汉族人多囊肾病1型致病基因突变检测体系的建立及初步应用

目的 建立适合筛查汉族人多囊肾病1型致病基因（PKD1）突变的检测体系。方法 利用设计的82对引物[8对针对PKD15′端多拷贝区的长链聚合酶链式反应（PCR）引物和57对巢式PCR引物，17对针对3′端单拷贝区PCR引物]分别对PKD1的46个外显子进行扩增，扩增产物通过单链构象多态性（SSCP）分析筛检出异常条带后，再经测序确定基因突变位点。利用建立的PCR－SSCP检测体系对汉族人2个常染色体显性遗传性多囊肾病患者家系进行PKDA1突变检测，健康献血员为对照。结果 用82对PCR引物，可成功扩增PKD1各个外显子区域，并经测序证实为PKD1目的片段。将建立的SSCP－PCR基因突变检测体系，分别从2个汉族人常染色体显性多囊肾病（ADPKD）家系检测出PKD1基因Del 3 bp(G49761-G49763)和C47629T2个突变，其可分别导致编码产物第3827位缺失谷氨酸（Glu3827)和第3555位丝氨酸，而产生由苯丙氨酸(S3555F)替代的改变。结论 本研究建立的PCR－SSCP检测体系，可完成PKD1各外显子区域特异性扩增，并成功检测出汉族人2个ADPKD家系基因突变位点，不仅为PKD1基因突变的致病机理研究提供宝贵资料，而且为下一步汉族人多囊肾病的大规模基因突变筛查和临床诊断试剂盒的研制奠定了基础。     

单链构象多态性分析检测脂蛋白脂酶基因变异

目的：建立聚合酶链反应-单链构象多态性(PCR-SSCP)技术检测脂蛋白脂酶(LPL)基因Hind Ⅲ多态性、Ser447Ter多态性及-93T／G基因突变的方法。方法：5μl Hind Ⅲ多态性或-93T／G基因突变的PCR产物和5μl甲酰胺变性液混匀变性，室温下用8％的聚丙烯酰胺凝胶电泳后银染。5μl Ser447Ter多态性的PCR产物和2μl甲酰胺变性液混匀变性，25℃左右用8％的聚丙烯酰胺凝胶电泳后银染。结果：PCR-SSCP成功检出LPL的Hind Ⅲ多态性、Ser447Ter多态性及-93T／G基因突变，并检出第9内含子50位一个G碱基缺失和启动子区域-85位一个C→T碱基置换即-85C／T。同时PCR—RFLP验证了200例标本的PCR-SSCP检测结果，两种方法检测已知基因变异时的结果完全一致。结论：PCR-SSCP法既能检测已知的L凡基因变异，也可检出未知的LPL基因变异，是临床实验室开展基因诊断的一种简便、有效的方法。     

目标序列捕获测序检出红细胞丙酮酸激酶缺乏症PKLR基因新的复合突变

目的:探讨红细胞丙酮酸激酶缺乏症(pyruvate kinase deficiency,PKD)的分子发病机制。方法:应用目标序列捕获和高通量二代测序技术(next-generation sequencing,NGS)对临床拟诊PKD患儿的PKLR基因12个外显子及其侧翼序列进行测序,采用SIFT及PolyPhen-2数据库预测突变对蛋白质功能的影响,在确定患者致病基因型后,应用Sanger测序技术对此基因型进行验证。结果:NGS结果显示,患儿PKLR基因存在罕见的双重杂合突变:第5外显子661GA(Asp221Asn)和第10外显子1528CT(Arg510Ter),导致该基因的第221位氨基酸由天冬氨酸突变为天冬酰胺,第510位氨基酸由精氨酸突变为终止密码子,使PKLR基因氨基酸链编码提前终止;Sanger测序技术进一步验证了该双重突变的存在。检索相关文献及数据库显示,这两种突变在人群中的发生率极低,蛋白质功能预测均显示为有害。结论:PKLR基因661 GA与1528 CT双重杂合突变是该PKD患儿的分子发病机制,PKD患者同时存在上述复合突变的报道在国内外尚属首次。     

