PBX1基因3''末端基因多态性rs3185695与系统性红斑狼疮发病相关研究

目的研究前B淋巴细胞白血病转录因子(PBX1)3’端单核苷酸多态性(SNP)与中国人系统性红斑狼疮(SLE)发病的相关性。方法对PBX1基因3’端下游区域通过测序发现SNP rs3185695, 再对发现的该SNP进行等位基因分型。以Haploview软件和延伸型传递不平衡试验(ETDT)分析SNP与中国人SLE发病的相关性。结果靠近PBX1 3’末端通过测序发现的SNP rs3185695进行家系传递不平衡检验(TDT)提示等位基因G优势传递给患者,传递:不传递(T:U)=62:41,P=0．0385。结论 PBX1基因可能为SLE的候选基因。     

系统性红斑狼疮患者16q12精细定位研究

目的预初研究提示16q12区与系统性红斑狼疮(SLE)存在关联,本研究旨在对该区域精细定位,以明确16号染色体与中国人SLE相关性。方法在16号染色体57.79~69.05cM(47218~52919kb)范围内选择8对微卫星标记(D16S540、D16S3044、D16S409、D16S517、D16S3136、D16S416、D16S419和D16S3034),扩增288个SLE患者家系DNA,产物经377DNA测序仪电泳,所得数据由Genescan软件收集进行基因分型,以ETDT及Genehunter进行连锁不平衡分析。结果D16S409及D16S517与SLE存在传递不平衡(P=0.0048和P<0.00001)。D16S517中,271bp等位基因优先传递给患病子代(P<0.00001),277bp等位基因则优先传递给正常子代(P<0.001)。结论研究结果证实人类SLE在16号染色体上存在疾病易感基因,中国人群16q12区(58.46cM,49Mb附近)与SLE发病相关联。     

1号染色体上高血压病易感基因的定位研究(大家系同胞对分析)

目的　定位和筛查高血压病 (EH)的易感基因位点。方法　应用染色体多点扫描策略和扩增片段长度多态性分析技术 ,在 1号染色体上选择 11个短串联重复序列 (shorttandemrepeat,STR) ,对一个典型的EH大家系进行同胞对连锁分析。结果　D1S165 6位点的统计量t值为 1.68,具有显著统计学意义 (t>1.64 )。统计每一位点在同胞组中共享最多的等位片段所占比例 ,观察到D1S165 6位点的 15 4bp等位片段与EH患者伴随频率最高 ,占 5 8.4 %。进一步以此片段作为特定因子进行传递不平衡分析 ,传递 15 4bp等位基因的频率显著高于期望值 (χ2td=6.0 0 ,P <0 .0 5 ) ,表明15 4bp片段在传递过程中存在显著连锁不平衡。结论　 1号染色体上的一个遗传标记 (D1S165 6)与EH连锁 ,提示该位点的附近可能存在EH的易感基因 ,这一结果为EH易感基因的进一步定位提供了重要的资料。     

肿瘤坏死因子-β基因多态与系统性红斑狼疮关系研究

目的探讨肿瘤坏死因子(TNF)-β的基因多态与华东地区汉族人系统性红斑狼疮(SLE)易感性的关系及其在SLE家系中的传递规律.方法运用聚合酶链式反应-限制性片段长度多态(PCR-RFLP)对99例SLE患者、116名正常人及12个SLE家系的48名成员进行了TNF-β基因分型.结果 SLE患者的TNF-β2等位基因频率高于对照组(62.6%/47.0%,OR＝1.89,P＝0.001 2),但可能样本量过小,在SLE家系研究中未发现TNF-β基因多态存在关联的证据以及传递/连锁不平衡的证据.结论 TNF-β2等位基因可能是SLE的易感等位基因.     

