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Variable Number Tandem Repeats (VNTRs) are tandem repeat (TR) loci that vary in copy number across a population. Using our program, VNTRseek, we analyzed human whole genome sequencing datasets from 2,770 individuals in order to detect minisatellite VNTRs, i.e., those with pattern sizes ≥7 bp. We detected 35,638 VNTR loci and classified 5,676 as commonly polymorphic (i.e., with non-reference alleles occurring in >5% of the population). Commonly polymorphic VNTR loci were found to be enriched in genomic regions with regulatory function, i.e., transcription start sites and enhancers. Investigation of the commonly polymorphic VNTRs in the context of population ancestry revealed that 1,096 loci contained population-specific alleles and that those could be used to classify individuals into super-populations with near-perfect accuracy. Search for quantitative trait loci (eQTLs), among the VNTRs proximal to genes, indicated that in 187 genes expression differences correlated with VNTR genotype. We validated our predictions in several ways, including experimentally, through the identification of predicted alleles in long reads, and by comparisons showing consistency between sequencing platforms. This study is the most comprehensive analysis of minisatellite VNTRs in the human population to date.

We found evidence of late-onset Alzheimer disease (LOAD)-associated genetic polymorphism within an exon of Mucin 6 (MUC6) and immediately downstream from another gene: Adaptor Related Protein Complex 2 Subunit Alpha 2 (AP2A2). PCR analyses on genomic DNA samples confirmed that the size of the MUC6 variable number tandem repeat (VNTR) region was highly polymorphic. In a cohort of autopsied subjects with quantitative digital pathology data (n = 119), the size of the polymorphic region was associated with the severity of pTau pathology in neocortex. In a separate replication cohort of autopsied subjects (n = 173), more pTau pathology was again observed in subjects with longer VNTR regions (p = 0.031). Unlike MUC6, AP2A2 is highly expressed in human brain. AP2A2 expression was lower in a subset analysis of brain samples from persons with longer versus shorter VNTR regions (p = 0.014 normalizing with AP2B1 expression). Double-label immunofluorescence studies showed that AP2A2 protein often colocalized with neurofibrillary tangles in LOAD but was not colocalized with pTau proteinopathy in progressive supranuclear palsy, or with TDP-43 proteinopathy. In summary, polymorphism in a repeat-rich region near AP2A2 was associated with neocortical pTau proteinopathy (because of the unique repeats, prior genome-wide association studies were probably unable to detect this association), and AP2A2 was often colocalized with neurofibrillary tangles in LOAD.

Variable number tandem repeats (VNTRs) are composed of consecutive repetitive DNA with hypervariable repeat count and composition. They include protein coding sequences and associations with clinical disorders. It has been difficult to incorporate VNTR analysis in disease studies that use short-read sequencing because the traditional approach of mapping to the human reference is less effective for repetitive and divergent sequences. In this work, we solve VNTR mapping for short reads with a repeat-pangenome graph (RPGG), a data structure that encodes both the population diversity and repeat structure of VNTR loci from multiple haplotype-resolved assemblies. We develop software to build a RPGG, and use the RPGG to estimate VNTR composition with short reads. We use this to discover VNTRs with length stratified by continental population, and expression quantitative trait loci, indicating that RPGG analysis of VNTRs will be critical for future studies of diversity and disease., Variable number tandem repeats (VNTRs) are difficult to analyze by short-read sequencing in disease studies. Here, the authors describe a VNTR mapping strategy for short-read analyses using a repeat pangenome graph. This method will help elucidate the contribution of VNTRs to diversity and disease.

The evolutionary conserved genomic sequences that have acquired significantly increased number of nucleotide substitutions specifically in the human lineage, called human accelerated regions (HARs), have been identified as candidate genomic regions that have contributed to the evolution of human-specific traits. A number of HARs were indeed shown to have novel enhancer activity and be associated with human-specific brain development and with cognition and social behavior. It is therefore of great importance to investigate the details of genomic function of each HAR to understand the roles of HARs as critical contributors to the genetic basis of human evolution. In this study, we identified a previously unannotated brain-expressed noncoding RNA gene, HSTR1, at a human-specific tandem repeat locus. Notably, the 5' flanking sequence of HSTR1 showed the signature of HARs and the dramatic human-specific enhancement of promoter activity, providing the evidence of positive selection to increase the expression level of HSTR1 during human evolution. We also revealed that the tandem repeat number in HSTR1 was highly variable among individual alleles and affected the stability of HSTR1 RNA, suggesting variation in the activity of HSTR1 between human individuals. Our work thus provides a novel candidate gene that potentially contributed to the evolution of the human brain. It may also underpin some of the variation between human brains.

Molecular epidemiological studies of Mycobacterium kansasii are hampered by the lack of highly-discriminatory genotyping modalities. The purpose of this study was to design a new, high-resolution fingerprinting method for M. kansasii. Complete genome sequence of the M. kansasii ATCC 12478 reference strain was searched for satellite-like repetitive DNA elements comprising tandem repeats. A total of 24 variable-number tandem repeat (VNTR) loci were identified with potential discriminatory capacity. Of these, 17 were used to study polymorphism among 67 M. kansasii strains representing six subtypes (I-VI). The results of VNTR typing were compared with those of pulsed-field gel electrophoresis (PFGE) with AsnI digestion. Six VNTRs i.e. (VNTR 1, 2, 8, 14, 20 and 23) allow to differentiate analyzed strains with the same discriminatory capacities as use of a 17-loci panel. VNTR typing and PFGE in conjunction revealed 45 distinct patterns, including 11 clusters with 33 isolates and 34 unique patterns. The Hunter-Gaston’s discriminatory index was 0.95 and 0.66 for PFGE and VNTR typing respectively, and 0.97 for the two methods combined. In conclusion, this study delivers a new typing scheme, based on VNTR polymorphism, and recommends it as a first-line test prior to PFGE analysis in a two-step typing strategy for M. kansasii.

