【作者】 易露茜；

【导师】 杨旭；

【作者基本信息】 中南大学 ， 内科学， 2007， 博士

【摘要】 肝豆状核变性又称威尔逊病(Wilson Disease,WD),是一种以铜代谢障碍为特征的常染色体隐性遗传病。其分子病因为ATP7B基因突变使其表达产物P型铜转运ATP酶功能缺陷而使胆汁中铜排泄减少,导致肝、脑等组织中铜沉积而产生毒性损伤。临床表现为广泛的、异质性的临床综合征,包括肝脏、神经、肾脏、角膜等多器官系统损害。暴发性肝豆状核变性(Fulminant Wilson Disease,FWD)是WD的一种少见而严重的临床类型,病情凶险,预后极差,如不进行肝移植治疗,病死率几乎为100%。其临床表现与其他原因引起的暴发性肝衰竭类似,极易误诊,国内外至今尚未见从临床、病理和分子生物学多个水平进行追踪观察的报道。目的:1、总结FWD患者的临床特点,探讨死亡原因和铜代谢指标的变化规律,为诊断和治疗提供科学依据。2、观察FWD患者的组织器官(特别是肝脏和肾脏)病理特征,进一步阐明发病机理,为诊断和治疗提供科学依据。3.分析FWD患者的分子生物学特点,检测基因突变,探讨分子病理学机制,为基因诊断提供科学依据。方法:1、系统观察临床症状与体征,定期检查三大常规、肝。肾功能、铜蓝蛋白、尿铜、血清铜和影像学检查等;用裂隙灯显微镜进行眼部K-F环检查;应用推荐的诊断标准进行诊断。2、通过尸体解剖或活体组织穿刺获取肝、肾、脑等重要器官组织,进行显微和超微结构检查;在特征性沉积颗粒上利用X-射线能谱仪进行点的元素分析。3、采用PCR扩增后直接测序法,检测患者及家系成员WD基因全长21个外显子及外显子/内含子交界区序列,进行在线序列分析。结果:1、两例患者均表现为铜蓝蛋白降低,24小时尿铜显著升高,血清游离铜极度升高;一例患者K-F环阳性,另一例未查,但肝铜显著升高;根据第八届WD国际研讨会提出的计分诊断标准,分别得5分和7分,符合WD的诊断。既往无肝病史,起病1月后出现严重肝功能衰竭,有中度至重度溶血,符合FWD的诊断。2、一例患者以肝病症状起病,一例以肾病症状起病,均于病后1个月进入肝衰竭末期后方确诊,失去了抢救的时机并死亡;两例患者病后均未及时治疗,病例二还使用了对肝脏有明显毒性的药物,是引起肝衰竭的重要诱因;两例患者均出现了感染、出血等多种严重并发症,是死亡的直接原因。3、两例患者尿铜分别增加67至252倍,血清游离铜显著升高。尿铜和血清游离铜显著升高对诊断FWD极有价值,也是判断病情轻重和预后的重要指标。4、尸体解剖发现患者肝脏外观呈典型的结节性肝硬化,直接证明至少部分FWD是在肝硬化基础发生的严重肝损伤。5、光镜下可见肝细胞大量变性、坏死,胞浆有大量颗粒沉积;电镜下胞浆有许多细胞器碎片和颗粒状高电子密度沉积物,线粒体大小、形态不一;说明铜对肝脏的损害相当严重,与目前国内外文献报道的基本吻合。肾、脑、肺、脾、胰腺、肠道、胆囊、心脏等器官组织均有明显的病理改变,有褐色颗粒沉积,说明铜对全身多器官系统均有严重损害。6、首次在超微病理检测的基础上,采用X-射线能谱仪对FWD肝脏组织进行了金属元素检测分析,直接证明肝细胞沉积的颗粒中含有大量铜。首次直接证实肝脏细胞沉积的颗粒中含有大量铁,与近年国外报道的WD患者存在铁代谢异常结论一致。7、采用21对引物和自行设计的实验条件,成功地扩增了ATP7B基因全部21个外显子及外显子/内含子交界区;琼脂糖凝胶电泳分析可见PCR产物大小与预期的相同,直接测序结果与基因库参照序列相同,说明引物设计正确,实验条件适当,实验结果可靠。8、与正常ATP7B基因比对分析表明,病例一ATP7B的21个外显子均未发现突变;病例二第13号外显子发现有一错义杂合突变:2975C→T突变(Pro992Leu)。这一突变位于ATP7B的通道/磷酸化区和第6跨膜功能区,该区域是ATP7B功能所必须的,如发生突变,势必影响ATP7B的功能。其父亲也有同样的突变,说明突变来源于父亲。该突变国内尚未见报道,提示我国WD的基因诊断不宜只检测第8、12号外显子等突变热区。该突变是否与暴发性病变有关,有待进一步证实。结论:1、至少部分FWD是在肝硬化基础上出现的严重肝损伤。误诊是WD发展成为FWD的重要原因;治疗不当,特别是使用肝毒性药物是加重肝损伤的重要诱因;感染、出血等多种严重并发症,是死亡的直接原因。为此,要高度重视WD的研究,加强学术交流和专业培训,提高认知性和诊疗水平。2、WD的临床表现复杂,起病形式多样,临床工作中诊断不明的肝病、肾病、溶血等要注意与WD的鉴别。3、尿铜和血清游离铜显著增高是FWD的重要临床特点,对诊断和预后判断有重要意义,降低血清游离铜水平可能是治疗的关键。4、FWD患者肝、肾、脑、肺、脾、胰腺、心脏等器官组织均有明显的病理改变和褐色(铜)颗粒沉积,全身多器官系统损害严重。5、X-射线能谱仪进行金属元素检测分析,不仅直接证明WD肝细胞沉积颗粒中含有大量铜和铁,与近年国外报道的WD患者存在铁代谢异常结论一致,也为WD的诊断提出了一种新的方法。6、病例二第13号外显子发现一错义杂合突变,来源于父亲,国内未见报道。突变位于ATP7B的通道/磷酸化区和第6跨膜功能区,可能对其功能有重要影响,是否与暴发性发病有关有待进一步证实。7、WD基因非常复杂,目前致病突变检出率不高,基因诊断仍有一定局限性,迫切需要建立简便、特异、经济、快速的基因诊断方法,并加强非编码区突变的研究。更多还原

【Abstract】 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.更多还原