粘多糖贮积症Ⅱ型患者艾杜糖-2-硫酸酯酶基因常见突变的检测

目的 探讨并建立粘多糖贮积症Ⅱ型（mucopolysaccharidosis Ⅱ,MPSⅡ)患者艾杜糖－2－硫酸酯酶（iduronate-2-sulphatase,IDS)基因常见突变的检测方法。方法 应用聚合酶链反应－单链构象多态性（polymerase chain reaction-single strand conformation polymorphism,PCR-SSCP）对IDS基因突变热点外显子3、8和9进行点突变检测；应用DNA测序对PCR－SSCP检出的突变进行序列分析；应用聚合酶链反应－限制性片段长度多态性（polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)对DNA测序的结果进行检测。结果 以PCR－SSCP发现该患者的IDS基因外显子9有明显异常泳动的条带；DNA测序发现患儿的外显子9发生点突变（C1672T），从而导致患者艾杜糖－2－硫酸酯酶蛋白发生氨基酸替换（R468W）；PCR－RFLP电泳检测结果显示粘多糖贮积症Ⅱ型患者仅出现554bp 1条带，而患儿父母出现257bp和297bp 2条带，进一步验证了序列分析的结果。结论 PCR－SSCP分析、DNA序列分析和PCR－RFLP分析是诊断MPSⅡ的有效方法，三者联合使用可以相互验证、互为补充，提高基因诊断的准确率和成功率。     

用RAPD技术分析2型糖尿病的遗传多态性

为探讨2型糖尿病和非糖尿病群体间的遗传多态性差异，寻找2型糖尿病的致病相关基因。本文应用随机扩增多态性DNA技术，从140个随机引物中筛选了25个扩增满意的引物，进行PCR扩增，研究2型糖尿病患者和非糖尿病者各10例。结果：1）5个RAPD图谱表现多态性改变；2）仅K－17的RAPD普的多态性在两组间存在显著性差异（P＜0.001)。表明K－17扩增的RAPD片段与糖尿病的遗传背景有关，可能是与2型糖尿病相关的RAPD标记。     

肌色素上皮源性因子基因与黑色素瘤的发生相关

目的：寻找与B16黑色素瘤发生相关的DNA序列或片段。方法：用105条引物对B16黑色素瘤及其来源小鼠C57BL／6J基因组DNA进行随机扩增多态DNA分析,比较肿瘤组织及其相应正常组织的DNA指纹,对差异很明显的片段收、克隆和测序,序列与GenBank数据库进行同源性分析。结果：105条引物中有24条引物扩增出的条带在肿瘤组织与其相应正常组织间存在差异。在6个差异很明显的回收DNA片段中引物AB8－5扩增后所得差异片段B8－5,肿瘤组织中此片段缺失,该片段序列长610bp,与GenBank序列数据库中鼠肌色素上皮源性因子基因具有99％（419／421）的同源性,与鼠肌色素上皮源性因子（PEDF）mRNA的同源性为100％（213／213）。可以认为该序列即为此基因,结论：黑色素瘤中存在PEDF基因缺失,提示PEDF基因与黑色素瘤发生相关。     

异常黑胆质成熟剂与清除剂对人Hela细胞凋亡基因表达的影响

目的：研究异常黑胆质成熟剂与清除剂两种复方作用前后,Hela细胞中凋亡基因p53,Fas及细胞增殖基因bcl-2的表达及它们之间的差异。方法应用体外细胞培养及RT－PCR对上述3中基因的表达及它们间的差异性进行分析,结果：用1g/L异常黑胆质成熟剂与清除剂处理24h,有大量p53基因表达,而Fas及bcl-2基因未见表达。结论,异常黑胆质成熟剂与清除剂可诱导野生型p53基因表达,这可能是它们清除体内恶变细胞及增强机体抗病功能的分生子生物学机制之一。     

