SLC22A12基因第8外显子和第8内含子多态与中国汉族人原发性高尿酸血症关联研究

目的 研究原发性高尿酸血症患者SLC22AI2基因第8内含子和第8外显子单核苷酸多态(single nucleotide polymorphism,SNP)位点与原发性高尿酸血症遗传易感性的关系.方法 选择山东沿海地区原发性高尿酸血症患者215例,正常对照人群323名.提取基因组DNA,PCR扩增SLC22A12基因第8内含子和第8外显子,对PCR扩增产物进行测序.结果 序列分析发现:(1)SLC22AI2基因第8外显子存在T1309C单核苷酸多态,第8内含子存在-103A＞G单核苷酸多态,这2个多态位点完全连锁.(2)高尿酸血症组-103A＞G G等位基因频率和T1309C C等位基因频率明显高于正常对照组(均为51.9%vs.42.4%,P＜0.01);(3)高尿酸血症组GG+GA基因型频率和CC+CT基因型频率显著高于正常对照组(均为80.0%vs.69.0%,P＜0.01).(4)-103 A＞G和T1309C基因多态中,含有等位基因G的基因型GG+GA及含有等位基因C的基因型CC+CT均使高尿酸血症的发病危险性上升了1.79倍(OR=1.79,95%CI:1.19～2.70).结论 SLC22A12基因第8外显子T130gC及第8内含子-103A＞G SNP与原发性高尿酸血症密切相关.     

葡萄糖醛酸转移酶1F基因多态性及与肝癌易感性研究

目的 探讨葡萄糖醛酸转移酶1F（UDP－glucuronosyltransferase 1F,UGT1F）基因的多态性及其与肝癌的关系。方法 应用聚合酶链反应、变性梯度凝胶电泳、单链构象多态和DNA测序技术，对84例肝癌患者和144名健康对照的UGT1F基因的多态性进行了研究。结果 在第1外显子和第2内含子发现3个新的单核苷酸多态。即第232位核苷酸T→G颠换，第528位核苷酸A→G转换和第376位核苷酸A→G转移。另外，754位点多态在我们的研究中得到了证实。分析病例组和对照组4个位点等位基因及基因型频率的分布，病例组UGT1F754位点G／G基因型频率（13.10%）和G等位基因频率（29.17%）均显著高于对照组（2.78%和19.44%）。其它位点，两组差异无显著性。结论 UGT1F基因的第2－5外显子高度保守，而第1外显子则呈高度多态。其754位点多态可能与肝癌有关。     

p53基因第8外显子在喉癌中的缺失和点突变

目的 探讨抑癌基因p53第8外显子突变在喉鳞癌分子发病机理中所起的作用。方法 应用聚合酶链反应－单链构象多态性（PCR－SSCP）银染技术和DNA直接测序法,检测喉鳞癌新鲜组织中抑癌基因p53基因第8外显子的突变情况。结果 60例喉鳞癌组织中,15例发生迁移率异常的SSCP区带。在SSCP阳性样本中随机抽出4例进行测序分析,发现两个标本在288位密码子缺失一个A,两个标本在292位密码子上缺失一个A。285、287密码子上共发生3次G→T的颠换,286密码子第3位碱基发生A→T的颠换。结论 喉鳞癌p53基因点突变与碱基缺失常常同时并存。p53基因第8外显子突变在喉癌的发生中可能起着重要作用。     

一种ABx变异型相关的B糖基转移酶基因新突变研究

目的 研究1例ABO血型系统ABx变异型的分子遗传背景.方法 用血型血清学技术鉴定1例ABO血型疑难样本的红细胞表型和唾液血型分泌物质,并用聚合酶链反应分别扩增先证者ABO基因全编码区共7个外显子及侧翼内含子序列,扩增产物经双酶切纯化后直接进行双向测序分析.进一步对存在突变位点的第6～7外显子扩增产物经进行TA克隆和单倍体序列分析.结果 先证者红细胞ABO抗原表达为A强B弱,结合其它血清学特征,鉴定其血型为罕见的ABx变异型.ABO基因全编码区测序发现9处核苷酸杂合位点,分别为第6外显子的297A/G杂合,第7外显子的467C/T、526C/G、657C/T、703G/A、796C/A、803G/C、808T/A、930G/A杂合.单倍体序列分析发现,其中一个等位基因为常见的A102,另一个等位基因除808T＞A突变外,其它变异位点与B101等位基因一致.808T＞A突变可导致B糖基转移酶第270位苯丙氨酸转变为异亮氨酸.结论 B糖基转移酶基因808T＞A突变可能引起酶活性减弱,并进而导致产生Bx变异型.     

