Study On The Relationship Of The ATP7B Gene Mutation And Clinical Phenotype In WD Patients

Background:Hepatolenticular degeneration (HLD), also called Wilson disease(WD), is an autosomal recessively inherited disorder of copper metabolism. The main clinical presentations are lower serum Cu and ceruloplasmin (CP) levels, cirrhosis, gradually serious extraparamidal neurological symptoms, Kayser-Fleischer ring (K-F ring) and psychiatric symptoms. WD gene mutations were highly genetic heterogeneity, the hot regions of mutations and frequent spots of mutations are different in different ethnic and regional populations. At present, there is great controversial on the genotype-phenotype correlation studies, so we aim to apply DHPLC to screen the ATP7B gene mutation in WD patients and explore the genotype-phenotype correlation.Objective:Applying rapid and efficient DHPLC to screen the gene of WD patients, to find out new hot regions or highly frequent spots of gene mutations for Chinese WD patients, attempt to establish a quick and convenient gene diagnosis method for clinical practice; and to analyze the relationships between the genotypes of highly frequent mutation spots, the types of gene mutation of Chinese WD patients and their clinical phenotypes.Methods:1. Eon 1～21 of ATP7B gene in 75 WD patients and their 171 relatives of 72 unrelated families which came from middle and easten China and 200 unrelated normal controls were detected by DHPLC.2. The clinical data and results of gene mutations of 75 WD patients which were detected by DHPLC were collected. Arg778Leu of exon8 and Ile1148Thr of exon16 were as the genotypes, missen mutations and non-missen mutations were as the mutation types, and clinical types, gender, age of onset, courses of disease, first symptoms and the detection results of copper metabolism, such as copper and CP were as clinical phenotypes. The relationships between the genotypes of highly frequent mutation spots of Chinese WD patients and their clinical phenotypes were analyzed.Results:1. 53 different melting profile shapes were found in ATP7B gene of 75 WD patients which came from middle and eastern China.2. It was proved by DNA sequencing, 24 missens mutations, 5 nonsens mutations, 3 splice-site mutations, 6 frameshift mutations and 13 polymorphism were found, including 2 novel polymorphism:Ile613Ile, Trp974Trp, and 10 novel mutations(not found in normal group): 1875-1876dupAATT, 2120A>G(Gln707Arg), 2121+3A>T, 2564C>A(Ser855Tyr), 2620G>C(Ala874Ser), 2761A>C(Ser921Arg), 3236G>T(Cys1039Phe), 3244-2A>C, 3446G>A(Gly1149Glu), 3682A>T(Arg1228stop).3. The rate of mutations of 75 WD patients was 90.67%(68/75),and its frequency of gene mutation was 68.67%(103/150). The rate of Arg778Leu mutation was 42.67%(32/75), and its frequency of chromosome mutation was 22.67%(34/150). The rate of Ile1148Thr mutation was 18.67% (14/75), and its frequency of chromosome mutation was 9.3%(14/150). The rate of mutation of exon8, 12 and 16 was 48%(72/150)。4. There were no relationships between the mutation types of ATP7B and the levels of CP and SC(P>0.05). The level of SC of Arg778Leu heterozygous mutation group was higher than no Arg778Leu mutation group (P=0.012), The level of SC of Arg778Leu homozygous mutation group was higher than no Arg778Leu mutation group (P=0.021); The level of CP of Ile1148Thr heterozygous mutation group was higher than no Ile1148Thr mutation group (P=0.000).5. There were no relationships between the genotypes of Arg778Leu mutation , Ile1148Thr mutation, mutation types and their clinical phenotypes of clinical types, gender, first symptoms, courses of disease, age of onset (P>0.05).Conclusion:DHPLC, which is a quick and easy method, can be used in clinical gene diagnosis. Ile1148Thr mutation is another highly frequent spot of ATP7B mutation in Chinese WD patients. There is a difficult relationship between genotype and phenotype, it is necessary to study further.