白细胞介素1基因多态性与维吾尔族群体慢性牙周炎易感性的关系

目的：探讨白细胞介素1（interleukin-1，IL-1）基因多态性与新疆地区维吾尔族居民慢性牙周炎（chronic periodontitis，CP）易感性的关系。方法：收集维吾尔族41例重度CP患者、43例中度CP患者、49例轻度CP患者和92名健康对照者的颊粘膜拭子，提取DNA。采用序列特异引物聚合酶链反应（sequence specific primers-polymerase chain reaction，SSP-PCR）和聚合酶链反应-限制性片段长度多态性（polymerase chain reaction-retriction fragment length pohymorphism，PCR-RFLP）方法对其进行IL-1A-889/NcoI和IL-1B 3954/TaqI位点的基因型测定，分析各基因型的分布。结果：IL-1A-889/NcoI基因型在重度CP、中度CP、轻度CP和对照组之间的分布差异无显著性；IL-1B 3954/TaqI等位基因2在重度CP中的检出率显著高于对照组，而在中度CP、轻度CP与对照组之间的分布差异无显著性。结论：IL-1B 3954/TaqI等位基因2可能与新疆维吾尔族重度CP遗传易感性相关。     

PAI-1基因和纤维蛋白原β链基因多态性与狼疮性肾炎肾小球微血栓的关系

目的 探讨纤溶酶原活化剂抑制物－1（plasminogen activator inhibitor-1,PAI-1）基因和纤维蛋白原β链基因多态性与狼生肾炎（lupus nephritis,LN）肾小球微血栓形成之间的相关性。方法 选取101例LN患者，依据肾活检组织肾小球微栓的有无，将患者分为两组：LN伴血栓（LN＋T）组46例；不伴血栓(LN-T)且55例。应用聚合酶链反应－限制性片段长度多态性和聚合酶链反应－序列长度多态性技术分别分析两修选基因的基因型，正常对照组为128名健康成人。结果 （1）PAI－1基因4G／4G基因型和4G等位基因与LN＋T组显著相关；LN中4G／4G型患者发生肾小球Pan内血栓的相对风险率的比值比（odds ratio,OR）为2.96,95%可信区间（confidence interval,CI）：1.26-6.92；92）纤维蛋白原β链基因G／A＋A／A基因型和A型等位基因与LN＋T组显著相关；LN中A型等位基因携带者发生肾小球Pan内血栓的相对风险率为OR＝2.44，95％CI：0.98－5.59；（3）LN患者若同时兼有上述两的血栓易感基因型，其发生肾小球微血栓的相对风险率明显增加，OR＝4.5,95%CI:1.34-15.12；血栓易感基因型的混合病因分值（45.98%）也高于各自单独的病因分值（PAI－1基因4G／4G型为31.67%、纤维蛋白原β链基因G／A＋A／A基因型为28.23%）。结论 LN肾小球微血栓的形成与PAI－1基因和纤维蛋白原β基因多态性有关，两基因的易栓基因型在LN肾小球微血栓这一病理表型的形成上具有协同效应。     

汉族人白细胞介素-18基因启动子多态性与慢性乙型肝炎的相关性研究

目的 探讨中国汉族人白细胞介素-18（interleukin-18,IL-18）基因启动子单核苷酸多态性及其与慢性乙型肝炎易感性之间的关系。方法 应用序列特异性引物一聚合酶链反应技术，检测231例慢性乙型肝炎患者和300名正常人儿．馏基因启动子-607C/A、-137G／C单核苷酸多态性位点基因型。结果 正常对照组和慢性乙型肝炎组中，IL-18基因启动子-607C/A位点3种基因型频率分别为CC型：0．22（66／300）和0．27（62／231），CA型：0．53（160／300）和0．50（116／231），AA型：0．25（74／300）和0．23（53／231）；IL-18基因启动子-137G／C位点3种基因型频率分别为GG型：0．67（202／300）和0．79（182／231），GC型：0．30（90／300）和0．19（45／231），CC型：0．03（8／300）和0．02（4／231）。经Y0检验，慢性乙型肝炎组IL-18基因启动子-137GG分布频率显著高于正常对照组（X^2=8．55，P=0．003），而-607C／-137C和-607A／-137C单倍型频率显著低于正常对照组。进一步比较慢性乙型肝炎患者儿．馏基因启动子多态性与乙型肝炎病毒（hepatitis Bvirus，HBV）DNA复制的关系，发现高水平HBV—DNA组-607位点AA基因型分布频率明显低于低水平HBV—DNA组（Y2=6．03，P=0．014）。结论 汉族人慢性乙型肝炎与IL-18基因启动子-607C/A、-137G／C单核苷酸多态性相关，其中IL-18基因启动子-137位点C等位基因可能对机体HBV感染有保护作用，而启动子-607位点AA型对感染后HBV—DNA的复制可能有抑制作用。     

