Nrf2基因启动子-653G/A,-651G/A和-617C/A位点单核苷酸多态性与帕金森病易感性的关联性研究

目的探讨陕西地区汉族人群中核因子E2相关因子2(Nrf2)基因多态性与帕金森病(parkinson's disease,PD)易感性的关系。方法选择2015年1月~2016年12月在安康市中医医院急诊科治疗的553例PD患者作为PD组,并按照患者发病年龄分为早发性帕金森病(EOPD)组186例和晚发性帕金森病(LOPD)组367例,同时随机选择同期在该院体检的健康体检人员350例作为对照组,采用聚合酶链反应(PCR)联合DNA直接测序法检测Nrf2基因启动子-653G/A,-651G/A和-617C/A位点单核苷酸多态性。结果 EOPD组与对照组比较,-653G/A位点基因型和等位基因分布频率差异均有统计学意义(χ~2=6.032,5.652,均P0.05)。PD组与对照组,LOPD组与对照组,EOPD组与LOPD组,三个SNP位点基因型和等位基因分布频率比较差异均无统计学意义(均P0.05)。EOPD组与对照组,EOPD组与LOPD组,单体型A-A-C[(-653G/A)-(-651G/A)-(-617C/A)]频率比较差异具有统计学意义(χ~2=6.566,8.350,P=0.011,0.004)。PD组与对照组,LOPD组与对照组,单体型频率比较差异均无统计学意义(均P0.05)。结论 Nrf2基因-653G/A位点单核苷酸多态性可能与PD遗传易感性相关。     

新疆维吾尔族单胺氧化酶B基因多态性与帕金森病的关系

目的 探讨新疆维吾尔族多巴胺代谢酶-单胺氧化酶B(monoamine oxidase B,MAOB)基因内含子13G/A多态性与帕金森病(Parkinson disease,PD)遗传易感性的关系.方法 采用聚合酶链反应-限制性片段长度多态性分析法(PCR-RLFP),经卡方检验,分析95例PD患者与104例健康对照的MAOB基因的等位基因和基因型频率分布情况.结果 PD组与健康对照组MAOB基因G/A等位基因频率、各基因型频率差异无统计学意义;男性PD组与男性健康对照组、女性PD组和女性健康对照组等住基因频率、各基因型频率差异无统计学意义;男性PD组与女性PD组等位基因、各基因型频率差异有统计学意义(x2=3.944,P＜0.05;x2=18.030,P＜0.01).结论 新疆维吾尔族MAOB基因内含子13 G/A多态性可能与PD遗传易感性无关,男性PD组与女性PD组基因型分布不一致.     

中国西南地区汉族人群肿瘤坏死因子基因多态性与鼻咽癌遗传易感性

目的:分析肿瘤坏死因子基因多态性分布与中国西南地区汉族人群鼻咽癌易感性的关系。方法:选择2000-10/2005-09在华西医科大学第一附属医院就诊的100例鼻咽癌患者为鼻咽癌组,均经过病理科活检确诊,其中包括未治疗44例,放疗后37例,放疗加化疗后19例,选择100名同期入院健康体检者为对照组。所有受试对象为中国西南地区汉族人,均对检测项目知情同意。采用聚合酶链反应-限制性片段长度多态性的方法检测100例中国西南地区汉族鼻咽癌患者和100名健康对照者肿瘤坏死因子α基因启动子区-308位点及肿瘤坏死因子β基因第一内含子252位点的等位基因以及基因型频率,分析两位点多态性与鼻咽癌遗传易感性的关系。结果:鼻咽癌组患者肿瘤坏死因子β( 252)位点G/A杂合子基因型频率显著高于对照组(57%,29%,P<0.01),野生型(G/G基因型)频率低于健康对照组(23%,51%,P<0.01),等位基因A的频率高于健康对照组(48.5%,13%,P<0.01);肿瘤坏死因子α(-308)位点基因型以及等位基因频率与对照组相比较无统计学差异。结论:本实验人群中未发现肿瘤坏死因子α(-308)位点多态性与鼻咽癌易感性无关;肿瘤坏死因子β( 252)位点基因多态性与鼻咽癌具有相关性,A等位基因可能是鼻咽癌的遗传易感基因,G/A杂合子基因型个体较易患鼻咽癌。     

