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.     

Novel repeat polymorphisms of the dopaminergic neurotransmitter genes among dogs and wolves

Genetic polymorphisms of the neurotransmission systems are intensively studied in the human because of a possible influence on personality traits and the risk of psychiatric disorders. The investigation of genetic variations of the dog genome has recently been a promising approach, as a considerable similarity can be observed between dogs and humans, in both genetic and social aspects, suggesting that the dog could become an appropriate animal model of human behavioral genetic studies. The aim of our study was the identification and analysis of variable number of tandem repeats polymorphisms (VNTRs) in the genes of the dopaminergic neurotransmitter system of dogs. The in silico search was followed by the development of PCR-based techniques for the analysis of the putative VNTRs. Highly variable repetitive sequence regions were found in the tyrosine hydroxylase (TH), dopamine transporter (DAT), and dopamine β-hydroxylase (DBH) genes. Allele frequency and genotype distribution of these novel polymorphisms together with the exon 3 and exon 1 VNTR of the dopamine D4 receptor gene were determined in a large sample involving four dog breeds (German Shepherd, Belgian Tervueren, Groenandael, and Malinois) and European Grey Wolves. A significant difference of allele and genotype frequencies was demonstrated among the analyzed breeds; therefore, an association analysis was also carried out between the activity–impulsivity phenotype and the described VNTRs. Preliminary findings are presented that polymorphisms of the DRD4, DBH, and DAT genes can be associated with attention deficit among Belgian Tervuerens.     

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/.     

Evolutionary structure of Plasmodium falciparum major variant surface antigen genes in South America: Implications for epidemic transmission and surveillance

Strong founder effects resulting from human migration out of Africa have led to geographic variation in single nucleotide polymorphisms (SNPs) and microsatellites (MS) of the malaria parasite, Plasmodium falciparum. This is particularly striking in South America where two major founder populations of P. falciparum have been identified that are presumed to have arisen from the transatlantic slave trade. Given the importance of the major variant surface antigen of the blood stages of P. falciparum as both a virulence factor and target of immunity, we decided to investigate the population genetics of the genes encoding “Plasmodium falciparum Erythrocyte Membrane Protein 1” (PfEMP1) among several countries in South America, in order to evaluate the transmission patterns of malaria in this continent. Deep sequencing of the DBLα domain of var genes from 128 P. falciparum isolates from five locations in South America was completed using a 454 high throughput sequencing protocol. Striking geographic variation in var DBLα sequences, similar to that seen for SNPs and MS markers, was observed. Colombia and French Guiana had distinct var DBLα sequences, whereas Peru and Venezuela showed an admixture. The importance of such geographic variation to herd immunity and malaria vaccination is discussed.     

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.     

Tandem repeat copy-number variation in protein-coding regions of human genes

Background

Tandem repeat variation in protein-coding regions will alter protein length and may introduce frameshifts. Tandem repeat variants are associated with variation in pathogenicity in bacteria and with human disease. We characterized tandem repeat polymorphism in human proteins, using the UniGene database, and tested whether these were associated with host defense roles.

Results

Protein-coding tandem repeat copy-number polymorphisms were detected in 249 tandem repeats found in 218 UniGene clusters; observed length differences ranged from 2 to 144 nucleotides, with unit copy lengths ranging from 2 to 57. This corresponded to 1.59% (218/13,749) of proteins investigated carrying detectable polymorphisms in the copy-number of protein-coding tandem repeats. We found no evidence that tandem repeat copy-number polymorphism was significantly elevated in defense-response proteins (p = 0.882). An association with the Gene Ontology term 'protein-binding' remained significant after covariate adjustment and correction for multiple testing. Combining this analysis with previous experimental evaluations of tandem repeat polymorphism, we estimate the approximate mean frequency of tandem repeat polymorphisms in human proteins to be 6%. Because 13.9% of the polymorphisms were not a multiple of three nucleotides, up to 1% of proteins may contain frameshifting tandem repeat polymorphisms.

Conclusion

Around 1 in 20 human proteins are likely to contain tandem repeat copy-number polymorphisms within coding regions. Such polymorphisms are not more frequent among defense-response proteins; their prevalence among protein-binding proteins may reflect lower selective constraints on their structural modification. The impact of frameshifting and longer copy-number variants on protein function and disease merits further investigation.     

Optimizing comparative genomic hybridization probes for genotyping and SNP detection in Plasmodium falciparum

Microarray-based comparative genomic hybridizations (CGH) interrogate genomic DNA to identify structural differences such as amplifications and deletions that are easily detected as large signal aberrations. Subtle signal deviations caused by single nucleotide polymorphisms (SNPs) can also be detected but is challenged by a high AT content (81%) in P. falciparum. We compared genome-wide CGH signal to sequence polymorphisms between parasite strains 3D7, HB3, and Dd2 using NimbleGen microarrays. From 23,191 SNPs (excluding var/rif/stevor genes), our CGH probe set detected SNPs with > 99.9% specificity but low (< 10%) sensitivity. Probe length, melting temperature, GC content, SNP location in the probe, mutation type, and hairpin structures affected SNP sensitivity. Previously unrecognized variable number tandem repeats (VNTRs) also were detected by this method. These findings will guide the redesign of a probe set to optimize an openly available CGH microarray platform for high-resolution genotyping suitable for population genomics studies.     

