Copper-chelating therapeutic effect in Wilson disease with different clinical phenotypes and polymorphisms of ATP7B gene

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慢性乙型肝炎拉米夫定耐药患者HBV基因型及ALT变化的观察

观察慢性乙型肝炎患者用拉米夫定治疗后HBVP基因变异与不同HBV基因型感染及HBV DNA复升水平和转氨酶变化.收集51例慢性乙型肝炎患者用拉米夫定治疗52-78周后发生YMDD变异的血清标本,对照组128例未用拉米夫定治疗的慢性乙型肝炎患者血清标本,应用聚合酶链反应方法,测定HBV DNA基因型;用限制性片段长度多态性分析方法(PCR RELP)测定HBV DNA YMDD变异;同时进行HBV DNA定量分析.结果显示51例拉米夫定治疗后HBV DNA基因变异患者以B型和C型为主,分别为10例(19.6%)和39例(76.47%),B C混和型2例(3.92%),未见其它基因型.拉米夫定治疗引起HBVDNAYMDD变异可以发生在不同HBV基因型感染的慢性乙型肝炎患者中,与对照组比较二者没有显著性差异.     

慢性乙型肝炎YMDD变异患者的临床与病理诊断分析

目的探讨慢性乙型肝炎发生YMDD变异时临床诊断与病理诊断的符合性,血清HBV DNA与病理分度的关系。方法对42例YMDD变异的慢性乙型肝炎患者进行测定HBV DNA、肝活检;进行临床诊断与病理诊断对比,作Kappa分析。设37例没有服用拉米夫定无YMDD变异患者为对照组,比较两组间病毒复制情况与病理分度。结果YMDD变异患者临床诊断与病理诊断总符合率为66.7%,慢性乙型肝炎轻度、中度、重度分别为83.3%、40.0%、66.7%,Kappa值=0.346,P<0.01。与对照组比较,肝组织病变程度轻,病理诊断中、重度肝炎少。结论发生YMDD变异时,慢性乙型肝炎病情活动的临床诊断与病理诊断一致性不强,因此,对没有禁忌证的患者应行肝活检,特别是中、重度肝炎患者更应该接受肝活检。慢性乙型病毒性肝炎抗病毒治疗是重要和有效的治疗手段。     

Genetic alterations in the S gene of hepatitis B virus in patients with acute hepatitis B,chronic hepatitis B and hepatitis B liver cirrhosis before and after liver transplantation

Abstract: Background: Several studies have shown that hepatitis B immunoglobulin (HBIG) imposes a selection pressure on the hepatitis B virus (HBV) S gene, and that the emergence of mutations in this region would make reinfection after orthotopic liver transplantation (OLT) possible. Aims: This study was undertaken to analyze the presence of HBV S-gene mutations in the different stages of HBV infection and the relationship between HBIG therapy and the emergence of mutations in liver transplant recipients. Methods: The frequency and location of mutations in the coding region of the HBV S gene were studied by PCR and direct sequencing in 30 patients (7 with acute self-limited hepatitis B, 16 with chronic hepatitis B and 7 recipients of (OLT) for HBV-related end stage liver disease who became reinfected). Results: The average number of ammo acid changes was higher in patients with a more advanced stage of disease, 0.57 mutations/100 positions in acute hepatitis B and 1.57 in chronic hepatitis B (1.28 in HBeAg-positive and 1.8 in anti-HBe-positive patients). The average number of substitutions in the transplanted patients was 2.7 before OLT and 3 after OLT. No amino acid substitutions were detected in the “a” determinant of HBsAg in acute hepatitis B, however, 8 substitutions were observed in 6 chronic patients. In 3 OLT patients, 4 substitutions were observed in samples before and after OLT. One of these patients, who had protective levels of anti-HBs, showed 3 additional new amino acid substitutions after OLT, suggesting escape mutant selection by the effect of HBIG therapy. No changes were observed between the consensus sequences obtained several years before and after transplantation, indicating consensus sequence stability. Conclusion: These results show that there is an accumulation of HBV S-gene mutations in HBV-related end-stage liver disease. Prophylaxis with HBIG mainly obtained from acute self-limited hepatitis patients who have a highly homogeneous viral population, may be one factor underlying the reinfection after liver transplantation.     

