STR在产前诊断中的应用

短串联重复序列(Short Tandem Repeat,STR)即微卫星位点，是一种广泛存在于人类基因组、以2～6个碱基为单位的串联重复排列的序列。STR具有高度多态性，可以作为一种理想的遗传标记，广泛应用于连锁图和单基因病等遗传病的相关基因定位。     

STR在产前诊断中的应用

短串联重复序列(Short Tandem Repeat,STR)即微卫星位点,是一种广泛存在于人类基因组、以2～6个碱基为单位的串联重复排列的序列.STR具有高度多态性,可以作为一种理想的遗传标记,广泛应用于连锁图和单基因病等遗传病的相关基因定位.     

STR在产前诊断中的应用

短串联重复序列(ShortTandemRepeatSTR)即微卫星位点,是一种广泛存在于人类基因组、以2～6个碱基为单位的串联重复排列的序列。STR具有高度多态性,可以作为一种理想的遗传标记,广泛应用于连锁图和单基因病等遗传病的相关基因定位。     

利用串联重复序列研究炭疽芽胞杆菌的基因分型

目的 应用基因组中多位点串联重复序列遗传标记对不同地区88株炭疽芽胞杆菌进行基因分型。方法炭疽芽胞杆菌染色体DNA基因组中存在着串联重复序列。在串联重复序列两侧设计引物，PCR扩增，琼脂糖和聚丙烯酰胺凝胶电泳，凝胶影像分析软件对PCR扩增产物碱基含量进行测算，并与测序结果进行比较，计算出串联重复拷贝数，对拷贝数进行聚类分析。结果 （1）聚类分析发现，88株菌株可分为三大群，45个基因型，基因型与生态环境存在一定的关系。就某一地区炭疽暴发而言，其可变数目串联重复序列遗传标记具有相似性。（2）研究发现，A16R疫苗株作为中国的疫苗株具有代表性。结论 炭疽芽胞杆菌基因组中的串联重复序列具有遗传稳定性和特异性，可作为炭疽芽胞杆菌基因分型的指标，在炭疽暴发和生物恐怖事件中的病原体溯源上具有重要的意义。     

人类短串联重复序列（STR）的研究进展

短串联重复序列（STR）作为第二代遗传标记,广泛的分布于人类基因组中。本文综述了STR及其分型特点,以及STR应用于遗传制图、法医学鉴定、人类学、群体遗传学、基因诊断、器官移植等研究方面的应用。     

Penta E基因座母系突变1例分析

<正>短串联重复序列(STR)是人类遗传学中重要的遗传标记,也是目前法医物证鉴定中应用最多的一类遗传标记。常染色体STR基因座的平均突变率为0.002~([1]),所以在用常染色体STR进行亲权鉴定时尤其在排除时需谨慎。目前关于常染色体STR突变在国内外报道较多为父系突变,母系突变较少见。本实验室鉴定617例亲子鉴定案例时,检出1例Penta E基因座不符合母系遗传关系。本文     

六例苯酮尿症产前基因诊断

联合应用短重复序列(STR)连锁分析与多重等位基因特异PCR(MASPCR)完成6例PKU风险胎儿的产前基因诊断。诊断结果:4例携带者,2例正常儿,产后基因验证结果一致。结果表明,以STR连锁分析为主,联合应用MASPCR是一个简便实用,诊断率高的基因诊断程序。     

汉族孤独症谱系障碍儿童脆性X基因突变研究

【目的】 采用优化的PCR体系对脆性X智力障碍基因-1(FMR-1)片段扩增,分析汉族孤独症谱系障碍(autism spectrum disorder, ASD)患儿FMR-1基因异常突变发生情况。 【方法】 以466例汉族ASD患儿为研究对象,其中典型孤独症433名,未分类广泛性发育障碍(PDD-NOS)33名。取患儿外周血3~5 mL提取DNA,采用优化的PCR体系对466例患儿DNA进行FMR-1基因片段扩增;对于扩增成功的PCR产物进一步采用短串联重复序列(short tanderm repeat, STR)荧光检测技术进行检测。 【结果】 466个ASD患儿中,464个ASD患者(99.57%)DNA样品PCR扩增成功;2个典型孤独症患者(0.43%)DNA样品PCR扩增失败(排除DNA质量和操作问题),推断FMR-1基因发生异常突变。对于扩增成功的PCR产物进一步采用STR荧光检测技术进行检测,结果显示PCR产物均小于500 bp,其(CGG)n重复个数属正常突变范围。 【结论】 研究采用优化的PCR体系对FMR-1基因片段扩增,进一步采用STR荧光检测技术准确推断出基因片段中三核苷酸(CGG)重复个数,优化后的PCR体系可适用于大样本孤独症谱系障碍儿童的FMR-1基因突变检测。检测结果显示汉族孤独症谱系障碍儿童FMR-1基因异常突变率为0.43%。     