肿瘤坏死因子—β基因多态与系统性红斑狼疮关节研究

目的：探讨肿瘤坏死因子（TNF）－β的基因多态与华东地区汉族人系统性红斑狼疮（SLE）易感性的关系及其在SLE家系中的传递规律。方法：运用聚合酶链式反应－限制性片段长度多态（PCR－RFLP）对99例SLE患者，116名正常人及12个SLE家系的48名成员进行了TNF－β基因分型。结果：SLE患者的TNF－β＊2等位基因频率高于对照组（62．2％／47．0，OR＝1．89，P＝0．0012），但可能样本量过小，在SLE家系研究中未发现TNF－β基因多态存在关联的证据以及传递／连锁不平衡的证据，结论：TNF－β＊2等位基因可能是SLE的易感等位基因。     

系统性红斑狼疮候选基因OAZ中单核苷酸多态性的系统筛选及相关分析

目的研究OLFl／EBF相关锌指蛋白基因（OAZ）上的单核苷酸多态性（SNP）与中国人群系统性红斑狼疮（SLE）发病的相关性。方法利用243个中国SLE患者核心家系DNA．依据http：／／www．hapmap．org/数据库中的信息，选取了OAZ基因上的35个SNP位点进行等位基因分型，以Haploview、Genehunter和Fbat生物信息学软件分析其与中国人SLE发病的相关性。结果对等位基因分型结果进行家系传递不平衡检验（TDT），提示rsl420683，rs6500240显示传递不平衡，rsl420683等位基因G优势传递给患者，传递：不传递=86：50，P=0．002；rs6500240等位基因C优势传递给患者，传递：不传递=88：56，P=0．008。结论OAZ基因上的SNP位点rs1420683和rs6500240与SLE发病相关．未发现这两个位点的突变影响转录因子的结合，并且相互之间并没有存在强的连锁关系，可能与邻近的某个影响蛋白结构或表达的功能性SNP存在连锁不平衡，需要进一步研究证实。     

山东省原发性高血压1号染色体基因扫描

目的 对原发性高血压患者及正常对照者的1号染色体进行扫描,查找与原发性高血压关联的遗传位点.方法 在1号染色体上间隔10 cM遗传距离选择31个微卫星遗传位点,用DNA混合池的方法 对原发性高血压患者450例和正常对照者450例的DNA样本进行基因扫描.采用CLUMP软件对患者组和对照组每个位点的等位基因频率进行比较.结果 在D1S196位点(1q24.2)、D1S249位点(1q32)和D1S2667位点(1p36.22)发现患者组与对照组的等位基因频率存在显著性差异(P＜0.01).结论 山东省原发性高血压患者在1号染色体的D1S196、D1S249与D1S2667位点附近可能存在原发性高血压易感基因,需要进行候选基因突变筛查.     

12个水盐代谢、离子转运候选基因与高血压病连锁分析

目的研究12个水盐代谢、离子转运调节基因与高血压病（EH）的关系，以筛选EH易感基因。方法在每一候选基因的染色体区域附近，选择微卫星DNA多态性位点作为遗传标记，采用微卫星引物荧光标记-基因扫描及分型技术，对95个EH核心家系的477个成员进行微卫星位点与EH连锁分析。遗传统计采用Genehunter软件包中两点非参数连锁（NPL）分析、最大LOD值及传递不平衡检验（TDT）。结果NPL检验显示，D12S398的Z=2.08,P＜0.05；LOD值=1.26,P＜0.01；TDT分析χ2=9.00,P＜0.005。其余位点NPL分析及TDT的P值均>0.05,LOD值<1。结论采用三种不同的遗传统计方法提示D12S398位点与EH存在连锁，并且在D12S398附近有血管加压素1A受体基因,该区域值得进一步研究。     

家族性混合型高脂血症与脂蛋白脂酶基因的连锁分析

目的 探讨脂蛋白脂酶基因与家族性混合型高脂血症是否连锁，以期发现家族性混合型高脂血症遗传易感位点。方法 从北京地区搜集12个（81人）家族性混合型高脂血症家系选择脂蛋白脂酶基因及其附近的微卫星遗传标记（LPLGZ14／15与D8S282）进行连锁分析。结果 CENEHUNTER软件包多点连锁分析显示微卫星遗传标记最大LOD score(HLOD)值如下：HLODLPLGZ14／15＝－8．9及HLODD8S282＝－10．5。结论 中国北京地区家族性混合型高脂血症家系提示，脂蛋白脂酶基因不是影响家族性混合型高脂血症表型的遗传易感基因。     