乙型肝炎病毒多聚酶变异与拉米夫定治疗反应

目的 探讨乙型肝炎病毒（HBV）多聚酶酪氨酸－蛋氨酸－天氧氨酸－天冬氨酸（YMDD）基序变异对拉米夫定抗病毒疗效的影响。方法 对19例拉米夫定治疗（100mg/d）48周时血清病毒核酸阳性的慢性乙型肝炎患者,采用聚合酶链反应（ PCR）产物直接测序技术,检测其血清中HBV多取和酶（YMDD）变异情况,并观察其血清HBV DNA和丙氨酸转氨酶（ALT）水平。结果 在检出YMDD变异株的10 例患者（4例为YV^550DD型,均伴L^526→M突变,6例为YI^550DD型）,至52周为止,共5例出现HBV DNA突发（分枝DNA信号扩增法）,其中2例伴ALT再高。追踪其治疗前血清均未发生上述变异。而在未检出YMDD变异的9例患者中,1例出现DNA突发后又阴转,而另1例治疗过程中HBV DNA未阴转、ALT始终波动于正常值上下的患者,发现多聚酶其他部位的变异^473→R,D^477→N,D^480→N,L^491→M)治疗前后相同。结论 乙型肝炎病毒多聚酶（ YMDD）变异与药物诱导相关,发生变异者较无变异者疗效降低;在拉米夫定治疗过程中,该变异的检测将有助于其疗效监测。     

Karyotypic and ras gene mutational analysis in idiopathic myelofibrosis

Summary. Karyotypic analysis was performed in a total of 69 patients with well-characterized idiopathic myelofibrosis. Karyotypic abnormalities were detected in 46% of cases examined during the chronic phase (29/63); with three abnormalities, del (13q), del(20q) and partial trisomy 1q, accounting for 75% of all abnormalities at diagnosis. The absence of del(5q), trisomy 8 and 21, as well as the rarity of monosomy 7, contrasts with pooled published data and may reflect our exclusion of closely related disorders, in particular MDS with fibrosis. Chromosomal aberrations increased to approximately 90% (8/9) in patients analysed during acute transformation. Mutational activation of codons 12, 13 and 61 of N-, Ha- and Ki-ras genes were assessed by polymerase chain reaction and hydridization with synthetic non-radioactive digoxigenin-labelled probes. Three mutations were detected in samples of peripheral blood DNA taken from 50 patients during the chronic phase of their disease: one N12 Asp (GGT GAT) and two N12 Ser (GGT AGT) mutations. The results from this study indicate that karyotypic abnormalities are present in at least 29% of cases at diagnosis and that del(13q), del(20q) and partial trisomy 1q are the most frequent findings. Ras mutations were relatively infrequent (6%) and appeared restricted to the N-ras gene. Karyotypic analysis at diagnosis was found to be of prognostic significance.     

Mutations in the thrombomodulin gene are rare in patients with severe thrombophilia

Because thrombomodulin plays a key role in the protein C pathway, we evaluated the contribution of thrombomodulin gene mutations to venous thrombosis. We examined 38 patients with recurrent, documented thrombotic events at a young age and a positive family history. Twelve individuals with low levels of soluble thrombomodulin in plasma were also studied. Finally, the allelic frequency of the Ala455Val polymorphism was estimated in 192 patients with at least one thrombotic event and in 369 age- and sex-matched asymptomatic controls. Two mutations were identified; G/A-201, in a severely thrombophilic patient and G/T 1456, in a patient with low soluble thrombomodulin levels. The first mutation has been reported by some, but not others, to be associated with moderately reduced levels of thrombomodulin. The second was identified previously in a patient with low soluble thrombomodulin, but expression studies failed to show functional changes in the mutant. Thrombomodulin gene mutations thus appear to be rare even in highly selected thrombophilic patients, and possibly functionally irrelevant. The allelic frequency of the Ala455Val polymorphism was identical in patients and controls. Considering the lack of a phenotype and the costly screening procedure, we recommend that thrombomodulin defects be sought only for research purposes.     

