Genome-wide prediction of human VNTRs

Polymorphic minisatellites, also known as variable number of tandem repeats (VNTRs), are tandem repeat regions that show variation in the number of repeat units among chromosomes in a population. Currently, there are no general methods for predicting which minisatellites have a high probability of being polymorphic, given their sequence characteristics. An earlier approach has focused on potentially highly polymorphic and hypervariable minisatellites, which make up only a small fraction of all minisatellites in the human genome. We have developed a model, based on available minisatellite and VNTR sequence data, that predicts the probability that a minisatellite (unit size > or = 6 bp) identified by the computer program Tandem Repeats Finder is polymorphic (VNTR). According to the model, minisatellites with high copy number and high degree of sequence similarity are most likely to be VNTRs. This approach was used to scan the draft sequence of the human genome for VNTRs. A total of 157,549 minisatellite repeats were found, of which 29,224 are predicted to be VNTRs. Contrary to previous results, VNTRs appear to be widespread and abundant throughout the human genome, with an estimated density of 9.1 VNTRs/Mb.     

Detection of species-specific long VNTRs in mitochondrial control region and their application to identifying sympatric Hong Kong grouper (Epinephelus akaara) and yellow grouper (Epinephelus awoara)

Length variation and heteroplasmy were observed in PCR products of the first half of mtDNA control region of both Hong Kong grouper (Epinephelus akaara) and yellow grouper (Epinephelus awoara). DNA sequencing unveiled the phenomena were caused by the presence of species-specific long variable number tandem repeats (VNTRs). This is the first report on the mtDNA VNTRs and their heteroplasmy in groupers. Moreover, these VNTRs are also the longest such structure found in teleost fish. Thereafter, we designed two species-specific PCR reverse primers according to the 3' end sequences of the VNTRs and successfully established assays for the identification of these two sympatric grouper species.     

Slipped-strand mispairing at noncontiguous repeats in Poecilia reticulata: a model for minisatellite birth

The standard slipped-strand mispairing (SSM) model for the formation of variable number tandem repeats (VNTRs) proposes that a few tandem repeats, produced by chance mutations, provide the "raw material" for VNTR expansion. However, this model is unlikely to explain the formation of VNTRs with long motifs (e.g., minisatellites), because the likelihood of a tandem repeat forming by chance decreases rapidly as the length of the repeat motif increases. Phylogenetic reconstruction of the birth of a mitochondrial (mt) DNA minisatellite in guppies suggests that VNTRs with long motifs can form as a consequence of SSM at noncontiguous repeats. VNTRs formed in this manner have motifs longer than the noncontiguous repeat originally formed by chance and are flanked by one unit of the original, noncontiguous repeat. SSM at noncontiguous repeats can therefore explain the birth of VNTRs with long motifs and the "imperfect" or "short direct" repeats frequently observed adjacent to both mtDNA and nuclear VNTRs.     

Five polymorphic microsatellite VNTRs on the human X chromosome

The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT.dG/dA.dC)n, where n = approximately 10-60. We and others have found that several of these repeats have variable lengths in different individuals, with allelic fragments varying in size by multiples of 2 bp. These "microsatellite" variable number of tandem repeats (VNTRs) may be scored by PCR, using unique flanking primers to amplify the repeat-containing regions and resolving the products on DNA sequencing gels. Since few VNTRs have been found on the X chromosome, we screened a flow-sorted X chromosome-specific genomic library for microsatellites. Approximately 25% of the phage clones hybridized to a poly (dT-dG).poly(dA-dC) probe. Of seven X-linked microsatellites present in positive phages, five are polymorphic and three have both eight or more alleles and heterozygosities exceeding 75%. Using PCR to amplify genomic DNAs from hybrid cell panels, we confirmed the X localization of these VNTRs and regionally mapped four of them. The fifth VNTR was regionally mapped by virtue of its tight linkage to DXS87 in Centre du Polymorphisme Humain families. We conclude that whatever factors limit the occurrence of "classical" VNTRs and RFLPs on the X chromosome do not appear to operate in the case of microsatellite VNTRs.     