中国汉族一个家系家族性颅内动脉瘤的候选基因位点连锁分析

目的对一个汉族家族性颅内动脉瘤(FIA)家系进行遗传连锁定位及基因突变分析。方法纳入19名家系成员,其中2例经DSA确诊为颅内动脉瘤。应用16个短串联重复序列微卫星标记物和3个单核苷酸多态性(SNP)标记物对19名成员进行了18个与FIA发病相关的候选基因/位点的参数连锁分析,并对其中的1个SNP标记物rs1333040进行直接测序,筛查是否存在突变。结果①ANIB1-8等候选基因位点与该家系不连锁,连锁值(LOD)均<1。②曾报道过的13q14.12-21.1上rs7983420和rs17054625之间的7-cM区域、17cen上的TNFRSF13B候选基因位点与该家系不连锁。③9p21上的rs1333040-T等位基因多态性与该FIA家系发病无关。结论排除ANIB1-8、7-cM、TNFRSF13B候选基因/位点与该家系的关系。     

A rapid one-stage whole-blood HPA-1a phenotyping assay using a recombinant monoclonal IgG1 anti-HPA-1a

Severe neonatal alloimmune thrombocytopenia and patients with HPA-1a-specific antibodies require transfusion of HPA-1a-negative platelets. Identifying HPA-1a-negative donors requires simple and reliable typing methods. Most existing techniques use polyclonal antibodies, are time consuming and involve platelet isolation. We have used a horseradish peroxidase (HRP)-conjugated recombinant IgG1 anti-HPA-1a (CAMTRAN007) to develop a rapid and reliable enzyme-linked immunosorbent assay (ELISA), which eliminates sample preparation and reduces the incubation and wash steps associated with traditional sandwich ELISAs. The assay uses simultaneous incubation of the monoclonal antibody RFGP56 to capture GPIIbIIIa from whole blood and the recombinant IgG1 antibody to detect captured HPA-1a antigen. It allows 96 samples to be typed in less than 1 h and can be used on stored samples. Initial testing of 85 samples of known HPA-1a genotype demonstrated that HPA-1a-negative samples had OD values of < 0.266, whereas HPA-1a-positive samples had OD values of > 0.6. Testing of 1862 random donor samples in two blood centres confirmed these OD cut-off values and identified 45 HPA-1a-negative samples (2.4%), all except one giving OD values of < 0.2. The remaining HPA-1a-negative sample had an OD value of 0.303. The HPA-1a status on all the negative samples and an equivalent number of randomly selected positive samples was confirmed by flow cytometry and polymerase chain reaction with sequence-specific primers (PCR- SSP).     

Allo-anti-P1 in a P1-Positive Person

An allo-anti-P1 antibody is reported in an elderly Caucasian male with P1+ red cells. There was no suggestion that the antibody was causing haemolysis. The case suggests that a slight structural variation may exist in the patient's B antigen giving a serological false-positive response when typed with reagent anti-P1, or that the patient's anti-P1 is directed against a determinant absent in the patient's own antigen structure.     

Acid-sensing ion channel (ASIC) 1a/2a heteromers have a flexible 2:1/1:2 stoichiometry

