与成人型多囊肾病基因位点1连锁的微卫星的遗传多态性研究

目的探讨广泛应用于高加索人多囊肾病（ＰＫＤ１）基因诊断的两个微卫星在中国汉族及壮族人群中的多态性及其等位基因频率是否存在群体差异。方法用聚合酶链反应（ＰＣＲ）扩增与ＰＫＤ１连锁的两个微卫星，１０％非变性聚丙烯酰胺凝胶电泳结合ＤＮＡ序列测定检测ＰＣＲ产物。结果ＡＣ２．５和ＳＭ７两个微卫星：（１）在汉族中分别观察到１０和１１个等位基因，期望杂合度分别为６８．４％及７７．４％，多态信息含量（ＰＩＣ）分别为０．６８及０．７７；（２）在壮族中分别观察到８及９个等位基因，期望杂合度分别６７．１％及５７．５％，ＰＩＣ分别为０．６７及０．５７；（３）６个ＡＣ２．５等位基因的频率及８个ＳＭ７等位基因的频率存在群体差异（Ｐ＜０．０５）。结论ＡＣ２．５和ＳＭ７在汉族和壮族中均高度多态（ＳＭ７在壮族中稍差），可用于汉、壮族人ＰＫＤ１基因诊断。同时，这两个微卫星的等位基因频率分布具有群体差异，在应用它们进行基因诊断和疾病关联性研究时应引起注意。     

苯丙酮尿症突变基因分析和产前诊断

应用多重等位基因特异ＰＣＲ方法检测分析了３０个苯丙酮尿症家系的５种苯丙氨酸羟化酶基因点突变：外显子７（Ａｒｇ２４３Ｇｌｎ），外显子１２（Ａｒｇ４１３Ｐｒｏ），外显子３（Ａｒｇ１１１Ｔｅｒｍ），外显子１１（Ｔｙｒ３５６Ｔｅｒｍ）和外显子６（Ｔｙｒ２０４Ｃｙｓ）。结果表明，这五种突变占ＰＫＵ基因的４６．６％，其中最常见的突变为前两种，分别占２３．３％和１０．０％。完成了１例ＰＫＵ风险胎儿的产前诊断。     

布氏菌544A基因片段的PCR扩增及其序列分析

利用ＰＣＲ和ＤＮＡ重组技术，成功的扩增了牛布氏菌５４４Ａ基因。回收含Ｂｒ．ａｂｏｒｔｕｓ５４４ＡＤＮＡ片段，插入ＳｍａＩ单酶切的ｐＵＣ９载体中，进行核苷酸序列分析，证实克隆的ＤＮＡ片段长度为２２４ｂｐ，在该序列中含有单个开放新闻记者框架。将重组用Ｋｐｎ１和ＢａｍＨ１双酶切下Ｂｒ．ａｂｏｒｔｕｓ５４４ａＤＮＡ片段标记探针，均与６种布氏菌杂交出现阳性结果。说明Ｂｒ．ａｂｏｒｔｕｓ５４４Ａ２２４ｂｐＤＮ     

血管紧张素Ⅱ受体Ⅰ型基因3′端CA重复多态性及与汉族人高血压病的关系

目的研究血管紧张素Ⅱ受体Ⅰ型基因（ＡＴ１Ｒ）３′端ＣＡ重复序列多态性在中国汉族人分布规律及与高血压病的关系。方法应用扩增片段长度多态性方法,对来自山东和西安两地的汉族人群进行血管紧张素Ⅱ受体Ⅰ型基因３′端重复多态性分析。结果在所研究人群中ＡＴ１Ｒ基因３′端重复序列存在９种等位基因（Ａ１～Ａ９）,扩增长度为１３０ｂｐ～１４６ｂｐ,其频率介于０．０１～０．３８,以扩增长度为１４０ｂｐ的Ａ４等位基因最为常见,其次为Ａ３和Ａ７,分布符合Ｈａｒｄｙ－Ｗｅｉｎｂｅｒｇ遗传平衡定律,ＣＡ重复多态杂合率为０．７８９,多态信息量（ＰＩＣ）为０．７４６。与白种人相比等位基因分布趋势无显著性差异,但个别等位基因相对频率存在不同程度的差异,白种人Ａ４等位基因频率高于中国人。原发性高血压人群ＡＴ１Ｒ基因ＣＡ重复多态分布与正常人群有显著性差异（Ｐ＜０．０１）。结论ＡＴ１Ｒ基因３′端ＣＡ重复多态性标志与中国汉族人高血压病相关。     