视网膜色素变性隐性遗传致病基因PDE6B的突变分析

目的 了解常染色体隐性遗传视网膜色素变性(autosomal recessive retinitis pigmentosa，ARRP)致病基因磷酸二酯酶β亚单位(phosphodiesterase β subunit,PDE6B)基因在中国视网膜色素变性(retinitis pigmentosa，RP)患者中的突变谱及突变率。方法 应用聚合酶链反应—单链构象多态性，对收集的35个常染色体隐性RP家系38例患者和55例散发RP患者进行PDE6B基因的22个外显子和5’端非翻译区突变筛选；对有变异条带者进行DNA序列分析。结果 测得一个常染色体隐性家系患者PDE6B基因第11外显子5’端上游第19位碱基(第10内含子内)发生G→A转换。1例散发RP患者同时检测到第6外显子第2492位点碱基T颠换为C和第10外显子5’端上游(第9内含子内)第27—28碱基之间有两个碱基TG插入。另两例散发RP患者分别发现第4外显子5’端上游30—31碱基处两个碱基GT插入和第18外显子3’端下游第15个碱基发生G→C颠换。结论 发现1名中国人的散发RP患者携带RP致病基因PDE6B基因的一种复合杂合突变。中国人的PDE6B基因内含子有多种变异。     

一个先天性白内障家系缝隙连接蛋白基因新突变

目的 鉴定一个中国常染色体显性遗传先天性白内障(autosomal dominant congenital cataract,ADCC)家系其致病与缝隙连接蛋白a3/a8(gap junction protein alpha3/alpha8,GJA3/GJA8)基因突变的关系.方法 对一个ADCC家系5名家系成员和100名正常健康人进行全面眼科检查.抽取外周血5 mL并提取基因组DNA.采用聚合酶链反应扩增GJA3/CJA8编码外显子及其侧翼内含子序列,纯化的PCR产物通过直接测序以筛查致病突变.结果 通过双向序列分析,发现GJA8基因第2外显子的第138位发生碱基G→A转换(c.138G》A,GGG→GGA),产生同义突变(G46G);第139个碱基因G→T颠换发生错义突变(c.139 G》T,GAT→TAT)导致第47位编码天门冬氨酸密码子突变为酪氨酸(D47Y),家系中非患者和100名对照者基因组序列均无此改变,而且生物信息检索显示GJAB基因第47位编码的天门冬氨酸具有高度的物种保守性.家系3例患者CJA3基因突变筛查未见任何碱基改变.结论 在一个中国人ADCC家系中发现GJAB基因新致病突变(D47Y).     

FKBP6基因278C/A单核苷酸多态性与原发无精症相关研究

目的对FKBP6基因第3、4外显子进行突变和多态性筛查,研究第3外显子278C/A位点及第2内含子C/T位点(rs7797242)在无精症患者和正常男性中的多态性,初步探讨与原发无精症的相关性。方法采用变性高效液相色谱和聚合酶链反应-限制性片段长度多态性方法,对第3、4外显子进行突变和多态性筛查,对177例无精症患者和231名正常男性的278C/A和C/T(rs7797242)多态性进行基因分型。结果278C和278A等位基因频率符合Hardy-Weinberg平衡。无精症患者278A显著低于正常对照,差异有统计学意义(P<0.05)。C/T多态性在两组中均未检出,第3、4外显子未筛查到新的变异。结论278A等位基因可能与原发无精症相关。C/T(rs7797242)及370G/A,430G/C,467T/C,468G/A在中国人群中非常罕见。     