Acid-sensing ion channels (ASICs) are widely expressed proton-gated Na⁺ channels playing a role in tissue acidosis and pain. A trimeric composition of ASICs has been suggested by crystallization. Upon coexpression of ASIC1a and ASIC2a in Xenopus oocytes, we observed the formation of heteromers and their coexistence with homomers by electrophysiology, but could not determine whether heteromeric complexes have a fixed subunit stoichiometry or whether certain stoichiometries are preferred over others. We therefore imaged ASICs labeled with green and red fluorescent proteins on a single-molecule level, counted bleaching steps from GFP and colocalized them with red tandem tetrameric mCherry for many individual complexes. Combinatorial analysis suggests a model of random mixing of ASIC1a and ASIC2a subunits to yield both 2:1 and 1:2 ASIC1a:ASIC2a heteromers together with ASIC1a and ASIC2a homomers.Acid-sensing ion channels (ASICs) are proton-gated Na⁺ channels, which are probably ubiquitously expressed in neurons, yet surprisingly little is known about their physiological function in the brain (1). It is believed that ASICs localize to the postsynapse and carry an excitatory postsynaptic current (2). ASICs in central neurons are mainly composed of homomeric ASIC1a and heteromeric ASIC1a/2a (3–6) and ASIC1a/2b (7). Genetic ablation of ASIC1a has indeed led to the conclusion that ASICs contribute to long-term potentiation and memory formation (2), but a more recent study challenged these findings (8). In contrast to our incomplete understanding of the physiological functions of ASICs, there is a comparatively large body of evidence that activation of ASIC1a-containing channels in pathophysiological states that are associated with sustained acidosis contributes to neuronal or axonal degeneration (9, 10). In addition, blockade of the ASIC1a/2a heteromer causes central analgesia (11). Together, these findings render ASIC1a-containing channels attractive drug target candidates.The crystal structure of chicken ASIC1 revealed an acidic pocket at subunit interfaces, which has been proposed to be the ligand-binding site of ASICs (12). In agreement, it has recently been shown that a gating modifier toxin of ASIC1, psalmotoxin 1, which behaves like an agonist (13), binds at the acidic pocket at subunit interfaces (14–16). Therefore, knowing the subunit composition of the ASIC1a/2a heteromer is of major interest, in particular for pharmacologically targeting this subtype.Fluorescence resonance energy transfer analysis suggested that the ASIC1a/2a heteromer contains at least two ASIC1a and two ASIC2a subunits (17). In agreement, several studies reported that the related epithelial Na⁺ channel (ENaC) is composed of four (18–21) or nine subunits (22–24). In strong contrast, however, the crystal structure of chicken ASIC1 revealed a number of three subunits (12, 25), suggesting that all ASICs and their relatives like ENaC are trimers. The trimeric structure of ASIC1a has in the meanwhile been confirmed by atomic force microscopy imaging (26). The stoichiometry of heteromeric ASICs, however, remains completely unknown.In this study, we first used electrophysiology to characterize mixtures of ASIC1a and ASIC2a at different expression ratios in Xenopus laevis oocytes to demonstrate that at least one heteromeric channel forms. Because we were unable to decide on the existence of a second heteromeric species by electrophysiology, we used a single-molecule photobleaching approach that resolves stoichiometries of membrane proteins with high accuracy. We tagged ASIC1a and ASIC2a with green and red fluorescent reporter proteins, which did not change the electrophysiological characteristics of homo- and heteromeric ASICs, suggesting that fusion of a fluorescent reporter has no impact on the molecular composition of ASICs. Colocalization of red and green reporter tags on a single-molecule level and counting of green bleaching steps confirms the trimeric nature of functional ASICs and shows that ASIC1a and ASIC2a randomly assemble into a complex with a flexible stoichiometry of either 1:2 or 2:1.     

Immunocytochemical demonstration of a1-Antitrypsin and a1-Antichymotrypsin in human gastrointestinal tract

a1-Antitrypsin and a1-Antichymotrypsin were demonstrated by the peroxidase--antiperoxidase (PAP) technique in paraffin sections from various parts of the normal adult gastrointestinal tract. Thus, positive reaction for these two proteinase inhibitors was given by the absorptive epithelial cells of the small intestine and epithelial cells of Brünner's duodenal glands and the glands of the pyloric antrum. Our findings suggest that the presence of a1-Antitrypsin and a1-Antichymotrypsin in epithelial cells of the small intestine, may represent a self-protecting mechanism against the pancreatic proteolytic enzymes of the intestinal juices. The significance of our observation and its possible role in the previously reported a1AT deficiency associated with coeliac-like disease is also discussed.     