Mutations, mutation rates, and evolution at the hypervariable VNTR loci of Yersinia pestis

VNTRs are able to discriminate among closely related isolates of recently emerged clonal pathogens, including Yersinia pestis the etiologic agent of plague, because of their great diversity. Diversity is driven largely by mutation but little is known about VNTR mutation rates, factors affecting mutation rates, or the mutational mechanisms. The molecular epidemiological utility of VNTRs will be greatly enhanced when this foundational knowledge is available. Here, we measure mutation rates for 43 VNTR loci in Y. pestis using an in vitro generated population encompassing approximately 96,000 generations. We estimate the combined 43-locus rate and individual rates for 14 loci. A comparison of Y. pestis and Escherichia coli O157:H7 VNTR mutation rates and products revealed a similar relationship between diversity and mutation rate in these two species. Likewise, the relationship between repeat copy number and mutation rate is nearly identical between these species, suggesting a generalized relationship that may be applicable to other species. The single- versus multiple-repeat mutation ratios and the insertion versus deletion mutation ratios were also similar, providing support for a general model for the mutations associated with VNTRs. Finally, we use two small sets of Y. pestis isolates to show how this general model and our estimated mutation rates can be used to compare alternate phylogenies, and to evaluate the significance of genotype matches, near-matches, and mismatches found in empirical comparisons with a reference database.     

Microevolution of pandemic Vibrio parahaemolyticus assessed by the number of repeat units in short sequence tandem repeat regions

The emergence of the pandemic strain Vibrio parahaemolyticus O3:K6 in 1996 caused a large increase of diarrhea outbreaks related to seafood consumption in Southeast Asia, and later worldwide. Isolates of this strain constitutes a clonal complex, and their effectual differentiation is possible by comparison of their variable number tandem repeats (VNTRs). The differentiation of the isolates by the differences in VNTRs will allow inferring the population dynamics and microevolution of this strain but this requires knowing the rate and mechanism of VNTRs' variation. Our study of mutants obtained after serial cultivation of clones showed that mutation rates of the six VNTRs examined are on the order of 10(-4) mutant per generation and that difference increases by stepwise addition of single mutations. The single stepwise mutation (SSM) was deduced because mutants with 1, 2, 3, or more repeat unit deletions or insertions follow a geometric distribution. Plausible phylogenetic trees are obtained when, according to SSM, the genetic distance between clusters with different number of repeats is assessed by the absolute differences in repeats. Using this approach, mutants originated from different isolates of pandemic V. parahaemolyticus after serial cultivation are clustered with their parental isolates. Additionally, isolates of pandemic V. parahaemolyticus from Southeast Asia, Tokyo, and northern and southern Chile are clustered according their geographical origin. The deepest split in these four populations is observed between the Tokyo and southern Chile populations. We conclude that proper phylogenetic relations and successful tracing of pandemic V. parahaemolyticus requires measuring the differences between isolates by the absolute number of repeats in the VNTRs considered.     

Variable Numbers of Tandem Repeats in Plasmodium falciparum Genes

Genome variation studies in Plasmodium falciparum have focused on SNPs and, more recently, large-scale copy number polymorphisms and ectopic rearrangements. Here, we examine another source of variation: variable number tandem repeats (VNTRs). Interspersed low complexity features, including the well-studied P. falciparum microsatellite sequences, are commonly classified as VNTRs; however, this study is focused on longer coding VNTR polymorphisms, a small class of copy number variations. Selection against frameshift mutation is a main constraint on tandem repeats (TRs) in coding regions, while limited propagation of TRs longer than 975 nt total length is a minor restriction in coding regions. Comparative analysis of three P. falciparum genomes reveals that more than 9% of all P. falciparum ORFs harbor VNTRs, much more than has been reported for any other species. Moreover, genotyping of VNTR loci in a drug-selected line, progeny of a genetic cross, and 334 field isolates demonstrates broad variability in these sequences. Functional enrichment analysis of ORFs harboring VNTRs identifies stress and DNA damage responses along with chromatin modification activities, suggesting an influence on genome mutability and functional variation. Analysis of the repeat units and their flanking regions in both P. falciparum and Plasmodium reichenowi sequences implicates a replication slippage mechanism in the generation of TRs from an initially unrepeated sequence. VNTRs can contribute to rapid adaptation by localized sequence duplication. They also can confound SNP-typing microarrays or mapping short-sequence reads and therefore must be accounted for in such analyses.     

