线粒体12S rRNA基因突变与2型糖尿病

目的 观察线粒体12S rRNA 1310、1438及1442位点在中国汉族2型糖尿病患者群体中的突变情况,同时筛查该区域与2型糖尿病发生有关的突变。方法 采用PDR－SSCP及PCR产物直接测序等技术对86例2型糖尿病患者及70名正常对照个体的血细胞线粒体DNA进行突变分析。结果 发现1例患者线粒体DNA 12S rRNA基因1438位点存在G→A的点突变,另1例存在1442位点G→A的点突变,所有对照个体均未发现该两位点的突变。未发现线粒体基因12S rRNA 1310位点C→T点突变。结论 1438位点G→A、1442位点G→A的突变可能与2型糖尿病的发生相关,该两位点突变的具体意义如何尚需进一步研究。1310位点C→T在血细胞中的突变率可能较低,进一步说明2型糖尿病的发生在线粒体遗传上具有一定的异质性。     

128例Ⅱ型糖尿病患者线粒体ND1基因突变位点的研究

目的通过对128例Ⅱ型糖尿病(Type 2 diabetes mellitus,T2DM)患者线粒体DNA ND1基因进行突变位点筛查,探索ND1基因点突变与山西人群T2DM的相关性。方法 PCR扩增患者ND1基因所在区段,PCR产物直接测序分析。结果128例患者共有38例患者检测到基因点突变,突变检出率为29.7%。38例患者共筛出22个突变位点,2种突变类型。其中31例存在单个位点突变,5例存在2个位点突变,2例存在3个位点突变。22个突变位点中,3552(T→A)突变频率最高,为40.9%(9/22);3394(T→C)、3435(C→T)、3497(C→T)、3316(G→A)、3571(C→T)、3537(A→G)的突变频率分别为22.7%(5/22)、22.7%(5/22)、18.2%(4/22)、13.6%(3/22)、13.6%(3/22)和10.1%(2/22);其余突变位点的突变率均为4.5%(1/22)。在所有突变位点中,除3688(G→C)为异质性突变外,其余突变均为同质性。此外,22个突变位点中存在一个新的突变位点3499(A→T),属首次报道。结论 3552(T→A)和3394(T→C)突变频率最高,可能与山西地区T2DM发病相关;新发现的突变位点3499A→T(Thr→Ser),其致病性需要进一步研究。     

基因芯片检测乙醇对慢性乙型肝炎患者乙肝病毒多位点基因突变的研究

目的：研究乙醇对HBV DNA多位点基因变异的影响，为慢性乙型肝炎的临床诊断和治疗提供依据。方法:选取85例慢性乙肝患者为研究对象，分为嗜酒组和非嗜酒组，采用DNA芯片技术, 检测HBV DNA前C区G1896A及A1814C位点、BCP区 A1762T及G1764A位点、P区C528A及T552C位点的基因突变。结果:嗜酒组在BCP区A1762T及G1764A位点的突变频率明显高于非嗜酒组(均P<0.05)。在前C区G1896A及A1814C位点，P区C528A及T552C位点突变频率无明显差异（均P>0.05）。结论:乙醇导致乙肝病毒BCP区A1762T及G1764A位点发生基因突变，增强HBV基因的表达和复制，加重慢性乙型肝炎患者的病情。     

寡核苷酸微阵列技术用于检测幽门螺杆菌克拉霉素耐药相关23S rRNA基因多态性

目的 幽门螺杆菌（Hp）克拉霉素耐药与23S rRNA基因A2142G、A2143G和C2182T点突变相关。本研究旨在探索建立一种快速、简便的检测方法，获取Hp克拉霉素耐药相关23S rRNA基因多态性的流行病学资料，为临床合理用药提供依据。方法收集胃黏膜标本，用于病理检查、提取DNA。根据核苷酸位点设计相应探针，探针3′端以氨基修饰，氨基与探针序列之间以间隔臂相连，将探针用芯片点样仪点至玻片上。下游引物5′末端用Cy3荧光标记，进行不对称PCR扩增。其产物与芯片杂交，杂交后洗涤、扫描芯片，观察信号强度。非荧光标记引物扩增PCR产物克隆至T载体，测序验证芯片结果。结果（1）寡核苷酸微阵列技术与测序检测Hb 23S rRNA基因多态性结果完全一致。（2）经病理和PCR证实为场阳性的54例标本，杂交结果显示A2142位点均为野生型（54/54）；A2143G突变率为11．11％（6/54），尚未发现A2143C和A2143T的突变；C2182T突变率为12．96％（7／54），尚未发现C2182A和C2182G的突变，其余均为野生型。结论（1）利用寡核苷酸微阵列技术检测坳克拉霉素耐药的23S rRNA基因多态性，快速、简便而准确，可以高通量并直接检测胃黏膜而不需进行细菌培养，为根除Hp选择用药提供科学依据，推动个体化治疗方案的实施。（2）本研究没有发现场23SrRNA基因2142点突变，A2143G和C2182T突变率分别为11．11％和12．96％。     

