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Allele and genotype frequencies of the three tetrameric short tandem repeat (STR) 1 Oci D351744 D1251090 and D185849 were analyzed in a German sample population using a multiplex PCR and electrophoresis of amplified products in denaturing gels followed by silver staining. Testing for Hardy-Weinberg equilibrium showed no significant deviation at the three loci.
The human cystatin B gene contains a variable number of 12-bp tandem repeats in its promoter region, of which the common alleles contain two or three copies and unusual expansion causes progressive myoclonus epilepsy of the Unverricht-Lundborg type. We undertook a comprehensive analysis of the genomic sequence to address the evolutionary events of this variable repeat. By examination of a contiguous genome sequence spanning 5.0 kb and linkage analysis of detected polymorphic changes, we identified six major intragenic haplotypes in unrelated Japanese subjects. The number of normal repeats was closely correlated with these alleles, indicating that changes in the array should be comparatively rare events during human evolution. To examine the origin of the repeat array further, we also analyzed five primate genomes. Repetitive polymorphism was unlikely in hominoids, and the array originated with the dodecamer itself in the course of primate evolution. The variability conceivably developed after the separation to humans.
The simian virus 40 (SV40) 72-base pair (bp) tandem-repeated sequences have a crucial role as an activator element in viral gene expression. We replaced the SV40 72-bp repeat with a 72-bp repeat derived from the long terminal repeat (LTR) of cloned Moloney murine sarcoma virus (MSV) DNA. Although there is no detectable sequence homology to SV40, the MSV repeats can substitute functionally for the SV40 repeats and generate a viable virus in monkey kidney cells.
The serine- and threonine-rich tandem repeat (TR) units that make up the characteristic feature of mucin glycoproteins are often polymorphic with substantial genetic variation in TR number. The precise effect of TR number on O-glycosylation is not fully understood, although the TR number of several mucins may be associated with apparent susceptibility to certain human diseases. To evaluate the contribution of TR number to O-glycosylation, we generated a series of chimeric mucins carrying increasing numbers of TR units from the MUC5B mucin in the context of an epitope-tagged MUC1 mucin backbone. These mucins were expressed in Caco2 colon carcinoma cell clones and purified by immunoprecipitation. O-Glycosylation was investigated by western blotting with antibodies to known carbohydrate structures and by fast atom bombardment-mass spectrometry. Additional carbohydrate epitopes were detected with antibodies on chimeric mucins with a higher TR number in comparison to those with fewer TRs. Using mass spectrometry, higher-molecular-weight glycans were detected more frequently on the mucins with extended TRs compared to those with fewer TRs. However no novel carbohydrate structures were seen, suggesting that TR number does not affect the specificity of O-glycosylation.
The tandem repeat of the MUC1 protein core is a major site of O-glycosylation that is catalyzed by several polypeptide GalNAc-transferases. To define structural features of the peptide substrates that contribute to acceptor substrate efficiency, solution structures of the 21-residue peptide AHGVTSAPDTRPAPGSTAPPA (AHG21) from the MUC1 protein core and four isoforms, glycosylated with alpha-N-acetylgalactosamine on corresponding Thr residues, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5,T17), were investigated by NMR spectroscopy and computational methods. NMR studies revealed that sugar attachment affected the conformational equilibrium of the peptide backbone near the glycosylated Thr residues. The clustering of the low-energy conformations for nonglycosylated and glycosylated counterparts within the VTSA, DTR, and GSTA fragments (including all sites of potential glycosylation catalyzed by GalNAc-T1, -T2, and -T4 transferases) showed that the glycosylated peptides display distinct structural propensities that may explain, in part, the differences in substrate specificities exhibited by these polypeptide GalNAc-transferases.
Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%--and by 23% in combination with spoligotyping--among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold--by threefold in combination with spoligotyping--under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.