VNTRseek—a computational tool to detect tandem repeat variants in high-throughput sequencing data

DNA tandem repeats (TRs) are ubiquitous genomic features which consist of two or more adjacent copies of an underlying pattern sequence. The copies may be identical or approximate. Variable number of tandem repeats or VNTRs are polymorphic TR loci in which the number of pattern copies is variable. In this paper we describe VNTRseek, our software for discovery of minisatellite VNTRs (pattern size ≥ 7 nucleotides) using whole genome sequencing data. VNTRseek maps sequencing reads to a set of reference TRs and then identifies putative VNTRs based on a discrepancy between the copy number of a reference and its mapped reads. VNTRseek was used to analyze the Watson and Khoisan genomes (454 technology) and two 1000 Genomes family trios (Illumina). In the Watson genome, we identified 752 VNTRs with pattern sizes ranging from 7 to 84 nt. In the Khoisan genome, we identified 2572 VNTRs with pattern sizes ranging from 7 to 105 nt. In the trios, we identified between 2660 and 3822 VNTRs per individual and found nearly 100% consistency with Mendelian inheritance. VNTRseek is, to the best of our knowledge, the first software for genome-wide detection of minisatellite VNTRs. It is available at http://orca.bu.edu/vntrseek/.     

Analysis of the novel VNTR polymorphisms ofMUC8 gene

Analysis of mucin genes has identified the presence of several features that may represent important functional domains in mucin glycoproteins. In the central region of each mucin, there are a variable number of tandem repeats (VNTR; minisatellites). However, their genomic levels are unclear because of complex genomic properties. We report here the distribution of VNTR and polymorphic analysis ofMUC8. We searched for VNTR ofMUC8 using the Tandem Repeat Finder program and found nine VNTR motif. Six (MUC8 MS1～MS6) among the nine VNTRs were evaluated in this study. Each VNTR inMUC8 region was analyzed in genomic DNA obtained from 200 unrelated individuals and multi-generational families. All VNTRs (MUC8 MS1, -MS2, -MS3, -MS4,-MS5 and -MS6) were genotyped as polymorphic. The degree of polymorphism within theMUC8-MS5 showed the highest heterozygosity (h = 0.786) in theMUC8 region. In order to perform a segregation analysis of the VNTRs inMUC8, we analyzed genomic DNA obtained from two generations of five families and from three generations of two families. Six of the polymorphic VNTRs were transmitted through meiosis following a Mendelian inheritance, which suggests that polymorphic VNTRs could be useful markers for paternity mapping and DNA fingerprinting.     

Genetic associations between ADHD and dopaminergic genes (<Emphasis Type="Italic">DAT1</Emphasis> and <Emphasis Type="Italic">DRD4</Emphasis>) VNTRs in Korean children

It is well known that dopaminergic genes affect the development of attention deficit hyperactivity disorder (ADHD) in various populations. Many studies have shown that variable number tandem repeats (VNTRs) located within the 3′-untranslated region of DAT1 and in exon 3 of DRD4 are associated with ADHD development; however, these results were inconsistent. Therefore, we investigated the genetic association between two VNTRs and ADHD in Korean children. We determined the VNTRs using PCR. We examined genotype and allele frequency differences between the experimental and control groups, along with the odds ratios, using Chi square and exact tests. We observed a significant association between the children with ADHD and the control group in the 10R/10R genotype of DAT1 VNTRs (p?=?0.025). In addition, the 11R allele of DAT1 VNTRs showed a higher frequency in the control group than in the ADHD group (p?=?0.023). Also, the short repeat (without 11R) and long repeat alleles (including 11R) were associated with ADHD (p?<?0.05). The analysis of DRD4 VNTRs revealed that the 2R allele is associated with ADHD (p?=?0.025). A significant result was also observed in long and short repeats (p?<?0.05). Additionally, ADHD subtypes showed that the DRD4 VNTRs are associated with combined and hyperactive-impulsive subtype groups (p?<?0.05). Therefore, our results suggest that DAT1 VNTRs and DRD4 VNTRs play a role in the genetic etiology of ADHD in Korean children.     