SUMMARYHuman tuberculosis (TB) caused byMycobacterium bovissurveillance is affected by a lack of data. The aims of the present study were: (i) to estimate the proportion of human TB caused byM. bovisover a period of 5 years in Bologna, Northern Italy, which, like most Western European countries, has been declared bovine TB-free; (ii) to compare the genetic profiles ofM. bovisstrains identified in humans with those circulating in cattle in the last 15 years in Italy. Among 511 TB patients, the proportion of human TB caused byM. boviswas 1·76%, significantly associated to extra-pulmonary localization (P= 0·004) and to being elderly (P< 0·001) and Italy-born (P= 0·036). The molecular epidemiology analysis by spoligotyping and Multilocus Variable Tandem Repeat Analysis confirmed that mostM. bovisstrains from Italy-born patients matched those circulating in cattle herds in Italy between 2001 and 2016. Two cases ofMycobacterium bovisBCG infection were also characterized. In conclusion, the rate of human TB caused byM. boviswas not negligible, highlighting the relevance of molecular typing in evaluating the effectiveness of programmes designed to eradicate TB in cattle in Italy.

We developed a novel method, PyroTyping, for discrimination of Mycobacterium tuberculosis isolates combining pyrosequencing and IS6110 polymorphism. A total of 100 isolates were analysed with IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, mycobacterial interspersed repetitive units – variable number tandem repeats (MIRU-VNTR), and PyroTyping. PyroTyping results regarding clustering or discrimination of the isolates were highly concordant with the other typing methods performed. PyroTyping is more rapid than RFLP and presents the same discriminatory power, thus, it may be useful for taking timely decisions for tuberculosis control.

// Philipp Hofer 1 , Cornelia Zochmeister 1 , Christian Behm 1 , Stefanie Brezina 1 , Andreas Baierl 2 , Angelina Doriguzzi 1 , Vanita Vanas 1 , Klaus Holzmann 1 , Hedwig Sutterluty-Fall 1 , Andrea Gsur 1 1 Medical University of Vienna, Institute of Cancer Research, A-1090 Vienna, Austria 2 University of Vienna, Department of Statistics and Operations Research, A-1010 Vienna, Austria Correspondence to: Andrea Gsur, email: andrea.gsur@meduniwien.ac.at Keywords: genetic variation, MNS16A, functional polymorphism, telomerase, TERT regulation Received: September 23, 2016 Accepted: February 21, 2017 Published: March 03, 2017 ABSTRACT MNS16A, a functional polymorphic tandem repeat minisatellite, is located in the promoter region of an antisense transcript of the human telomerase reverse transcriptase gene. MNS16A promoter activity depends on the variable number of tandem repeats (VNTR) presenting varying numbers of transcription factor binding sites for GATA binding protein 1. Although MNS16A has been investigated in multiple cancer epidemiology studies with incongruent findings, functional data of only two VNTRs (VNTR-243 and VNTR-302) were available thus far, linking the shorter VNTR to higher promoter activity. For the first time, we investigated promoter activity of all six VNTRs of MNS16A in cell lines of colorectal, lung and prostate cancer using Luciferase reporter assay. In all investigated cell lines shorter VNTRs showed higher promoter activity. While this anticipated indirect linear relationship was affirmed for colorectal cancer SW480 ( P = 0.006), a piecewise linear regression model provided significantly better model fit in lung cancer A-427 ( P = 6.9 × 10 –9 ) and prostate cancer LNCaP ( P = 0.039). In silico search for transcription factor binding sites in MNS16A core repeat element suggested a higher degree of complexity involving X-box binding protein 1, general transcription factor II–I, and glucocorticoid receptor alpha in addition to GATA binding protein 1. Further functional studies in additional cancers are requested to extend our knowledge of MNS16A functionality uncovering potential cancer type-specific differences. Risk alleles may vary in different malignancies and their determination in vitro could be relevant for interpretation of genotype data.

Introduction: Characterizing Mycobacterium intracellulare responsible for nontuberculous mycobacterial (NTM) infections may aid in controlling outbreaks. This study aimed to compare 16S ribosomal ribonucleic acid (rRNA) sequencing and variable-number tandem repeat (VNTR) genotyping of M. intracellulare strains isolated from clinical samples, and to characterize VNTR clusters associated with NTM infections or cavity formation. Methodology: Sputum samples were obtained from 77 HIV-negative patients with pulmonary disease between 2009 and 2013. One M. intracellulare strain was isolated from each patient and genotyped using 16S rRNA and eight loci VNTR sequencing. Results: Single nucleotide polymorphism (SNP) genotyping identified seven point mutations at nucleotide positions 101, 178, 190, 252, 382, 443, and 490 in 16S rRNA, and four SNP patterns were identified: type 1 (16 strains), 2 (41 strains), 3 (11 strains), and 4 (1 strain); 5 strains had unique SNP patterns. VNTR genotyping identified VNTR12 as the most discriminating marker (allelic diversity 0.692). VNTR3 was the most homogeneous marker (allelic diversity 0.518), but each locus had high discriminating ability. The 77 strains were clustered according to the unpaired group method using arithmetic averages: cluster 1 (17 strains), 2 (43 strains), 3 (9 strains), and 4 (4 strains); 4strains had unique SNP patterns. Overall, over 90% strains were matched to similar SNP and VNTR groupings. VNTR clusters were associated with NTM infection (p =0.007) and presence of a cavity (p =0.042). Both methods distinguished four subtypes of M. intracellulare, which corresponded. Conclusions: VNTRs may represent an effective, user-friendly, low-cost typing technique.

Background & objectives: A complicated epidemiological situation characterized by significantly high tuberculosis (TB) morbidity is observed in West Siberia. This study was aimed to investigate the genetic characteristics of Mycobacterium tuberculosis circulating in the southern part of West Siberia (in the Omsk region). Methods: From March 2013 to January 2015, 100 isolates of M. tuberculosis were obtained from patients with pulmonary TB living in the Omsk region. Drug susceptibility testing was performed on Lowenstein-Jensen medium (absolute concentration method). Genetic typing of isolates was carried out by variable number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) typing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The genetic types and characteristics of cluster strains were determined using 15 MIRU-VNTR loci. Results: Thirty six VNTR types were found. Twenty six (26.0%) isolates had a unique profile, and the remaining 74 were grouped in 10 clusters containing from 2 to 23 isolates. The Beijing genotype was found in 72 isolates, 61 (85.0%) of which were part of five clusters that included two large clusters containing 23 isolates. Other genetic families, such as Latin-American Mediterranean (LAM, 11.0%), S family (2.0%) and Haarlem (4.0%), were also detected. The genetic family of 11 isolates could not be determined. Six different VNTR profiles were found in these non-classified isolates. Only 16 per cent of isolates were sensitive to anti-TB drugs. The katG315 (94.8%) and rpoB531 (92.2%) mutations were identified in 77 multidrug-resistant M. tuberculosis isolates. Interpretation & conclusions: This study showed that the M. tuberculosis population in the Omsk region was heterogeneous. The Beijing genotype predominated and was actively spreading. The findings obtained point to the need for the implementation of more effective preventive measures to stop the spread of drug-resistant M. tuberculosis strains.

