| BackgroundHepatolenticular degeneration(HLD), also called Wilson disease(WD), is an autosomal recessively inherited disorder of copper metabolism. But the mechanism of copper metabolism is still not completely clear. Because of the basal metabolic disorder, the excretion of copper and the synthesis of ceruloplasmin is decreased.Excess toxic copper accumulate predominantly in the liver, brain, kidney, skin,pancreas, and cormea, which lead to the damage of organs.WD is a treatable inherited neurological disease. The progression of the disease could be controled if patients accepted theraoy in time. Because the early symptoms are complicated and variable, many WD patients are often misdiagnosed as other diseases, so it is important to summarize the clinical characteristics of WD.WD was divided into four types(liver types; brain types; other types; mixed types) according to the guide of diagnosis and treatment of He Patolenticular degeneration formulated by chinese medical association in 2008.The analyse of WD according to clinical classification is helpful to the diagnosis and treatment of the disease.The pathogenesis of WD is the gene mutation of copper-transporting P-type ATPase(ATP7B) which located in 13ql4.3. The synthesis of ceruloplasmin in liver is decreased because of the gene mutation of ATP7 B. Then the copper in vivo can’t be transported out of the body through ATP7 B. Excess toxic copper accumulate predominantly in the liver, brain, kidney, skin, and cormea, which lead to the damage of organs. According to the development of molecular biology, the gene diagnose technology is being used more and more widely.Due to its complex and diverse clinical symptom early diagnosis is difficult. The hot spots are different between various areas and ethnic groups in China.Identifying charaeteristics of ATP7 B mutations in Chinese will help us to find the hot spots of ATP7 B. The hot spots are different between various areas and ethnic groups in China.There is no common opinion on genotype-phenotype. Identifying on genotype-phenotype will provide guidance for more efficient early diagnosis and prognosis.Penicillamine is mainly used for the treatment of WD, because it is curative effective, cheap and easy to abtain in China. According to the research of the gene mutation, the observation of different gene mutations in patients with type using penicillamine to treat WD, may lay a foundation for the individualized treatment of WD.Objectives1.This research is to analyse the clinical characteristics of WD according to clinical classification.2.To investigate the mutations of ATP7 B and find out new hot regions or high frequency mutation sports in Chinese WD patients.3. To analyze the relationships between the genotypes of high frequeney mutation spots and their clinical phenotypes.4. Get insight into the role of gene mutation of ATP7 B, its pathogenesis,and the relationship between gene mutation and treatment effect of penicillamine.MethodsThe experiment was composed of four Parts.Chapter 1: Clinical analysis of WD related clinical classificationCollect basic clinical data of patients diagnosed as WD and divide the WD into 4types according to clinical manifestations(liver types; brain types; other types; mixed types). first symptoms of the disease, gender, serum copper and serum copper blue protein, 24 h urine copper, Alamine aminotransferase( ALT), hyaluronic acid(HA),laminin(LN), precollagen type Ⅲ(PC Ⅲ), collagen Ⅳ(C Ⅳ) were discussed according to the clinical classification of WD.Chapter 2: Detection for hot regions or high frequeney sports of gene mutations of ATP7 B.Polymerase chain reaction and direct DNA sequencing were used to detect ATP7 B mutations of 52 WD patients.Chapter 3:The relationships between the genotypes of high frequency mutation spots and clinical phenotypes in patients with WD.The clinical data and results of gene mutations of 52 WD Patients were collected.Arg778 Leu mutation of exon 8 and Pro992 Leu mutation of exon 13 were as the genotyes, and clinical types, gender, age, first symptoms, K- F ring, serum copper and copper blue protein, 24-hour urine copper, ALT and liver fibrosis serological results as clinical phenotypes. The relationships between the genotypes of high frequency mutation spots and their clinical Phenotp es were analyzed.Chapter 4: Curative effect of penicillamine on different types of gene mutations in ATP7BA total of 40 cases were colected all of which had done gene sequencing. The patients were divided into Arg778 Leu mutation group and non Arg778 Leu mutation group. Penicillamine were used for the treatment of WD. The content of serum copper and serum copper blue protein, 24-hour urine copper, ALT, HA, PCIII, LN and CⅣwere detected in 1, 3, 6 and 12 months after treatment.Results:Chapter 1: Clinical analysis of WD related clinical