山西汉族遗传性多发性骨软骨瘤家系EXT1及EXT2基因突变研究

目的 对山西一个汉族遗传性多发性骨软骨瘤家系的EXT1和EXT2基因的全部外显子序列进行分析,以寻找致病突变.方法 用PCR扩增先证者EXT1和EXT2基因的全部外显子,将PCR产物送直接测序分析.结果 发现EXT1基因2种同义突变(P477P、E587E)、3种内含子突变(c.1537-48A＞G、c.1721 +203 A＞G、c.1722-103 C＞G).EXT2基因共发现5种内含子突变(c.-29-148 A＞T、c.1080-18 T＞A、c.1336-93 C＞T、c.1526-166 C＞T、c.1526-195C＞T).其中,EXT1 P477P、EXT1 E587E和EXT2 c.1080-18 T＞A为多发性骨软骨瘤突变数据库已收录的多态位点,其余7个位点尚未见报道.结论 对该家系EXT1、EXT2基因全部外显子的测序分析未发现明确的致病突变,该家系遗传性多发性骨软骨瘤的发生是否由除EXT1、EXT2外的其它EXT相关基因引起尚需进一步的连锁定位分析.     

非缺失/重复型Duchenne肌营养不良症患者的致病点突变分析

目的检测非缺失／重复突变型Duchenne肌营养不良症（Duchenne muscular dystrophy，DMD）的致病点突变。方法对6个家系的6个无关DMD男性患者的DMD基因的79个外显子及5′-3′-非翻译序列进行PER扩增，产物通过变性高效液相色谱（denaturing high performance liquid chromatography，DHPLC）技术进行突变筛查。结果6例非缺失／重复突变型Duchenne肌营养不良症患者，检测出了5例患者的致病点突变，即697-698insGT，C616T，G1255T，C4279T和C2302T。第1个点突变引起移码突变，后4个致病点突变引起翻译的提前终止，最终导致Duchenne肌营养不良症。患者3除致病点突变外，在第39内含子还发现1个T5586＋61A点突变；患者5还检测出了一个位于第8外显子的错义突变；而没有检出致病点突变的患者6，发现了2个外显子突变及2个内含子序列点突变，即C2168＋13T、G5234A、C5280T和5740-13dupG。所有检出的突变有7个点突变未见报道。结论变性高效液相色谱技术结合测序，可用于检测DMD患者的点突变．该方法具有准确，灵敏的特点。     

Hallervorden-Spatz综合征 PANK2基因的突变

目的探讨泛酸激酶2(pantothenate kinase 2,PANK2)基因突变与中国人Hallervorden-Spatz综合征(Hallervorden-Spatz syndrome,HSS)的关系.方法应用聚合酶链反应、DNA直接测序、PCR产物限制性内切酶酶切和聚合酶链反应-单链构象多态性等技术检测5例HSS患者、3名家系成员及51名正常人 PANK2基因的碱基序列.结果检测出 PANK2基因一个新的复合杂合突变位于第3外显子的A803G和第5外显子的T1172A;同时检测出3个单核苷酸多态, 位于5'-UTR区的-38 t>a,第1内含子区的IVS1+42 c>a和第1外显子区的G77C,其中-38 t>a,IVS1+42 c>a为首次报道.结论中国人HSS患者存在 PANK2基因突变.     

浙江地区汉族人群caspase-8、-10基因四个单核苷酸多态性位点的单倍型研究

目的分析汉族人群caspase-8、-10基因的单核苷酸多态性（single nucleotide polymorphisins，SNPs）位点及其构成的单倍型，为研究caspase-8、-10基因与多基因复杂疾病的关联分析奠定基础。方法采用PCR、变性高效液相色谱技术和DNA测序技术检测caspase-10基因的第2．5外显子，caspase-8基因的第8-10外显子及其部分侧翼序列的多态性位点；分析配对位点的连锁不平衡关系，最大期望值法估算它们构成的单倍型。结果（1）caspase-10基因的第2、5外显子分别检出一个SNP位点A2823G和A12799G，其中A12799G是新发现的低信息度的SNP；caspase-8基因中检测到3个SNP位点A43466G、G51484A、G52951A，分别位于第8、9外显子和第9内含子；它们均未改变所编码蛋白的一级结构；（2）caspase-10基因中A2823G位点与caspase-8基因中3个位点间已达到连锁平衡，caspase-8基因中A43466G与G52951A、G51484A与G52951A也达到连锁平衡，连锁不平衡系数分别接近于0；只有A43466G与G51484A存在强的连锁不平衡，连锁不平衡系数接近于1；（3）caspase-10基因的A2823G位点与caspase-8基因的3个位点预计产生11种单倍型，其中A-2823／A-43466／G-51484／G-52951是主要单倍型，频率为0．381l，其次是A．2823／A-43466／G-51484／A-52951，频率为0．2536；这4个SNP位点联用，多态信息含量可达到0．7106。结论浙江地区汉族人群caspase-10、-8基因中的SNP位点至少处于3个不同的单倍型块；联合多个相邻的位点构成单倍型，可以弥补单个SNP信息度较低的不足。     