神经丝轻链基因在腓骨肌萎缩症中的突变分析

目的：探讨神经丝轻链基因（neurofilament-light gene,NF-L）在中国人腓骨肌萎缩症（Charcot-Marie-Tooth disease,CMT）中的突变特点。方法：应用聚合酶链反应－单链构象多态性技术结合DNA序列分析方法，对32个来自全国5省汉族的CMT家系先证者进行了NF－L基因的突变分析。结果：32例先证者中只有1例患者出现异常条带，经DNA测序证实该患者在NF－L基因的外显子3发生了1329C→T碱基改变，由于编码的氨基酸未改变，均为酪氨酸（Tyr），为一种同义突变。结论：NF－L基因突变可能在中国人的腓骨肌萎缩症患者中少见。     

载脂蛋白E基因多态性与新疆维吾尔族自然长寿的相关性研究

目的 探讨载脂蛋白E（apolipoproteinE，apoE）基因多态性与新疆维吾尔族自然长寿的关系。方法 应用聚合酶链反应-限制性片段长度多态性方法检测百岁组42名，90岁组102名，65～70岁组70名和对照组53名的apoE基因多态性。结果 百岁组apoE的ε3／3、ε2／3和ε3／4基因型频率分别为69．0％、23．8％和2．4％，其ε3、ε2和ε4等位基因频率分别为82．1％、16．7％和1．2％，百岁组ε3／4基因型及ε4、ε3等位基因频率显著低于对照组（P〈0．01），ε2／3基因型及ε2等位基因频率则显著高于对照组（P〈0．01）。百岁与opoE基因的ε2等位基因呈正关联，与ε4等位基因呈负关联。结论在新疆维吾尔族，opoE基因多态性与个体寿命密切相关，同时也应考虑到长寿是年龄依赖的多种因素影响的结果。     

正常人群E2F-4基因（AGC）n重复多态性研究

目的： 探讨正常人群E2F-4基因（AGC）n重复序列的多态性。方法: 收集100例健康查体者外周血样品，提取基因组DNA，针对E2F-4基因AGC 三核苷酸重复区段设计引物，PCR方法扩增后采用直接测序法对扩增产物测序分析。结果: E2F-4基因（AGC）n序列在正常人群中以（AGC）13最为多见，占91%，另外9例E2F-4基因出现5种情况的AGC重复数目变异，表现为13/14、13/15、13/16、13/17和13/18基因型。结论: E2F-4基因AGC重复序列在正常人群中呈现重复多态性，为进一步研究其病理学意义及与疾病发生关系奠定了分子遗传学基础。     

Mutation analysis of the KAL gene in female patients with gonadotropin-releasing hormone deficiency