肥胖症患者瘦素受体基因外显子4变异特征

目的：探讨瘦素受体基因外显子4变异情况及是否与肥胖症相关。方法：选择肥胖症患者[体质量指数(BMI)≥28kg／m^2]和正常人(18．5≤BMI&;lt;23ks／m^2)各50例，运用PCR-SSCP方法，检测瘦素受体基因外显子4的变异情况。结果：50例肥胖症患者瘦素受体基因外显子4未发现碱基改变。结论：瘦素受体基因外显子4中519位点变异不是肥胖症的遗传因素。     

两种新的无义突变Trp228stop和Tp383stop导致的遗传性凝血在子Ⅺ缺陷症

目的 检测遗传性凝血因子Ⅺ（FⅪ）缺陷症患家系中FⅪ基因的突变。方法 检测先证及其家系成员血浆FⅪ：C及FⅪ：Ag，并以其外周血单个核细胞中提取的基因组DNA为模板，PCR扩增FⅪ基因的所有外显子及其侧翼内含子序列，用DNA测序仪检测FⅪ的基因突变。结果 FⅪ基因第7号外显子和11号外显子编码228位和383位氨基酸的碱基发生无义突变TGG→TGA（Trp228stop),TGG→TAG(Trp383stop) ,均为杂合型。结论 此两种基因突变可能是导致患FⅪ缺陷的分子发病机制。     

β链基因突变导致遗传性无纤维蛋白原血症一例报告

目的检测1例遗传性无纤维蛋白原血症患者家系纤维蛋白原(Fg)基因突变。方法检测先证者及其家系成员血浆Fg^2 C及Fg的水平，从外周血单个核细胞中提取基因组DNA，PCR扩增Fg基因的所有外显子及侧翼内含子序列，检测其基因突变。结果发现先证者Fg基因共2处突变，FGB基因7972碱基处缺失G以及FGG基因2543碱基处的纯合T→A。结论FGG基因2543碱基处为多态位点，形成1144位氨基酸的I／K多态性。FGB基因7972碱基处缺失G，导致β链419位氨基酸之后的移码突变，形成了缺少最后27个氨基酸的截短的B链，后者可能是导致遗传性无纤维蛋白原血症的病理机制之一。FGB基因7972碱基处缺失G为国际首次发现的Fg基因突变。     

Landsteiner-Wiener血型系统测序分型技术的建立

目的建立Landsteiner-Wiener血型系统的测序分型技术。方法(1)随机采集45名非血缘关系无偿志愿捐献者外周血样,用EDTA抗凝。(2)从外周抗凝血样中提取总RNA,应用逆转录-聚合酶链反应(RT-PCR)技术,反转录产生LW基因的cDNA(包括3个外显子),采用ABI PrismTM 3100 DNA测序仪对RT-PCR产物中LW基因的第1外显子进行测序。(3)提取基因组DNA,采用一对引物扩增LW基因的第1外显子、第1内含子和第2外显子,全长1224bp,对第1外显子PCR产物直接进行DNA测序分析。结果45例捐血者的样本,分别经RT-PCR产物测序和PCR产物直接测序,均取得了成功,LW基因第1外显子多态性位置第308碱基均为腺嘌呤。结论本研究在国内率先建立了LW血型基因的分子测序方法,可为进一步研究中国人群LW血型基因多态性提供有效方法。     

糖尿病视网膜病变与候选基因(LW、IN)相关性的研究

目的探讨糖尿病视网膜病变与Landsteiner-Wiener、Indian血型基因的相关性。方法将53名临床诊断为糖尿病视网膜病变患者的血样作为实验组,同时随机采集160名非血缘关系无偿志愿捐献者外周血样作为对照组;以逆转录-聚合酶链反应(RT-PCR)和DNA直接序列测定技术同时检测Landsteiner-Wiener血型系统基因,以DNA直接序列测定技术检测Indian血型系统基因。采用Woolf法计算相对危险度值。结果实验组53份糖尿病视网膜病变患者样本均为LWa/LWa纯合型和INb/INb基因纯合型;对照组160份正常样血者的样本中,LW基因第1外显子多态性位置第308位碱基均为A,定为LWa/LWa基因型;IN基因第2个外显子多态性位置第252位碱基均为G,定为INb/INb基因型。结论糖尿病视网膜病变与LW、IN基因未发现有关联。     