Background: Mycobacterium tuberculosis genotyping is an essential step for understanding the epidemiology of tuberculosis. Mycobacterial-interspersed-repetitive-units (MIRU)-variable-number-of-tandem-repeats (VNTR) typing is an important method for this purpose. Objectives: The study aimed to determine the reproducibility of 15-loci MIRU-VNTR method. Methods: DNA extraction and 15-loci MIRU-VNTR were carried out for genotyping of 60 M. tuberculosis isolates collected from different clinical samples of 27 M. tuberculosis patients, each with two to four clinical isolates of M. tuberculosis. The similarity of M. tuberculosis isolates of the same patient was investigated by MIRU-VNTRplus. Results: The patterns of MIRU-VNTR were identical in 43 M. tuberculosis isolates collected from 20 tuberculosis patients, giving the repeatability of 71.6% for the test. In 12 isolates of M. tuberculosis belonging to five patients, the variable-number tandem repeat (VNTR) in only one locus was different. In three M. tuberculosis isolates of one patient, two different genotypes were identified. Conclusions: This study confirms the high reproducibility of 15-loci MIRU-VNTR, except for limited cases.

Summary Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing is widely used to genotype Mycobacterium tuberculosis complex in epidemiological studies for tracking tuberculosis transmission. Recent long-read sequencing technologies from Pacific Biosciences and Oxford Nanopore Technologies can produce reads that are long enough to cover the entire repeat regions in each MIRU-VNTR locus which was previously not possible using the short reads from Illumina high-throughput sequencing technologies. We thus developed MIRUReader for MIRU-VNTR typing directly from long sequence reads. Availability and implementation Source code and documentation for MIRUReader program is freely available at https://github.com/phglab/MIRUReader. Supplementary information Supplementary data are available at Bioinformatics online.

Mycobacterium africanum is an important cause of human tuberculosis and is found almost exclusively in West Africa. We identified a cluster of patients in Montreal, Canada, with M africanum disease that share identical genotypic signatures by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and a putative epidemiological link, thus providing evidence of possible local transmission of M africanum in Montreal over a 10-year period.

A monophyletic group of Drosophila hAT transposable elements, referred to as Herves-like, was characterized and found to be present in 46% of 57 screened Drosophila species. A remarkable characteristic of these elements is the presence of a long array of minisatellite repeats (MnRs) in both subterminal extremities of the elements. The copy number of these minisatellites was highly variable between and within populations. Twenty-three strains of Drosophila willistoni, covering its geographic distribution, were screened for polymorphism in the copy number of 5' MnRs, showing a variation from 7 to 20 repeat copies. These MnRs are well conserved among Drosophila species and probably function as transposase binding sequences, as provided by short subterminal repeats in other hAT elements. Miniature inverted repeat transposable elements were found in 27% of species carrying Herves-like elements. Phylogenetic analysis showed incongruences between transposable elements and species phylogenies, suggesting that at least four horizontal transfer events have occurred.

Variable number tandem repeat (VNTR) is a frequently employed typing method of Mycobacterium avium paratuberculosis (MAP) isolates. Based on whole genome sequencing in a previous study, allelic diversity at some VNTR loci seems to over- or under-estimate the actual phylogenetic variance among isolates. Interestingly, two closely related isolates on one farm showed polymorphism at the VNTR 7 locus, raising concerns about the misleading role that it might play in genotyping. We aimed to investigate the underlying basis of VNTR 7-polymorphism by analyzing sequence data for published genomes and field isolates of MAP and other M. avium complex (MAC) members. In contrast to MAP strains from cattle, strains from sheep displayed an "imperfect" repeat within VNTR 7, which was identical to respective allele types in other MAC genomes. Subspecies- and strain-specific single nucleotide polymorphisms (SNPs) and two novel (16 and 56 bp) repeats were detected. Given the combination of the three existing repeats, there are at least five different patterns for VNTR 7. The present findings highlight a higher polymorphism and probable instability of VNTR 7 locus that needs to be considered and challenged in future studies. Until then, sequencing of this locus in future studies is important to correctly assign the underlying allele types.(1).

Molecular typing of Mycobacterium tuberculosis(MTB) has greatly enhanced the understanding of the population structure of MTB isolates and epidemiology of tuberculosis (TB). To characterize prevalent genotypes of MTB, microarrays‑based spoligotyping and mycobacterial interspersed repetitive unit‑variable number of tandem repeats (MIRU‑VNTR) were applied on 80 isolates collected from primary health care facilities in Tanga, North‑eastern Tanzania. A total of 18 distinct spoligotypes were identified. The lineages by order of their predominance were EAI and CAS families (26.25%, 21 isolates each), LAM family and T super‑family (10%, 8 isolates each), MANU family (3.75%, 3 isolates), Beijing family (2.5%, 2 isolates) and S family (1.25%, 1 isolate). Overall, sixteen (20%) strains could not be allocated to any lineage according to the SITVIT_WEB database. The allelic diversity (h) for specific MIRU‑VNTR loci showed a considerable variation ranging from 0.826 of VNTR locus 3192 to 0.141 of VNTR locus 2059. The allelic diversity for 11 loci (VNTR 3192, 2996, 2165, 960, 4052, 424, 4156, 2531, 1644, 802 and 3690) exceeded 0.6, indicating highly discriminatory power. Seven loci (VNTR 2163b, 2401, 1955, 577, 4348, 2687 and 580) showed moderate discrimination (0.3 ≤ h ≥ 0.6), and three loci (VNTR3007, 154 and 2059) were less polymorphic. The present study suggests that the TB cases in Tanga might be caused by a diverse array of MTB strain families that may be indicative of a cosmopolitan population with frequent migration and travel. Microarray‑based spoligotyping and MIRU‑VNTR could be reliable tools in detecting different MTB genotypes in high burden settings.