Short tandem repeat (STR) variation is an often overlooked source of variation between genomes. STRs comprise about 3% of the human genome and are highly polymorphic. Some cause Mendelian disease, and others affect gene expression. Their contribution to common disease is not well-understood, but recent software tools designed to genotype STRs using short read sequencing data will help address this. Here, we compare software that genotypes common STRs and rarer STR expansions genome-wide, with the aim of applying them to population-scale genomes. By using the Genome-In-A-Bottle (GIAB) consortium and 1000 Genomes Project short-read sequencing data, we compare performance in terms of sequence length, depth, computing resources needed, genotyping accuracy and number of STRs genotyped. To ensure broad applicability of our findings, we also measure genotyping performance against a set of genomes from clinical samples with known STR expansions, and a set of STRs commonly used for forensic identification. We find that HipSTR, ExpansionHunter and GangSTR perform well in genotyping common STRs, including the CODIS 13 core STRs used for forensic analysis. GangSTR and ExpansionHunter outperform HipSTR for genotyping call rate and memory usage. ExpansionHunter denovo (EHdn), STRling and GangSTR outperformed STRetch for detecting expanded STRs, and EHdn and STRling used considerably less processor time compared to GangSTR. Analysis on shared genomic sequence data provided by the GIAB consortium allows future performance comparisons of new software approaches on a common set of data, facilitating comparisons and allowing researchers to choose the best software that fulfils their needs.
OBJECTIVE: To investigate whether DRD4 exon III48 bp variant number tandem repeat(VNTR) polymorphism is associated with tic disorder. METHODS: One hundred and twenty-two nucleus families were collected using Structured clinical interview for genetic study of Tourette syndrome and related disorders for family-based association analysis of tic disorder and DRD4 exon III 48bp VNTR polymorphism. One hundred and twenty-two trios were divided into two groups: tic disorder group (82 trios of Tourette syndrome or chronic tic disorder, TS&CT) and tic disorder accompanied with attention deficit and hyperactivity disorder (ADHD) group (40 trios of Tourette syndrome or chronic tic disorder accompanied with ADHD, TS&ADHD). Transmission disequilibrium test (TDT), in addition to polymerase chain reaction and VNTR technique were conducted in 122 trios. RESULTS: There exist 5 alleles at this polymorphic locus in this sample including DRD4 exon III 48bp 2-6 repeats. No transmission disequilibrium was found between DRD4 exon III 48 bp VNTR and tic disorder (chi square=7.44, P 0.12); however, when the sample was divided into two groups, transmission disequilibrium was noticed between the cases of TS&ADHD and this locus by overall allele-wise analysis (chi square=11.74, P 0.02), and there exists transmission disequilibrium exclusively between 5 or 6 repeats of 48bp VNTR(longer alleles) by allele-wise analysis (chi square=10.57, P 0.032, chi square=6.13, P 0.01). No transmission disequilibrium was seen between TS&CT and DRD4 exon III 48bp VNTR(chi square=3.38, P 0.50). CONCLUSION: The results of this study have revealed an association between the longer alleles of DRD4 exon III 48bp VNTR polymorphism and tic disorder accompanied with ADHD, thus suggesting a possible genetic risk factor of tic disorder accompanied with ADHD in Chinese.
This paper presents allele frequencies of two short tandem repeats (CD4 and F13A1) in three anthropologically defined populations: Sardinians (Italy), Corsicans (France) and Piaroa Indians (Venezuela). The comparison shows some relevant differences both in number and distribution of the CD4 and F13A1 alleles.
Two short tandem repeat (STR) systems, HumTPO and HumLPL, were investigated in 218 unrelated Japanese living in Gunma and Fukui Prefectures by means of discontinuous, horizontal polyacrylamide gel electrophoresis and silver staining. Five and six alleles were identified at the HumTPO and HumLPL loci, respectively. No deviations from the Hardy-Weinberg equilibrium or differences in genotype frequency were found between the two Japanese populations in the two systems. Our detection methods offered several advantages including time-saving and safe electrophoretic procedures, and fast and easy silver-staining, in comparison with standard and conventional STR analysis using vertical polyacrylamide gel electrophoresis.
