Study On Hot Regions And High Frequency Spots Of WD Gene Mutations And Gene Diagnosis For Special Types Of WD

BackgroundHepatolenticular degeneration (HLD), also called Wilson disease (WD), is an autosomal recessively recessively inherited disorder of copper metabolism, with an incidence of 15～30 in 1,000,000 in different populations. WD gene, which located in 13q14.3, encoded a 165Kda copper-transporting P-type ATPase (ATP7B) containing 1465 amino acids, and also called ATP7B gene. ATP7B gene mutations can lead to functional deficit of ATP7Base in hepatocytes, which located in trans-Golgi network (TGN) and membrane of bile vessel, and result in transmembrane transport disturbance of copper ion, dyssynthesis of ceruloplasmin (CP) and impaired biliary excretion of copper in hepatocytes. Finally, excess toxic copper accumulate predominantly in the liver, brain, kidney and comea, and caused lower serum CP levels, cirrhosis, neurological/psychiatric symptoms and Kayser-Fleischer ring (K-F ring) in WD patients.WD is a treatable inherited neurological disease. Most of WD patients can obtain eusemia if they accepted therapy in time. Otherwise, their conditions would be worse or even died. However, many WD patients are often misdiagnosed as other diseases because their early symptoms are complicated and variable and the laboratory copper metabolism tests are often acquired false positive or false negative results in some patients. So it is difficult to make early diagnosis, especially presymptomatic diagnosis and antenatal diagnosis for WD patients before WD gene was cloned.Many data showed that WD gene mutations were highly genetic heterogeneity. There are more than 300 mutations were found in WD patients and most of them are compound heterozygous mutations. The common mutation types are rare and most of them are scarce mutations. The features of mutations are obviously distinct in different populations. The highly genetic heterogeneity can explain the reasons of complicated and variable symptoms in these patients, but it takes many difficulties for gene diagnosis of WD patients. For this reason, it is a fast and high-performance gene diagnosis strategy for WD patients that identification hot regions of mutations in different ethnic populations, and using techniques such as PCR or multiplex PCR to amplify these regions and scan these regions by sequencing.Because copper deposition in the liver is usually experienced accumulation stage and saturation stage for WD patients, most of them would be onset between 5 and 35 years. However, recent epidemiological investigations showed that not a few patients present illness after 40 years old. These patients are not typically and can be misdiagnosed as other diseases more often. For WD patients, low serum CP level is a distinctive biochemistry change, and it is the most important evidence for diagnosis too. However, there are 5 percent WD patients with normal CP levels. These 2 special types of WD patients are more often misdiagnosed as other diseases, and gene diagnosis can provide more powerful evidences for them. But few research data was found for these patients. Sera CP in the body are combined with Holo-CP (conjugated with copper ion) and Apo-CP (not conjugated with copper ion). Recent research showed that transform disturbance from Apo-CP to Holo-CP in WD patients, and their sera Holo-CP expression levels were decreased compared with normal heath person. There have been no report about Holo-CP expression levels in WD patients with normal CP levels from now on.Based on the progress of gene diagnosis for WD and the foundation of our research on diagnosis and treatment for WD during past decades years, this project plan to find out new hot regions or high frequency spots of gene mutations for Chinese WD patients by restriction mapping analysis and DNA sequencing, and attempt to establish a quick and convenient gene diagnosis method for clinical practice. At the same time, this project plan to collect clinical data and genomic DNA samples of WD patients with late onset or with normal sera CP levels, analyze their clinical features and characters of gene mutations, detect their sera Holo-CP expression levels and attempt to study their molecular pathogenesis.Objeetives1. To find out new hot regions or high frequency spots of gene mutations for Chinese WD patients, attempt to establish a quick and convenient gene diagnosis method for clinical practice.2. To analyze the relationships between the genotypes of high frequency mutation spots of Chinese WD patients and their clinical phenotypes, attempt to study their molecular pathogenesis.3. To analyze clinical features and characters of gene mutations of Chinese WD patients with late onset or with normal sera CP levels, attempt to study their molecular pathogenesis.4. To detect sera Holo-CP expression levels