白细胞介素-18基因多态性与肺结核病易感性的关系

目的：通过病例-对照研究,探讨白细胞介素-18（IL-18）基因启动子区-607C/A、-137G/C位点单核苷酸多态性（SNP）与肺结核病的关系。方法：采用序列特异性引物PCR（PCR-SSP）及测序技术检测深圳地区汉族人群肺结核患者200例及健康对照者197例IL-18启动子区-607 C/A 、-137G/C位点多态性基因型。采用直接计数法计算各组基因型频率及等位基因频率,进行χ2 检验。以P值＜0.05为具有统计学意义。结果：肺结核患者IL-18启动子区-607位点A/A纯合子、A/C杂合子、C/C纯合子基因型频率分别为19.5%、55.0%、25.5% ;A、C等位基因频率分别为47.0%、53.0%。健康对照者A/A纯合子、A/C杂合子、C/C纯合子基因型频率分别为22.8%、46.7%、30.5%;A、C等位基因频率分别为46.2%、53.8%。两组人群-607位点基因型及等位基因分布无明显差异(P>0.05)。肺结核患者IL-18启动子区-137G/C位点C/C纯合子、C/G杂合子、G/G纯合子基因型频率分别为4.5%、18.5%、77.0%;C、G等位基因频率分别为13.8%、86.2%。健康对照者C/C纯合子、C/G杂合子、G/G纯合子基因型频率分别为5.6%、25.9%、68.5%;C、G等位基因频率分别为18.5%、81.5%。两组人群-137位点基因型分布无明显差异(P>0.05)。结论：IL-18启动子区-607、-137位点基因多态性与中国汉族人群肺结核病易感性无关。     

骨髓增殖性肿瘤患者TET2基因SNP rs3733609 C/T与JAK2V617F突变负荷关系的研究

目的:探讨骨髓增殖性肿瘤(myeloproliferative neoplasms,MPN)患者中TET2基因SNP rs3733609 C/T多态性与JAK2V617F基因突变负荷的关系。方法:采用RT-PCR法扩增TET2基因9号外显子,基因测序法分析SNP rs3733609位点碱基序列;应用MGB Taqman探针PCR法定量检测JAK2V617F突变负荷,分析TET2基因SNP rs3733609 C/T与JAK2V617F突变负荷及临床参数之间的相关性。结果:85例JAK2V617F+MPN患者中19例(22.4%)检出TET2基因rs3733609 C/T杂合多态性(正常为T/T),106例健康志愿者中仅有9例检测出,发生率仅为8.5%(9/106)。与阴性组(TET2 rs3733609 T/T)相比,TET2基因SNP rs3733609(CT/TC)+患者在中位年龄、血红蛋白水平、血小板计数无显著差异,而外周血白细胞数、JAK2V617F突变负荷显著增高。JAK2V617F高突变负荷组TET2基因SNP rs3733609(CT/TC)+7例(36.8%,7/19),其比例高于低突变负荷组(18.2%,12/66)。结论:TET2 SNP rs3733609 C/T可能是一个新的MPN易感等位基因,影响MPN患者的临床特点及克隆演化。     

葡萄糖-6-磷酸脱氢酶缺乏症基因诊断方法的建立

目的建立有效可行的高通量葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PD)基因诊断新技术。方法建立单管三重PCR扩增G6PD基因外显子2~13目的片段,构建双管25重SNaPshot反应体系检测25个已报道的中国人群的G6PD基因突变位点。用20份已知基因型的样品进行重复性试验。对60例未知样品同时用多重SNaPshot基因分型技术和DNA测序法进行双盲试验。结果建立的多重SNaPshot技术可同时检测25个G6PD基因突变位点,且可区分正常、女性杂合子与男性半合子/女性纯合子,有效检出复合突变。批内及批间的重复性均达到100%。60例未知样本检出23例突变,SNaPshot基因分型结果与测序结果完全一致,特异性和准确性均为100%。结论建立的单管三重PCR结合双管25重SNaPshot技术是一种有效可行的G6PD缺乏症基因诊断的新方法。     