Objective: To investigate the association of CAG repeat numbers in the androgen receptor (AR) gene with female pattern hair loss (FPHL) in a Chinese population. Methods: A total of 200 Han Chinese patients with FPHL (142 Ludwig II and 58 Ludwig III cases) and 200 healthy controls were enrolled in this study. The polymorphism of CAG repeat numbers was analyzed by the fluorescent amplified fragment length polymorphism technique. Results: The CAG biallelic mean length was 23.73 ± 2.04 repeats in Han Chinese FPHL patients and 23.90 ± 2.13 repeats in healthy controls, without any significant difference between the two groups (p = 0.481). In addition, neither the shorter nor the longer CAG repeat numbers were significantly different between FPHL and control subjects (p = 0.726, p = 0.383). Conclusion: The polymorphism of CAG repeat numbers of the AR gene may not be the genetic marker of FPHL in a Chinese population.

We developed a new method of typing for anginosus group streptococci (SAG). It is the first SAG-dedicated, PCR-based method, which allows to determine the relationship between strains. The method is based on the detection of tandem repeats among 9 genomic loci and is classified as multilocus variable number tandem repeats fingerprint (MLVF) type of analysis. Using the described method, it is possible to detect over half million MLVF patterns, which correlate with pulsed-field gel electrophoresis profiles. The other advantage of the method is relatively short time from "cell to data", low costs, and easy application for epidemiological and evolutionary studies.

The possible origin of Escherichia coli found on cuts and trimmings in the breaking facility of a beef packing plant was examined using multiple-locus variable-number tandem repeat analysis. Coliforms and E. coli were enumerated in samples obtained from 160 carcasses that would enter the breaking facility when work commenced and after each of the three production breaks throughout the day, from the conveyor belt before work and after each break, and from cuts and trimmings when work commenced and after each break. Most samples yielded no E. coli, irrespective of the surface types. E. coli was recovered from 7 (5%) carcasses, at numbers mostly ≤1.0 log CFU/160,000 cm(2). The log total numbers of E. coli recovered from the conveyor belt, cuts, and trimmings were mostly between 1 and 2 log CFU/80,000 cm(2). A total of 554 E. coli isolates were recovered. Multiple-locus variable-number tandem repeat analysis of 327 selected isolates identified 80 distinct genotypes, with 37 (46%) each containing one isolate. However, 28% of the isolates were of genotypes that were recovered from more than one sampling day. Of the 80 genotypes, 65 and 2% were found in one or all four sampling periods throughout the day. However, they represented 23 and 14% of the isolates, respectively. Of the genotypes identified for each surface type, at least one contained ≥9 isolates. No unique genotypes were associated with carcasses, but 10, 17, and 19 were uniquely associated with cuts, trimmings, and the belt, respectively. Of the isolates recovered from cuts, 49, 3, and 19% were of genotypes that were found among isolates recovered from the belt, carcasses, or both the belt and carcasses, respectively. A similar composition was found for isolates recovered from trimmings. These findings show that the E. coli found on cuts and trimmings at this beef packing plant mainly originated from the conveyor belt and that small number of E. coli strains survived the daily cleaning and sanitation process, thus persisting in the plant.

Torulaspora delbrueckii yeast strains are being increasingly applied at the industrial level, such as in the winemaking process, and so their identification and characterisation require effective, fast, accurate, reproducible and reliable approaches. Therefore, the development of typing techniques that allow discrimination at the strain level will provide an essential tool for those working with T. delbrueckii strains. Here, 28 T. delbrueckii strains from various substrates were characterised using different PCR-fingerprinting molecular methods: random amplified polymorphic DNA with polymerase chain reaction (RAPD-PCR), minisatellites SED1, AGA1, DAN4 and the newly designed T. delbrueckii (Td)PIR, and microsatellites (GAC)5 and (GTG)5. The aim was to determine and compare the efficacies, reproducibilities and discriminating powers of these molecular methods. RAPD-PCR using the M13 primers and the newly designed TdPIR3 minisatellite primer pair provided discrimination of the greatest number of T. delbrueckii strains. TdPIR3 clustered the 28 strains into 16 different groups with an efficiency of 100%, while M13 clustered the strains into 17 different groups, although with a lower efficiency of 89%. Moreover, the TdPIR3 primers showed reproducible profiles when the stringency of the PCR protocol was varied, which highlighted the great robustness of this technique. In contrast, variation of the stringency of the M13 PCR protocol resulted in modification of the amplified profiles, which suggested low reproducibility of this technique.

Cryptosporidium chipmunk genotype I is an emerging zoonotic pathogen in humans. The lack of subtyping tools makes it impossible to determine the role of zoonotic transmission in epidemiology. To identify potential subtyping markers, we sequenced the genome of a human chipmunk genotype I isolate. Altogether, 9,509,783 bp of assembled sequences in 853 contigs were obtained, with an N50 of 117,886 bp and >200-fold coverage. Based on the whole-genome sequence data, two genetic markers encoding the 60-kDa glycoprotein (gp60) and a mucin protein (ortholog of cgd1_470) were selected for the development of a subtyping tool. The tool was used for characterizing chipmunk genotype I in 25 human specimens from four U.S. states and Sweden, one specimen each from an eastern gray squirrel, a chipmunk, and a deer mouse, and 4 water samples from New York. At the gp60 locus, although different subtypes were seen among the animals, water, and humans, the 15 subtypes identified differed mostly in the numbers of trinucleotide repeats (TCA, TCG, or TCT) in the serine repeat region, with only two single nucleotide polymorphisms in the nonrepeat region. Some geographic differences were found in the subtype distribution of chipmunk genotype I from humans. In contrast, only two subtypes were found at the mucin locus, which differed from each other in the numbers of a 30-bp minisatellite repeat. Thus, Cryptosporidium chipmunk genotype I isolates from humans and wildlife are genetically similar, and zoonotic transmission might play a potential role in human infections.