of WD patients with normal CP levels, attempt to study their molecular pathogenesis and provide a more sensitive protein marker for them.MethodsThe experiment was composed of four parts.Chapter 1:Detection for hot regions or high frequency spots of gene mutations of WD.1. Exon 13 of ATP7B gene in 139 unrelated WD patients which came from middle and eastern China and 52 unrelated normal controls were detected by PCR-DNA sequencing.2. Exon8,12,13 of ATP7B gene in 142 unrelated WD patients and 52 unrelated normal controls were detected by restriction mapping analysis.Chapter 2: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 142 WD patients which were detected by restriction mapping analysis were collected. Arg778Leu of exon8 and Pro992Leu mutations of exon13 were as the genotypes, and clinical types, gender, age of onset, courses of disease, first symptoms and the detection results of copper metabolism, such as CP and serum copper oxidase (Sco)were as clinical phenotypes. The relationships between the genotypes of high frequency mutation spots of Chinese WD patients and their clinical phenotypes were analyzed.Chapter 3:Gene diagnosis and the clinical features of WD patients with late-onset and normal sera CP levels.The clinical data and genomic DNA samples of 13 cases of WD patients with late-onset and 19 cases of WD patients with normal sera CP levels were collected. Their clinical features were analyzed and their mutations were detected by restriction mapping analysis and DNA sequencing including promoter and all exons. Chapter 4:Detection of sera Holo-CP expression levels of WD patients with normal CP levels and study on their pathogenesis.Sera samples were collected in 19 WD patients with normal CP levels,30 normal controls and 30 WD patients with lower CP levels. Holo-CP expression levels were detected by native polyacrylamide gel electrophoresis (native-PAGE) and Western blot.ResultsChapterⅠ:Detection for hot regions or high frequency spots of gene mutations of WD.1. No abnormality was founded in 52 controls. In 139 patients,6 mutations and 1 polymorphism were found, including 1 novel mutation (Gly988Val). The novel mutation was deposited in GeneBank with the Accession Numbers FJ705811. The compound heterozygous mutation of Arg969Gln/Gly988Val corresponds to severious clinical phenotype. The rate of mutations and polymorphisms of Exon13 was 29.49% (41/139), and its frequency of chromosome mutation was 16.19%(45/278). The rate of Pro992Leu homozygous/heterozygous mutations was 23.02%(32/139), and its frequency of chromosome mutation was 12.59%(35/278).2. Arg778Leu of exon8 was detected in 48.59%(69/142) patients.13 patients were homozygous mutation and another 56 patients were heterozygous mutation. Thr935Met of exonl2 was detected in 5.63%(8/142) patients and 8 cases were all heterozygous mutation. Pro992Leu of exonl3 was detected in 24.65%(35/142) patients.3 patients were homozygous mutation and 32 patients were heterozygous mutation. Among them,12 cases were compound heterozygous mutation with Arg778Leu/Pro992Leu,1 case was compound heterozygous mutation with Arg778Leu/Thr935Met, and 1 case was Arg778Leu homozygous mutation which combined with Pro992Leu heterozygous mutation. Mutations were detected in 69.01% (98/142) patients totally. The results of this method were confirmed by DNA sequencing.Chapter 2: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 Arg778Leu mutation, Pro992Leu mutation and their clinical phenotypes of clinical types, gender, first symptoms, courses of disease and the levels of CS (P>0.05).2. The average ages of onset of Arg778Leu homozygous mutation group, Pro992Leu homozygous mutation group and Arg778Leu-Pro992Leu compound heterozygous mutation group were lower than heterozygous mutation group and no Arg778Leu or Pro992Leu mutation group, but there were no statistical significance(P>0.05).3. The levels of Sco and CP of Arg778Leu homozygous mutation group were lower than no Arg778Leu mutation group significantly (P=0.019, and P=0.011). The levels of Sco and CP of Arg778Leu homozygous mutation group were lower than Arg778Leu heterozygous mutation group, the levels of Sco was no statistical significance(P=0.081) and the levels of CP was statistical significance(P=0.042). The levels of Sco and CP of Arg778Leu heterozygous mutation group were lower than no Arg778Leu mutation group, but no statistical significance(P=0.328, and P= 0.408).4. The levels of Sco and CP of Pro992Leu homozygous mutation group and Pro992Leu heterozygous mutation group were lower than no Pro992Leu mutation group, and the levels of Sco and CP of Pro992Leu homozygous mutation group were lower than Pro992Leu heterozygous mutation group, but there were all no statistical significance (P>0.05).5. The levels of Sco and CP of Arg778Leu-Pro992Leu homozygous mutation group and Arg778Leu-Pro992Leu