MUC20, an upregulated novel gene in the renal tissues of patients with IgA nephropathy (IgAN), was recently identified. The variable number of tandem repeats (VNTR) polymorphism of the MUC20 gene was detected in several cell lines. In the present study, we investigated a possible association of MUC20 VNTR polymorphism with the clinical manifestations and progression in patients with IgAN. A total of 1,147 Chinese subjects, including 657 patients with IgAN and 490 geographically matched healthy controls, were involved in this investigation. One hundred and thirty-seven patients had been followed up for 60.6 +/- 22.4 months. MUC20 VNTR polymorphism was genotyped by polymerase chain reaction amplification and confirmed by sequencing. The alleles were divided into two groups according to the repeat times of MUC20 VNTR, i.e. small alleles (VNTR repeat times < or = 3) and large alleles (VNTR repeat times >3), and the genotypes of subjects were classified into SS, SL and LL groups. The frequencies of the alleles and genotypes of MUC20 VNTR polymorphisms did not differ between patients with IgAN and healthy controls. Additionally, there was no significant difference in the clinical features. Furthermore, IgAN patients with SL/LL genotypes had a higher risk of decline in renal function (odds ratio 20.9; 95% confidence interval 2.6-168.1; p = 0.004) than those with SS genotypes. The present study revealed that there is no association between the VNTR polymorphism of the MUC20 gene and the clinical manifestations in IgAN patients at the time of renal biopsy. However, IgAN patients with SL/LL genotypes had a higher risk of the progression to end-stage renal disease.
Thirteen tetranucleotide and 2 pentanucleotide repeat units were analyzed in 120 unrelated individuals of Patel and other communities of Gujarat, India. Allele frequency data obtained from the analysis of 15 short tandem repeat markers of the population were found to be satisfying Hardy-Weinberg equilibrium, with marginal deviations. Departures from Hardy-Weinberg equilibrium were observed in Patel communities at locus vWA and for that of the other communities at locus D7S820 and at locus TPOX. The power of discrimination values on an average fall within the range of 0.718 and 0.870, with deviations at locus D3S1358 showing a value of 0.400 for Patels. The value ranged between 0.709 and 0.869, with slight variations among the studied alleles in the other group. Thus, the 15 markers selected for this study were found to be highly suitable in human identification and for providing information on genetic polymorphism of the population of Gujarat.
OBJECTIVE: To get preliminary genotype and allele frequency distributions of D6S366, D6S477, D12S375, D12S391 and PLA2A loci in Chinese Han population in Chengdu area and to validate more short Tandem repeat (STR) systems for forensic application. METHODS: The polymerase chain reaction (PCR) was used. Five STR systems (D6S366, D6S477, D12S375, D12S391 and PLA2A) were amplified on DNA samples, which were extracted from 108 unrelated individuals EDTA-blood by Phenol/Chloroform method. The PCR products were analyzed by PAG vertical electrophoresis. RESULTS: Seven alleles were found at D6S366 locus, nine alleles at D6S477 locus, five alleles at D12S375 locus, nine alleles at D12S391 locus and seven alleles at PLA2A locus. No deviations from Hardy-Weinberg equilibrium were observed. The heterozygosities observed were 0.69, 0.78, 0.81, 0.68 and 0.79 for D6S366, D6S477, D12S375, D12S391 and PLA2A respectively. The chances of exclusion were 0.3621, 0.6004, 0.4581, 0.7014 and 0.5589 and the discriminating powers were 0.79, 0.93, 0.86, 0.96 and 0.91. CONCLUSION: All of the five loci in this study were useful markers for individual identification and paternity test and for genetics purposes.
The purpose of this study was to evaluate the relationship between attention deficit hyperactivity disorder (ADHD) and polymorphism of the two regions of the 5-HTT gene [variable number of tandem repeats (VNTR) and 5-HTTLRR] in a sample of Turkish children. Using the PCR technique, these polymorphisms were assessed in 71 patients with ADHD and 128 healthy controls. The 5-HTTLPR S/S genotype was significantly lower in the patients than in the controls (p = 0.018). Homozygous and heterozygous L variant predominated in the ADHD group. But the VNTR STin2.12/12 genotype was significantly less found in the patients than in the controls (p = 0.001). There was no significant difference between the frequency of the short (S), long, 10, and 12 alleles of both groups. The lack of an S/S variant of 5-HTTLPR polymorphism of the STin2.12/12 variant of VNTR polymorphism appears to be associated with an increased risk of ADHD.
Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. To establish an efficient, accurate and fast diagnostic method for carrier detection and presymptomatic identification of WD in Chinese population, we studied haplotypes of short tandem repeat (STR) polymorphisms flanking the WD gene in 40 Chinese WD families. The results suggested that this genetic diagnosis system based on the four STR polymorphisms is of high value for the detection of potential carriers and WD homozygotes in families with at least one previously affected child. It is an efficient, accurate and fast diagnostic method that can be well suited for routine use in clinical laboratories.