中国人膜联蛋白A1基因单核苷酸多态性及其与2型糖尿病的相关性

目的　筛查上海地区汉族人群中膜联蛋白 A1(annexin A1,ANXA1)基因的单核苷酸多态性 (single nucleotide polymorphisms,SNPs) ,并通过关联分析研究其与 2型糖尿病的相关性。方法　选取2 4例 2型糖尿病患者的 DNA样本 ,采用直接测序法对 ANXA1基因的启动子区、全部外显子及其临近内含子区作 SNPs筛查 ,并在其余的 171例 2型糖尿病和 189名正常对照间作进一步的基因分型。结果ANXA1基因测序长度 6 798bp,共检出 7个 SNPs,其中启动子区 2个 (- 7974 C>T,- 70 4 0 G>T) ,内含子区 3个 ( 90 5 9A>G, 92 0 4 C>T, 10 4 86 A>G) ,5′-非翻译区 1个 (- 6 6 14 A>G) ,编码区 1个( 1784 A>G)。进一步的基因分型后显示这些 SNPs的等位基因频率在 2型糖尿病和正常对照组之间差异无显著性 (P>0 .0 5 )。结论　ANXA1基因多态性与上海地区汉族人群中 2型糖尿病无显著相关性。     

Detection and assignment of mutations and minihaplotypes in human DNA using peptide mass signature genotyping (PMSG): application to the human RDS/peripherin gene

Peptide mass-signature genotyping (PMSG) is a scanning genotyping method that identifies mutations and polymorphisms by translating the sequence of interest in more than one reading frame and measuring the masses of the resulting peptides by mass spectrometry. PMSG was applied to the RDS/peripherin gene of 16 individuals from a family exhibiting autosomal dominant macular degeneration. The method revealed an A-->T transversion in the 5' splice site of intron 2 that is the likely cause of the disease. It also revealed four different minihaplotypes in exon 3 that represent particular combinations of SNPs at four different locations. This study demonstrates the utility of PMSG for identifying and characterizing point mutations and local minihaplotypes that are not readily analyzed by other approaches.     

No role for estrogen receptor 1 gene intron 1 Pvu II and exon 4 C325G polymorphisms in migraine susceptibility

Background  

We have previously reported an association between the estrogen receptor 1 (ESR1) gene exon 8 G594A polymorphism and migraine susceptibility in two independent Australian cohorts. In this paper we report results of analysis of two further single nucleotide polymorphisms (SNPs) in the ESR1 gene in the same study group, the T/C Pvu II SNP in intron 1 and the C325G SNP in exon 4, as well as results of linkage disequilibrium (LD) analysis on these markers.     