compound heterozygous mutation group were lower than no Arg778Leu and no Pro992Leu mutation group significantly (P values were 0.011 and 0.049 for Sco, P values were 0.003 and 0.055 for CP). The levels of Sco and CP of Arg778Leu-Pro992Leu homozygous mutation group were lower than only Arg778Leu or Pro992Leu heterozygous group significantly (P value was 0.049 for Sco, P value was 0.009 for CP). The levels of Sco and CP of Arg778Leu-Pro992Leu compound heterozygous mutation group were lower than only Arg778Leu or Pro992Leu heterozygous group, but there were no statistical significance (P>0.05). The levels of Sco and CP of Arg778Leu-Pro992Leu homozygous mutation group were lower than Arg778Leu-Pro992Leu compound heterozygous mutation group, but there were no statistical significance (P>0.05).Chapter 3:Gene diagnosis and the clinical features of WD patients with late-onset and normal sera CP levels.1. In 13 WD patients with late-onset,6 cases were male and 7 cases were female. Ages of onset were from 40 to 60 years, and their average age of onset was (2.61±2.21) years. Courses of disease were from 0.3 to 8 years and their average course of disease was (2.61±2.21) years.3 cases were patients with hepatic type,7 cases were patients with cerebral type and 3 cases were patients with cerebral-visceral type.6 cases were onset with hepatic symptoms,7 cases were onset with neurological symptoms. Their detection results of Scu, CP and SC were consistent with WD, and 5 of them had abnormal detection results of liver function. They all had comea K-F rings except 1 patient which age of onset was 60 years. The results of abdominal ultrasound examination showed that they all were cirrhosis. Cranium MRI showed abnormal signal in basal ganglia. Compared with patients who were onset with hepatic symptoms, the patients who were onset with neurological symptoms were slow onset and their conditions were less severiouly. 2. There were 6 mutations and 11 polymorphisms in promoter region and all exons of ATP7B gene for these 13 WD patients with late-onset, and 11 cases of them had mutations in exon8,12 and 13. They were all heterozygous mutations or combined heterozygous mutations. The frequency of Arg778Leu was 46.15%(6/13) and Pro992Leu was 30.77%(4/13) in these WD patients. Thr935Met mutation was not found, but 2 cases were detected Arg919Gly heterozygous mutation.3. In 19 WD patients with normal sera CP levels,9 cases were male and 10 cass were female. Ages of onset were from 4 to 31 years and their average age of onset was (14.68±7.72) years. Courses of disease were from 0.1 to 15 years and their average courses of disease was (3.89±4.09) years.16 cases were patients with hepatic type,1 case was patient with cerebral type,1 case was patient with cerebral-visceral type and 1 case was patient with presymptomatic type.17 cases were onset with hepatic symptoms,2 cases were onset with neurological symptoms. Their detection results of Sco, CP and SC were normal, and 9 of them had abnormal detection results. 12 cases of them had comea K-F rings. The results of abdominal ultrasound examination showed that they all were cirrhosis or acute hepatic injury. Cranium MRI showed abnormal signal in basal ganglia in 2 cases of patients with neurological symptoms.4 cases of them were diagnosed by liver biopsy, and all patients were had final diagnosis by penicillamine challenge test (PCT).4. There were 11 mutations and 14 polymorphisms in promoter region and all exons of ATP7B gene for these 19 WD patients with normal CP levels.4 mutations including 1870-65G>A, Met645Leu,2575+81A>G and Ala874Pro were novel. In these 19 WD patients,16 cases of them were detected mutations, and any mutations were not found in 3 cases of them. The rate of mutations detection was 84.21% (16/19)。For 16 patients who were detected mutations,3 cases were homozygous mutation,2 cases were compound heterozygous mutation, and only 1 mutation were found in the others cases. The missense mutations is the major mutation types, and 3 kinds of splice junction mutations were found. The frequency of Arg778Leu was 15.79% (3/19), Thr935Met and Pro992Leu mutation were not found.Chapter 4:Detection of sera Holo-CP expression levels of WD patients with normal CP levels and study on their pathogenesis.The expression levels of sera Holo-CP of normal controls and WD patients with normal CP levels were higher than WD patients with low CP levels (P=0.000), and the expression levels of sera Holo-CP of WD patients with normal CP levels was lower than normal controls (P=0.013).Conclusions1.6 mutations and 1 polymorphism were found in ATP7B gene of 139 WD patients which came from middle and eastern China.2. Gly988Val, which was a novel mutation, was found in ATP7B gene of 139 WD patients which came from middle and eastern China. Arg969Gln mutation was found first time in Chinese