we revealed R778L and P992L, two common ATP7B mutations in the Chinese WD population, are serious mutation type, while the T935M and R919G are modest mutation types. These finding provides guidance for more efficient early diagnosis, treatment and prognosis in Chinese WD population.