Oncogenic chimeric transcription factors drive tumor-specific transcription, processing, and translation of silent genomic regions.

Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). Here, we find that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neogenes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, we show that hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, this study identifies the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer.

STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.

STAG2, a cohesin family gene, is among the most recurrently mutated genes in cancer. STAG2 loss of function (LOF) is associated with aggressive behavior in Ewing sarcoma, a childhood cancer driven by aberrant transcription induced by the EWSR1-FLI1 fusion oncogene. Here, using isogenic Ewing cells, we show that, while STAG2 LOF profoundly changes the transcriptome, it does not significantly impact EWSR1-FLI1, CTCF/cohesin, or acetylated H3K27 DNA binding patterns. In contrast, it strongly alters the anchored dynamic loop extrusion process at boundary CTCF sites and dramatically decreases promoter-enhancer interactions, particularly affecting the expression of genes regulated by EWSR1-FLI1 at GGAA microsatellite neo-enhancers. Down-modulation of cis-mediated EWSR1-FLI1 activity, observed in STAG2-LOF conditions, is associated with enhanced migration and invasion properties of Ewing cells previously observed in EWSR1-FLI1low cells. Our study illuminates a process whereby STAG2-LOF fine-tunes the activity of an oncogenic transcription factor through altered CTCF-anchored loop extrusion and cis-mediated enhancer mechanisms.

Suitability of IS6110-RFLP and MIRU-VNTR for differentiating spoligotyped drug-resistant mycobacterium tuberculosis clinical isolates from Sichuan in China.

Genotypes of Mycobacterium tuberculosis complex (MTBC) vary with the geographic origin of the patients and can affect tuberculosis (TB) transmission. This study was aimed to further differentiate spoligotype-defined clusters of drug-resistant MTBC clinical isolates split in Beijing (n = 190) versus non-Beijing isolates (n = 84) from Sichuan region, the second high-burden province in China, by IS6110-restriction fragment length polymorphism (RFLP) and 24-locus MIRU-VNTRs. Among 274 spoligotyped isolates, the clustering ratio of Beijing family was 5.3% by 24-locus MIRU-VNTRs versus 2.1% by IS6110-RFLP, while none of the non-Beijing isolates were clustered by 24-locus MIRU-VNTRs versus 9.5% by IS6110-RFLP. Hence, neither the 24-locus MIRU-VNTR was sufficient enough to fully discriminate the Beijing family, nor the IS6110-RFLP for the non-Beijing isolates. A region adjusted scheme combining 12 highly discriminatory VNTR loci with IS6110-RFLP was a better alternative for typing Beijing strains in Sichuan than 24-locus MIRU-VNTRs alone. IS6110-RFLP was for the first time introduced to systematically genotype MTBC in Sichuan and we conclude that the region-adjusted scheme of 12 highly discriminative VNTRs might be a suitable alternative to 24-locus MIRU-VNTR scheme for non-Beijing strains, while the clusters of the Beijing isolates should be further subtyped using IS6110-RFLP for optimal discrimination.

Molecular epidemiology of Mycobacterium tuberculosis isolates from Kerala, India using IS6110-RFLP, spoligotyping and MIRU-VNTRs.

Tuberculosis (TB) continues to be a major health problem in India, and there is very little information about the prevalent genotypes of tubercle bacilli that cause TB in India, especially in Kerala. Our aim was to study the different circulating strains of Mycobacterium tuberculosis (MTB) that are prevalent in Kerala, India. We analyzed 168 MTB isolates from as many pulmonary TB patients using IS6110-RFLP, spoligotyping and MIRU-VNTRs. The results of IS6110-RFLP revealed that majority of isolates had null copy (10.89%) or single copy (44.87%) of IS6110 insertion. Low copy (<6) isolates accounted for 71.5% in the isolates studied. Genotypic clade designations were done by comparing with the SITVIT2 database which showed 68 patterns; of which 51 corresponded to different shared types whereas 17 patterns were orphans. Among the 51 SITs recorded, 42 SITs matched a preexisting SIT in the SITVIT2 database, whereas 9 SITs were newly-created. Majority of the isolates (64.28%) belonged to the ancestral East-African Indian (EAI) lineage. MIRU-40 and 31 (HGDI=0.6555 and 0.6524) showed highest discrimination, while MIRU-2 and 20 (HGDI=0.0354 and 0.0696) had the least discriminatory power. ETR-A and B (HGDI 0.7382 and 0.6743) discriminated better as compared to other MIRU loci. The overall HGDI for MIRU-VNTRs at 0.9735 (calculated for 166 isolates) showed a better discriminatory power than spoligotyping used alone. This study of MTB genotypic diversity was useful by providing a first snapshot of circulating MTB genotypic clones in Kerala.

