广东地区汉族人MBL基因点突变的研究

目的：研究我国汉族人群甘露聚糖结合凝集素（MBL）结构基因外显子1第52、54、57位密码点突变（CGT52TGT、GGC54GAC和GGA57GAA）。方法：收集广东地区汉族普通人群的血标本，提取白细胞基因组DNA，以PCR扩增目的基冈片段，应用荧光探针杂交可视技术检测其MBL基因的点突变。结果：从202份样本中，检出GGC54GAC点突变纯合子6例和杂合子44例（等位基因频率为0．139），GGA57GAA点突变杂合子1例（频率为0．002），未发现CGT52TGT点突变（频率为0）。结论：广东地区汉族人群MBL基因点突变的频率较高，为防治MBL缺损病提供了一定的实验依据。     

RET基因功能性单核苷酸多态与先天性巨结肠风险

目的先天性巨结肠是一种肠神经系统发育异常导致的先天性消化道畸形。RET基因是其主要致病基因，本研究探讨RET启动子区的两个功能性单核苷酸多态-5G／A和-1A／C与先天性巨结肠遗传易感性的关系。方法以聚合酶链反应（PCR）和直接测序（direct-squencing）分析方法，检测了52例先天性巨结肠病人和120例正常对照者RET-5G／A和-1A／C的基因型。比较不同基因型与先天性巨结肠风险的相关性。结果RET-5AA和-1CC基因型频率在先天性巨结肠患者和正常对照中的分布有显著性差异（P值分别为〈0．001和0．003），携带RET-5AA和-1CC基因型者罹患先天性巨结肠的风险分别是携带RET-5GG和-1AA基因型者的11．40倍（95％CI，2．89-53．09）和4．65倍（95％CI，0．98-32．30）。此外，单倍型分析发现，同时携带两种风险等位基因的A-C单倍型者的患病风险比携带G-A型者增高了4．38倍（95％CI，2．53-11．90）。结论RET基因功能性单核苷酸多态-5G／A和-1A／C多态可能是中国人先天性巨结肠的遗传易感因素。     

DNA修复基因：核苷酸切除修复基因单核苷酸多态与癌症发生风险的研究进展

单核苷酸多态(single nucleotide polymorphism，SNP)是目前受到高度重视的全新一代遗传标记，将是今后基因组学研究的一大主要工具。DNA修复系统基因在维持基因组功能整体性，修复致癌因素所致的损伤及抗癌过程中有着重要作用。一些普通的及新的DNA修复基因单核苷酸多态与癌症发生风险的关联已经被验证或正在被验证，本文仅就核苷酸切除修复基因单核苷酸多态与癌症发生风险的研究进展作一综述。     

DNA修复基因:核苷酸切除修复基因单核苷酸多态与癌症发生风险的研究进展

单核苷酸多态(singIe nucleotide polymorphism,SNP)是目前受到高度重视的全新一代遗传标记,将是今后基因组学研究的一大主要工具。DNA修复系统基因在维持基因组功能整体性,修复致癌因素所致的损伤及抗癌过程中有着重要作用。一些普通的及新的DNA修复基因单核苷酸多态与癌症发生风险的关联已经被验证或正在被验证,本文仅就核苷酸切除修复基因单核苷酸多态与癌症发生风险的研究进展作一综述。     