Combinatorial interaction between two human serotonin transporter gene variable number tandem repeats and their regulation by CTCF

Two distinct variable number tandem repeats (VNTRs) within the human serotonin transporter gene ( SLC6A4 ) have been implicated as predisposing factors for CNS disorders. The linked polymorphic region in the 5'-promoter exists as short ( s ) and long ( l ) alleles of a 22 or 23 bp elements. The second within intron 2 (Stin2) exists as three variants containing 9, 10 or 12 copies of a 16 or 17 bp element. These VNTRs, individually or in combination, supported differential reporter gene expression in rat neonate prefrontal cortical cultures. The level of reporter gene activity from the dual VNTR constructs indicated combinatorial action between the two domains. Chromatin immunoprecipitation demonstrated that both these VNTR domains can bind the CCCTC-binding factor and this correlated with the ability of exogenously supplied CCCTC-binding factor to modulate the expression supported by these reporter gene constructs. We suggest that the potential for interaction between multiple polymorphic domains should be incorporated into genetic association studies.     

A Computer Simulation Study of Vntr Population Genetics: Constrained Recombination Rules Out the Infinite Alleles Model

Extensive allelic diversity in variable numbers of tandem repeats (VNTRs) has been discovered in the human genome. For population genetic studies of VNTRs, such as forensic applications, it is important to know whether a neutral mutation-drift balance of VNTR polymorphism can be represented by the infinite alleles model. The assumption of the infinite alleles model that each new mutant is unique is very likely to be violated by unequal sister chromatid exchange (USCE), the primary process believed to generate VNTR mutants. We show that increasing both mutation rates and misalignment constraint for intrachromosomal recombination in a computer simulation model reduces simulated VNTR diversity below the expectations of the infinite alleles model. Maximal constraint, represented as slippage of single repeats, reduces simulated VNTR diversity to levels expected from the stepwise mutation model. Although misalignment rule is the more important variable, mutation rate also has an effect. At moderate rates of USCE, simulated VNTR diversity fluctuates around infinite alleles expectation. However, if rates of USCE are high, as for hypervariable VNTRs, simulated VNTR diversity is consistently lower than predicted by the infinite alleles model. This has been observed for many VNTRs and accounted for by technical problems in distinguishing alleles of neighboring size classes. We use sampling theory to confirm the intrinsically poor fit to the infinite alleles model of both simulated VNTR diversity and observed VNTR polymorphisms sampled from two Papua New Guinean populations.     

甜菜属厚皮菜SWTY-1细胞质VNTRs多态性分析

利用数目可变串联重复序列(Variable Number of Tandem Repeats,VNTRs)微卫星标记方法,对重庆厚皮菜甜菜材料SWTY-1群体中100个单株的细胞质线粒体DNA片段中TR2位点VNTRs片段多态性进行分析。结果显示97个单株线粒体TR2位点微卫星串联重复序列均为3拷贝,与普通糖甜菜一致;3个单株线粒体TR2位点微卫星串联重复序列为6拷贝,发现甜菜属厚皮菜细胞质TR2位点VNTRs存在多态性,在该群体中发现了不同于甜菜栽培种新的细胞质单株。对该群体材料100个单株的抽薹及结籽进行观测,结果显示微卫星串联重复序列为6拷贝的变异植株中2个单株花期未抽苔开花,1株抽苔晚未形成正常种子;细胞质TR2位点VNTRs片段拷贝数为3的植株中2个单株未能正常抽薹,其他植株均正常抽薹结籽。     

Variable-number tandem repeats as molecular markers for biotypes of Pasteuria ramosa in Daphnia spp

Variable-number tandem repeats (VNTRs) have been identified in populations of Pasteuria ramosa, a castrating endobacterium of Daphnia species. The allelic polymorphisms at 14 loci in laboratory and geographically diverse soil samples showed that VNTRs may serve as biomarkers for the genetic characterization of P. ramosa isolates.     