Isolated gonadotropin-releasing hormone (GnRH) deficiency, including Kallmann's syndrome (KS) and idiopathic hypogonadotropic hypogonadism (IHH), is a congenital disorder, which is characterized by a functional deficit in hypothalamic GnRH secretion. Despite recent advances in the understanding of the pathogenesis of the X-linked form of KS as the identification of the KAL gene (Xp22.3), the genetic basis of the sporadic form in female patients remains unclear. Although most searches for mutations in X chromosome have been reported in males, the newly recognized phenomenon of inheritance, such as genomic imprinting and uniparental disomy, raises the possibility of a female phenotype in the X- linked genetic defect. Here, the molecular study of the coding region of the KAL gene (exon 5 to 14) in 10 unrelated females with KS (n=6) or IHH (n=4) is reported. None of the subjects had familial histories of delayed puberty or hypogonadism. Samples from 4 healthy, unrelated female volunteers were used for identification of polymorphisms. PCR of the 10 exons of the KAL gene was performed on genomic DNA. The PCR products of the 10 exons were subject to single strand conformation polymorphism (SSCP) analysis to identify possible mutations. In an SSCP analysis of the amplified fragments (fragment size: 147 to 302 bp), no mutations or polymorphisms were found in any of the 10 patients and 4 controls. In conclusion, it is unlikely that KAL gene mutations are a clinically significant cause of sporadic GnRH deficiency in female patients, indicating the existence of defects in unidentified genes that result in the expression of the phenotypes in females.     

Polymorphisms in the 5-lipoxygenase activating protein (ALOX5AP) gene are not associated with asthma in an Australian population

BACKGROUND: The cysteinyl-leukotrienes (cys-LTs) are important pro-inflammatory mediators in asthma, and have been shown to have a role in specific disease subtypes, including asthma severity. Few studies have investigated the role of polymorphisms in the ALOX5AP gene, encoding 5-lipoxygenase activating protein (FLAP), and asthma. We hypothesized that polymorphisms in this gene are associated with asthma and in particular, with asthma severity, in an Australian population. OBJECTIVE: To screen the coding region of the ALOX5AP gene for polymorphisms and to determine the association between previously described polymorphisms and asthma and asthma severity in an Australian population. METHODS: We used PCR-SSCP and PCR-RFLP analysis to examine a previously described promoter polyA variable repeat polymorphism and two intronic polymorphisms (IVS2+12C>A, IVS2+105T>C), and to screen all five exons of the gene for new polymorphisms, in a large Australian population of randomly selected, non-asthmatic controls (n=457), mild asthmatics (n=274), moderate asthmatics (n=231) and severe asthmatics (n=79). RESULTS: We confirmed the presence of two polymorphisms in intron 2 and found no association between these polymorphisms and asthma or asthma severity, nor between a promoter polymorphism in the ALOX5AP gene and asthma or asthma severity. Gene fragment analysis of the promoter polymorphism revealed novel, conserved repeat numbers in our population, and no new polymorphisms were found in the coding region of the gene. CONCLUSION: These findings in a large, well characterized asthma population, reveal that, while FLAP is an important enzyme in cys-LTs biosynthesis, polymorphisms in the ALOX5AP gene are not likely to be functionally associated with the asthma phenotype.     

新疆维、汉两民族健康人群KCNE1基因多态性分布

目的 研究心肌钾离子通道β亚单位基因(potassium voltage-gated channel,Isk-related family,member 1,KCNE1)标签SNP rs2834497和rs4817656在新疆维吾尔族和汉族健康人群中的分布情况。方法选择新疆地区409名维吾尔族和406名汉族,均为健康人群,采用等位基因特异性PCR(allele-spocific polymerase chain reaction,AS-PCR)方法进行基因分型。结果 ①rs2834497三种基因型AA型、AG型和GG型在维吾尔族人群中的分布频率分别为：65.5％、29.8％和4.6％;在汉族人群中的分布频率分别为：55.4％、36.9％和7.6％,两组基因型分布比较有统计学意义(x2= 9.92,P＜0.01);②rs4817656三种基因型CC型、CT型和TT型在维吾尔族人群中的分布频率分别为：26.4％、53.5％和20.0％;在汉族人群中的分布频率分别为：22.2％、49.0％和28.8％。两组基因型分布比较有统计学意义(x2=8.74,P＜0.05);③rs4817656和rs2834497共构建4个单体型,其中CG和TA单体型在维族中的分布明显高于汉族(x2= 37.83,P＜0.01;x2=4.13,P＜0.05),TG单体型在汉族中的分布明显高于维族(x2= 30.77,P＜0.01)。结论 KCNE1基因标签SNP rs2834497和rs4817656在新疆汉族和维吾尔族健康人群中的分布差异具有统计学意义。     