Htra2基因内含子5-59A/G位点多态性与帕金森病的相关性研究

目的探讨粤西地区汉族人群中Htra2（又被称作Omi）基因内含子5-59A/G位点(rs2241027)的单核苷酸多态性（SNP）与帕金森病（PD）的相关性。方法采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）技术,检测56例PD患者和109例健康人的Htra2基因内含子5-59A/G位点多态性的基因型。结果病例组A等位基因频率(46.4%)倾向高于对照组（36.7%）（P=0.073）;AA基因型频率（21.4%）亦倾向高于对照组（11.0%）（P=0.072）。经性别分层分析发现,男性AA基因型频率（25.7%）高于对照组（10.3%）(P=0.041);病例组A等位基因频率（48.6%）倾向高于对照组（34.6%）(P=0.051)。结论5-59A/G位点等位基因A和AA基因型均可能增加PD的发病风险,特别是男性。     

PRKCG基因多态性扫描及其与帕金森病的相关性研究

目的寻找蛋白激酶Cγ(PKCγ)基因PRKCG的变异位点 ,探讨其与帕金森病 (PD)发病的关系。方法取PD患者和正常对照人群的DNA ,用高压液相色谱分析 (DHPLC)做PRKCG全编码区 18个外显子和外显子 内含子交界区扫描 ,比较变异位点上早发PD、晚发PD和正常对照的基因型频率、基因频率。结果共扫描到 7个单核苷酸多态性 (SNP)位点和 1个重复序列。其中有 5个多态性位点属首次发现。 8个位点中有 3个变异位点 (IVS11 2 6G ,IVS13 76C ,14 97C)完全连锁。相关性分析显示这些位点与散发性PD之间没有统计学相关性。结论PRKCG基因可能不是散发PD的易感基因。     

影响亚甲基四氢叶酸还原酶和甲硫氨酸合成酶还原酶基因多态性的因素分析

目的了解影响女性亚甲基四氢叶酸还原酶(MTHFR)C677T、A1298C位点和甲硫氨酸合成酶还原酶(MTRR)A66G位点基因多态性的因素。方法采用横断面调查研究方法,以湖北省内1 902例女性为对象,采集口腔黏膜上皮细胞,提取DNA,用实时荧光定量PCR技术检测MTHFR和MTRR基因,统计分析不同民族MTHFR和MTRR基因多态性的差异,与其他地区人群的MTHFR和MTRR基因多态性进行比较,并分析不同位点基因多态性之间的关联关系。结果 (1)湖北省汉族女性和少数民族女性在MTHFR C677T和MTRR基因多态性构成差异无统计学意义(P>0.05),而MTHFR A1298C位点基因多态性构成差异有统计学意义(P<0.05)。(2)MTHFR C677T位点的纯合突变基因型TT的频率从南到北呈上升趋势,MTHFR A1298C位点的纯合突变基因型CC的频率从南到北呈下降趋势,而MTRR A66G位点基因多态性在不同地区差异不大。(3)MTHFR C677T位点的纯合突变基因型TT的频率在A1298C位点的正常型、杂合型、纯合突变型中分别为15.9%、0.1%、0.0%,差异有统计学意义(P<0.01);MTHFR C677T位点的纯合突变基因型TT的频率在MTRR A66G位点的正常型、杂合型、纯合突变型中分别为9.4%、5.9%、0.7%,差异无统计学意义(P>0.05)。结论 MTHFR A1298C位点基因多态分布存在民族差异;而MTRR C677T、A1298C位点基因多态分布存在地区差异;同一基因不同位点的基因多态性有关联。     