河北省鼠疫耶尔森菌多位点串联重复序列分析

目的对分离自河北省长爪沙鼠鼠疫疫源地的116株鼠疫菌株进行基因分析研究。方法根据鼠疫基因组比对,选择15对引物对测试菌株进行PCR扩增,并对扩增结果进行分析。结果 15对引物对116株实验菌株均得到相应的扩增结果,同一引物测试菌株扩增结果目标条带长度均一致,串联重复序列的重复数相同。不同引物扩增目标条带长度不同,串联重复序列的重复数也不同,如引物M52对所有菌株扩增产物长度均为153 bp,串联重复序列的重复数均为3,而引物M59的扩增产物长度为250 bp,重复数均为7。结论多位点串联重复序列分析(MLVA)证实河北省鼠疫自然疫源地的鼠疫菌基因稳定,鼠疫菌MLVA数据库将对未来的鼠疫菌变异和溯源提供技术支持。     

联合应用常染色体STR、X-STR和线粒体SNP鉴别缺失双亲姐妹同胞关系

目的对双亲缺失的姐妹进行同胞鉴定。方法采用chelex-100法提取血液DNA,通过检测常染色体短串联重复序列(short tandem repeat,STR)位点、X染色体STR位点和线粒体单核苷酸多态性(single nucleotide polymorphism,SNP)进行基因分型,分型结果通过判别函数、ITO法、X染色体STR位点和线粒体SNP分析的方法进行同胞关系判定。结果 39个常染色体等位基因位点的共享基因数为27个,用辨别函数判别归于无关个体;用ITO法计算亲缘关系系数(FSI)为6.95003×10^(-44),判别为无关个体;12个X染色体STR等位基因位点中,有3个X染色体STR位点不匹配,排除姐妹俩来自同一父亲;线粒体SNP的检测显有3个基因座不同,两人来源于不同母系。结论通过联合利用常染色体STR、X染色体STR和线粒体SNP检测技术进行确认两人不是同胞姐妹关系。     

广西巴马瑶族STR基因座遗传多态性分析

目的 分析广西壮族自治区巴马瑶族人群15个短串联重复序列基因座(STR)遗传多态性.方法 采用聚合酶链反应-短串联重复序列(PCR-STR)技术及遗传分析仪,对巴马瑶族114名个体血样DNA样品进行各基因座等位基因频率及杂合度(H)、个体识别力(DP)、非父排除率(EP)、多态信息含量(PIC)等群体遗传学指标分析.结果 15个基因座共检出等位基因120种,频率为0.004 4～0.530 7.除TPOX基因座外,其余14个位点的H为0.6228～0.868 4,PIC为0.592 9～0.844 1,DP为0.817 6～0.959 1,EP为0.319 1～0.731 5;累积个体识别力达0.999 999 999,累积非父排除率达0.999 953 702.巴马瑶族分别与金秀瑶族、临桂瑶族、清远瑶族比较,15个STR基因座大部分等位基因频率分布差异均有统计学意义(P<0.05).结论 广西巴马瑶族的14个基因座属于高度多态性遗传标记,可应用于民族群体遗传学研究.     

Instability at Short Tandem Repeats in Lymphoblastoid Cell Lines

ObjectivesEpstein Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) are a useful biological resource, however, genomic variations can happen during the generation and immortalization processes of LCLs. The purpose of this study was to identify genomic variations in LCL DNA compared with matched blood DNA using short tandem repeats (STRs) analysis.MethodsWe analyzed 15 STRs with blood DNA and their matched LCL DNA samples from 6645 unrelated healthy individuals.ResultsMutations (such as repeat variations and triallelic patterns) of 15 STR loci were detected in 612 LCL DNAs (9.2% of total) without mutations in their matched blood DNA. The repeat variations of 15 STRs were detected in 526 LCL DNAs (mutation rate = 0.0792) and triallelic patterns were identified in 123 (mutation rate = 0.0185). Among 15 STRs, the most common repeat variations (n = 214, mutation rate = 0.0322) and triallelic patterns (n = 17, mutation rate = 0.0026) were found at FGA locus.ConclusionOur study shows that mutations in STRs can occur during generation and immortalization of LCLs.     