OBJECTIVE: In order to determine the status of engraftment following allogeneic bone marrow transplantation (allo-BMT) and predict the outcome, short tandem repeats (STR) were analysed by fluorescence labeling polymerase chain reaction (PCR). METHOD: Fluorescence labeling PCR detecting STR 9 loci was established. DNA was extracted by monoclonal magnetic beads from recipients peripheral blood nucleated cells and labeled with four color fluorescences before and from 7 days to six months after allogeneic bone marrow transplantation. RESULTS: 1. STR and clinical feature of 12 patients after allogeneic bone marrow transplantation were analysed. In the ten patients with full donor chimeras, 8 received marrow grafts from HLA identical sibling donors, 1 from HLA identical unrelated donor and 1 from HLA haploidentical mother. All of the 10 patients had durable engraftment and survived disease-freely for 3-25 months of follow-up. 2. One patient received a HLA-AB loci mismatched graft and his STR showed mixed chimeras at day 30, and converted into full donor chimeras at day 60 after transplantation. 3. One patient received HLA haploidentical sibling donor stem cells failed to be engrafted and died 4 months later. CONCLUSION: Analysis of multi-loci of STR after allo-BMT provided a real indication of engraftment. Mixed chimeras after transplantation suggested that donor lymphocytes or bone marrow cells be re-infused to enhance the conversion from mixed chimeras to full donor chimeras. For the documentation of engraftment, analysis of STR was better than any other available methods.
BACKGROUND: Precise chimerism monitoring is important for the prediction of the success of allogeneic bone marrow transplantation (BMT). Most of the current procedures employed for chimerism follow-up with short tandem repeat (STR) markers are either time-consuming, labor-intensive, or use expensive assays, making it burdensome to perform large-scale studies of transplanted patients. AIM: To set-up a simple nonradioactive method to investigate a set of STR markers that could be used in the evaluation of chimerism status after allogeneic BMT. METHOD: Six dinucleotide STRs (D2S123, D5S107, CRTL1, D7S500, D11S1356, and TP53) were analyzed by touchdown (TD)-PCR followed by medium size non-denaturing polyacrylamide gel electrophoresis and silver staining. The sensitivity of the approach was evaluated by dilution competition assays. Peripheral blood samples were taken from a group of 50 healthy Argentinean donors, two transplanted patients, and their respective bone marrow donors. Buccal mucosa samples were also obtained from the BMT recipients. RESULTS: Four markers, D2S123, D7S500, D11S1356, and TP53, presented the highest heterozygosities (0.67-0.88) under our experimental system. A sensitivity of 0.8-1.6% for chimerism detection was consistently found for the different STR. The usefulness of these STR in chimerism analysis was illustrated with the screening of related siblings analyzing two transplanted patients with persistent mixed chimerism, which were previously studied by fluorescence in situ hybridization (FISH). Similar proportions of mixed chimerism were obtained with STR analysis compared with those estimated by FISH. DISCUSSION: To our knowledge, this was the first study of mixed chimerism using TD-PCR to achieve a highly specific STR amplification. This approach allows simple and accurate chimerism quantification because it avoids slippage of Taq polymerase on repeat stretches and prevents the differential amplification of the shorter allele. STR heterozygosities and the high level of sensitivity of this method demonstrated that this approach is not only very informative in this population, but is also rapid (taking less than 14 hours) and cost-efficient. CONCLUSION: The data confirms that this method is a useful tool applicable to routine large-scale STR genotyping and mixed chimerism analysis in low-complexity laboratories worldwide.
Blood samples from 489 unrelated Caucasian Mestizo and 252 individuals of African descent in Colombia were amplified and typed for three short tandem repeat (STR) markers (D12S1090, D3S1744, and D18S849). All markers conformed to Hardy-Weinberg equilibrium expectations in both populations studied. In addition, heterozygosity, mean exclusion chance, polymorphism information content, discrimination power, and the assumption of independence within and between loci were determined. The mean exclusion chance for all three STR markers is 0.9750 in the Caucasian Mestizo population and 0.9731 in the African Colombian Population. The discrimination power is 0.999925 and 0.999911 in the Caucasian Mestizo and African Colombian respectively.