Endothelial nitric oxide synthase Glu 298 Asp (G894T) and Apolipoprotein E gene polymorphism as possible risk factors for coronary heart disease among Egyptians

In Egypt, The prevalence of chronic heart disease (CHD) is 8.3%. It is the principal cause of death and is responsible for 22% of total mortality. The age-adjusted mortality rate is 174 per 100,000 of population. There are many studies on traditional risk factors and CHD in Egypt but the study of novel risk factors is deficient.

Copper-chelating therapeutic effect in Wilson disease with different clinical phenotypes and polymorphisms of ATP7B gene

AIM To investigate the copper-chelating therapeutic effect in Wilson disease (WD) with different clinical phenotypes and polymorphisms of ATP7B gene.METHODS One hundred and twenty-two WD patients with different clinical phenotypes were given DMPS intravenously and Gandou copper-chelating tablet orally for one month. The therapeutic effect was judged by modified Goldstein mothod. Exon 18 of ATP7B gene extracted from the DNA of patients and 20 healthy volunteers was amplified with PCR mutation and polymorphism were screened with SSCP technique.RESULTS Four kinds of abnormal migration bands in PCR-SSCP were observed in 37 WD patients, mutation frequencies of three different disease phenotypes, and curative effect between mutation group and non-mutation group showed no statistically significant difference (P＞0.05), but the total effectiveness rates in patients with Wilson type or pseudosclerosis type were significantly higher than those of patients with hepatic type (X2=6.17, P＜0.05).CONCLUSION Most WD patients are compound heterozygotes, the patients with different clinical phenotypes have different response to copper-chelating therapy. Specific mutation, at least in part, plays a role in influencing the disease phenotypes and therapeutic effect.     

APOE polymorphism and lipid profile in three ethnic groups in the Singapore population

Background: Serum lipid concentrations are modulated by environmental factors such as exercise, alcohol intake, smoking, obesity and dietary intake and genetic factors. Polymorphisms at the Apolipoprotein E (APOE) locus have consistently shown a significant association with total and LDL-cholesterol (LDL-C). However, their impact on HDL-cholesterol (HDL-C) may be population dependent. Having three major ethnic groups within a similar social environment allows us to study the role of genetics and their interactions with lifestyle factors on the serum lipid profile and coronary risk in Asians. Methods: This study included 1740 males (1146 Chinese, 327 Malays and 267 Asian Indians) and 1950 females (1329 Chinese, 360 Malays and 261 Asian Indians) with complete data on anthropometric indices, fasting lipids, smoking status, alcohol consumption, exercise frequency and genotype at the APOE locus. Results: Malays and Asian Indians were more obese compared with the Chinese. Smoking was uncommon in all females but Malay males had significantly higher prevalence of smokers. Malays had the highest LDL-C whilst Indians had the lowest HDL-C, The 3 allele was the most frequent allele in all three ethnic groups. Malays had the highest frequency of 4 (0.180 and 0.152) compared with Chinese (0.085 and 0.087) and Indians (0.108 and 0.075) in males and females, respectively. The 2 allele was the least common in Asian Indians. Total cholesterol (TC) and LDL-C was highest in 4 carriers and lowest in 2 carriers. The reverse was seen in HDL-C with the highest levels seen in 2 subjects. The association between ethnic group and HDL-C differed according to APOE genotype and gender. Asian Indians had the lowest HDL-C for each APOE genotype except in Asian Indian males with 2, where HDL-C concentrations were intermediate between Chinese and Malays. Conclusion: Ethnic differences in lipid profile could be explained in part by the higher prevalence of 4 in the Malays. Ethnicity may influence the association between APOE genotypes and HDL-C. APOE genotype showed no correlation with HDL-C in Malay males whereas the association in Asian Indians was particularly marked. Further studies of interactions between genes and environmental factors will contribute to the understanding of differences of coronary risk amongst ethnic groups.     