中国汉族人HIV-1协同受体CXCR4基因编码区新的突变位点研究

目的 对中国汉族人群HIV—1协同受体CXCR4编码区的基因多态性进行研究，为中国的获得性免疫缺陷综合征(AIDS)防治提供依据。方法 CXCR4(cDNA编号AFl47204)编码区用2对引物进行PCR扩增，然后分别测序，测序样本数为48例。用DNAstar分析测序结果，寻找SNP位点。结果 在编码区发现了7个SNP位点，其中3个同义突变(129位C→T、426位C→T，968C→T)，3个有义突变(38位C→T、90位A→T、712位A→C)，1个终止突变(106位C→T使谷氨酸密码子变成终止密码子)。其中38位C→T、90位A→T、712位A→C、106位C→T，基因突变频率为4．2％、4．2％、9、4％和3．1％。结论 在CXCR4编码区找到的7个SNP位点中，4个引起氨基酸改变，1个已有报道，对HIV—1感染和AIDS病程的影响值得研究。     

乙型肝炎病毒核心启动子及前C区突变检测基因芯片的制备及临床应用

目的 建立检测乙型肝炎病毒核心启动子及前C区突变基因芯片并探讨其临床应用的价值.方法 设计并合成针对核心启动子1762/1764、1814及前C区1896位点突变的特异性探针,制备寡核苷酸芯片.采用不对称PCR对该区域进行扩增,扩增产物与芯片杂交后分析结果,并评价该方法的特异性、灵敏度.采用该方法检测138例HBV DNA阳性血清标本.结果 该方法能够特异地检测乙型肝炎病毒核心启动子1762/1764、1814及前C区1896位点,灵敏度达1×101拷贝/μl.138例HBV DNA阳性血清标本中,T1762/A1764突变40例（28.99%）,C1814突变11例（7.97%）,A1896突变16例（11.59%）.前C区A1896突变在高拷贝组中明显高于低拷贝组（P＜0.01）.结论 本研究建立的基因芯片能够准确同时检测HBVV核心启动子区突变和前C区突变,前C区A1896突变可能与HBV复制状态有关.     

线粒体糖尿病家系基因突变的遗传学筛查

目的 探索与糖尿病发病相关的线粒体基因突变位点。方法 采用PCR、DNA直接测序技术对28个临床疑似线粒体基因突变糖尿病家系(mitochondrial diabetes mellitus，MDM)进行线粒体基因突变高发区域(tRNA^Leu(UUR)基因及NADH脱氢酶1基因)的筛查。结果 两个家系发现与糖尿病发病有关的突变位点。其中1个家系(16号)同时携带nt3243A→G突变和16S rRNA3205C→T突变。该家系两例患者均有消瘦、耳聋、β细胞功能低下、发病年龄低的特点，先证者血乳酸水平在正常高限。在另1先证者患耳聋的家系(28号)发现NDl基因3434A→G突变，导致氨基酸改变，与家系中糖尿病呈共同分离。上述突变均未在正常人中检测到。结论 3243位点突变在本组家系中的发现率为3．08％。16S rRNA3205C→T突变可能与糖尿病发病有关或与nt3243A→G突变协同作用。NDl基因3434A→G突变与28号家系糖尿病的发生有关。     

沈阳地区家庭聚集性感染HBV的家庭HBV前C区基因突变率及其临床意义的研究

目的 研究沈阳地区家庭聚集性感染乙型肝炎病毒的家庭HBV前C区1896位G→A基因突变率及其临床意义。方法 采用PCR—RFLP法检测HBV前C区1896位G→A基因突变。结果 患者及其家庭成员HBV前C区1896位G→A基因突变发生率分别为56．3％和40．5％，明显高于患者配偶25．0％的突变发生率，且配偶中抗-HBs阳性率为26．3％。同时，这种突变在慢性乙型肝炎患者中的发生率为52．4％，在HBV携带者中的发生率为44．4％，在慢性重型肝炎患者中的发生率仅为20．0％。结论 HBV前C区1896位G→A基因突变的发生，可能与HBV的持续感染有关。     

HBV DNA的前C区1896 G/A变异与血清HBV DNA水平

为探讨乙型肝炎病毒（HBV）前C区1896G／A变异对血清HBV DNA水平复制及临床表现的影响，本研究报道采用酶联免疫吸附试验（EHSA）和荧光定量PCR方法（FQ-PCR）及突变特异引物PCR方法（AS-PCR）对95例诊断符合2000年修订的病毒性肝炎防治方案的HBV感染者进行了HBV血清免疫标志物和HBV DNA定量及HBV DNA1896位点G／A变异检测的结果。     