Theory and methodology for utilizing genes as biomarkers to determine potential biological mixtures

PURPOSE: Genetically determined mixture information can be used as a surrogate for physical or behavioral characteristics in epidemiological studies examining research questions related to socially stigmatized behaviors and horizontally transmitted infections. A new measure, the probability of mixture discrimination (PMD), was developed to aid mixture analysis that estimates the ability to differentiate single from multiple genomes in biological mixtures. METHODS: Four autosomal short tandem repeats (STRs) were identified, genotyped and evaluated in African American, European American, Hispanic, and Chinese individuals to estimate PMD. Theoretical PMD frameworks were also developed for autosomal and sex-linked (X and Y) STR markers in potential male/male, male/female and female/female mixtures. RESULTS: Autosomal STRs genetically determine the presence of multiple genomes in mixture samples of unknown genders with more power than the apparently simpler X and Y chromosome STRs. Evaluation of four autosomal STR loci enables the detection of mixtures of DNA from multiple sources with above 99% probability in all four racial/ethnic populations. CONCLUSIONS: The genetic-based approach has applications in epidemiology that provide viable alternatives to survey-based study designs. The analysis of genes as biomarkers can be used as a gold standard for validating measurements from self-reported behaviors that tend to be sensitive or socially stigmatizing, such as those involving sex and drugs.     

Complex preimplantation genetic diagnosis for beta-thalassaemia,sideroblastic anaemia,and human leukocyte antigen (HLA)-typing

Preimplantation genetic diagnosis (PGD) to select histocompatible siblings to facilitate curative haematopoeitic stem-cell transplantation (HSCT) is now an acceptable option in the absence of an available human leukocyte antigen (HLA) compatible donor. We describe a case where the couple who requested HLA-PGD, were both carriers of two serious haematological diseases, beta-thalassaemia and sideroblastic anaemia. Their daughter, affected with sideroblastic anaemia, was programmed to have HSCT. A multiplex-fluorescent-touchdown-PCR protocol was optimized for the simultaneous amplification of: the two HBB-gene mutated regions (c.118C?>?T, c.25-26delAA), four short tandem repeats (STRs) in chr11p15.5 linked to the HBB gene, the SLC25A38 gene mutation (c.726C?>?T), two STRs in chr3p22.1 linked to the SLC25A38 gene, plus eleven informative STRs for HLA-haplotyping (chr6p22.1-21.3). This was followed by real-time nested PCR and high-resolution melting analysis (HRMA) for the detection of HBB and SLC25A38 gene mutations, as well as the analysis of all STRs on an automatic genetic analyzer (sequencer). The couple completed four clinical in vitro fertilization (IVF)/PGD cycles. At least one matched unaffected embryo was identified and transferred in each cycle. A twin pregnancy was established in the fourth PGD cycle and genotyping results at all loci were confirmed by prenatal diagnosis. Two healthy baby girls were delivered at week 38 of pregnancy. The need to exclude two familial disorders for HLA-PGD is rarely encountered. The methodological approach described here is fast, accurate, clinically-validated, and of relatively low cost.     

Identification and characterization of microsatellite markers in the Chagas disease vector Triatoma dimidiata.

Triatoma dimidiata, one of the major vectors of Chagas disease in Central America, is found in both domestic and peri-domestic habitats. Questions concerning population boundaries, infestation rates, insecticide resistance, and geographic dispersal of triatomine bugs persist and may be resolved using genetic markers such as microsatellites. Microsatellites are short tandem repeats found dispersed throughout a genome and can be useful for genotypic identification. We developed a plasmid library from the genomic DNA isolated from a single T. dimidiata adult collected in Guatemala. Ten thousand clones were screened using a probe consisting of nine microsatellite oligonucleotides. Eight loci appear polymorphic among populations found in Guatemala, Honduras, and Mexico, and thus are potentially useful for population genetic applications.     

Concordance of variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses of Mycobacterium tuberculosis strains

Variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses were compared to determine whether VNTR analysis was effective for population genetic analysis of Mycobacterium tuberculosis strains. A total of 682 strains, 510 Beijing genotype and 172 non-Beijing genotype strains, were studied.The number of repeats was investigated for 24 VNTR loci: the 15 loci of “optimized miru”, the 8 loci of “Beijing option”, and 1 locus for “JATA12”. Six loci (miru31, Mtub4, QUB4156c, QUB3232, VNTR3820, and VNTR4120) showed significantly different median numbers of repeats in strains belonging to different lineages defined by LSP (P < 0.01, Mann–Whitney U test). When a minimum-spanning tree (MST) was reconstructed using these 6 loci, most strains clustered in the expected branches in the MST branches. However, topology of the MST was not congruent with the evolutional hypothesis of M. tuberculosis, indicating that MST analysis using VNTR data should not use for phylogeny of the organism.When the standardized index of association (sI_A) was calculated using data for the 6 VNTR loci, the value of sI_A was significantly different from zero (Monte Carlo simulation with 10,000 resamplings) in every lineage, indicating the linkage disequilibrium in different lineage strains of M. tuberculosis. These results were consistent with the hypothesis that clonal evolution of lineages of the organism has occurred.Therefore, the 6 loci identified in this study would be effective for M. tuberculosis population genetic analysis due to their significantly different median numbers of repeat and linkage disequilibrium though VNTR data was not effective for phylogeny of the organism.     