Landsteiner-Wiener血型系统测序分型技术的建立

目的 建立Landsteiner-Wiener血型系统的测序分型技术.方法 (1)随机采集45名非血缘关系无偿志愿捐献者外周血样,用EDTA抗凝.(2)从外周抗凝血样中提取总RNA,应用逆转录-聚合酶链反应(RT-PCR)技术.反转录产生LW基因的cDNA(包括3个外显子),采用ABI PrismTM 3100 DNA测序仪对RT-PCR产物中LW基因的第1外显子进行测序.(3)提取基因组DNA,采用一对引物扩增LW基因的第1外显子、第1内含子和第2外显子,全长1224 bp,对第1外显子PCR产物直接进行DNA测序分析.结果 45例捐血者的样本,分别经RT-PCR产物测序和PCR产物直接测序,均取得了成功,LW基因第1外显子多态性位置第308碱基均为腺嘌呤.结论 本研究在国内率先建立了LW血型基因的分子测序方法,可为进一步研究中国人群LW血型基因多态性提供有效方法.     

应用聚合酶链反应技术构建人血小板反应素1基因第13外显子编码钙结合域突变体

目的：利用聚合酶链反应定点突变技术构建人血小板反应素1基因第13外显子编码钙结合域突变体。 方法：实验于2005—03／06在南京医科大学第一附属医院心血管病研究所完成。选择南京医科大学第一附属医院体检中心健康体检者血标本提取人基因组DNA，设计、合成两对引物，将突变位点设计在引物内，突变位点包含限制性内切酶BseNI酶切位点，采用聚合酶链式反应定点突变技术，应用高保真TaqDNA聚合酶，通过三轮聚合酶链扩增反应对血小板反应素1基因的第13外显子进行扩增，将第13外显子碱基8831A置换为G，对最终扩增产物进行酶切鉴定和测序。 结果：获得人血小板反应素1第13外显子编码钙结合域突变体，经酶切鉴定和测序分析，除引入突变点产生预期A8831→G突变外，其余碱基序列无改变。 结论：采用聚合酶链反应体外定点突变技术，成功构建人血小板反应素1基因第13外显子编码钙结合域突变体，为研究血小板反应素1基因该位点突变导致血小板反应素1蛋白的结构、功能变化奠定了基础。     

人类凝血因子Ⅻ基因一种新突变的发现与确证

目的 对一例凝血因子Ⅻ缺陷症患者进行凝血因子Ⅻ（Factor Ⅻ,F Ⅻ）基因序列分析,以发现可能存在的基因变异.方法 PCR结合直接测序方法对该患者F Ⅻ基因全部14个外显子及其侧翼序列进行分析,应用PCR产物反向测序法或等位基因特异PCR技术对突变位点进行确证,随机选择100名体检健康人作为正常对照.结果 该患者F Ⅻ基因第14外显子存在G1706A错义突变,5′非翻译区的46位核苷酸为TT纯合子;等位基因特异PCR证实在100名体检健康人中未发现F Ⅻ G1706A突变.结论 F Ⅻ基因的G1706A错义突变及5′非翻译区的46TT多态性可能与本例患者F Ⅻ缺陷有关.     

轻型β-珠蛋白生成障碍性贫血患者β-珠蛋白基因单核苷酸多态性分析

目的对临床诊断为轻型β-珠蛋白生成障碍性贫血患者进行β-珠蛋白基因单核苷酸多态性分析.方法用PCR法对β-珠蛋白基因进行扩增,扩增产物经纯化后测序,确定其单核苷酸多态性.结果β-珠蛋白基因分析片段中共发现3个位点存在单核苷酸多态性,分别是外显子1第59位的T/C多态性、内含子2第-16位的G/C多态性及内含子2第-74位的T/G多态性.结论同国外报道的正常人群相比,轻型β-珠蛋白生成障碍性贫血患者的β-珠蛋白基因单核苷酸多态性位点显著减少,各位点的碱基频率也有不同.     

A novel missense mutation C127R (FH Zagreb) in the LDL-receptor gene.