Efficient discrimination by MIRU-VNTRs of Mycobacterium tuberculosis clinical isolates belonging to the predominant SIT11/EAI3-IND ancestral genotypic lineage in Kerala, India.

The present study evaluated the ability of MIRU-VNTRs to discriminate Mycobacterium tuberculosis (MTB) clinical isolates belonging to the SIT11/EAI3-IND ancestral genotypic lineage, which is highly prevalent in Kerala, India. Starting from 168 MTB clinical isolates, spoligotyping (discriminatory index of 0.9113) differentiated the strains into 68 distinct patterns, the biggest cluster being SIT11/48 SIT11 (n=48). The present study shows that 12-loci MIRUs and 3 ETRs allowed an efficient discrimination of these isolates (discriminatory indexes of 0.7819 and 0.5523, respectively).

MLVA based classification of Mycobacterium tuberculosis complex lineages for a robust phylogeographic snapshot of its worldwide molecular diversity.

Multiple-locus variable-number tandem repeat analysis (MLVA) is useful to establish transmission routes and sources of infections for various microorganisms including Mycobacterium tuberculosis complex (MTC). The recently released SITVITWEB database contains 12-loci Mycobacterial Interspersed Repetitive Units--Variable Number of Tandem DNA Repeats (MIRU-VNTR) profiles and spoligotype patterns for thousands of MTC strains; it uses MIRU International Types (MIT) and Spoligotype International Types (SIT) to designate clustered patterns worldwide. Considering existing doubts on the ability of spoligotyping alone to reveal exact phylogenetic relationships between MTC strains, we developed a MLVA based classification for MTC genotypic lineages. We studied 6 different subsets of MTC isolates encompassing 7793 strains worldwide. Minimum spanning trees (MST) were constructed to identify major lineages, and the most common representative located as a central node was taken as the prototype defining different phylogenetic groups. A total of 7 major lineages with their respective prototypes were identified: Indo-Oceanic/MIT57, East Asian and African Indian/MIT17, Euro American/MIT116, West African-I/MIT934, West African-II/MIT664, M. bovis/MIT49, M.canettii/MIT60. Further MST subdivision identified an additional 34 sublineage MIT prototypes. The phylogenetic relationships among the 37 newly defined MIRU-VNTR lineages were inferred using a classification algorithm based on a bayesian approach. This information was used to construct an updated phylogenetic and phylogeographic snapshot of worldwide MTC diversity studied both at the regional, sub-regional, and country level according to the United Nations specifications. We also looked for IS6110 insertional events that are known to modify the results of the spoligotyping in specific circumstances, and showed that a fair portion of convergence leading to the currently observed bias in phylogenetic classification of strains may be traced back to the presence of IS6110. These results shed new light on the evolutionary history of the pathogen in relation to the history of peopling and human migration.

SITVITWEB--a publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology.

Among various genotyping methods to study Mycobacterium tuberculosis complex (MTC) genotypic polymorphism, spoligotyping and mycobacterial interspersed repetitive units-variable number of DNA tandem repeats (MIRU-VNTRs) have recently gained international approval as robust, fast, and reproducible typing methods generating data in a portable format. Spoligotyping constituted the backbone of a publicly available database SpolDB4 released in 2006; nonetheless this method possesses a low discriminatory power when used alone and should be ideally used in conjunction with a second typing method such as MIRU-VNTRs for high-resolution epidemiological studies. We hereby describe a publicly available international database named SITVITWEB which incorporates such multimarker data allowing to have a global vision of MTC genetic diversity worldwide based on 62,582 clinical isolates corresponding to 153 countries of patient origin (105 countries of isolation). We report a total of 7105 spoligotype patterns (corresponding to 58,180 clinical isolates) - grouped into 2740 shared-types or spoligotype international types (SIT) containing 53,816 clinical isolates and 4364 orphan patterns. Interestingly, only 7% of the MTC isolates worldwide were orphans whereas more than half of SITed isolates (n=27,059) were restricted to only 24 most prevalent SITs. The database also contains a total of 2379 MIRU patterns (from 8161 clinical isolates) from 87 countries of patient origin (35 countries of isolation); these were grouped in 847 shared-types or MIRU international types (MIT) containing 6626 isolates and 1533 orphan patterns. Lastly, data on 5-locus exact tandem repeats (ETRs) were available on 4626 isolates from 59 countries of patient origin (22 countries of isolation); a total of 458 different VNTR patterns were observed - split into 245 shared-types or VNTR International Types (VIT) containing 4413 isolates) and 213 orphan patterns. Datamining of SITVITWEB further allowed to update rules defining MTC genotypic lineages as well to have a new insight into MTC population structure and worldwide distribution at country, sub-regional and continental levels. At evolutionary level, the data compiled may be useful to distinguish the occasional convergent evolution of genotypes versus specific evolution of sublineages essentially influenced by adaptation to the host. This database is publicly available at: http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE.