用Amp－FLP技术研究中国汉族群体D4S95和DXS52位点的遗传多态性

为研究中国汉族群体Ｄ４Ｓ９５和ＤＸＳ５２位点的遗传多态性，应用改进的Ｄ４Ｓ９５和ＤＸＳ５２位点的扩增片段长度多态性（Ａｍｐ－ＦＬＰ）分析技术，检测了中国汉族个体共２２２名。结果分别为，（１）Ｄ４Ｓ９５位点：在１０８名无关个体中发现了７个等位基因（片段长度为９１０～１１５０ｂｐ），１８种基因型，杂合性为０．７６，个人鉴别力为０．８７１（２）ＤＸＳ５２位点：在１１４名无关个体中（男性７０人，女性４４人）发现了１４个等位基因（片段长度为６９５～２４００ｂｐ），在４４名女性个体中发现了２２种基因型，杂合性为０．７７．个人鉴别力为男性０．８９，女性０．９３１检测了两个家系两代３口之家的两位点的基因型，证明该两位点的基因按Ｍｅｎｄｅｌ定律遗传，该技术在法医科学鉴定中具有重要的应用价值。     

联合应用STR与A／C连锁分析进行PKU的产前基因诊断

目的：对经典ＰＫＵ快速,高效易推广的产前基因诊断方法进行探讨。方法：用ＳＴＲ－Ａｍｐ－ＦＬＰ和ＰＣＲ－ＳＳＣＰ对云南１３个ＰＫＵ家系的ＰＫＵ家系的ＰＡＨ基因内与Ａ／Ｃ位点进行连锁分析。结果：检出２４２－２５２ｂｐ的ＳＴＲ８个等位片段,ＰＩＣ为０．６９８,杂合率是５７．６９％,用ＳＴＲ和ＳＴＲ加Ａ／Ｃ的１００％基因诊断率是４６．１５％和６１．５４％,且ＳＴＲ加入Ａ／Ｃ对所有家系均能提供信息。完成１例     

血管紧张素Ⅱ受体Ⅰ型基因3‘端CA重复多态性及汉族人高血 …

目的 研究血管紧张素Ⅱ受体Ⅰ型基因（ＡＴ１Ｒ）３‘邓列多太 在中国汉族人分布规律及与主血压病的关系。方法 应用扩增片段长度多态性方法,对来自山东和西安两地的汉族人群进行血管紧张素Ⅱ受体Ⅰ型基因３’端重复性多态性分析。结果 在所研究人群中ＡＴＲ基因３‘端重复序列存在９种等位基因（Ａ１－Ａ９）,扩增长度为１３０ｂｐ－１４６ｂｐ,其频率介于０．０１－０．３８,以扩增长度为１４０ｂｐ的Ａ４等位基因最为常见     

聚合酶链反应快速诊断结核病的应用研究

应用聚合酶链反应（ＰＣＲ）技术建立了扩增结核杆菌特异重复序列ＩＳ６１１０部分基因的方法。对９种抗酸分枝杆菌、３种普通菌进行检测，结果仅人型结核杆菌、牛型结核杆菌及ＢＣＧ扩增出１２３ｂｐ特异性条带，ＰＣＲ产物经ＳａｌⅠ酶切后产生７０ｂｐ与５３ｂｐ两个片段。ＰＣＲ检测人型结核杆菌的敏感性达１０ｆｇＤＮＡ或５个菌体。应用ＰＣＲ检测了１０９份结核临床标本，其总阳性率为７２．５％，明显高于抗酸染色涂片（２．８％）与细菌培养（９．２％）的阳性率（Ｐ＜０．００１）。ＰＣＲ的检出率也较ＥＬＩＳＡ法检测抗ＰＰＤ－ＩｇＧ的阳性率（６３．３％）为高，但尚无统计学差异（ｐ＞０．０５），但是ＥＬＩＳＡ检测抗体有１９．０％的假阳性。研究表明，ＰＣＲ是一种特异、敏感、快速诊断结核病的方法。     