Toxoplasma gondii is an intracellular protozoan which is widely distributed. Infection occurs as a result of ingestion of uncooked meat and exposure to cat feces. Immunocompetent individuals are generally asymptomatic, while severe disease may occur in immunocompromised subjects and in congenital toxoplasmosis, which is caused by transplacental acquisition of T. gondii. Genetic diversity of T. gondii has often been studied using a PCR-RFLP scheme based on nine molecular markers. These studies led to the description of a clonal population structure with three main lineages (I, II and III) in North America and Europe and higher genetic diversity in South America. The aim of this study was to develop molecular markers that could allow the discrimination of genetic variants within each clonal lineage. We analyzed the genome of T. gondii to identify genes containing variable number tandem repeats (VNTRs). The coding sequences of T. gondii ME49 genome were processed with Tandem Repeat Finder software. A panel of candidate markers was selected based on the following parameters: the repeat unit size (>9 bp) and composition (to avoid single and dinucleotide runs), the number of copies (

Background Combining different molecular typing methods for Mycobacterium tuberculosis complex (MTBC) can be a powerful tool for molecular epidemiology-based investigation of TB. However, the current standard method that provides high discriminatory power for such a combination, mycobacterial interspersed repetitive units-variable numbers of tandem repeats typing (MIRU-VNTR), is laborious, time-consuming and often too costly for many resource-limited laboratories. We aimed to evaluate a reduced set of loci for MIRU-VNTR typing in combination with spoligotyping and SNP-typing for routine molecular epidemiology of TB. Method Spoligotyping and SNP-typing, in combination with the 15 loci MIRU-VNTR typing, were first used to type clinical MTBC isolates (n = 158) from Madagascar. A step by step reduction of MIRU-VNTR loci number was then performed according to the Hunter and Gaston Discriminatory Index (HGDI) and to the Principal component analysis (PCA) correlation with the spoligotype profiles to evaluate the discrimination power inside the generated spoligotype clusters. The 15 MIRU-VNTR was used as reference and SNP-typing was used to determine the main MTBC lineages. Results Of the 158 clinical isolates studied, the SNP-typing classified 23 into Lineage 1 (14.6%), 31 into Lineage 2 (19.6%), 23 into Lineage 3 (14.6%) and 81 into Lineage 4 strains (51.3%). 37 different spoligotypes profiles were obtained, 15 of which were unique and 20 in clusters. 15-loci MIRU-VNTR typing revealed 144 different genotypes: 132 isolates had a unique MIRU-VNTR profile and 27 isolates were grouped into 12 clusters. After a stepwise reduction of the MIRU-VNTR loci number within each main spoligotype families, three different sets composed of 5 customised MIRU-VNTR loci had a similar discrimination level to the reference 15 loci MIRU-VNTR in lineage 1, lineage 2 and lineage 3. For lineage 4, a set of 4 and 3 MIRU-VNTR loci were proposed to subtype the Harleem and LAM spoligotype families, respectively. For the T spoligotype family, a set of 5 MIRU-VNTR loci was proposed. Conclusion According to the lineages and the spoligotype families, the number of MIRU-VNTR loci can be reduced to get an optimal classification of MTBC. These customized sets of MIRU-VNTR loci reduce workload and save resources while maintaining optimal discriminatory power.

Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing.

The U.S. Centers for Disease Control and Prevention (CDC) uses a combination of spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) analyses as part of the National TB Genotyping Service (NTGS). The NTGS expansion from 12-locus MIRU-VNTR (MIRU12) to 24-locus MIRU-VNTR (MIRU24) in 2009 enhanced the ability to discriminate Mycobacterium tuberculosis strains. In the current study, we investigated the MIRU24 concordance among epidemiologic-linked tuberculosis (TB) patients in four U.S. health jurisdictions. We also evaluated the programmatic benefits of combining MIRU24 and spoligotyping with epidemiologic evidence in identifying potential recent TB transmission. We examined 342 TB patients in 42 spoligotype/MIRU12 (PCRType) clusters (equivalent to 46 spoligotype/MIRU24 [GENType] clusters) to identify epidemiologic links among cases. GENType clusters, when compared to PCRType clusters, had 12 times higher odds of epidemiologic links being identified if patients were younger than 25 years and 3 times higher odds if patients resided in the same zip code, or had HIV infection. Sixty (18%) fewer PCRType-clustered patients would need investigations if clusters are defined using GENType instead of PCRType. An important advantage of defining clusters by MIRU24 is resource savings related to the reduced number of clustered cases needing investigation.

Fragile sites represent regions of chromatin that fail to compact during mitosis. Based on the prevalence and pattern of inheritance they are classified as rare fragile sites or common fragile sites. Rare fragile sites either occur spontaneously or can be induced by certain AT-specific binding chemicals namely distamycin, Hoechst 33258, Berenil and others. The most common of all rare autosomal fragile sites is fra(16)(q22) with a heterozygote frequency of ~5%. FRA16B results from an expansion of a 33 bp AT-rich Minisatellite repeat. These rare forms are usually heritable and segregate in a Mendelian fashion. The proband who was referred for secondary amenorrhoea, revealed 46,XX,fra(16)(q22.1)pat karyotype. Her father and younger sibling were also found to be carriers. This study aimed to delineate the genotypic and phenotypic features exhibited by these carriers and to evaluate FRA16B expression using AT-specific binding chemicals. The additives employed were Berenil, BrdU and Hoechst 33258. Berenil at a concentration of 150 µg/ml showed the highest expression of FRA16B. Although the recent breakthrough in molecular characterization of fragile sites plays a critical role in comprehending their association with various diseases, the physiological link between them and amenorrhoea is not clearly understood.

Objectives.We studied the genetic diversity of clinical isolates from patients with tuberculosis in the multiethnic area of Xinjiang autonomous region in China. A total of 311 clinicalM. tuberculosisisolates were collected in 2006 and 2011 and genotyped by two genotyping methods. All isolates were grouped into 68 distinct spoligotypes using the spoligotyping method. The Beijing family was dominant, followed by T1 and CAS. MIRU-VNTR results showed that a total of 195 different VNTR types were identified. Ten of the 15 loci were highly or moderately discriminant according to their HGDI scores, and 13 loci had good discriminatory power in non-Beijing family strains, whereas only two loci had good discriminatory power in Beijing family strains. Chi-square tests demonstrated that there were no correlations between four characteristics (sex, age, type of case, and treatment history) and the Beijing family. In summary, Beijing family strains were predominant in Xinjiang, and theVNTR-15Chinalocus-set was suitable for genotyping all Xinjiang strains, but not for the Beijing family strains. Thus, these data suggested that different genotype distributions may exist in different regions; MLVA locus-sets should be adjusted accordingly, with newly added loci to increase resolution if necessary.