Mycobacterium tuberculosis spoligotypes that may derive from mixed strain infections are revealed by a novel computational approach.

Global control of tuberculosis is increasingly dependent on rapid and accurate genetic typing of Mycobacteriumtuberculosis. Spoligotyping is a first-line genotypic fingerprinting method for M.tuberculosis isolates. An international online database (SpolDB4) of spoligotype patterns has been established wherein a clustered pattern (shared by ≥2 isolates) is designated a shared international type (SIT). Dual infections of single patients by distinct strains of M. tuberculosis is increasingly reported in high tuberculosis incidence areas, raising the possibility of false composite spoligotype patterns if performed upon mixed strain samples. A computational approach was applied to SpolDB4 and found that of the reported 1939 SITs, 54% could be a composite of two other SITs. Although many of the spoligotypes listed in SpolDB4 may be the product of admixing, the majority of patterns were reported with a corresponding low case frequency and so the effect of misclassification upon database integrity with these is likely minimal. Phylogenetic analysis of the five SITs most prone to be a composite demonstrated that these patterns designate nodes from which the ramifications of large families T, MANU, LAM, and EAI emerged. We illustrate how geographic context may indicate when an observed pattern could be the product of mixed infection. Importantly, when one of the most composite-prone SITs is obtained, further genetic testing by alternate methods is prudent to rule-out mixed infection, especially in high tuberculosis prevalence areas. These findings have broad practical implications for tuberculosis control and surveillance, as well as highlight the utility of a computational approach in providing solutions to biological questions in which the information can be digitalized.

Molecular epidemiology and genetic diversity of Mycobacterium tuberculosis complex in the Cross River State, Nigeria.

This study provides with a first insight on Mycobacterium tuberculosis complex epidemiology and genetic diversity in the Cross River State, Nigeria. Starting with 137 smear positive patients recruited over a period of 12months (June 2008 to May 2009), we obtained 97 pure mycobacterial isolates out of which 81 (83.5%) were identified as M. tuberculosis complex. Genotyping revealed a total of 27 spoligotypes patterns with 10 clusters (n=64% or 79% of clustered isolates, 2-32 isolates/cluster), with patients in the age group range 25-34 years being significantly associated with shared-type pattern SIT61 (p=0.019). Comparison with SITVIT2 database showed that with the exception of a single cluster (SIT727/H1), all other clusters observed were representative of West Africa; the two main lineages involved were LAM10-CAM (n=42/81% or 51.8%) of M. tuberculosis and AFRI_2 sublineage of Mycobacterium africanum (n=27/81% or 33.3%). Subsequent 12-loci MIRU typing resulted in a total of 13 SIT/MIT clusters (n=52 isolates, 2-9 isolates per cluster), with a resulting recent n-1 transmission rate of 48.1%. Available drug-susceptibility testing (DST) results for 58/81 clinical isolates revealed 6/58% or 10.4% cases of multiple drug-resistance (MDR); 5/6 MDR cases were caused by strains belonging to LAM10-CAM lineage (a specific cluster SIT61/MIT266 in 4/6 cases, and an orphan spoligotype pattern in 1/6 case). Additionally, MIT266 was associated with streptomycin resistance (p=0.016). All the six MDRTB isolates were concomitantly resistance to streptomycin and ethambutol; however, 4/6 MDR strains with identical MIRU patterns were characterized by consecutive strain numbers hence the possibility of laboratory cross contamination could not be excluded in 3/4 serial cases. The present preliminary study underlines the usefulness of spoligotyping and 12-loci MIRU-VNTRs to establish a baseline of circulating genotypic lineages of M. tuberculosis complex in Nigeria.

The Guinea-Bissau family of Mycobacterium tuberculosis complex revisited.