单管双向等位基因专一性扩增的单核苷酸多态分型的新方法

目的：建立一种基于等位基因专一性PCR原理的单核苷酸多态（single nucleotidepolymorphism,SNP)分型新方法：单管双向等位基因专一性扩增（single-tube bi-directional allele specificamplification,SB－ASA）,并考察专一性引物的3’端第3位碱基不配对对特异延伸的影响。方法：一个PCR反应体系包含两个3’末端分别与SNP两个等位基因特异结合的引物,它们延伸方向相反,产生长度不同的等位基因专一扩增产物,同时在两个等位基因特异性引物的3’端第3位碱基引入不配对以增加特异性。PCR产物经琼脂糖凝胶电泳后分析确定样本的基因型。观察在不同的温度条件下,近3’末端引入与不引入碱基不配对时两种引物特异延伸的情况,比较两种引物能特异延伸的退火温度（annealingtemperature,Ta)范围。结果：对于4个不同类型的SNP位点,SB－ASA都成功地分型了36个样本,与直接测序的结果完全一致。两条专一性引物3’物第3位碱基引入不配对后,能特异延伸的退火温度Ta范围分别从64℃～69℃、60℃～62℃扩大到46℃～66℃、56℃～61℃。结论：SB－ASA是一种简单快速而有效的SNP分型新方法;在等位基因专一性PCR体系中,专一性引物3’端第3位碱基引入不配对能增加引物对两个等位基因的区分能力。     

TG相互作用因子基因编码区域的单核苷酸多态在中国高度近视人群与正常人群中的分布

目的 筛查TG相互作用因子(TG interacting factor，TGIF)基因编码区域变异与中国高度近视人群的相关性及单核苷酸多态(single nucleotide polymorphism，SNP)在中国人群中的分布特征。方法 采用聚合酶链反应-单链构象多态性检测204例中国人高度近视先证者及112名正常人的TGIF基因所有编码外显子及两侧序列有无突变；对有突变的外显子区域进行克隆测序。结果 在TGIF基因3个外显子编码区域及两侧序列发现3个SNP和1个单核苷酸突变，分别是IVS-2 nt350G→T(36／204)、密码子140CCA→CCG，Prol40Pro、密码子163CCG→CTG，Pro163Leu及密码子126GTG→GCG，Vall26Ala(1／204)；其中密码子140CCA→CCG、密码子163 CCG→CTG两个SNP在中国人群中形成3个等位基因和5种基因型，符合Hardy-Weinberg平衡定律。结论 TGIF基因编码区域的变异与中国高度近视人群无相关性；TGIF基因编码区域密码子140和163的SNP的基因频率和基因型频率在中国人群的分布达到了遗传平衡．     

GLI3基因与单纯性马蹄内翻足的关联分析及其突变筛查

目的对GLU基因与单纯性马蹄内翻足进行关联分析和突变筛查，探讨GLU基因与单纯性马蹄内翻足的相关性。方法应用限制性片段长度多态性分析技术，分析84个单纯性马蹄内翻足核心家系中GLI3基因内两个单核苷酸多态（single nucleotide polymorphisms, SNP）位点的基因型，并应用ETDT软伯统计分析各SNP位点基因型与单纯性马蹄内翻足的关联；应用变性梯度凝胶电泳技术对103例单纯性马蹄内翻足患者GLI3基因的第9至12外显子进行突变筛查。结果经ETDT分析，位于GLI3基因第4外显子的cSNP rs846266差异无统计学意义（χ^2=3．3582，P〉0．05）；第14外显子的cSNP rs929387差异有统计学意义（χ^2=7．2466，P〈0．05），在单纯性马蹄内翻足核心家系中存在传递不平衡；发现1例患者及其母亲的第9外显子有108（G→A）的同义点突变。结论GLI3基因与单纯性马蹄内翻足相关，其第9至12外显子可能并非该病的突变热点。     

共济失调毛细血管扩张症基因研究进展

共济失调毛细血管扩张症（Ａｔａｘｉａ－ｔｅｌａｎｇｉｅｃｔａｓｉａ，ＡＴ）是一种常染色体隐性遗传病，累及多个系统。１９９５年，ＡＴ的致病基因（ＡＴＭ）被克隆。该基因全长约１５０ｋｂ，编码序列１２ｋｂ，由６６个外显子组成。目前尚不清楚ＡＴ基因产物的确切功能，但由于其序列与多种蛋白质有同源性，因此推测其功能可能与细胞周期调控，ＤＮＡ修复，以及保持端粒长度等有关。目前发现ＡＴＭ与ＡＴ病人及ＡＴＭ携带者常见的淋巴瘤和白血病有明确的相关性。ＡＴＭ携带者的肿瘤发病率也明显高于常人，其中乳腺癌与ＡＴＭ的相关性相对最密切，但仍有待于进一步论证。     