Two high-resolution strain discrimination methods based on variable number tandem repeat (VNTR) analysis were devised for the plant soft-rotting pathogens Dickeya dianthicola and Dickeya solani and used to investigate their diversity and epidemiology. Analysis of D. dianthicola identified 19 VNTR profiles (P1-P19). Diversity was observed in the earliest strains analysed (from 1957), which included four profiles from European strains isolated from Dianthus caryophyllus (P4, P7, P8 and P11) and one from Chrysanthemum morifolium (also P7) originating from the USA. Eleven D. dianthicola VNTR profiles were identified from potato dating from 1984. The time period between which specific D. dianthicola VNTR profiles were first isolated from ornamental hosts and then from potato, was between 8 and 16 years respectively. Identification of two profiles (P2 and P8) that occurred in both ornamental hosts and potato supported cross-infection between these hosts. Limited variation in VNTR profile was identified from strains of D. solani, from which only three VNTR profiles were discriminated by variation at only one of five loci, which may reflect the short time period and/or limited number of D. solani introductions since the pathogen’s first emergence in Europe. Shared profiles between D. solani isolates from hyacinth and potato strains, is consistent with cross infection between hosts grown for horticultural production and potato.

Bradykinin B2 receptor is involved in many processes, including the regulation of blood pressure and smooth muscle contraction, vasodilation, inflammation, edema, cell proliferation, and pain. This receptor attracts special attention as one of the factors that have cardioprotective and infarct-limiting effects. Certain genetic variants of the coding and noncoding regions of the bradykinin B2 receptor gene (BDKRB2) may play a role in modulating its expression. The 3′-untranslated region of BDKRB2 exon 3 harbors a minisatellite repeat (B2-VNTR), which affects the mRNA stability. Hence, it is of interest to study a possible association of B2-VNRT alleles with various forms of coronary heart disease (CHD). In our work the allele and genotype frequency distributions of B2-VNTR were compared between healthy individuals and patients with CHD (angina pectoris or myocardial infarction (MI)) of the Russian ethnic group. Based on its length polymorphism, B2-VNTR was classed with low-polymorphic non-hypervariable minisatellites. Three B2-VNTR alleles, which consisted of 43, 38, and 33 repeats, were observed in all investigated cohorts. The alleles with 43 and 33 repeats were the most prevalent. The allele and genotype frequencies of B2-VNTR did not significantly differ between males and females in control group, and also between healthy males and males with angina pectoris or MI. Thus, B2-VNTR length polymorphism was not associated with these clinical forms of CHD in males. However, we do not exclude the possibility of an association of the short B2-VNTR alleles (38 and 33 repeats) with a cardioprotective effect in females with CHD. This hypothesis requires further investigation.

Among non-Saccharomyces wine yeasts, Candida zemlpinina is one of the frequently isolated and oenologically important species. It is mostly known from European winemaking areas and it has become one of the key species of non-Saccharomyces wine yeasts to study. Investigating the diversity of C. zemplinina isolates is important for a deeper understanding of the non-Saccharomyces wine yeasts and for the yeast starter industry, as numerous researches have pointed to the potential use of this species in winemaking. For assessing the biodiversity of a larger number of strains, RAPD and micro/minisatellite PCR is often the method of choice, however, this technique is often unstandardized. Whereas some laboratories use these methods for species identifications, others apply RAPD primers for determining intraspecies diversity. In this study, we have tested 5 different RAPD and micro/minisatellite primers on strains of C. zemplinina isolated from different locations. We show that after a rigorous PCR-optimization aimed at reproducibility and comparability of band patterns with these PCR-reactions, diversity of different strains from a wide range of geographic locations is relatively low. The analysis of several oenologically important physiological traits of the strains showed a relatively low level of diversity as well. We also demonstrate that the intraspecific diversity of C. zemplinina observable with different techniques (RAPD, micro/minisatellite or physiological analysis) may be fairly different and not necessarily comparable.

The human genome contains numerous large tandem repeats, many of which remain poorly characterized. Here we report a novel transfer RNA (tRNA) tandem repeat on human chromosome 1q23.3 that shows extensive copy number variation with 9-43 repeat units per allele and displays evidence of meiotic and mitotic instability. Each repeat unit consists of a 7.3 kb GC-rich sequence that binds the insulator protein CTCF and bears the chromatin hallmarks of a bivalent domain in human embryonic stem cells. A tRNA containing tandem repeat composed of at least three 7.6-kb GC-rich repeat units reside within a syntenic region of mouse chromosome 1. However, DNA sequence analysis reveals that, with the exception of the tRNA genes that account for less than 6% of a repeat unit, the remaining 7.2 kb is not conserved with the notable exception of a 24 base pair sequence corresponding to the CTCF binding site, suggesting an important role for this protein at the locus.

Multiple locus variable number tandem repeats (VNTRs) analysis (MLVA) has become a reliable tool, able to establish genetic relationships for epidemiological surveillance and molecular subtyping of pathogens such as verotoxigenic Escherichia coli (VTEC). This emerging pathogen whose primary reservoir is the cattle causes severe disease in humans, such as hemorrhagic colitis and hemolytic uremic syndrome. With the aim of comparing a recently proposed MLVA assay with that used routinely in our laboratory, we analyzed a set of VTEC isolates (n = 72) obtained from meat using both assays. All samples could be typed by the new MLVA assay, and an increase in the number of distinct profiles (31–43) was observed. However, intraserotype resolution was not significantly enhanced; thus, the incorporation of more VNTR loci is still needed to achieve a greater discrimination among non-O157:H7 serotypes. Fil: González, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Sanso, Andrea Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Lucchesi, Paula Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Bustamante, Ana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina

DNA tandem repeats (TRs), also designated as satellite DNA, are inter- or intragenic nucleotide sequences that are repeated two or more times in a head-to-tail manner. Because TR tracts are prone to strand-slippage replication and recombination events that cause the TR copy number to increase or decrease, loci containing TRs are hypermutable. An increasing number of examples illustrate that bacteria can exploit this instability of TRs to reversibly shut down or modulate the function of specific genes, allowing them to adapt to changing environments on short evolutionary time scales without an increased overall mutation rate. In this review, we discuss the prevalence and distribution of inter- and intragenic TRs in bacteria and the mechanisms of their instability. In addition, we review evidence demonstrating a role of TR variations in bacterial adaptation strategies, ranging from immune evasion and tissue tropism to the modulation of environmental stress tolerance. Nevertheless, while bioinformatic analysis reveals that most bacterial genomes contain a few up to several dozens of intra- and intergenic TRs, only a small fraction of these have been functionally studied to date.