The Guinea-Bissau family of strains is a unique group of the Mycobacterium tuberculosis complex that, although genotypically closely related, phenotypically demonstrates considerable heterogeneity. We have investigated 414 M. tuberculosis complex strains collected in Guinea-Bissau between 1989 and 2008 in order to further characterize the Guinea-Bissau family of strains. To determine the strain lineages present in the study sample, binary outcomes of spoligotyping were compared with spoligotypes existing in the international database SITVIT2. The major circulating M. tuberculosis clades ranked in the following order: AFRI (n = 195, 47.10%), Latin-American-Mediterranean (LAM) (n = 75, 18.12%), ill-defined T clade (n = 53, 12.8%), Haarlem (n = 37, 8.85%), East-African-Indian (EAI) (n = 25, 6.04%), Unknown (n = 12, 2.87%), Beijing (n = 7, 1.68%), X clade (n = 4, 0.96%), Manu (n = 4, 0.97%), CAS (n = 2, 0.48%). Two strains of the LAM clade isolated in 2007 belonged to the Cameroon family (SIT61). All AFRI isolates except one belonged to the Guinea-Bissau family, i.e. they have an AFRI_1 spoligotype pattern, they have a distinct RFLP pattern with low numbers of IS6110 insertions, and they lack the regions of difference RD7, RD8, RD9 and RD10, RD701 and RD702. This profile classifies the Guinea-Bissau family, irrespective of phenotypic biovar, as part of the M. africanum West African 2 lineage, or the AFRI_1 sublineage according to the spoligtyping nomenclature. Guinea-Bissau family strains display a variation of biochemical traits classically used to differentiate M. tuberculosis from M. bovis. Yet, the differential expression of these biochemical traits was not related to any genes so far investigated (narGHJI and pncA). Guinea-Bissau has the highest prevalence of M. africanum recorded in the African continent, and the Guinea-Bissau family shows a high phylogeographical specificity for Western Africa, with Guinea-Bissau being the epicenter. Trends over time however indicate that this family of strains is waning in most parts of Western Africa, including Guinea-Bissau (p = 0.048).

Spoligotype profile of Mycobacterium tuberculosis complex strains from HIV-positive and -negative patients in Nigeria: a comparative analysis.

We ran a comparative analysis of all patients for whom a positive culture of Mycobacterium tuberculosis complex was available between April 2004 and October 2005 and whose HIV serology results were known, with spoligotyping results (n = 163) split into 49 HIV-positive patients and 114 HIV-negative patients. Spoligotype international type 373 (SIT373) (T1 lineage), which was highly prevalent among the HIV(+) patients, was totally absent from the HIV(-) population, suggesting that we had a specific clone affecting nearly 1/3 of all HIV-tuberculosis (TB)-coinfected patients. Among the LAM10-CAM sublineage strains, we had only a single strain of SIT403 among HIV(-) patients (0.88%), as opposed to 12.25% of the HIV(+) population (χ(2) = 10.77; P < 0.01), indicating a strong association between the strain and the HIV(+) population. The LAM10-CAM lineage spoligotype SIT61 was prevalent among the 2 subsets (37.72% in HIV(-) versus 12.24% in HIV(+) populations), though, with a significant difference between the 2 groups (χ(2) = 10.53; P < 0.01). However, there was no significant difference for SIT53 (T1 lineage) in the 2 subsets: 6.14 versus 8.2% (χ(2) = 0.22; P > 0.05). A total of 7/49, or 14.3%, other SITs among HIV(+) patients were not found among the HIV(-) patients. When added to the most prevalent SIT among HIV(+) patients (SIT373; n = 16), 23/49, or 47%, isolates among HIV-TB-coinfected patients were unique. We conclude that further studies should be carried out to investigate the evolution of these genotypes and others in the emergence of multidrug resistance and control of tuberculosis in Nigeria.

Molecular diversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Mozambique.

BACKGROUND: Mozambique is one of the countries with the highest burden of tuberculosis (TB) in Sub-Saharan Africa, and information on the predominant genotypes of Mycobacterium tuberculosis circulating in the country are important to better understand the epidemic. This study determined the predominant strain lineages that cause TB in Mozambique. RESULTS: A total of 445 M. tuberculosis isolates from seven different provinces of Mozambique were characterized by spoligotyping and resulting profiles were compared with the international spoligotyping database SITVIT2.The four most predominant lineages observed were: the Latin-American Mediterranean (LAM, n = 165 or 37%); the East African-Indian (EAI, n = 132 or 29.7%); an evolutionary recent but yet ill-defined T clade, (n = 52 or 11.6%); and the globally-emerging Beijing clone, (n = 31 or 7%). A high spoligotype diversity was found for the EAI, LAM and T lineages. CONCLUSIONS: The TB epidemic in Mozambique is caused by a wide diversity of spoligotypes with predominance of LAM, EAI, T and Beijing lineages.