血管紧张素原基因的六种单核苷酸多态与原发性高血压的相关性

目的　研究血管紧张素原 (angiotensinogen,AGT)基因 6个位点的单核苷酸多态及其构成的单倍型与中国汉族人原发性高血压的相关性。方法　采用多重SNa Pshot反应 ,在 185例原发性高血压患者和185名健康对照者中 ,对 AGT基因启动子区域的 G- 2 17A、G- 15 2 A、A- 2 0 C、G- 6 A及第 2外显子的T174 M和 M2 35 T多态进行基因分型。结果　 6种单核苷酸多态的基因型分布及其等位基因频率在原发性高血压组和对照组中差异无显著性 (P>0 .0 5 )。单倍型分析提示由 - 15 2 A,- 2 0 C,- 6 A和 2 35 T等位基因构成的 H4单倍型在原发性高血压组中明显增加 ,与对照组相比差异有显著性 (P<0 .0 5 )。结论　AGT基因G- 15 2 A,A- 2 0 C,G- 6 A和 M2 35 T多态可能对中国汉族人原发性高血压的发病起了重要作用。     

parkin基因的一个新的点突变

目的：研究parkin基因外显子2-10点突变与散发性早发帕金森病发病的关系。方法：应用聚合酶链反应（polymerase chain reaction ，PCR）、琼脂糖电泳、单链构象多态性（single strand conformation polymorphism，SSCP）、DNA测序及限制性核酸内切酶酶切方法，检测了60例散发性早发帕金森病患者以及120名正常人外周血白细胞DNA的parkin基因外显子2-10点突变。结果：发现1例患者的parkin基因外显子2存在纯合突变（G237→C），限制性内切酶酶切证实，其它外显子未见突变，120名正常对照也未见突变。结论：parkin基因外显子存在点突变，可能与部分散发性早发帕金森病发病有关。     

Single nucleotide polymorphisms of ataxia telangiectasia mutated and the risk of papillary thyroid carcinoma

Genetic factors associated with susceptibility to papillary thyroid carcinoma (PTC) are not well known. We evaluated the association between single nucleotide polymorphisms (SNPs) of ataxia telangiectasia mutated (ATM) and the risk of PTC. A total of 437 histologically confirmed PTC cases and 184 cancer‐free controls without thyroid nodules were recruited. Genotypes with respect to five ATM SNPs (rs189037, rs664677, rs373759, rs664143, and rs4585) were determined by the TaqMan assay, and odds ratios and 95% confidence intervals were obtained by logistic regression analysis. Linkage disequilibria and haplotypes were examined from the genotype data. When evaluated separately the genotype distributions of the five ATM SNPs were similar in the PTC cases and controls. Three ATM SNPs (rs373759, rs664143, and rs4585) were found to be in strong linkage disequilibrium (D′ = 1.00, P < 0.001). When the three haplotypes (C‐A‐G), (T‐G‐T), and (C‐G‐T) of these three ATM SNP sites were analyzed, ATM haplotype (C‐G‐T) +/? was associated with a lower risk of PTC than ATM haplotype (C‐G‐T) ?/? (P = 0.03) after adjusting for age and gender. Our results suggest that genetic polymorphisms of ATM may play an important role in the development of thyroid cancer in the Korean population. Environ. Mol. Mutagen. 56:70–76, 2015. © 2014 Wiley Periodicals, Inc.     