Differentiation of tuberculosis strains in a population with mainly Beijing-family strains

A high prevalence of tuberculosis (TB) isolates that are genetically homogenous and from the Beijing family has been reported in Russia. To map TB transmission caused by these strains, new genotyping systems are needed. Mycobacterial interspersed repetitive units (MIRUs) offer the possibility of rapid PCR-based typing with comparable discrimination to IS6110 restriction fragment length polymorphism techniques. Spoligotyping and detection of IS6110 insertion in the dnaA-dnaN region were used to identify Beijing strains in 187 Mycobacterium tuberculosis isolates from Samara, Russia. The Beijing isolates were analyzed by using 12-MIRU and 3-exact tandem repeats (ETR) loci and by an expanded set of 10 additional variable number tandem repeats loci. The expanded set of 25 MIRUs provided better discrimination than the original set of 15 (Hunter-Gaston diversity index 0.870 vs. 0.625). Loci MIRU 26, 1982, and 3232 were the most polymorphic in Beijing isolates.     

福建畲族X染色体12个STR基因座的群体遗传学研究

目的:对福建畲族人群X染色体上12个STR基因座遗传多态性进行分析,探讨其种族特异性及其在法医学中的应用。方法:应用InvestigatorArgusX-12试剂盒对102例福建畲族无关个体的血样进行复合扩增及基因分型。结果:共检出122对等位基因,基因频率范围为0.007～0.575,12个位点的女性基因频率符合Hardy-Weinberg平衡。结论:12个STR位点除DX7423外,均具高度的多态信息含量,在个体识别和亲权鉴定案件,尤其是特殊亲权鉴定案件中具有应用价值。     

Molecular characterization of Neisseria gonorrhoeae isolates in Almaty, Kazakhstan, by VNTR analysis, Opa-typing and NG-MAST

In the present study, new variable number tandem repeats (VNTR) loci in the Neisseria gonorrhoeae genome were identified in silico. VNTR analysis scheme using PCR and agarose or polyacrylamide gel electrophoresis was developed based on nine VNTR loci with various degrees of polymorphism. The method was used to genotype a collection of 48 isolates, obtained from patients with gonorrhea in Almaty, Kazakhstan during the period from December 2008 to November 2009. This collection of isolates was also characterized by the opa-typing and multiantigen sequence typing (NG-MAST). The discriminatory power of the VNTR analysis translated by Hunter-Gaston Discrimination Index (HGDI) was similar to that of opa typing (HGDI=0.98 versus 0.97) and slightly higher than that of NG-MAST (HDGI=0.95). The adjusted Rand (AR) coefficients and Wallace coefficients showed that the overall concordance between the typing methods was not high. VNTR analysis described here is simple, inexpensive, easy to interpret, and it would be reliable for the comparison of data obtained in different laboratories. The proposed VNTR loci might be used for epidemiological studies of gonococcal infections.     

Differential evolution of repetitive sequences in Cryptosporidium parvum and Cryptosporidium hominis.

Cryptosporidium parvum and Cryptosporidium hominis are two morphologically identical species of Apicomplexan protozoa infecting humans. Although the genomes of these species are 97% identical, their host range is strikingly different. C. parvum infects humans and animals and is primarily a zoonotic infection, whereas C. hominis is typically not detected in animals. The extent of genetic polymorphism in both species has been surveyed locally, but not on a larger geographical scale. Herein, a collection of unrelated C. parvum and C. hominis isolates was genotyped using multiple, randomly distributed micro- and minisatellites. In average, minisatellites, consisting of tandemly repeated sequence motifs of 6-24 basepair, were more polymorphic than microsatellites. When the average number of micro- and minisatellite alleles per locus was used as a measure of heterogeneity, no difference between C. parvum and C. hominis was found. However, the frequency distribution of alleles in both species was significantly different and in 6 of the 14 loci the size of the C. parvum and C. hominis repeats did not overlap. Assuming that C. parvum and C. hominis evolved from a common ancestor, these observations suggest a differential evolution of repeat length at these loci.