乙型肝炎病毒e系统血清转换中HBV前C、BCP基因检测及临床意义

目的：探讨乙型肝炎患者HBeAg和HBeAb双阳性状态下HBV前C区基因及BCP区基因变异情况,探讨前C区A1896及BCP区T1762与A1764变异与HBe转换的关系。方法：采用时间分辨荧光免疫分析方法定量检测乙肝“二对半”,对HBeAg／HBeAb双阳性标本采用基于膜显色的DNA芯片检测HBV nt1896、nt1762、nt1764位基因。结果：35例HBeAg、HBeAb、HBVDNA阳性的患者均存在nt1762和nt1764的突变,有14例患者出现了nt1896的突变。结论：在乙型肝炎HBe转换过程中均伴有BCP区T1762和A1764的突变,部分存在A1896位点的突变,T1762和A1764的突变早于A1896的突变,A1896的突变主要在e抗体产生过程中或产生以后。     

Detection of hepatitis B virus X gene and PreC promoter mutations from chronic hepatitis B patients in the south of Turkey

Hepatitis B virus (HBV) infection is a global health problem with more than 2 billion infected individuals. HBV infection leads to diverse outcomes ranging from acute to chronic hepatitis, which may result in severe complications as liver cirrhosis and hepatocellular carcinoma (HCC). HBV is one of the most important human DNA viruses having strong oncogenic potential. Recently, many studies have reported on HBV X gene and PreC promoter mutations associated with HCC. In order to detect the prevalence of HBx gene and PreC promoter mutations possibly related to HCC, we have analyzed sera samples collected from 61 patients with chronic hepatitis B. We have detected T1653 mutation in 1 of 61 (1.63%), A1896 mutation in 10 of 61 (16.39%), and T1762-A1764 dual mutation in 4 of 61 (6.55%). T1653 and T1762-A1764 dual mutations were suggested significantly related to HCC in earlier reported studies. Our findings demonstrate that HBx gene and PreC promoter mutations related to HCC are present in our region and prospective clinical chord studies would be useful for better patient management and of early diagnosis of possible HCC cases.     

DNA芯片检测乙型肝炎病毒基因多态性

目的　建立DNA芯片检测乙型肝炎病毒 (hepatitisBvirus,HBV)基因多态性的研究方法并对实验条件进行优化。方法　设计多条寡核苷酸探针 ,在硅烷化芯片的特定位置上 ,用点样仪将探针固定 ,并与PCR扩增的HBV基因相应区段杂交 ,杂交结果影印至硝酸纤维素膜 ,经BCIP NBT避光显色 ,用放大镜观察杂交信号呈暗紫色圆点 ,根据特定位置上杂交信号的有无和与之相应的探针序列来判定基因突变的类型。结果　通过 1次杂交反应可检测HBV前C C区 (nt 1896 1814 )、BCP区 (nt1762 1764)和P区 (nt 52 8 552 )等多个位点的变异 ,与测序分析结果完全一致 ,具有较好的检测灵敏度和重复性。结论　DNA芯片检测HBV基因常见突变位点多态性 ,操作简便易行 ,技术要求不高 ,具有临床推广应用价值 ,而且可以方便地通过向寡核苷酸探针阵列中添加相应探针 ,扩大基因芯片的检测应用范围 ,为临床检测提供了新的方法     

A sensitive method to detect the hepatitis B virus mutations by using solid phase PCR on oligonucleotide array

A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutations were immobilized on modified glass slides through 5' terminal linker, while the 3' terminal was made to be free. The mutated loci were designed to locate on the last nucleotides of 3' terminal respectively, and the positive control probes lacked the last nucleotide of 3' terminal in comparison with the probes used for detection. Probes fixed on oligonucleotide array were also the solid phase amplification primers. One pair of liquid primers was used to amplify the short template product from whole HBV DNA. Using target DNA as template, the solid primers were extended under the action of Taq DNA polymerase and incorporated with Cy-3dCTP as marker. During the thermal cycling reaction, probes served as solid phase amplification primers and amplification products bound to the oligonucleotide array, which could be visualized by incorporation with fluorescent dyes. After amplification, the oligonucleotide array was washed, performed with laser scanning, and then used for quantitative analysis to obtain the information for mutations. The hybridization results were compared with DNA sequencing. It was demonstrated that in case of sample A, the ratios of fluorescence intensities in wide type and in the mutated types of 1896G-A and 1901G-A mutations in HBV were 3.81:1 and 1:3.79 respectively, while, in case of sample B, those were 1:2.89 and 1:3.03 respectively, indicating the presence of point mutations in these two loci. These results correlated to those obtained from DNA sequencing analysis in which the fluorescence intensity ratios in wide type and in the mutated types of 1996G-A and 1901D-A mutations in HBV were 1.26:1 and 1.67:1 respectively. From the above observations, it is evident that the method using solid phase PCR based on oligonucleotide array appears to be a sensitive and promising way to detect mutations with low-density.     