子宫内膜癌PTEN基因突变研究

目的探讨PTEN基因突变与子宫内膜癌临床病理特征及发生发展的关系。方法以聚合酶链反应—单链构象多态性方法（PCR-SSCP）检测31例子宫内膜癌和10例正常子宫内膜组织的PTEN基因突变。结果子宫内膜癌组的突变率为32.3%,正常组无突变,二者比较有统计学差异（P〈0.05）。子宫内膜癌组G1级突变率（45.0%）明显高于G2-G3级（9.1%）。结论PTEN突变与子宫内膜癌的发生发展密切相关。     

重组人乙型肝炎病毒S基因表达的体内外评价

目的 构建能表达乙型肝炎病毒（HBV）S基因的DNA疫苗，并在离体与活体条件下评价重组S基因的表达。方法 将克隆的S-X基因片段插入真核细胞表达载体。获得重组质粒，在离体转录系统与转染细胞系中表达重组S基因。并在小鼠中评价HBsAg激发抗-HBs的效果。结果 S基因的表达已通过Northern印迹杂交，Western印迹杂交和ELISA（抗原及抗体检测）予以证实。结论 S基因已成功地实现体外重组与表达。     

乙型肝炎病毒基因变异与免疫调节的关系

目的探讨乙型肝炎病毒（HBV）基本核心启动子（BCP）及前C基因变异与慢性乙型肝炎患者外周血CD4^＋CD25^＋调节性T细胞水平的关系。方法应用基因芯片技术检测HBV BCP及前C基因T1762、A1764、A1896位碱基变化;应用流式细胞仪检测外周血CD4^＋CD25^＋调节性T细胞表达水平。结果急性乙型肝炎、慢性活动性乙型肝炎、肝硬化、肝癌患者血清中HBV DNA T1762、A1764、A1896位碱基突变率分别为0、41.2%、69.4%、100%;急性乙型肝炎、慢性活动性乙型肝炎、肝硬化、肝癌患者外周血CD4^＋CD25^＋调节性T细胞水平高于正常对照;HBeAg阳性感染者高于HBeAg阴性感染者;变异株感染者低于非变异株感染者（P均〈0.05）。结论前C/C基因变异与HBV感染后肝病的慢性化及临床病情加重有关,与CD4^＋CD25^＋调节性T细胞的表达水平相关。     

早发性癫痫脑病男性患儿 ARX 基因突变及其临床特点

目的：探讨早发性癫痫脑病男性患儿ARX基因突变及其临床表型特点。方法收集42例男性早发性癫痫脑病患儿详细的临床资料,采集患儿及家长外周血,应用PCR、直接测序和多重连接依赖的探针扩增（ MLPA）技术对ARX基因第二外显子进行突变分析。对患儿的临床表型特点进行分析。结果42例早发性癫痫脑病患儿中3例患儿存在ARX基因第二外显子大片段缺失,检出率为7．14％（3/42）。其中1例为非特异性癫痫脑病,2例为婴儿痉挛症。3例患儿均于出生后6个月内出现难以控制的癫痫发作,2例患儿在脑电监测中发现高度失律,3例患儿对多种抗癫痫手段反应差,且均表现为严重的智力运动发育落后或倒退。未检出缺失的39例患儿发作形式呈现更多样化的特点,其中5例患儿大运动发育基本正常,3例患儿对ACTH或生酮饮食手段控制癫痫有效。结论早发性癫痫脑病患儿中部分存在ARX基因第二外显子大片段缺失。具有ARX基因突变的早发性癫痫脑病患儿,其临床表现为6个月内出现难治性癫痫发作,以部分性发作及痉挛发作为主,同时有显著的智力运动发育落后、甚至倒退;少数未检出缺失的患儿的大运动发育可基本正常,对ACTH或生酮饮食反应良好。     