早衰蛋白-1基因内含子多态性与Alzheimer's病的关系

为探讨早衰蛋白－１（ＰＳ－１）基因多态性与Ａｌｚｈｅｉｍｅｒ’ｓ病（ＡＤ０的关系,用聚合酶链反应－限制性片段长度多态性（ＰＣＲ－ＲＦＬＰ）方法检测５１例散发性ＡＤ患者ＰＳ－１外显子８的３‘端内含子基因型,其中４０例晚发性ＡＤ患者,同时检测８７６例健康对照者ＰＳＤ－１基因型。结果发现散发性ＡＤ组１／１基因型和１等位基因频率分别为０．５１０和０．６９６,显著高于对照组的０．２９９和０．５２２;晚发性Ａ     

冠心病、原发性高血压人群ACE基因多态性分布及序列测定

目的研究血管紧张素转换酶（ＡＣＥ）基因插入／缺失（Ｉ／Ｄ）多态性在冠心病（ＣＡＤ）、原发性高血压（ＥＨ）和健康人群中的分布,并进行序列分析。方法以多聚酶链反应（ＰＣＲ）方法检测了１３７例ＣＡＤ患者、４２例ＥＨ患者和６３例健康人群的ＡＣＥ基因型,以荧光标记自动测序法测定Ｄ和Ｉ等位基因序列。结果ＣＡＤ组ＤＤ型ＡＣＥ基因出现频率显著高于对照组（０．４５对０．２１,Ｐ＜０．０１）,而ＥＨ组与对照组比较无显著差异。Ｄ等位基因长１９１ｂｐ,Ｉ等位基因长４７９ｂｐ,其核苷酸序列与国外文献报道略有不同。结论ＡＣＥ基因Ｉ／Ｄ多态性是ＣＡＤ发病的独立危险因素,与ＥＨ无相关,ＡＣＥ基因１６内含子中一段２８８ｂｐ的插入片段造成Ｉ／Ｄ多态性。     

三个新的PAH基因短串联重复序列多态性及其在苯丙酮尿症产前诊断中的应用

目的提高经典型苯丙酮尿症的产前诊断的成功率。方法在苯丙氨酸羟化酶（phenylalanine hydroxylase，PAH）基因附近选择了3个新的短串联重复序列（short tandem repeat，STR）位点（PAH26、PAH32和PAH9），进行扩增长度片段多态性分析，确定它们在中国人群的分布及在诊断中的应用价值。结果3个新的STR位点的多态信息含量分别为0．518（PAH26）、0．413（PAH32）和0．362（PAH9）。这3个位点之间，PAH9与第3内含子中的STR位点（TCTA）n之间存在连锁不平衡，其他位点组合不存在连锁不平衡。联合第3内含子中的STR位点（TCTA）n和2个新的位点，可以对家系中突变基因标记进行95％N断，并成功地用于4例产前诊断中。结论选择性地应用PAH基因中的3个STR位点组合，可以95％地区分经典型苯丙酮尿症家系中父母的两个基因，从而准确地进行快速产前诊断。     

Variation of short tandem repeats within and between populations

Using the polymerase chain reaction (PCR) the frequency distributionsof three short tandem repeats (STR) were investigated in fivepopulations: North European, Cypriot, Pakistani, Gujaratl andVietnamese. Each STR ls situated within an intron; the markersare in the genes for human coagulation factor XIII (4bp repeat),llpoprotein llpase (4bp repeat) and CD4 (5bp repeat). Populationdata were generated for each STR and allele frequencies calculated.A calculation of the level of population substructuring forthe three systems was also made. The llpoprotein lipase STRdata showed no evidence for population substructuring, but therewas a significant level of substructuring in the other two systems.This lnitial pilot study demonstrates the need to validate eachmarker used for DNA profiling in different human populations,and that some markers (such as LPL) can be used with confidencein widely differing ethnic groups, while others (such as CD4and F13A) may be of value In distinguishing sub-groups.     