Major histocompatibility complex class I chain related (MIC) A gene, TNFa microsatellite alleles and TNFB alleles in juvenile idiopathic arthritis patients from Latvia

In order to analyze involvement of major histocompatibility complex class I chain-related gene A (MICA) and tumor necrosis factor a (TNFa) microsatellite polymorphisms as well as TNFB gene in juvenile idiopathic arthritis (JIA), we studied 128 patients divided into groups according to clinical features [monoarthritis (n = 14), oligoarthritis (n = 58), polyarthritis (n = 50), and systemic (n = 6)], and 114 age- and sex-matched healthy controls from Latvia. DNA samples were amplified with specific primers and used for genotyping of MICA and TNFa microsatellite. Typing for a biallelic NcoI polymerase chain reaction RFLP polymorphism located at the first intron of TNFB gene was done as follows: restriction digests generated fragments of 555bp and 185bp for TNFB*1 allele, and 740bp for TNFB*2 allele. The results were compared between cases and controls. We found significant increase of MICA allele A4 (p = 0.009; odds ratio [OR] = 2.3) and allele TNFa2 (p = 0.0001; OR = 4.4) in patients compared with controls. The frequency of allele TNFa9 was significantly decreased (p = 0.0001; OR = 0.1) in patients with JIA. No significant differences of TNFB allele frequency were found. Our data suggest that MICA and TNFa microsatellite polymorphisms may be used as markers for determination of susceptibility and protection from JIA.     

中国不同民族人群中PrP蛋白基因第129位氨基酸多态性分析

目的 研究人类PrP基因（PRNP）第129位氨基酸多态性在中国汉族及维吾尔族人群中的分布。方法 从抽样人群外周血中提取白细胞DNA，进行聚合酶链反应－限制性片段长度多态性分析（PCR－RFLP），使用SAS for Windows 6.12对结果进行分析。结果 83例汉族人群129 Met基因频率为97．0％，129 Val为3．0％；35例维吾尔族人群129 Met基因频率为91．4％，129 Val为8．6％。分别比较不同人群PRNP第129位氨基酸基因型的分布情况，结果显示汉族人群和维吾尔族人群（P＝0．0490）、汉族人群和高加索人群（P＝0．0005）、维吾尔族人群和高加索人群间（P＝0．0010）差异有显著性，而汉族人群与大和民族人群间差异无显著性（P＝0．5040）。结论 中国汉族人群PRNP第129位氨基酸基因型的分布与大和民族人群相似，但与维吾尔族人群有差异。     

新疆维、汉两民族健康人群KCNEl基因多态性分布

目的研究心肌钾离子通道B亚单位基因（potassiumvoltage．gatedchannel,Isk—relatedfamily,member1,KCNEl）标签SNPrs2834497和rs4817656在新疆维吾尔族和汉族健康人群中的分布情况。方法选择新疆地区409名维吾尔族和406名汉族,均为健康人群,采用等位基因特异性PCR（allele—specificpolymerasechainreaction,AS—PCR）方法进行基因分型。结果①rs2834497三种基因型AA型、AG型和GG型在维吾尔族人群中的分布频率分别为：65．5％、29．8％和4．6％;在汉族人群中的分布频率分别为：55．4％、36．9％和7．6％,两组基因型分布比较有统计学意义（χ^2=9．92,P〈0．01）;②rs4817656三种基因型cc型、cT型和TT型在维吾尔族人群中的分布频率分别为：26．4％、53．5％和20．0％;在汉族人群中的分布频率分别为：22．2％、49．0％和28．8％。两组基因型分布比较有统计学意义（χ^2=8．74,P〈0．05）;③rs4817656和rs2834497共构建4个单体型,其中CG和删单体型在维族中的分布明显高于汉族（χ^2=37．83,P〈0．01;,=4．13,P〈0．05）,％单体型在汉族中的分布明显高于维族（χ^2=30．77,P〈0．01）。结论KCNEl基因标签SNPrs2834497和rs4817656在新疆汉族和维吾尔族健康人群中的分布差异具有统计学意义。     