Genetic polymorphisms of ataxia telangiectasia mutated affect lung cancer risk

The ataxia telangiectasia mutated (ATM) gene is known to be activated by DNA damage and involved in cell cycle arrest, apoptosis and DNA repair. Therefore, ATM gene polymorphisms may act as important factors predicting individual susceptibility to lung cancer. To evaluate the role of ATM gene polymorphisms in lung cancer development, genotypes of the ATM polymorphisms, -4518A>G, IVS21-77C>T, IVS61-55T>C, and IVS62+60G>A, were determined in 616 lung cancer patients and 616 cancer-free controls. When the effects of selected ATM genotypes were evaluated separately, only one ATM genotype (IVS62+60G>A) showed an association with lung cancer risk. Subjects with the A allele at the site (IVS62+60G>A) have significantly higher risk of lung cancer than those with the G allele [odds ratio (OR)=1.6, 95% confidence interval (CI) 1.1-2.1]. When the haplotypes of four ATM single nucleotide polymorphism sites (-4518A>G, IVS21-77C>T, IVS61-55T>C and IVS62+60G>A) were studied, the ATTA haplotype showed significantly increased risk of lung cancer compared with the GCCA haplotype, the most common haplotype (OR=7.6, 95% CI 1.7-33.5). Furthermore, subjects with the (NN)TA haplotype showed highly significant and increased risk of lung cancer when compared with those without the (NN)TA haplotype (OR=13.2, 95% CI 3.1-56.1). Therefore, our results suggest that polymorphisms or haplotypes of the ATM gene play an important role in the development of lung cancer.     

Five novel single-nucleotide polymorphisms of human interferon gamma identified by sequencing the entire gene

Interferon gamma (IFNG) plays important roles in the regulation of bone remodelling. We describe here six single-nucleotide polymorphisms (SNPs) in the IFNG gene, five of which are novel, and their allelic frequencies in the Japanese population, as determined by sequencing 48 alleles of the entire gene. Four of these polymorphisms were identified inside the third intron, at nucleotide (nt) positions 2459 (A/G), 2671 (T/C), 3177 (T/G), and 3273 (G/A). In exon 4, SNPs were identified at nt positions 5199 (A/T) and 5272 (A/G). These polymorphic sites will be useful for genetic studies of disorders that affect the inflammatory process or calcium metabolism. Received: September 18, 2000 / Accepted: October 2, 2000     

Mutation analysis of synapsin III gene in schizophrenia

Synapsin III is a new synapsin family gene with the putative function of synaptogenesis regulation and neurotransmitter release in the brain. The gene was mapped to 22q12-q13, a schizophrenia susceptible region gene as suggested by several linkage studies. Hence, the synapsin III gene is considered a candidate gene of schizophrenia. We systematically sequenced the protein coding and 5'-promoter regions of the synapsin III gene to look for mutations in 62 Han Chinese schizophrenic patients from Taiwan with positive family history. Further case-control association study was performed among 163 patients and 151 controls using the genetic polymorphic markers identified from these 62 patients. Three single nucleotide polymorphisms (SNPs) were identified: g.-631C > G and g.-196G>A at 5'-promoter region, and g.69G>A at exon 1. Besides, no other mutations were identified in these patients. The g.69G>A polymorphism does not alter the amino acid threonine at codon 23 (ACG>ACA). Further case-control association studies also did not find significant differences of genotype or allele frequency distributions of these three polymorphisms between 163 patients and 151 non-psychotic comparison individuals. Hence, our data are not in favor of a large effect of synapsin III gene in the pathogenesis of schizophrenia.     

The association of SERPINE2 gene with COPD in a Chinese Han population

Purpose

Polymorphisms of several candidate genes have been studied and associated with the development of chronic obstructive pulmonary disease (COPD). One such candidate is the SERPINE2 (Serpin peptidase inhibitor, clade E member 2) gene.

Materials and Methods

To assess whether the SERPINE2 gene is associated with COPD in a Chinese Han population. Samples were collected from a Chinese Han population and analyzed for the association of single nucleotide polymor phisms (SNPs) or haplotypes of SERPINE2 gene with COPD in a case-control study. Three SNPs including rs840088 G/A in intron 1, rs1438831 A/G in 5'' upstream sequence and rs3795879 G/A in intron 3 were detected using the polymerase chain reaction (PCR)-based restriction fragment length polymorphism technique in 409 COPD subjects and 411 controls. Genotyping of the SREPINE2 polymorphisms at positions rs840088, rs1438831and rs3795879 was performed.

Results

We found that none of the rs840088G/A, rs1438831G/A and rs3795879 G/A polymorphisms were associated with the disease. The p-values were 0.630, 0.208 and 0.398 respectively.

Conclusion

Our data suggested that there was no significant association between SERPINE2 polymorphism and COPD susceptibility in the Chinese Han population.