Immunodeficiency in ataxia telangiectasia is correlated strongly with the presence of two null mutations in the ataxia telangiectasia mutated gene

Immunodeficiency affects over half of all patients with ataxia telangiectasia (A-T) and when present can contribute significantly to morbidity and mortality. A retrospective review of clinical history, immunological findings, ataxia telangiectasia mutated (ATM) enzyme activity and ATM mutation type was conducted on 80 consecutive patients attending the National Clinic for Ataxia Telangiectasia, Nottingham, UK between 1994 and 2006. The aim was to characterize the immunodeficiency in A-T and determine its relationship to the ATM mutations present. Sixty-one patients had mutations resulting in complete loss of ATM kinase activity (group A) and 19 patients had leaky splice or missense mutations resulting in residual kinase activity (group B). There was a significantly higher proportion of patients with recurrent sinopulmonary infections in group A compared with group B (31 of 61 versus four of 19 P = 0.03) and a greater need for prophylactic antibiotics (30 of 61 versus one of 19 P = 0.001). Comparing group A with group B patients, 25 of 46 had undetectable/low immunoglobulin A (IgA) levels compared with none of 19; T cell lymphopenia was found in 28 of 56 compared with one of 18 and B cell lymphopenia in 35 of 55 compared with four of 18 patients (P = 0.00004, 0.001 and 0.003 respectively). Low IgG2 subclass levels and low levels of antibodies to pneumococcal polysaccharide were more common in group A than group B (16 of 27 versus one of 11 P = 0.01; 34/43 versus six of 17 P = 0.002) patients. Ig replacement therapy was required in 10 (12.5%) of the whole cohort, all in group A. In conclusion, A-T patients with no ATM kinase activity had a markedly more severe immunological phenotype than those expressing low levels of ATM activity.     

Hepatitis B virus X stimulates redox signaling through activation of ataxia telangiectasia mutated kinase

Hepatitis B virus X (HBX) protein plays a crucial role in carcinogenesis, but its mechanism is unclear. The involvement of ataxia telangiectasia mutated (ATM) kinase in the enhanced redox system was investigated by examining the phosphorylation level of ATM in HBX gene-transfected cells and in transgenic mice following redox system manipulation by treatment with hydrogen peroxide (H₂O₂) or antioxidant. Western blotting and immunostaining showed that phospho-ATM was significantly increased by HBX both in vitro (3.2-fold; p<0.05) and in vivo (4-fold; p<0.05), and this effect was abrogated by antioxidant treatment. The level of PKC-δ in HBX-expressing cells was increased 3.5-fold compared to controls. Nuclear localized NF-E2-related factor 2 (Nrf2) was increased in HBX-expressing cells exposed to H₂O₂, but remained at lower levels after the treatment with rottlerin, KU55933, or caffeine. The levels of anti-oxidant molecules were increased in HBX expressing cells and in transgenic mice, indicating that HBX stimulates the Nrf2-mediated redox system. The levels of intracellular reactive oxygen species (ROS) were significantly increased in HBX-expressing cells treated with hydrogen peroxide in the presence of ATM inhibitor KU55933 or caffeine. Treatment of HBX-expressing cells with KU55933 or caffeine before the exposure to H₂O₂ increased the ratio of cell apoptosis to 33 ± 4% (p<0.05) and 22 ± 4% (p<0.05), respectively. Collectively, HBX stimulates the ATM-mediated PKC-δ/Nrf2 pathway, and maintains the enhanced activity of the redox system. Therefore, manipulating ATM kinase activity might be a useful strategy for treating HBX-induced carcinogenesis.     

中国人共济失调毛细血管扩张症ATM基因突变研究

目的　探讨中国人共济失调毛细血管扩张症(ataxia- telangiectasia,AT) ATM基因的突变特点。方法　应用聚合酶链反应、逆转录-聚合酶链反应、聚丙烯酰胺凝胶电泳结合DNA序列分析方法对2例中国人临床诊断AT的患者ATM基因进行突变的筛选与检测。结果　在1例患者中发现第11外显子的1346 (G>C)的错义突变,为一种纯合突变;在另1例患者中发现第6外显子的6 10 (G>T)的无义突变和第4 7外显子的6 6 79(C>T)的错义突变,为一种复合性杂合突变。突变位点均位于ATM基因功能域。结论　在2例中国人AT患者中发现了3种新的ATM基因突变。     

