The Mutation Of ATP7B Gene And Clinical Correlation In Chinese WD Patients

Background:Hepatolenticular Degeneration （HLD）, also known as Wilson’s Disease （WD）, is an autosomal recessive disorder caused by abnormal copper metabolism. The gene is located on13q14.3. The mutations of WD gene, which cause abnormal ceruloplasmin synthesis,disordered biliary excreting copper and copper deposition in the body,result in cirrhosis,neurologic manifestation,corneal K-F ring and high urinary copper.Few mutation hot spots predominate the cases of ATP7B gene mutation, accompanied by various rare mutations.The hot spots are different between China and European countries. And they vary be-tween various areas and ethnic groups in China. There is no common opinion on genotype-phenotype, involving further research in future.Aims:This thesis is to investigate the mutations of ATP7B and their correlation with clinical manifestations of the WD patients in China. Based on the investigation, the thesis will get insight into the role of genetic test in the clinical diagnosis of WD and its pathogenesis. In the long run, this research is to provide the scientific evidence for setting up a China’s specific genetic test for WD.Methods:1. For103WD patient and34controls,21exons of ATP7B gene are amplified with Polymerase Chain Reaction, and the correspond-ing DNA products are sequenced. The sequencing results are compared with that of the normal ATP7B gene and the results are documented.2. The clinical manifestations of the103WD patients are docu-mented in detail. In turn, the correlations between genotype and phenotype are extensively investigated.Results:1.15polymorphisms are identified, including Ala971Ala（2913T>A） and Gly1266Gly（3798G>T） which have not been reported in the existing literature.32pathogenic mutations are identified which consist of32missense mutations,3nonsense mutation-s and1frame-shift mutation.13novel mutations are identified, comprising （1510₁511insA, Leu692Pro （2075T>C）, Gln680Stop （2038C>T）, Leu745Ile （2233C>A）, Thr850Ile （2549C>A）, Ala874Pro （2620G>C）, Pro1070Arp （3209C>G）, Trp1153Cys （3460C>T）, Gly1149Glu （3446G>A）, Ser1226P （3677C>T）, Gly1265Cy s （3793G>T）, Leu1275Ser （3824T>C）, Ala1295Val （3884C>T））.2. The frequency of the Arg778Leu mutation on exon8is18.93%while it is13.10%for Pro992Leu mutation on exon13. The indi-vidual frequency of other mutation is less than5%. So Arg778Leu mutation and Pro992Leu mutation are the hot spots in our re-search.3. The mutation on four exons （8,12,13,16） account for54.37%of all of the mutation cases. There is no pathogenic mutation on five exons （1,4,5,6,21） at all.4. The mutation rate detected by direct sequencing among103WD patients is92.23%. We find three kind of mutations in three peo-ple out of34normal controls, includingThr977Met（2930C>T）, Arg1319Stop （3955C>T）, Arg778Leu（2333C>T）mutationso 5. Arg778Leu mutation and Pro992Leu mutation has no correlation with clinical types, gender, K-F ring, serum ceruloplasmin, ALT,24h urinary copper and age of onset（P>0.05）.Conclusion:Arg778mutation on exon8and Pro992Leu mutation on exon13are the hot spots mutation. Four exons （8,12,13,16） should be considered with high priority for the testing in China. The correlation between genotype and phenotype should be investigated further in the future. The technique of DNA detection is of high sensibility and specificity for WD diagnosis. It can be used in the clinical gene screening in the future.