Mutation and haplotype analysis of phenylalanine hydroxylase alleles in classical PKU patients from the Czech Republic: identification of four novel mutations.

Mutations, haplotypes, and other polymorphic markers in the phenylalanine hydroxylase (PAH) gene were analysed in 133 unrelated Czech families with classical phenylketonuria (PKU). Almost 95% of all mutant alleles were identified, using a combination of PCR and restriction analysis, denaturing gradient gel electrophoresis (DGGE), and sequencing. A total of 30 different mutations, 16 various RFLP/VNTR haplotypes, and four polymorphisms were detected on 266 independent mutant chromosomes. The most common molecular defect observed in the Czech population was R408W (54.9%). Each of the other 29 mutations was present in no more than 5% of alleles and 13 mutations were found in only one PKU allele each (0.4%). Four novel mutations G239A, R270fsdel5bp, A342P, and IVS11nt-8g-->a were identified. In 14 (5.1%) alleles, linked to four different RFLP/VNTR haplotypes, the sequence alterations still remain unknown. Our results confirm that PKU is a heterogeneous disorder at the molecular level. Since there is evidence for the gene flow coming from northern, western, and southern parts of Europe into our Slavic population, it is clear that human migration has been the most important factor in the spread of PKU alleles in Europe.     

Phenylketonuria screening registry as a resource for population genetic studies

Background: Neonatal screening for metabolic diseases, involving samples stored on filter paper (Guthrie spots), provides a potential resource for genetic epidemiological studies. Objective: To develop a method to make these dried blood spots available for large scale genetic epidemiology. Methods: DNA from untraceable Guthrie spots was extracted using a saponin and chelex-100 based method and preamplified by improved primer preamplification. Analyses were done on 38 samples each of fresh, 10, and 25 year old Guthrie spots and the success rate determined for PCR amplification for five amplicon lengths. Results: The method was applicable even on 25 year old samples. The success rate was 100% for 100 bp amplicons and 80% for 396 bp amplicons. Ninety four Guthrie samples were genotyped, including carriers of two different PKU mutations; all carriers were found (six R158Q, four R252W), with no false positives. Finally, 2132 anonymous samples from the Swedish PKU registry were extracted and preamplified and the allele frequencies of APOε4, PPARγ Pro12Ala, and the CCR5 32 bp deletion determined. Local variations in allele frequencies suggested subpopulation structuring. There was a significant difference (p<0.01) in regional allele frequencies for the CCR5 32 bp deletion in the Swedish population. Conclusion: Whole genome amplification makes it feasible to conduct large genetic epidemiological studies using PKU screening registries.     

The molecular basis of phenylketonuria in Latvia

Characterization of the molecular basis of phenylketonuria (PKU) in Latvia has been accomplished through the analysis of 96 unrelated chromosomes from 50 Latvian PKU patients. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed through a combined approach in which R158Q, R252W, R261Q, G272X, IVS10-11G>A and R408W mutations were first screened for by PCR or restriction generating PCR amplification of PAH gene exons 5, 7, 11 and 12 followed by digestion with the appropriate diagnostic enzyme. Subsequently 'broad range' denaturing gradient gel electrophoresis analysis of the 13 PAH gene exons has been used to study uncharacterized PKU chromosomes. A mutation detection rate of 98% was achieved. 12 different mutations were found, with the most frequent mutation, R408W, accounting for 76% of Latvian PKU alleles. Six mutations (R408W, E280K, R158Q, A104D, R261Q and P281L) represent 92% of PKU chromosomes. PAH VNTR and STR alleles have been also identified and minihaplotype associations with PKU mutations were also determined.     