Analysis of the precore and core promoter DNA sequence in liver tissues from patients with hepatocellular carcinoma.

To investigate the role of mutant hepatitis B virus (HBV) in the development of hepatocellular carcinoma (HCC), 20 patients with HCC were studied for precore and core promoter mutations in tumorous and nontumorous tissues. The precore and core promoter region was amplified and analyzed by direct sequencing. Among the 20 tumorous and nontumorous tissues, precore mutant HBV was found in 12 (60%) and 18 (90%), respectively. Of the 12 tumorous tissues with precore mutant, nine tissues had a single mutation (1896) and one tissue had another single mutation (1899). The remaining two tissues had a double mutation (1896 and 1899). A single mutation (1896) and a single mutation (1899) were found in 11 and two of the 18 nontumorous tissues with precore mutant, respectively. Among 20 tumorous and nontumorous tissues, HBV with a C to T mutation at nucleotide (nt) 1846 was detected in six and eight, respectively, and was associated with the virus carrying a mutation (1896 or 1899) except in two tumorous tissues. Mutations at nt 1762 and 1764 in core promoter were observed in 16 (80%) tumorous tissues and 18 (90%) nontumorous tissues. Mutations in the precore and core promoter region were found frequently in nontumorous tissue and in tumorous tissue (18/20 and 12/20 in precore region, 18/20 and 16/20 in core promoter respectively). The high prevalence of precore and core promoter mutations in liver tissue from patients with HCC suggests that these mutations may contribute to the development of HCC.     

Molecular analysis of hepatitis B virus genomes isolated from black African patients with fulminant hepatitis B

To investigate further the possible role of mutant hepatitis B viruses in the pathogenesis of fulminant hepatitis B, the genomic sequence of hepatitis B virus isolates from 9 South African blacks with this disease, including 5 entire genomes, was analysed. Seven of the isolates were genotype A. The mutation most often reported in patients with fulminant hepatitis B, the G1896A precore stop-codon substitution, was, as expected, not present in the genotype A isolates with the exception of one in which it was accompanied by a compensatory C1858T substitution. G1896A was, however, present in the one genotype D isolate. No other precore-defective mutants were detected. The other mutation commonly found in patients with fulminant hepatitis B, the paired A1762T, G1764A substitution in the basic core promoter, was present in only one patient and G1764A in one other. The pre-surface initiation-codon mutation documented in a number of patients with fulminant hepatitis B was not found in our isolates. An 18-amino acid deletion present in the pre-surface region of one isolate has not previously been described in fulminant hepatitis B. Variations within the surface region were mainly genotype specific and not previously described. A relatively large number of mutations were present in the middle region of the core gene in those isolates without G1896A or A1762T, G1764A mutations, although the pattern was not consistent with those in published studies. Thus, as in other published series in which the entire genome of hepatitis B virus responsible for fulminant hepatitis was sequenced, we detected many mutations in different genes, but none was common to all the reported isolates.     

Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis

The association of precore stop codon mutation (A1896), dinucleotide mutation (T1762/A1764) in the basic core promoter of hepatitis B virus (HBV) genome, and genotype of HBV with fulminant or subfulminant hepatitis remains controversial. We studied HBV genotypes as well as mutations in the precore and basic core promoter regions in 18 hepatitis B carriers with fulminant or subfulminant hepatitis. Genotyping of HBV was performed by polymerase chain reaction-restriction fragment length polymorphism. The presence of A1896 in the precore gene and T1762/A1764 in the basic core promoter gene was determined by the polymerase chain reaction and by direct sequencing. Eighteen age- and sex-matched patients with chronic active hepatitis B served as controls. The HBV was of genotype B in 14, genotype C in 3, and unclassified in 1. Precore A1896 mutation occurred in 12 (67%) of the 18 patients. In contrast, the prevalence of basic core promoter mutation was only 17%. Nevertheless, the distribution of HBV genotype and the prevalence of precore A1896 mutation in the fulminant and subfulminant hepatitis patients were similar to those in 18 control patients. In conclusion, the genomic variability of HBV does not seem to contribute to the fulminant and subfulminant exacerbation of chronic hepatitis B in Taiwanese HBV carriers.