Conserved sequences flank variable tandem repeats in two S-antigen genes of Plasmodium falciparum

We describe the isolation of two chromosomal DNA fragments from Plasmodium falciparum. These fragments encode the antigenically distinct S antigens of two different P. falciparum isolates, namely FC27 from Papua New Guinea and NF7 from Ghana. The complete nucleotide sequences of both fragments are presented. The fragments are homologous over most of their lengths, including the entire regions flanking the protein coding sequences. Whereas the N- and C-terminal portions of sequences encoding the S antigens are homologous, major portions of the coding sequences are not. The nonhomologous regions are comprised of tandemly repeated sequences, of 33 bp in FC27 and predominantly of 24 bp in NF7. The 33 bp tandem repeats encoded by the FC27 S-antigen gene could not be detected in the NF7 genome. Conversely, the 24 bp tandem repeats encoded by the NF7 S-antigen gene could not be detected in the FC27 genome. The pattern of sequence variation within the repeats of both genes suggests a mechanism for the generation of S-antigen diversity.     

Toward a High-ResolutionPlasmodium falciparumLinkage Map: Polymorphic Markers from Hundreds of Simple Sequence Repeats

A total of 507 simple sequence repeats (SSRs or “microsatellites”) were identified fromPlasmodium falciparumsequences in GenBank and from inserts in a genomic DNA library. Oligonucleotide primers from sequences that flank 224 of these SSRs were synthesized and used in PCR assays to test for simple sequence length polymorphisms (SSLPs). Of the 224 SSRs, 188 showed SSLPs among 12 differentP. falciparumlines; 116 of these SSLPs were assigned to chromosome linkage groups by physical mapping and by comparing their inheritance patterns against those of restriction fragment length polymorphism markers in a genetic cross (HB3×Dd2). The predominant SSLPs inP. falciparumwere found to contain [TA]_n, [T]_n, and [TAA]_n, a feature that is reminiscent of plant genomes and is consistent with the proposed algal-like origin of malaria parasites. Since such SSLPs are abundant and readily isolated, they are a powerful resource for genetic analysis ofP. falciparum.     

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.     

PCR primed with VNTR core sequences yields species specific patterns and hypervariable probes.

The use of genomic DNA-based techniques in ecological and evolutionary studies has been limited by the availability of suitable probes for species of interest due to the technical difficulty of isolating and applying such probes. We have developed a simple technique that directs polymerase chain reaction (PCR) amplification to regions rich in variable number of tandem repeats (VNTRs). By using published VNTR core sequences as primers in PCRs, fragments were amplified that showed little variation within a species, but did show differences between species. When the amplified fragments were used as probes with genomic DNA Southern blots they produced hypervariable single-locus or few-locus patterns in fish, birds, and humans. We have named this procedure as Directed Amplification of Minisatellite-region DNA (DAMD).     

Prediction of tandem repeat polymorphisms in the coding region of dog genome

After the dog genome was sequenced, an increasing number of studies involving genetic research of dogs have been conducted to understand gene functions and mammalian evolution. To study the genetic diversity in dogs and other mammals, genetic markers linked to function and conserved in wide lineages are necessary. Thus far, few polymorphic markers have been used in dogs. In this study, we surveyed the entire dog genome and predicted a total of 109 tandem repeats (TRs) located on the protein coding region that may be polymorphic by our prediction model. We selected 10 TRs that may be related to neurophysiology and neural developments, and tested them in 167 individuals of 8 dog breeds: 5 European dog breeds (Beagle, Golden Retriever, Labrador Retriever, German Shepherd, and Toy Poodle) and 3 Japanese dog breeds (Japanese Spitz, Shiba, and Shikoku). Among the tested TRs, nine were polymorphic indicating that 90% of the TRs were successfully predicted to be polymorphic. PCR fragments of the TRs were amplified from dog brain cDNA, showing their expression in the dog brain. Our results provide abundant opportunities for the study of phenotypic variations in dogs, and our prediction method for variable number of tandem repeats (VNTRs) can be applied to any other animal genome sequences for the survey of functional and polymorphic markers.     

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.     

Identification of complex DNA polymorphisms based on variable number of tandem repeats (VNTR) and restriction site polymorphism

Summary Restriction fragment length polymorphisms (RFLPs) of genomic DNA are generally attributable to base changes that create or abolish restriction endonuclease sites or to nucleotide sequence insertions or deletions that alter the distance separating two restriction sites. Minisatellite or variable number of tandem repeats (VNTR) markers are prominent examples of the latter type of polymorphism. In this report, we describe complex DNA polymorphisms that are due both to the presence of VNTRs as well as to altered restriction endonuclease sites. A strategy for identifying such polymorphisms and resolving their component allelic fragments is demonstrated.     

Influence of interleukin-4 gene polymorphisms and interleukin-4 serum level on susceptibility and severity of rheumatoid arthritis in Egyptian population

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease in which interleukin-4 (IL-4) plays an important role. This study aimed to investigate the influence of IL-4 variable number of tandem repeats (VNTRs) and IL-4-590 promoter polymorphisms on RA susceptibility, activity and severity in Egyptian population.Materials and methodsOne hundred and seventy-two RA patients and 172 controls were enrolled in this study. IL-4 VNTR and IL-4-590 promoter polymorphisms were genotyped using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Serum IL-4 and anti-cyclic citrullinated peptides (anti-CCPs) antibody concentrations were measured by enzyme linked immunosorbent assay (ELISA).ResultsSubjects with IL-4-590 TT genotype were significantly more likely to develop RA. IL-4 VNTR 1/1 genotype, IL-4-590 TT and CT genotypes were significantly more associated with erosive RA and positive anti-CCP antibody. RA severity parameters were significantly increased, while, IL-4 level was significantly decreased in RA patients with IL-4 VNTR 1/1 and IL-4-590 TT genotypes. Only patients with IL-4-590 TT genotype showed a significant increase of all RA activity parameters.ConclusionIL-4 VNTR and IL-4-590 promoter polymorphisms may be helpful for assessing RA severity in Egyptian population. Moreover, IL-4-590 promoter polymorphism may be associated with increased risk and activity of RA.     

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.