H1oo: a pre-embryonic H1 linker histone in search of a function

The mouse oocyte-specific linker histone H1oo (1) constitutes a novel mammalian homologue of the oocyte-specific linker histone B4 of the frog and of the cs-H1 linker histone of the sea urchin; (2) is expressed as early as the germinal vesicle (PI) stage oocyte, persisting into the MII stage oocyte, the oocytic polar bodies, and the 2-cell embryo, extinction becoming apparent at the 4-8 cell embryonic stage; and (3) may play a key role in the control of gene expression during oogenesis and early embryogenesis, presumably through the perturbation of chromatin structure.     

Physiological properties of hERG 1a/1b heteromeric currents and a hERG 1b-specific mutation associated with Long-QT syndrome

Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long-QT syndrome (LQTS). Biochemical and functional studies have demonstrated that I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homooligomers of the original hERG 1a isolate. Here, we examine currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We find that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a 2-fold increase in the apparent rates of activation and recovery from inactivation, thus reducing rectification and facilitating current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the 2 channel types. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding 4-fold shift in the IC50. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients that was absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells dramatically reduced 1b protein levels. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr and enhance drug sensitivity, and represent a potential mechanism underlying inherited or acquired LQTS.     

一例Rotor综合征SLCO1B1和SLCO1B3基因突变分析

目的初步探讨1例Rotor综合征患者的临床特点和SLCO1B1和SLCO1B3基因突变情况,从分子遗传学角度分析该疾病的发生机制。方法收集患者临床资料,从外周血白细胞提取基因组DNA,采用二代测序进行四千种已知单基因遗传性疾病筛查,用Sanger测序法分析验证二代测序发现的突变位点。结果患儿主要临床表现为反复皮肤及巩膜黄染,实验室检查示高胆红素血症,直接、间接胆红素双向增高。高通量测序发现患儿携带SLCO1B1基因c.1738 CT纯合突变和SLCO1B3基因c.360_481 del纯合突变。c.1738 CT突变为无义突变,已有文献报道,推测导致蛋白的第580位氨基酸密码子由精氨酸变为终止密码子。c.360_481 del突变为移码突变,蛋白编码区的第360至481位碱基缺失。该变异未见文献报道,也未见SNP数据库收录。此变异使蛋白缺失40个氨基酸的同时还造成开放阅读框移码,可能造成蛋白功能丧失。结论 SLCO1B1和SLCO1B3基因突变导致的有机阴离子转运多肽OATP1B1和OATP1B3功能缺陷是该Rotor综合征患者临床表现的分子遗传基础。     

Protection of telomeres by a conserved Stn1-Ten1 complex

Telomeres are specialized chromatin structures that protect chromosome ends. Critical among telomere proteins are those that bind the telomeric single-strand DNA (ssDNA) overhangs. These proteins are thought to differ among eukaryotes. Three interacting proteins (Cdc13, Stn1, and Ten1) associate with the telomeric overhang in budding yeast, a single protein known as Pot1 (protection of telomeres-1) performs this function in fission yeast, and a two-subunit complex consisting of POT1 and TPP1 associates with telomeric ssDNA in humans. Cdc13 and Pot1 have related oligonucleotide/oligosaccharide-binding fold (OB-fold) domains that bind the telomeric ssDNA overhang. Here we show that Schizosaccharomyces pombe has Stn1- and Ten1-like proteins that are essential for chromosome end protection. Stn1 orthologs exist in all species that have Pot1, whereas Ten1-like proteins can be found in all fungi. Fission yeast Stn1 and Ten1 localize at telomeres in a manner that correlates with the length of the ssDNA overhang, suggesting that they specifically associate with the telomeric ssDNA. Unlike in budding yeast, in which Cdc13, Stn1, and Ten1 all interact, fission yeast Stn1 and Ten1 associate with each other, but not with Pot1. Our findings suggest that two separate protein complexes are required for chromosome end protection in fission yeast. Structural profiling studies detect OB-fold domains in Stn1 and Ten1 orthologs, indicating that protection of telomeres by multiple proteins with OB-fold domains is conserved in eukaryotic evolution.