组织型激肽释放酶基因多态性与长沙地区汉族人群脑出血的关联研究

目的 分析长沙地区汉族人群脑出血与组织型激肽释放酶(kallikrein 1,KLK1)基因多态性的关系.方法 收集273例散发性脑出血患者和140名正常对照者的外周血标本.采用多重单碱基延伸单核苷酸多态分型技术和DNA测序法检测KLK1基因rs5516及rs5517多态性位点在两组人群中的分布.结果 (1)脑出血组及对照组KLK1基因rs5516多态和等位基因频率分布差异无统计学意义(P>0.05);脑出血组组织型KLK1基因rs5517多态A等位基因频率显著高于对照组(P<0.05).(2)对照组rs5517多态AA及GA基因型携带者舒张压水平显著高于GG基因型携带者(P<0.05);而rs5516位点各基因型亚组间血压水平差异无统计学意义(P>0.05).结论 组织型激肽释放酶基因rs5516多态性与脑出血无关,而组织型激肽释放酶基因rs5517多态性与脑出血存在关联,可能通过影响血压水平而参与脑出血的发生发展.     

韩国人calpain-10基因单核苷酸多态性及其组合型的分析

目的分析发现于墨西哥裔美国人群中的2型糖尿病易感基因calpain-10单核苷酸多态性（single nucletide polymorphism。SNP）及其组合型在韩国人群中的分布。方法采用聚合酶链式反应．限制性片段长度多态性技术，分析312名健康韩国人calpain-10基因UCSNP-43、-19、-63位点的基因型及其组合型，计算基因型、等位基因和组合型频率。结果UCSNP-43位点基因型频率为1／1型的是86．2％、1／2型的是13．5％、2／2型的是0．3％，等位基因频率为G的是0．930、A0．070。UCSNP-19位点基因型频率为1／1型的是9．9％、1／2型的是44．6％、2／2型的是45．5％，等位基因频率为D的是0．322、I的是0．678。UCSNP-63位点基因型频率为1／1型的是57．4％、1／2型的是35．9％、2／2型的是6．7％，等位基因频率为C的是0．754、T的是0．246。各基因型分布匀符合Hardy-Weinberg平衡定律。韩国人中上述3个单核苷酸多态性基因型组合类型共见12种，75．6％由3种基因型组合构成。按UCSNP-43，-19，-63排列，分别为GG-DI-CC（单倍型组合111／121）、GG-DI-CT（112／121）、GC-II-CC（121／121），其频率分别为10．6％、28．8％、36．2％。结论韩国人calpain-10基因SNP分布与白人、美籍墨西哥人及美籍Pima印第安人等种族间存在较大差异，与中国和日本人群较为相近，其112／121单倍型组合频率显著高于美籍墨西哥人。     

Investigation of tRNALys/Leu and ATPase 6/8 gene mutations in Iranian ataxia telangiectasia patients

Introduction

Ataxia telangiectasia (AT) is a rare human neurodegenerative autosomal recessive multisystem disease. AT is the result of mutations in the AT-mutated (ATM) gene. ATM protein is required for radiation-induced apoptosis and acts before mitochondrial collapse. The tRNA genes are considered one of the hot spots for mutations causing mitochondrial disorders. Due to the important role of ATM in apoptosis and its effect on the cell cycle it might be possible that it has a central role in mtDNA mutations. On the other hand, the tRNA^Lys/Leu gene and also ATPase6 and ATPase8 genes are important for many mitochondrial diseases and many causative mutations have been reported from these genes.

Material and methods

In the present research, we performed mutation screening for these genes in 20 patients who were diagnosed with ataxia telangiectasia by a PCR sequencing method.

Results

The results showed a significant level of mtDNA variations in AT patients. Among 20 patients in this study, 12 patients (60%) were detected with point mutations, among which 8 mutations (40%) belonged to the MT-ATP6 gene. There was probably a second effect of mtDNA mutations in AT disease and mtDNA plays a main role in establishment of AT.

Conclusions

MtDNA mutations might be responsible for the decline of mitochondrial function in AT patients. Mitochondrial investigation can help to understand the mechanism of damage in AT disease.     