We employed the analysis of single-strand conformation polymorphisms to identify mutations in exon 4 of the low density lipoprotein receptor gene causing familial hypercholesterolemia. Three familial hypercholesterolemia heterozygotes had abnormal single-strand conformation polymorphism patterns. DNA sequencing revealed that the abnormal pattern of exon 4A was due to heterozygosity (T/C) at nucleotide 442. Nucleotide 442 is the first base of codon 127, and the T-->C mutation (C127R) changes this codon from CysTGT to ArgCGT. Abnormal patterns of exon 4B were due to heterozygosity (A/G) at nucleotide 662: nucleotide 662 is the second base of codon 200, and the A-->G mutation (D200G) changes this codon from AspGAC to GlyGGC. Mutation D200G was previously described as FH Padova, but mutation C127R (FH Zagreb) has not been reported previously. This novel mutation was confirmed by restriction endonuclease analysis with Dsa I. The screening of 420 familial hypercholesterolemia heterozygotes suggests that C127R and D200G account for about 0.7% of mutations causing familial hypercholesterolemia in Croatia.     

遗传性凝血因子Ⅶ缺陷症家系中R152Q及IVS6+1G→T突变的鉴定

目的检测1个遗传性凝血因子Ⅶ缺陷症家系中因子Ⅶ基因的突变。方法应用PCR结合直接测序的方法对先证者的因子Ⅶ基因9个外显子及其侧翼序列进行分析，鉴别其中可能存在的基因变异。应用PCR产物反向测序法或PCR限制性内切酶分析技术对突变位点进行确证。随机选取100名健康体检者作为正常对照。结果先证者FⅦ基因第6外显子和第6内含子分别存在R152Q和IVS6＋1G→T杂合突变，家系分析表明这2个突变是双重杂合子型，前者遗传自父亲，后者遗传自母亲。100名健康对照者均未发现R152Q错义突变。结论FⅦ基因第6外显子R152Q错义突变和第6内含子供体剪接位点IVS6＋1G→T突变可能是先证者因子Ⅶ先天性缺陷的原因。     

Glu29→Gly纯合子突变导致的遗传性凝血酶原缺乏症

目的 对1个遗传性凝血酶原缺乏症家系进行凝血酶原(FⅡ)基因突变的检测。方法 用活化的部分凝血活酶时间（APTT)，凝血酶原时间(PT)及FⅡ促凝活性(FⅡ：C)、FⅡ抗原(FⅡ：Ag)测定进行表型诊断；用PCR法对先证的FⅡ基因14个外显子及其侧翼序列和5′端非翻译区(5′UTR)、3′端非翻译区(3′UTR)序列进行扩增，PCR产物纯化后直接测序，检测其基因突变。家系成员DNA在先证FⅡ基因突变区域扩增后测序。突变位点经限制性内切酶分析证实。103名健康献血作对照。结果 先证表型诊断为凝血酶原缺乏症(Ⅰ型)；FⅡ外显子区共发现3个与献报道的FⅡ基因序列不同的位点，其中位于第2外显子区的为纯合突变A601G。家系分析表明先证父亲、母亲和外祖母均为A601G杂合子。结论 纯合错义突变A601G引起的Glu29→Gly是导致该例遗传性凝血酶原缺乏的原因。     

Simple genotype analysis of the Asp299Gly polymorphism of the Toll-like receptor-4 gene that is associated with lipopolysaccharide hyporesponsiveness

A nonsynonymous single nucleotide polymorphism (Asp299Gly) in the Toll-like receptor-4 (TLR-4) gene affects the responsiveness to lipopolysaccharide in humans. To analyze this important polymorphism more efficiently, we developed a simple polymerase chain reaction (PCR) restriction length fragment polymorphism (RFLP) assay and examined the Asp299Gly allele frequency in a Japanese population. The PCR primer was designed with 1- or 2-bp mismatches, creating the recognition sequence for restriction enzyme BsaBI or BstXI, allowing RFLP analysis of the digested products. Genotyping was carried out with this assay for 275 DNA specimens from 107 healthy volunteers and 168 patients with various diseases, including ulcerative colitis (n = 86). The Asp299Gly allele of the TLR-4 gene was not detected in any of the specimens, suggesting that it is very rare in Japanese.     