西北汉族女性ACTN3基因多态性频率分布特征

目的调查汉族普通女性人群ACTN3基因多态性频率分布特征。方法选择18～22岁西北籍汉族健康女学生226例作为研究对象,用荧光实时定量PCR法分析ACTN3基因的多态性频率分布特点。结果 ACTN3基因R577X多态位点分布频率符合Hardy-Weinberg平衡,ACTN3基因R577X杂合子RX基因型频率为22.1%,纯合子RR为40.4%,XX为37.5%,RX基因型频率多于XX基因型(P<0.05),ACTN3基因R577X多态位点R等位基因频率为51.3%,高于X等位基因频率48.7%(P<0.05)。结论从种族单一性、样本数量和年龄段的选择等因素考虑,本研究结果可以作为西北籍汉族女性ACTN3基因R577X多态性频率分布的代表。     

真性红细胞增多症患者T细胞的克隆特点

目的　了解真性红细胞增多症（ＰＶ）患者外周血Ｔ细胞受体（ＴＣＲ）Ｖβ２４ 个亚家族基因的表达及其克隆特点。方法　采用逆转录多聚酶链反应（ＲＴＰＣＲ） 检测３ 例ＰＶ 患者外周血单个核细胞ＴＣＲＶβ２４ 个亚家族基因表达情况;采用基因扫描分析Ｔ细胞的克隆特点。结果　３ 例ＰＶ患者外周血仅表达４ ～１４ 个Ｖβ亚家族,主要为Ｖβ２ 、Ｖβ３ 、Ｖβ１６ 、Ｖβ２１ 和Ｖβ２３ ;２ 例患者表达的Ｖβ３ 和Ｖβ２３Ｔ细胞为克隆性增殖。结论　ＰＶ 患者外周血ＴＣＲＶβ亚家族的优势表达和克隆性增殖可能是患者体内对抗恶性细胞的免疫反应特征。     

Use of polymerase chain reaction for determining T-cell clonality

AIM: To distinguish T-cell lymphomas and reactive T-cell proliferation it is important to confirm the ability of T-cells to be cloned. Conventional histological and immunophenotypic methods fail to determine the ability of T-cells to be cloned. An experience in the use of detection of T-cell receptor gene gamma-chain (TCRy) rearrangement for determining T-cellular clonality is described. MATERIAL AND METHODS: Polymerase chain reaction (PCR) and single strand conformational polymorphism (SSCP) were used to determine T-cell clonality. Twenty healthy donors, 28 patients with T-lymphomas, and 26 patients with various non-T-cell lymphoproliferative disorders or reactive processes were studied. RESULTS: T-cell monoclonality was detected in 23/28 (82%) T-cell lymphoma cases, whereas in all the samples from normal subjects a polyclonal pattern of rearrangements TCRy was found. The sensitivity of the method was estimated as 2.5%, 7%, and 10% was demonstrated for bone marrow, spleen, and peripheral blood, respectively. CONCLUSION: PCR-SSCP for TCRy was found to be a useful supplement to routine histological and immunophenotypic methods in the diagnosis of T-cell lymphomas.     

抗原受体基因重排克隆性检测在淋巴瘤诊断中的应用

本研究旨在探讨抗原受体基因重排克隆性检测在淋巴瘤诊断中的意义。收集分析了31例淋巴瘤组织,石蜡包埋切片,HE染色后观察形态,免疫组织化学分析免疫表型。采用BIOMED-2标准化试剂盒检测抗原受体基因重排克隆性。结果表明,31例病例中形态学及免疫组织化学分析疑似T细胞淋巴瘤12例,T细胞反应性增生1例,B细胞淋巴瘤16例,B细胞反应性增生2例。基因重排克隆性检测免疫球蛋白(Ig)阳性率94.44%(17/18),T细胞抗原受体(TCR)阳性率92.31%(12/13),2例阴性。最终12例确诊为T细胞淋巴瘤,B细胞淋巴瘤17例,反应性增生2例,阳性检出率为93%。结论:抗原受体重排基因克隆性分析是诊断淋巴瘤的一种有效辅助手段。     

Assessment of clonality in gastrointestinal cancer by DNA fingerprinting.

