Clinical Analysis, Pathologic Study And Gene Mutation Detection Of Patients With Fulminant Wilson Disease

Wilson Disease(WD)is an autosomal recessive inherited disorder featured by copper metabolic dysfunction.It's molecular etiological factor is ATP7B gene mutation,which prevents its expression product, P-type copper transmitting ATP enzymes.This malfunction reduces copper excretion in the bile,which causes toxicity to liver,brain and other organ tissues with excessive copper accumulation.Extensive and heterogeneous in clinical symptoms,WD induces multi-systemic lesions to the liver,nerve,kidney,cornea and other organs.Fulminant Wilson Disease(FWD),a rare but grave clinical type of WD,is known for its severity with extremely poor prognosis.Its mortality is almost 100% without liver transplant.As its clinical manifestations are similar to those of fulminant hepatic failure caused by other factors,misdiagnosis of FWD is very common.Up to now,no long-term integrated study of FWD involving clinical,pathology and molecular biology has been reported either in domestic or abroad.Objectives:1.To review the clinical characteristics of FWD patients to discuss the death causes of the disease and the changing patterns of copper metabolism so as to provide reliable data for diagnosis and treatment of FWD. 2.To observe the pathological features of FWD patients' organ tissues(particularly the liver and kidney)and further to reveal its pathological mechanism,providing scientific evidence for diagnosis and treatment plan for FWD.3.To analyze FWD patients' molecular biological traits,detect its gene mutation,and explore its molecular pathological mechanism,which serve as scientific evidences for genetic diagnosis and treatment of FWD.Methods:1.Systematic observation of FWD patients' clinical symptoms and physical signs were conducted;the patients also received regular check-ups of the three routines,hepatic and renal function, copper-protein,urine copper,serum copper,and imagingstudy;the slit-lamp biomicroscope was used to check their eye K-F link;and diagnosis was made in accordance with the recommended criteria.2.Micro-and ultramicrostructural examinations were carried out of liver,kidney and brain tissues obtained via autopsy or through in vivo tissue puncture;spot elemental analysis was done on marked granular sediments with the x-ray power spectrometer.3.PCR-amplified direct sequencing(DS)was used to check the patients and their family members'21 WD gene total-length exons and the sequence of the exon/intron junctional zones,followed by on-line sequence analysis. Results:1.The two subjects in the study both showed a decline in copper-protein,a remarkable increase in 24-hour urine copper and a sharp rise in dissociated serum copper;one of them was found positive in the K-F link and the other didn't take such a check but showed a clear growth in the level of liver copper,and they scored 5 and 7 respectively,which was in accordance with diagnostic criteria suggested by the 8th International WD Conference(2002,Germany)and in consistence with WD diagnosis.The patient had no medical record of any liver disease,but, one month later after the onset of the disease,suffered hepatic failure complicated by medium or even severe haematolysis,which was also consistent with FWD diagnosis.2.One patient started with hepatopathic symptoms while the other began with renal disorder;correct diagnosis was not made until one month later when both victims developed late-phase hepatic failure and died of delayed diagnosis;neither of them received on time treatment.In the second case,doctors used drugs which clearly caused hepatic toxicity. This was a major cause of hepatic failure;a number of serious complications(e.g.infection and hemorrhage)occurred,directly causing the death.3.In both cases,urine copper increased 67 and 252 times respectively,and dissociated plasma copper grew significantly. Remarkable increase in urine copper and dissociated serum copper is of great value for FWD diagnosis and it is also an important index for the judgment of severity and prognosis of the disease.4.Autopsy showed typical tuberous sclerosis on the surface of the patient's liver,directly suggesting that at least with some FWD patients severe hepatic failure occurs as a result of hepatic cirrhosis.5.The light microscope showed degeneration and necrosis of a large number of liver cells and a great many granular sediments in kytoplasm; the electron microscope showed a lot of cell organelle fragments and granulo-electron-dense sediments in kytoplasm,and the mitochondria varied in size and form,suggesting great damage of copper to the liver, which confirmed what had been reported in China and overseas.Evident pathological changes were detected in tissues of the kidney,brain,lung, spleen,pancreas,intestinal tract and gallbladder,and brown granular sediments were also found,suggesting that copper caused severe damage to many organ systems of the body.6.For the first time,on the basis of ultramicropathological analysis, the x-ray power spectrometer was used to conduct tungsten detection in FWD hepatic tissues,directly confirming the existence of large amounts of copper in hepatic cells.Also,for the first time worldwide,it was directly proved that there existed large quantifies of iron in granular sediments of hepatic cells,a finding consistent with the conclusion reported in recent years both in China and overseas that iron dysfunction existed with WD patients.7.Using 21 primers under the self-designed experiment condition, we successfully amplified all the 21 ATP7B gene exons and exon/intron junctional zones;Agarose gel electrophoresis(AGE)showed that the straps of PCR products were as large as those expected,and the result of direct sequencing was the same as the reference sequence of the gene bank,indicating the accuracy of the primer design,the adequacy of the test condition and the reliability of the test result.8.Compared with the normal ATP7B gene,the 21 ATP7B exons of the first patient showed no mutation while a missense heterozygosis mutation occurred at the 13th exon of the second patient:2975C→T mutation(Pro992Leu).The mutation took place in the ATP7B tunnel/phosphorylation domain and the 6th transmembrane domain,which are indispensable for ATP7B function,and mutation in these areas is bound to affect ATP7B function.The mutation was passed on from the patient's father,with whom mutation of this type was also detected.No report on such a mutation was reported,warning that in China WD genetic diagnosis should not be confined to such high frequency zones of mutation as the 8th and 12th exons.Further study is required to check whether such a mutation is related to fulminant pathological changes. Conclusions:1.At least with some FWD patients,their severe hepatic damage is a result of hepatic cirrhosis.Misdiagnosis is a the major factor that causes WD to develop into FWD;Inappropriate therapies,particularly the use of drugs of hepatotoxicity,aggravates hepatic damage;A number of severe complications,such as infection and haemorrhage,are the direct causes of death.Therefore,we should put effort in WD research,academic exchange and the training of WD specialists,in order to improve doctor's knowledge of diagnosis and treatment skill of WD.2.As WD demonstrates complicated clinical manifestations and takes on varied forms of onset,clinical doctors should be able to distinguish it from DU hepatic and renal diseases and hematolysis.3.Significant increase in urine copper and dissociated serum copper is a vital clinical feature of FWD and is crucial for its treatment and prognosis judgment.Reducing dissociated serum copper may play a key role in FWD treatment.4.Sufferings of FWD patients include obvious pathological changes and brown copper granular sediments in the liver,kidney,brain,lung, spleen,pancreas and heart,and severe damage of many organ systems of the body.5.Tungsten detection with the x-ray power spectrometer not only directly proves the existence of large quantities of copper and iron in the granular sediments of WD patients' hepatic cells,a finding in consistence with the lately reports in China and overseas that iron metabolism in WD patients tends to be abnormal,but also serves as a new technique for WD diagnosis.6.It is the first report in China that missense heterozygosis mutation, of the paternal source,occurs at the 13th exon of the second patient.The mutation is detected in the ATP7B tunnel/phosphorylation domain and the 6th transmembrane domain,which may have serious effects on ATP7B function.Further study is necessary to determine whether such a mutation is connected with fulminating pathological changes.7.Due to the genetic complicatedness of WD,the current detection rate of its pathopoiesis mutation remains low,which,to certain extent, limits the genetic diagnosis of the disease.It is urgent to develop convenient,specialized,economical and instant methods of genetic diagnosis and more effort should be put on the research of mutation.