classification1. Included in this study cases, 245 cases of liver type(47.8%), 180 cases of brain type(35.1%), and other types of 35 cases(6.8%), mixed type 53 cases(10.3%).Liver symptoms as the first symptoms : 271 cases(52.8%); nervous system as the first symptoms: 201 cases(39.2%); other symptoms as the first symptoms: 41 cases(8%).2. Liver type and brain type compared with other types of the change of serum copper blue protein is statistically significant; Liver type 、bain type and other types compared with mixed type the change of serum copper blue protein has statistical significance.3. The liver type of serum ALT was higher compared with the brain type and mixed type. The difference is statistically significant.4. The differences of HA, PC Ⅲ, LN,and C Ⅳ were statistically significant in the different clinical classifications.5. The incidence of liver fibrosis or cirrhosis is higher in liver type than other types. The difference is statistically significant.Chapter 2: Detection for hot regions or high frequeney sports of gene mutations of ATP7 B.1.62 polymorphism loci were found, of which 23 were the known disease-causing mutations, 6 of which were first found.2.Exon 8, 12,13 were hot point mutations of Chinese ATP7 B gene. The frequency of the Arg778 Leu mutation on exon 8 is 47% while it is 22% for Pro992 Leu mutation on exon 13. So Arg778 Leu mutation and Pro992 Leu mutation are the hot spots in our research.Chapter 3:The relationships between the genotypes of high frequency mutation spots and clinical phenotypes in patients with WD.1.There were no relationships between the genotypes of Arg778 Leu mutation,no Arg778 Leu mutation patients and their clinical phenotypes of clinical types, gender,age, first symptoms, K-F ring, serum copper and copper blue protein,,ALT and liver fibrosis serological results(P>0.05).2. There were no relationships between the genotypes of Pro992 Leu mutation,no Pro992 Leu mutation patients and their clinical phenotypes of clinical types, gender, age, first symptoms, K-F ring, serum copper and copper blue protein,24-hour urine copper,and liver fibrosis serological results(P>0.05).3. The level of 24-hour urine copper of Arg778 Leu mutation is lower than no Arg778 Leu mutation group significantly(P <0.05).4. The level of ALT of Pro992 Leu mutation is lower than no Pro992 Leu mutation group significantly(P <0.05).Chapter 4: Curative effect of penicillamine on different types of gene mutations in ATP7B1. The decline of 24-hour urine copper of Arg778 Leu mutation group is more significant than no Arg778 Leu mutation group after treatment of 12 mounth with Penicillamine(P <0.05).2. The decline of IV-C of Arg778 Leu mutation group is more significant than no Arg778 Leu mutation group after treatment of 12 mounth with Penicillamine(P <0.05).3.There were no relationships between Arg778 Leu mutation group and no Arg778 Leu mutation group about the serum copper and serum copper blue protein,24-hour urine copper, ALT, HA, PCIII, LN and C Ⅳ after treatment of 1,3,6,12 mounth with Penicillamine.Conclusions:1. The liver type of serum ALT was higher compared with the brain type and mixed type.;The differences of HA, PC Ⅲ, LN and C Ⅳ were statistically significant in the different clinical classifications.2.62 polymorphism loci were found, of which 23 were the known disease-causing mutations, 6 of which were first found.3. Exon 8, 12 and 13 were hot point mutations of Chinese ATP7 B gene.4. The level of 24-hour urine copper of Arg778 Leu mutation is lower than no Arg778 Leu mutation group significantly. The level of ALT of Pro992 Leu mutation is lower than no Pro992 Leu mutation group significantly.5.There were no relationships between the genotypes of Arg778 Leu mutation,no Arg778 Leu mutation patients and their clinical phenotypes of clinical types, gender,age, first symptoms, K-F ring, serum copper and copper blue protein,,ALT and liver fibrosis serological results.6. There were no relationships between the genotypes of Pro992 Leu mutation,no Pro992 Leu mutation patients and their clinical phenotypes of clinical types, gender, age, first symptoms, K-F ring, serum copper and copper blue protein,24-hour urine copper,and liver fibrosis serological results.7. The decline of 24-hour urine copper of Arg778 Leu mutation group is more significant than no Arg778 Leu mutation group after treatment of 12 mounth with Penicillamine. The decline of CⅣ of Arg778 Leu mutation group is more significant than no Arg778 Leu mutation group after treatment of 12 mounth with Penicillamine.8. There were no relationships between Arg778 Leu mutation group and no Arg778 Leu mutation group about the serum copper and serum copper blue protein,24-hour urine copper, ALT, HA, PCIII, LN and C Ⅳ after treatment of 1,3,6,12 mounth with Penicillamine. |
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