广西人群S100B基因多态性与系统性红斑狼疮的相关性研究

目的:探讨S100B基因rs9984765、rs2839356和rs2186358遗传多态性与系统性红斑狼疮(SLE)的相关性。方法:选取313例SLE患者作为病例组,年龄和性别匹配的396例正常人作为对照组,采用单碱基延伸PCR技术(SBE-PCR)和DNA测序法对S100B基因3个位点进行基因分型检测。结果:rs9984765和rs2186358位点的基因型和等位基因频率在SLE患者组和对照组间分布差异均无统计学显著性,而rs2839356位点的C等位基因在两组间比较差异有统计学意义(P=0. 040)。进一步分析rs2839356位点的等位基因与SLE患者临床表现的关系,发现rs2839356位点的C等位基因在伴有神经系统病变的SLE患者中高于不伴有神经系统病变的SLE患者(P=0. 023)。结论:在广西人群中,S100B基因rs9984765和rs2186358位点的基因多态性可能与SLE的遗传易感性无关,而携带rs2839356的C等位基因可能具有增加SLE及其并发神经系统病变的发病风险。     

Detection of microsporidia (Enterocytozoon bieneusi) in intestinal biopsy specimens from human immunodeficiency virus-infected patients by PCR.

Intestinal microsporidiosis has been implicated as a major cause of chronic diarrhea in human immunodeficiency virus (HIV)-infected patients. So far diagnosis depends on direct visualization of the parasites by light and transmission electron microscopy. We evaluated the diagnostic value of microsporidian DNA amplification by PCR on duodenal biopsy specimens obtained from patients with and without intestinal microsporidiosis caused by Enterocytozoon bieneusi. Thirteen HIV-infected patients (all CDC stage C3) were studied. Eight patients had intestinal microsporidiosis caused by E. bieneusi (n = 6), Septata intestinalis (n = 1), and Encephalitozoon cuniculi (n = 1); microsporidioses were diagnosed by light microscopy of stool samples and confirmed by light and electron microscopy of intestinal biopsy specimens. Five patients had no microsporidia in their stool samples or in their intestinal biopsy specimens, as examined by light and electron microscopy. Additionally, DNA prepared from Toxoplasma gondii derived from mouse ascites was used as a further control. A 353-bp DNA fragment of the small-subunit rRNA gene could be amplified from all six biopsy specimens infected with E. bieneusi, and the nature of the PCR products was confirmed by Southern blot hybridization. No amplification of DNA fragments was seen by using DNA extracted from biopsy specimens with S. intestinalis or E. cuniculi infection or without microsporidian infection and with template DNA extracted from T. gondii. The results suggest that PCR testing of intestinal biopsy specimens may be a useful approach to diagnosing microsporidiosis in HIV-infected patients.     

一个表皮松解性掌跖角化病维吾尔家系致病基因研究

目的 对一个维吾尔族表皮松解性掌跖角化病(epidermolytic palmoplantar keratoderma,EPPK)家系角蛋白9基因(keratin 9 gene,KRT9)进行测序,以检测其是否为该病的致病基因.方法 提取新疆地区一个维吾尔族EPPK家系外周血基因组DNA,针对已知候选基因KRT9和KRT1,分别对其所在染色体位置17q12-q21和12q13选取遗传标记进行连锁分析研究,确定连锁区域后,对区域内KRT9基因所有外显子进行测序分析.结果 分别得到48个家庭成员遗传标记的基因型和单倍型,经Linkage软件计算分析,发现标记D17S1787在=0时Lod值达到8.65,并最终将该病候选区域定位于遗传标记17/TG/36620115-D17S846之间约1 Mb范围内.排除该病与位于染色体12q13上的遗传标记DI2S96(θ=0时Lod=-∞)连锁.未发现KRT9基因存在致病性突变.结论 提示该表皮松解性掌跖角化病家系的致病基因位于染色体17q21.2上(chr17:36620083-37146934)约1 Mb区域内,且突变位点不位于KRT9基因编码区.