Characterization of a set of variable number of tandem repeat markers conserved in bovidae.

Screening purpose-built libraries with minisatellite probes, we have isolated 36 bovine variable number of tandem repeat markers (VNTRs) characterized by a mean heterozygosity of 59.3 within the American Holstein breed. Matching probabilities and exclusion powers were estimated by Monte-Carlo simulation, showing that the top 5 to 10 markers could be used as a very efficient DNA-based system for individual identification and paternity diagnosis. The isolated VNTR systems should contribute significantly to the establishment of a bovine primary DNA marker map. Linkage analysis, use of somatic cell hybrids, and in situ hybridization demonstrate that these bovine VNTRs are scattered throughout the bovine genome, without evidence for proterminal confinement as in the human, and that at least some of them are organized as clusters. Moreover, Southern blot analysis and in situ hybridization demonstrate conservation of sequence and map location of minisatellites within Bovidae.     

Polymerase chain reaction amplification of two polymorphic simple repeat sequences within the von Willebrand factor gene: application to family studies in von Willebrand disease

Summary We have used the polymerase chain reaction to amplify two variable number of tandem repeats (VNTRs) within a region of repetitive DNA located in intron 40 of the von Willebrand factor (vWf) gene. Heterozygosity for VNTR I was observed in 30 out of 39 normal unrelated individuals tested (77%), and for VNTR II in 29 out of 44 (66%) similar individuals. Family studies were carried out on 11 kindreds with von Willebrand disease (vWD). Ten of these families were found to be informative for one or other of the VNTRs or for a combination of data from both VNTRs. This method can be used for antenatal diagnosis and for carrier diagnosis in recessive forms of vWD. It is also useful for tracking the gene associated with vWD in type I families where there may be one or more individuals with a phenotypically uncertain diagnosis.     

应用变性高效液相色谱技术建立胸苷酸合成酶基因多态检测平台

TS基因5′非翻译区(5′ untranslation region, 5′UTR)增强子区域(TS enhancer region, TSER)存在28 bp的2次(2R)、3次(3R)的串联重复多态, 在3R等位基因第二次重复中还存在一个G→C的单核苷酸多态性(single nucleotide polymorphisms, SNPs), 同时在3′非翻译区(3′ untranslation region, 3′ UTR)存在6个碱基片段缺失/插入多态。这些多态形式的存在影响了TS基因mRNA的稳定和翻译效率, 并可导致不同TS基因型肿瘤患者对以5-fuorouracil (5-FU)为基础的化疗疗效产生差异。为提高TS基因型临床检测的效率和准确性, 方便、快捷、准确和自动化区分各种纯合及杂合基因型, 设计多重PCR反应, 同时扩增TS基因5′ 和3′ 非翻译区多态所处片段。利用DHPLC技术建立TS基因多态性检测平台, 在非变性条件下, 通过优化DHPLC 洗脱梯度, 同时检测5′ TSER区的串联重复多态和3′ UTR片段长度多态; 在变性条件下, 检测5′ TSER区单核苷酸多态。同时采用PCR-RFLP和DNA 测序方法, 验证DHPLC分析结果。     

Polymerase chain reaction (PCR) amplification of hypervariable genomic sequences.

This paper describes a rapid method for VNTRs (variable number of tandem repeats) typing using polymerase chain reaction (PCR). Three VNTRs (YNZ22, Apo B, MCT118) were amplified and alleles mendelian segregation was confirmed. We also demonstrate their applicability to paternity testing and forensic purposes.