From gels to chips: “Minisequencing” primer extension for analysis of point mutations and single nucleotide polymorphisms

In the minisequencing primer extension reaction, a DNA polymerase is used specifically to extend a primer that anneals immediately adjacent to the nucleotide position to be analyzed with a single labeled nucleoside triphospate complementary to the nucleotide at the variant site. The reaction allows highly specific detection of point mutations and single nucleotide polymorphisms (SNPs). Because all SNPs can be analyzed with high specificty at the same reaction conditions, minisequencing is a promising reaction principle for multiplex high‐throughput genotyping assays. It is also a useful tool for accurate quantitative PCR‐based analysis. This review discusses the different approaches, ranging from traditional gel‐based formats to multiplex detection on microarrays that have been developed and applied to minisequencing assays. Hum Mutat 13:1–10, 1999. © 1999 Wiley‐Liss, Inc.     

Underexpression and abnormal localization of ATM products in ataxia telangiectasia patients bearing ATM missense mutations

Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immune defects and predisposition to malignancies. A-T is caused by biallelic inactivation of the ATM gene, in most cases by frameshift or nonsense mutations. More rarely, ATM missense mutations with unknown consequences on ATM function are found, making definitive diagnosis more challenging. In this study, a series of 15 missense mutations, including 11 not previously reported, were identified in 16 patients with clinical diagnosis of A-T belonging to 14 families and 1 patient with atypical clinical features. ATM function was evaluated in patient lymphoblastoid cell lines by measuring H2AX and KAP1 phosphorylation in response to ionizing radiation, confirming the A-T diagnosis for 16 cases. In accordance with previous studies, we showed that missense mutations associated with A-T often lead to ATM protein underexpression (15 out of 16 cases). In addition, we demonstrated that most missense mutations lead to an abnormal cytoplasmic localization of ATM, correlated with its decreased expression. This new finding highlights ATM mislocalization as a new mechanism of ATM dysfunction, which may lead to therapeutic strategies for missense mutation associated A-T.     

Sequenom质谱法分析细胞毒性T淋巴细胞相关分子4基因多态性与宫颈癌易感性的关系

目的 探索细胞毒性T淋巴细胞相关分子4(cytotoxic T-lymphocyte-associated protein 4,CTLA4)基因单核苷酸多态性(single nucleotide polymorphism,SNPs)与宫颈癌易感性的关系.方法 应用sequenom MassARRAY时间飞行质谱系统对100例宫颈癌及100名健康对照者 CTLA4基因20个多态位点(CTLA4_1～CTLA4_20)进行基因型分型,统计分析基因型频率和肿瘤易感性的关系.结果 与正常人群中最常见的 CTLA4基因单倍型-1576A、-318C和1402G相比,带有单倍型-1576G、-318T或1402A的个体均显著增加宫颈癌的风险(P<0.05),相对风险度的比值比及其95%可信区间分别为2.87(1.75～4.76),4.02(1.72～9.09)和4.51(1.46～13.88),而其余基因型与宫颈癌发病风险没有显著的相关性;其中rs5742909易感位点与先前的报道相一致.而双荧光素酶报告基因实验进一步证明,位于基因启动子区域的rs11571316多态位点能显著影响报告基因的表达活性.结论 CTLA4基因启动子区域的SNP可能通过影响 CTLA4基因的表达水平来影响个体对宫颈癌的易感性.

Abstract：

Objective To evaluate the association between single nucleotide polymorphisms (SNPs) of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene and susceptibility to cervical cancer. Methods One hundred patients and 100 healthy controls from Hubei province were genotyped for 20 polymorphic loci using Sequenom. Results The frequency of rs11571316 G allele and rs5742909 T allele, which are localized in the promoter region, and rs11571319 A allele, which is downstream of the gene, were significantly higher in patients than in controls. Luciferase assay showed that, as the previously reported rs5742909 T allele, rs11571316 G allele could significantly increase the expression of the reporter gene. Conclusion SNPs in the promoter region of CTLA4 gene might increase the susceptibility to cervical cancer by increasing CTLA4 gene expression.