WD patients. The compound heterozygous mutation of Arg969Gln/Gly988Val corresponds to severious clinical phenotype.3. Exon 13 of ATP7B gene is one of hot mutation regions in Chinese WD patients.4. The detection rates of gene mutations can be significantly improved by analysis exon8,12 and 13 of WD gene with the method of restriction mapping. The method can help to diagnose suspicious WD cases and their family members in clinical.5. Pro992Leu mutation is one of high frequency mutation spots in Chinese WD patients.6. There were no relationships between the genotypes of Arg778Leu mutation, Pro992Leu mutation, Arg778Leu-Pro992Leu compound heterozygous mutation and the clinical phenotypes of clinical types, first symptoms, genders, courses of disease and average ages of onset in 142 Chinese WD patients..7. The levels of Sco and CP of WD patients with Arg778Leu homozygous mutation, Pro992Leu homozygous mutation, Arg778Leu-Pro992Leu homozygous/ compound heterozygous mutation were lower than those with no Arg778Leu mutation or no Pro992Leu mutation and only Arg778Leu mutation mutation or only Pro992Leu heterozygous mutation.8. Arg778Leu mutationand Pro992Leu mutation are pathogenic mutations which affected the function of WD protein seriously.9. Extrapyramidal symptoms were mainly clinical manifestations in 13 WD patients with late-onset which were onset with neurological symptoms. Their liver damage was mild and good effect can be acquired for them by de-copper treatment.10. Compared with WD patients with late-onset which were onset with neurological symptoms. The patients which were onset with liver symptoms were serious condition. Some of them were accompanied with extrapyramidal symptoms and parts of symptoms can be improved after de-copper treatment.11.6 mutations and 11 polymorphisms were found in ATP7B gene of 13 WD patients with late-onset.12. The mutations of 13 WD patients with late-onset were all heterozygous mutations or compound heterozygous mutations, the detection rates of mutations were 46.15% for Arg778Leu heterozygous mutation and 30.77% for Pro992Leu heterozygous mutation in these patients.13. Liver damage symptoms were mainly clinical manifestations in 19 WD patients with normal CP levels. However, there were patients who were onset with neurological symptoms or without any symptoms.14.11 mutations and 14 polymorphisms were found in ATP7B gene of 19 WD patients with normal CP levels.15. The detection rate of Arg778Leu mutation was 15.79% in 19 WD with normal CP levels. Thr935Met mutation and Pro992Leu mutation were not found in these patients. The total detection rate of mutations in exon8,12 and 13 was 42.11%.16.4 novel mutations were found in ATP7B gene of 19 WD patients with normal CP levels.17. Low expression of Holo-CP and transform disturbance from Apo-CP to Holo-CP were found in typical WD patients with low CP levels by native-PAGE and Western blot.18. Low expression of Holo-CP and transform disturbance from Apo-CP to Holo-CP were found in WD patients with mormal CP levels by native-PAGE and Western blot.The detection of Holo-CP expression levels is a sensitive diadynamic criteria for skeptical WD patients in clinical practice.The originalities of this current project lie in:1. Gly988Val, which was a novel mutation, was found in ATP7B gene of 139 WD patients which came from middle and eastern China. Arg969Gln mutation was found first time in Chinese WD patients.2. Exon 13 of ATP7B gene is one of hot mutation regions in Chinese WD patients.3. Pro992Leu mutation is one of high frequency mutation spots in Chinese WD patients.4. The levels of Sco and CP of WD patients with Arg778Leu homozygous mutation, Pro992Leu homozygous mutation, Arg778Leu-Pro992Leu homozygous/ compound heterozygous mutation were lower than those with no Arg778Leu mutation or no Pro992Leu mutation and only Arg778Leu mutation mutation or only Pro992Leu heterozygous mutation. 5.6 mutations and 11 polymorphisms were found in ATP7B gene of 13 WD patients with late-onset. These mutations were all heterozygous mutations or compound heterozygous mutations, the detection rates of mutations were 46.15% for Arg778Leu heterozygous mutation and 30.77% for Pro992Leu heterozygous mutation.6. Liver damage symptoms were mainly clinical manifestations in 19 WD patients with normal CP levels. However, there were patients who were onset with neurological symptoms or without any symptoms.7.11 mutations and 14 polymorphisms were found in ATP7B gene of 19 WD patients with normal CP levels.4 novel mutations were found in ATP7B gene of these WD patients.8. Low expression of Holo-CP and transform disturbance from Apo-CP to Holo-CP were found in WD patients with mormal CP levels by native-PAGE and Western blot.The detection of Holo-CP expression levels is a sensitive diadynamic criteria for skeptical WD patients in clinical practice.