TP53 Gene Mutations Are Rare in Nondysplastic Barrett's Esophagus

In search of potential prognostic markers, we analyzed a large series of tissues of Barrett's esophagus and samples of adenocarcinomas arising in the terrain of Barrett's esophagus for TP53 gene mutations by direct sequencing of exons 5 to 9 of the TP53 gene. While 9 of 21 adenocarcinomas tested (42.9%) contained a TP53 mutation, none of 24 samples from Barrett's esophagus were mutated. This observation suggests that TP53 gene mutation may be a relatively late event in the progression from nondysplastic Barrett's esophagus to adenocarcinoma of esophagus. Therefore, TP53 gene mutations alone are not likely to represent a widely useful prognostic marker of the risk of progression to malignancy, at least not in Barrett's esophagus without dysplasia. This work was supported by grant ND/7009-3 from the Internal Grant Agency of the Ministry of Health of the Czech Republic.     

Microsomal triglyceride transfer protein (MTP) -493G/T gene polymorphism contributes to fat liver accumulation in HCV genotype 3 infected patients

Summary. (A) A reduced activity of microsomal triglyceride transfer protein (MTP), a key enzyme of assembly/secretion of lipoproteins, is related to HCV steatosis. Host genetic background may influence development of steatosis. The aim of the study was to investigate the association between MTP‐493 G/T gene polymorphism, fat liver accumulation and fibrosis progression in HCV infected patients. A total of 102 naïve patients with liver biopsy proven chronic hepatitis C were evaluated for MTP‐493 G/T gene polymorphism, HCV RNA, HCV genotype, HOMA‐IR, serum adiponectin, TNF‐α and serum lipid levels. HCV genotype 3 infected patients carrying the T allele of the MTP gene polymorphism showed higher degree of steatosis than those carrying GG genotype (3.45 ± 0.37 vs 1.30 ± 0.45, respectively; P < 0.001). MTP‘T’ allele carriers also had higher HCV RNA serum levels (P < 0.01) and hepatic fibrosis (P < 0.001). Irrespective of MTP genotype, patients with HCV genotype 3 had lower levels of cholesterol, ApoB, HDL and LDL. In HCV genotype non‐3 infected patients no parameters were associated with MTP gene polymorphism. In conclusion the presence of T allele of MTP‐493G/T gene polymorphism predisposes patients infested with HCV genotype 3 to develop higher degree of fatty liver accumulation.     

Two novel factor VII gene mutations in a Chinese family with factor VII deficiency

We report two novel factor VII (FVII) gene mutations in a Chinese family with FVII deficiency. The proband, a 55-year-old woman. was incidentally found to have right shoulder arthritis consistent with chronic haemophilic arthropathy. FVII studies showed a FVII activity of 0.02 iu/ml and a FVII antigen of 49%. Molecular analysis showed a double heterozygous state, with an exon 4 nonsense mutation (C6003-->A; Cys61-->Term) and an exon 8 missense mutation (T10902-->G; Cys329-->Gly) that disrupted a Cys310/Cys329 disulphide bond. The genotypes and phenotypes were correlated in the patient's daughters. Two daughters were heterozygous for the Cys61-->Term mutation and showed a type 1 FVII gene mutation phenotype consistent with a nonsense mutation. One daughter was heterozygous for the Csy329-->Gly mutation and showed a type 2 mutation phenotype consistent with a missense mutation. These are the first reported FVII gene mutations in the Chinese people.