DNA fingerprinting with three different probes (33.15, 33.6, and alpha-globin 3'HVR) was investigated as a method for the determination of clonality in gastrointestinal tumors. In 29/44 carcinomas the tumor DNA showed clonal somatic mutations that were not seen in the corresponding peripheral blood and normal mucosa samples. The changes consisted of either novel fingerprint bands, losses of bands, or both. The probe 33.15 yielded the highest rate of abnormal DNA fingerprints (21/44 carcinomas). Sequential use of the probes increased the number of cases where clonal fingerprint markers could be detected. One out of five colorectal adenomas also showed a clonal loss of a fingerprint band. In two cases of gastric cancer, DNA from the metastatic tumor had a different DNA fingerprint from that found in the primary carcinoma. DNA fingerprinting offers a novel approach to determining clonality in tumors and may prove useful for the study of tumor progression.     

Molecular genetic approaches for the diagnosis of clonality in lymphoid neoplasms

Rearrangements of immunoglobulin (Ig) and T cell receptor (TCR) genes provide a highly sensitive molecular marker for the detection of clonality in lymphoid lesions and allow the pathologist to (1) distinguish polyclonal from monoclonal lymphoid proliferations, (2) provide corroborative evidence for lineage when used in conjunction with immunophenotypic techniques, (3) differentiate clonal lymphoid lesions from poorly differentiated nonlymphoid neoplasms, and (4) assess residual disease at the molecular level. The use of these probes in conjunction with morphology and immunohistochemistry or flow cytometry allows the pathologist to assign virtually all lymphoid neoplasms to the B or T cell lineage. The cloning of Ig and TCR genes also has led to the identification of new protooncogenes that reside at the breakpoints of chromosomal translocations frequently observed in lymphoid neoplasms. Molecular probes for these new genes involved in the pathogenesis of lymphoid neoplasms may be used as additional molecular markers for the determination of clonality, lineage, and even histologic subtype of lymphoid neoplasms. Finally, the development of new molecular technologies such as polymerase chain reaction and pulsed-field gel electrophoresis has provided new tools for the highly sensitive detection of genetic rearrangements in human tumors and will greatly enhance the ability of the pathologist to monitor minimal residual disease and detect early relapse.     

An automated semiquantitative B and T cell clonality assay.

BACKGROUND: Rearrangements of the antigen receptor genes in B and T cells generate products of unique length and sequence. Polymerase chain reaction (PCR) assays are routinely used to identify clonal lymphocyte populations by detecting clonal V-J rearrangements or chromosomal translocations within these antigen receptor loci. Multiple primer sets are, however, required to detect the majority of clonal B- and T-cell malignancies. Products from the individual reactions must be analyzed separately to avoid misinterpretation. Moreover, small clonal populations remain difficult to identify. To address these difficulties, we propose that an integrated fluorescence-based approach to clonal B- and T-cell detection would simultaneously identify both B- and T-cell neoplasia; increase amplicon resolution, analytic sensitivity, and assay throughput; produce more comprehensive and semiquantitative data useful for evaluation of hematologic malignancies; and eliminate labor intensive agarose and polyacrylamide gel electrophoresis. METHODS AND RESULTS: Samples were genomic DNA and cDNA. Differentially labeled primers were used to amplify regions diagnostic for B- and T-cell clonality in a single plate with a single thermocycler program. Combined amplicon products underwent capillary electrophoresis for high resolution fractionation and differential fluorescence detection and quantification. Data were automatically analyzed and archived. In a comparative analysis of a variety of clinical samples, this automated and integrated B- and T-cell assay showed >94% agreement (33 of 35 results) with individual B- and T-cell PCR assays. Furthermore, this assay had an overall monoclonality detection rate of 100%, and as little as 100 ng of sample DNA yielded complete B- and T-cell clonality test results. The limit of detection was approximately 10-2 cells, and amplicons were sized to within 0.1 basepair. Serial dilutions of clonal B- and T-cell lines comprising a coded proficiency panel were identified and correctly ranked. Specificity was 100% as determined by analysis of 18 control samples that were all negative for B- and T-cell clonality. CONCLUSIONS: Our data show that this automated and integrated B- and T-cell clonality assay system is a sensitive and specific tool useful for rapid identification of clonal lymphocyte populations and will likely have broad clinical applications.