Y染色体特异短串联重复序列初步研究

目的了解人类Ｙ染色体特异短串联重复序列等位片段结构特征,揭示成都汉族群体５个Ｙ染色体特异ＳＴＲ基因座的遗传多态性。方法无血缘关系样本采自成都地区汉族群体。ＰＣＲ扩增５个Ｙ染色体特异ＳＴＲ基因座。ＰＣＲ产物由非变性聚丙烯酰胺凝胶不连续缓冲系统水平电泳分型和自动激光荧光测序仪分析。结果观察到人类Ｙ染色体特异ＳＴＲ等位片段具有复杂的串联重复结构。制备了５个Ｙ染色体特异ＳＴＲ基因座分型标准物用于分型。观察到我国人群５个Ｙ染色体特异ＳＴＲ基因座均具有遗传多态性。获得了成都汉族群体５个Ｙ染色体特异ＳＴＲ基因座的等位基因频率。结论本研究为群体遗传亲缘关系分析提供了高效遗传标记,为研究我国人群Ｙ染色体特异ＳＴＲ的群体遗传提供了具有可比性的资料,为研究人类近期进化事件提供了新的可能性     

有汗型外胚层发育不良一家系的Cx30基因突变检测及产前诊断

目的对一个中国汉族有汗型外胚层发育不良（hidrotic ectodermal dysplasis，HED）家系进行了突变筛查，并在此基础上对该家系中已孕5个月的胎儿进行了产前诊断。方法共收集了该家系2例患者及4名正常人的外周血标本，抽取了胎儿的脐血标本。扩增Cx30基因的整个编码区序列，直接双向测序，突变进一步经内切酶酶切分析验证，在成功地获得基因诊断结果后，进一步进行产前诊断。首先从脐血DNA标本中扩出Cx30基因的整个编码区序列，经内切酶酶切分析检测突变，并进一步将整个编码区序列克隆入T载体，测序验证突变。结果在两个受累患者中检测了相同的突变，即在Cx30基因存在一个263C→T的点突变，该突变导致了在GJB6蛋白第2个跨膜区中氨基酸残基改变（A88V）。胎儿的检测结果表明其基因组中同样存在该致病突变，因此是1个受累胎儿。结论实验数据表明Cx30基因中错义突变A88V也是中国汉族人群中有汗型外胚层发育不良的致病原因之一，可以通过基因诊断和产前诊断阻止致病基因的传递。     

中国广州汉族人群六个STR位点的调查

目的 了解广州汉族人在ｖＷＡ３１Ａ等６个短串联重复序列（ｓｈｏｒｔ ｔａｎｄｅｍ ｒｅｐｅａｔ,ＳＴＲ）位点法医学的有关数据。方法 运用ＳＴＲ－ＰＣＲ、４％变性聚丙烯酰胺凝胶电池,结合荧光ＤＮＡ自动测技术,对汉族人群６个ＳＴＲ位点的等位基因频率和基因型频率进行了调查,并与其他种族或人群的等位基因频率进行了比较。结果 ６个ＷＴＲ位点是ｖＷＡ３１Ａ、ＴＨ０１、Ｆ１２Ａ０１、Ｆ１３Ａ０１、ＦＥＳ、ＴＰＯ     

中国汉族人群四个STR的多态性及其与IDDM的相关性研究

目的 获得Ｄ１５Ｓ６５７、Ｄ１１Ｓ１３６９、Ｄ６Ｓ２４２０、Ｄ６Ｓ５０３位点在中国成都汉族人群中的群体遗传数据，并研究上述４种短串联重复序列（ｓｈｏｒｔ ｔａｎｄｅｍ ｒｅｐｅａｔｓ，ＳＴＰ）的多态性与胰岛依赖型糖尿病（ｉｎｓｕｌｉｎ－ｄｅｐｅｎｄｅｎｔ ｄｉａｂｅｔｅｓ ｍｅｌｌｉｔｕｓ，ＩＤＤＭ）的关系。方法 应用ＰＣＲ－ｐｏｌｙａｃｒｙｌａｍｉｄｅ ｇｅｌ ｌｅｃｔｒｏｐｈｏｒｅｓｉｓ（ＰＡ