Rapid detection of the SPINK5 polymorphism Glu420Lys by real-time PCR technology

BACKGROUND: The SPINK5 gene, encoding the serine protease inhibitor LEKTI maps to chromosome 5q32, and has been suggested to be a locus predisposing to atopic diseases in general. The Glu420Lys variant showed significant association with atopy, asthma and atopic dermatitis in recent studies. AIMS OF THE STUDY: Development of a high throughput assay to analyse the polymorphism G1258A (Glu420Lys) in exon 14 of the SPINK5 gene followed by the validation using samples of 235 latex-allergic health care workers (HCWs) with (N=63) and without asthma (N=172), and 80 non-atopic controls. METHODS: Twenty DNA samples were first analysed by a polymerase chain restriction fragment analysis (RFLP) using Hph I to generate defined control DNAs which were used for the development of the assay suitable for the detection of the Glu420Lys variant by LightCycler technology. RESULTS AND CONCLUSIONS: 315 samples were successfully screened with this new assay. The temperatures in the melting analysis of the SPINK5 exon 14 PCR product were characteristic to the probes hybridised to the mutant (AA) at 51.5 degrees C and to the wild-type (GG) at 59.5 degrees C. The fast and reliable mutation detection in the tested samples makes this high-speed method suitable for larger epidemiological studies.     

Single nucleotide polymorphisms for DNA repair genes in breast cancer patients

BACKGROUND: The incidence rate for breast cancer (BC) has been increasing in many countries and BC still remains the most common form of cancer in female and continues to be a major health problem worldwide. We explored the association of single nucleotide polymorphisms (SNPs) in DNA repair genes with breast cancer. METHODS: SSCP and RFLP were used to analyze genotypes of DNA repair genes for NBS1, XPC, XPD and XRCC3. RESULTS: T/C in XRCC3 exon 7 had a somewhat deviation from HWE in BC group (P=0.08). The genotype frequency for heterozygote A/C in XPC exon 15 and T/C in XRCC3 exon 7, homozygote A/A in XPD exon 10 were significantly different between BC group and control group in Chinese population (P<0.05, OR=1.47, 95% CI, 1.00-2.16 for A/C in XPC exon 15; P<0.05, OR=1.79, 95% CI, 0.98-3.26 for T/C in XRCC3 exon 7; P<0.05, OR=0.51, 95% CI, 0.27-0.94 for G/A in XPD exon 10). For the SNPs in NBS1 exon 5 (Glu185Gln, G/C) and XPD exon 23 (Lys751Gln, A/C), no remarkable difference for genotype distributions and allele frequencies was observed between BC group and control group in the study. CONCLUSIONS: The genotypes of A/C in XPC exon 15, T/C in XRCC3 exon 7 and A/A in XPD exon 10 studied were significantly different between BC group and control group in Chinese population.     

Genotyping with a dried blood spot method: a useful technique for application in pharmacogenetics

Several commercial DNA isolation kits are available for extracting the genomic DNA from the ethylene diamine tetra-acetic acid (EDTA) whole blood samples. To obtain DNA from whole blood these DNA isolation procedures require quite some hands on time and are rather expensive. An alternative technique could be dried blood spot (DBS) sampling, with which DNA isolation is faster, cheaper and logistics are easier. We have developed a non-commercial DBS method and examined its performance in practice. DNA isolation from EDTA blood samples and made blood spots on filter paper from 106 renal transplant recipients were compared. Additionally, DNA isolation with a column method and two different DBS method was performed for 10 healthy volunteers and compared. Also DNA isolation with only capillary blood using both DBS methods from another 100 healthy volunteers has been investigated. Real-time PCR FRET assays for the CYP3A4 A-392G, CYP3A5 A6986G, ABCB1 C1236T, G2677T/A and C3435T polymorphisms were used and the melting curves of both DNA isolation methods were compared. In all cases DNA extracted with the column method corresponded completely with the results of the DNA isolated with the DBS procedure. Hence, DNA isolation from filter paper appears to be a useful alternative for the commercially available DNA isolation kits.