The repetitive DNA content of the stick insect species Bacillus rossius (facultative parthenogenetic), Bacillus grandii (gonochoric), and Bacillus atticus (obligate parthenogenetic) was analyzed through the survey of random genomic libraries roughly corresponding to 0.006% of the genome. By repeat masking, 19 families of transposable elements were identified (two LTR and six non-LTR retrotransposons; 11 DNA transposons). Moreover, a de novo analysis revealed, among the three libraries, the first MITE family observed in polyneopteran genomes. On the whole, transposable element abundance represented 23.3% of the genome in B. rossius, 22.9% in B. atticus, and 18% in B. grandii. Tandem repeat content in the three libraries is much lower: 1.32%, 0.64%, and 1.86% in B. rossius, B. grandii, and B. atticus, respectively. Microsatellites are the most abundant in all species. Minisatellites were only found in B. rossius and B. atticus, and five monomers belonging to the Bag320 satellite family were detected in B. atticus. Assuming the survey provides adequate representation of the relative genome, the obligate parthenogenetic species (B. atticus), compared with the other two species analyzed, does not show a lower transposable element content, as expected from some theoretical and empirical studies.

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

Canine and human behavior are shaped by similar evolutionary processes, yet the identification of the behavioral phenotype is often difficult. A widely used method relies on breed stereotypes provided by experts such as dog trainers. To reveal a valid association between behavior and genetic factors, an association study of behavioral phenotyping and genotyping is essential. We screened for variable number of tandem repeat (VNTR) polymorphisms in intron 4 of the tyrosine hydroxylase gene in military working dogs (belonging to pass and fail groups based on the results of an in-training examination conducted by the drillmaster), which were scored based on possessiveness, audaciousness, concentration, and motor ability by qualification examination. We first characterized each genotype by sequencing, in which the 1/1 type consists of a single copy of a 36-bp sequence and the 2/2 type is a duplicated form of the 36-bp repeat unit. The 1/2 alleles showed a single nucleotide change as a heteroduplex, which generated a PCR product of similar size as that of the 1/1-182-bp. The military working dogs showed the 2/2 type of VNTR and heteroduplex. For the pass group, two dogs possessed 2/2 type (40 %), whereas three dogs were of the heteroduplex type (60 %). However, all members of fail group showed the 2/2 type (100 %). These data indicate that repeat polymorphisms with behavioral phenotyping can identify military working dogs that would pass or fail the in-training examination.

Sporadic and epidemiologically linked Yersinia enterocolitica strains ( n = 379) isolated from fecal samples from human patients, tonsil or fecal samples from pigs collected at slaughterhouses, and pork samples collected at meat stores were genotyped using multiple-locus variable-number tandem-repeat analysis (MLVA) with six loci, i.e., V2A, V4, V5, V6, V7, and V9. In total, 312 different MLVA types were found. Similar types were detected (i) in fecal samples collected from human patients over 2 to 3 consecutive years, (ii) in samples from humans and pigs, and (iii) in samples from pigs that originated from the same farms. Among porcine strains, we found farm-specific MLVA profiles. Variations in the numbers of tandem repeats from one to four for variable-number tandem-repeat (VNTR) loci V2A, V5, V6, and V7 were observed within a farm. MLVA was applicable for serotypes O:3, O:5,27, and O:9 and appeared to be a highly discriminating tool for distinguishing sporadic and outbreak-related strains. With long-term use, interpretation of the results became more challenging due to variations in more-discriminating loci, as was observed for strains originating from pig farms. Additionally, we encountered unexpectedly short V2A VNTR fragments and sequenced them. According to the sequencing results, updated guidelines for interpreting V2A VNTR results were prepared.

Significant evidence supports a genetic contribution to the development of posttraumatic stress disorder (PTSD). Three previous studies have demonstrated an association between PTSD and the nine repeat allele of the 3' untranslated region (3'UTR) variable number tandem repeat (VNTR) in the dopamine transporter (DAT, rs28363170). Recently a novel, functionally significant C/T single-nucleotide polymorphism (SNP) in the 3'UTR (rs27072) with putative interactions with the 3'VNTR, has been identified. To provide enhanced support for the role of DAT and striatal dopamine regulation in the development of PTSD, this study examined the impact of a haplotype defined by the C allele of rs27072 and the nine repeat allele of the 3'VNTR on PTSD diagnosis in young trauma-exposed children.DAT haplotypes were determined in 150 trauma-exposed 3-6 year-old children. PTSD was assessed with a semistructured interview. After excluding double heterozygotes, analysis was performed on 143 total subjects. Haplotype was examined in relation to categorical and continuous measures of PTSD, controlling for trauma type and race. Additional analysis within the two largest race categories was performed, as other means of controlling for ethnic stratification were not available.The number of haplotypes (0, 1, or 2) defined by the presence of the nine repeat allele of rs28363170 (VNTR in the 3'UTR) and the C allele of rs27072 (SNP in the 3'UTR) was significantly associated with both the diagnosis of PTSD and total PTSD symptoms. Specifically, children with one or two copies of the haplotype had significantly more PTSD symptoms and were more likely to be diagnosed with PTSD than were children without this haplotype.These findings extend previous findings associating genetic variation in the DAT with PTSD. The association of a haplotype in DAT with PTSD provides incremental traction for a model of genetic vulnerability to PTSD, a specific underlying mechanism implicating striatal dopamine regulation, and insight into potential future personalized interventions.

Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.

In order to switch from IS 6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% ( n = 3,123). Of the remaining 855 cases, 12% ( n = 479) of the cases were clustered only by VNTR, 7.7% ( n = 305) only by RFLP typing, and 1.8% ( n = 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% ( n = 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS 6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLP-clustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems.

New cases of leprosy are still being detected in Colombia after the country declared achievement of the WHO defined 'elimination' status. To study the ecology of leprosy in endemic regions, a combination of geographic and molecular tools were applied for a group of 201 multibacillary patients including six multi-case families from eleven departments. The location (latitude and longitude) of patient residences were mapped. Slit skin smears and/or skin biopsies were collected and DNA was extracted. Standard agarose gel electrophoresis following a multiplex PCR-was developed for rapid and inexpensive strain typing of Mycobacterium leprae based on copy numbers of two VNTR minisatellite loci 27-5 and 12-5. A SNP (C/T) in gyrA (SNP7614) was mapped by introducing a novel PCR-RFLP into an ongoing drug resistance surveillance effort. Multiple genotypes were detected combining the three molecular markers. The two frequent genotypes in Colombia were SNP7614(C)/27-5(5)/12-5(4) [C54] predominantly distributed in the Atlantic departments and SNP7614 (T)/27-5(4)/12-5(5) [T45] associated with the Andean departments. A novel genotype SNP7614 (C)/27-5(6)/12-5(4) [C64] was detected in cities along the Magdalena river which separates the Andean from Atlantic departments; a subset was further characterized showing association with a rare allele of minisatellite 23-3 and the SNP type 1 of M. leprae. The genotypes within intra-family cases were conserved. Overall, this is the first large scale study that utilized simple and rapid assay formats for identification of major strain types and their distribution in Colombia. It provides the framework for further strain type discrimination and geographic information systems as tools for tracing transmission of leprosy.

Although genetic lesions responsible for some mendelian disorders can be rapidly discovered through massively parallel sequencing of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing and de novo assembly did we find that each of six families with MCKD1 harbors an equivalent but apparently independently arising mutation in sequence markedly under-represented in massively parallel sequencing data: the insertion of a single cytosine in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5–5 kb), GC-rich (>80%) coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1. These results provide a cautionary tale about the challenges in identifying the genes responsible for mendelian, let alone more complex, disorders through massively parallel sequencing., National Institutes of Health (U.S.) (Intramural Research Program), National Human Genome Research Institute (U.S.), Charles University (program UNCE 204011), Charles University (program PRVOUK-P24/LF1/3), Czech Republic. Ministry of Education, Youth, and Sports (grant NT13116-4/2012), Czech Republic. Ministry of Health (grant NT13116-4/2012), Czech Republic. Ministry of Health (grant LH12015), National Institutes of Health (U.S.) (Harvard Digestive Diseases Center, grant DK34854)

Listeria monocytogenes is an important worldwide foodborne pathogen. For listeriosis outbreak investigations, an optimal multiple-locus variable-number-tandem-repeat analysis typing panel was identified from 4 previously reported schemes. The optimal combination of loci consisted of 9 loci (LMV6, LMV1, LMV2, Lm11, Lm10, LMV7, Lm32, LM-TR6, and Lm23), which produced the same level of differentiation ability Simpson index of 0.9914 as that of all 14 loci combined in the previous 4 reports. This panel provided higher differentiation ability than the individual of the 4 previously reported schemes, suggesting it would be useful for future surveillance and outbreak investigations.

Genetic variations of functional genes in various animal species have been previously examined to understand the relationship between genotypes and phenotypes. Repeat polymorphism can be found in not only coding sequences but also untranslated regions of the protein-coding genes, affecting gene regulation via a variety of mechanisms. In this respect, repeat polymorphisms including microsatellites, variable number of tandem repeats (VNTRs), and short interspersed nuclear elements (SINEs) in relation to functional genes contribute to recognition of genetic or phenotypic variation and individual identification. These elements are biologically valuable markers for studies on association between genetic make-up and behavioral patterns in dog breeds. Hence, to examine the effect of genotype on behavior, studies on this combination are certainly important. Hence, this review could cast light on the functional roles of repeat polymorphisms in dog behavior and breed variation.

The fungus Mycosphaerella fijiensis is the causative agent of black sigatoka, which is one of the most destructive diseases of banana plants. Infection with this pathogen results in underdeveloped fruit, with no commercial value. We analyzed the distribution of the M. fijiensis mating-type system and its genetic variability using M13 phage DNA markers. We found a 1:1 distribution of mating-type alleles, indicating MAT1-1 and MAT1-2 idiomorphs. A polymorphism analysis using three different primers for M13 markers showed that only the M13 minisatellite primers generated polymorphic products. We then utilized this polymorphism to characterize 40 isolates from various Brazilian states. The largest genetic distances were found between isolates from the same location and between isolates from different parts of the country. Therefore, there was no correlation between the genetic similarity and the geographic origin of the isolates. The M13 marker was used to generate genetic fingerprints for five isolates; these fingerprints were compared with the band profiles obtained from inter-simple sequence repeat (UBC861) and inter-retrotransposon amplified polymorphism analyses. We found that the M13 marker was more effective than the other two markers for differentiating these isolates. The fungus Mycosphaerella fijiensis is the causative agent of black sigatoka, which is one of the most destructive diseases of banana plants. Infection with this pathogen results in underdeveloped fruit, with no commercial value. We analyzed the distribution of the M. fijiensis mating-type system and its genetic variability using M13 phage DNA markers. We found a 1:1 distribution of mating-type alleles, indicating MAT1-1 and MAT1-2 idiomorphs. A polymorphism analysis using three different primers for M13 markers showed that only the M13 minisatellite primers generated polymorphic products. We then utilized this polymorphism to characterize 40 isolates from various Brazilian states. The largest genetic distances were found between isolates from the same location and between isolates from different parts of the country. Therefore, there was no correlation between the genetic similarity and the geographic origin of the isolates. The M13 marker was used to generate genetic fingerprints for five isolates; these fingerprints were compared with the band profiles obtained from inter-simple sequence repeat (UBC861) and inter-retrotransposon amplified polymorphism analyses. We found that the M13 marker was more effective than the other two markers for differentiating these isolates.

Double-stranded break (DSB) repair during meiotic recombination in yeast Saccharomyces cerevisiae leads to the formation of heteroduplex DNA, a hybrid DNA molecule composed of single strands from two homologous chromosomes. Differences in sequence between the strands within heteroduplex DNA generate mismatches or large unpaired loops that are substrates for repair. At least two pathways function to repair large loops that form within heteroduplex DNA: the RAD1-dependent large loop repair (LLR) pathway and another as yet uncharacterized RAD1-independent LLR pathway. Repair of large loops during meiotic recombination is especially important for the genomic stability of the repetitive DNA sequences known as minisatellites. Minisatellite DNA tracts are generally stable during mitotic cell divisions but frequently alter in length during meiosis. Using a yeast minisatellite system in which the human minisatellite associated with the HRAS1 proto-oncogene has been inserted into the recombination hotspot region upstream of HIS4 in S. cerevisiae, our lab previously showed that the RAD1-dependent LLR pathway controls minisatellite length expansions, but not contractions. Here we show that minisatellite length expansions are controlled by the products of the CSM3 and TOF1 genes, while contractions are controlled by MRC1. By examining meiotic segregation patterns in yeast strains heterozygous for the 26bp his4-lopd insert, we found that deleting CSM3 caused a loss of LLR activity similar to that seen in a RAD1 mutant. Double mutant analysis revealed that failure to repair loops is exacerbated upon deleting both RAD1 and CSM3 - specifically the type of repair that fills in loops, which would generate minisatellite length expansions. A model for minisatellite length alteration based on these results is presented.