Phylogenomic exploration of the relationships between strains of Mycobacterium avium subspecies paratuberculosis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (Map) is an infectious enteric pathogen that causes Johne's disease in livestock. Determining genetic diversity is prerequisite to understanding the epidemiology and biology of Map. We performed the first whole genome sequencing (WGS) of 141 global Map isolates that encompass the main molecular strain types currently reported. We investigated the phylogeny of the Map strains, the diversity of the genome and the limitations of commonly used genotyping methods. RESULTS: Single nucleotide polymorphism (SNP) and phylogenetic analyses confirmed two major lineages concordant with the former Type S and Type C designations. The Type I and Type III strain groups are subtypes of Type S, and Type B strains are a subtype of Type C and not restricted to Bison species. We found that the genome-wide SNPs detected provided greater resolution between isolates than currently employed genotyping methods. Furthermore, the SNP used for IS1311 typing is not informative, as it is likely to have occurred after Type S and C strains diverged and does not assign all strains to the correct lineage. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) differentiates Type S from Type C but provides limited resolution between isolates within these lineages and the polymorphisms detected do not necessarily accurately reflect the phylogenetic relationships between strains. WGS of passaged strains and coalescent analysis of the collection revealed a very high level of genetic stability, with the substitution rate estimated to be less than 0.5 SNPs per genome per year. CONCLUSIONS: This study clarifies the phylogenetic relationships between the previously described Map strain groups, and highlights the limitations of current genotyping techniques. Map isolates exhibit restricted genetic diversity and a substitution rate consistent with a monomorphic pathogen. WGS provides the ultimate level of resolution for differentiation between strains. However, WGS alone will not be sufficient for tracing and tracking Map infections, yet importantly it can provide a phylogenetic context for affirming epidemiological connections

Busulfan and subsequent malignancy: An evidence-based risk assessment

BACKGROUND: The incidence of secondary malignancies associated with busulfan exposure is considered low, but has been poorly characterized. Because this alkylating agent is increasingly utilized as conditioning prior to gene therapy in nonmalignant hematologic and related disorders, more precise characterization of busulfan's potential contribution to subsequent malignant risk is warranted. PROCEDURE: We conducted a literature-based assessment of busulfan and subsequent late effects, with emphasis on secondary malignancies, identifying publications via PubMed searches, and selecting those reporting at least 3 years of follow-up. RESULTS: We identified eight pediatric and 13 adult publications describing long-term follow-up in 570 pediatric and 2076 adult hematopoietic cell transplant (HCT) recipients. Secondary malignancies were reported in 0.5% of pediatric HCT recipients, with no cases of myelodysplastic syndrome (MDS) or acute myelocytic leukemia (AML). Fatal secondary malignancies were reported in 0.8% of 1887 evaluable adult HCT recipients, and an overall incidence of secondary malignancies of 4.8% was reported in a subset of 389 evaluable adult patients. We also reviewed long-term results from eight publications evaluating lentiviral- and human promotor-based HSC-targeted gene therapy in 215 patients with nonmalignant conditions, in which busulfan/treosulfan monotherapy or busulfan/fludarabine was the only conditioning. Two malignancies were reported in patients with sickle cell disease (SCD), one of which was potentially busulfan-related. No additional malignancies were reported in 173 patients with follow-up of 5-12 years. CONCLUSION: The incidence of busulfan-related secondary malignancies is low, and likely to be substantially less than 1% in pediatric transplant recipients, especially those receiving busulfan monotherapy for nonmalignant conditions other than SCD

An Esrrb and nanog cell fate regulatory module controlled by feed forward loop interactions

Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory 'dimensions' coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module

Next-generation sequencing in bone marrow failure syndromes and isolated cytopenias: experience of the spanish network on bone marrow failure sundromes

© 2021 the Author(s).Inherited bone marrow failure syndromes (IBMFSs) are a group of congenital rare diseases characterized by bone marrow failure, congenital anomalies, high genetic heterogeneity, and predisposition to cancer. Appropriate treatment and cancer surveillance ideally depend on the identification of the mutated gene. A next-generation sequencing (NGS) panel of genes could be 1 initial genetic screening test to be carried out in a comprehensive study of IBMFSs, allowing molecular detection in affected patients. We designed 2 NGS panels of IBMFS genes: version 1 included 129 genes and version 2 involved 145 genes. The cohort included a total of 204 patients with suspected IBMFSs without molecular diagnosis. Capture-based targeted sequencing covered > 99% of the target regions of 145 genes, with more than 20 independent reads. No differences were seen between the 2 versions of the panel. The NGS tool allowed a total of 91 patients to be diagnosed, with an overall molecular diagnostic rate of 44%. Among the 167 patients with classified IBMFSs, 81 patients (48%) were diagnosed. Unclassified IBMFSs involved a total of 37 patients, of whom 9 patients (24%) were diagnosed. The preexisting diagnosis of 6 clinically classified patients (6%) was amended, implying a change of therapy for some of them. Our NGS IBMFS gene panel assay is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders

Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling

SummaryMany signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A (Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming

Dual latent tuberculosis screening with tuberculin skin tests and QuantiFERON-TB assays before TNF-α inhibitor initiation in children in Spain

Tumor-necrosis-factor-α inhibitors (anti-TNF-α) are associated with an increased risk of tuberculosis (TB) disease, primarily due to reactivation of latent TB infection (LTBI). We assessed the performance of parallel LTBI screening with tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube assays (QFT-GIT) before anti-TNF-α treatment in children with immune-mediated inflammatory disorders in a low TB-burden setting. We conducted a multicenter cohort study involving 17 pediatric tertiary centers in Spain. LTBI was defined as the presence of a positive TST and/or QFT-GIT result without clinical or radiological signs of TB disease. A total of 270 patients (median age:11.0 years) were included, mainly with rheumatological (55.9%) or inflammatory bowel disease (34.8%). Twelve patients (4.4%) were diagnosed with TB infection at screening (LTBI, n = 11; TB disease, n = 1). Concordance between TST and QFT-GIT results was moderate (TST+/QFT-GIT+, n = 4; TST-/QFT-GIT+, n = 3; TST+/QFT-GIT-, n = 5; kappa coefficient: 0.48, 95% CI: 0.36-0.60). Indeterminate QFT-GIT results occurred in 10 patients (3.7%) and were associated with young age and elevated C-reactive protein concentrations. Eleven of 12 patients with TB infection uneventfully completed standard LTBI or TB treatment. During a median follow-up period of 6.4 years, only 2 patients developed TB disease (incidence density: 130 (95% CI: 20-440) per 100,000 person-years), both probable de novo infections. Conclusion: A substantial number of patients were diagnosed with LTBI during screening. The dual strategy identified more cases than either of the tests alone, and test agreement was only moderate. Our data show that in children in a low TB prevalence setting, a dual screening strategy with TST and IGRA before anti-TNF-α treatment is effective

Genetic analyses of aplastic anemia and idiopathic pulmonary fibrosis patients with short telomeres, possible implication of DNA-repair genes

Background: Telomeres are nucleoprotein structures present at the terminal region of the chromosomes. Mutations in genes coding for proteins involved in telomere maintenance are causative of a number of disorders known as telomeropathies. The genetic origin of these diseases is heterogeneous and has not been determined for a significant proportion of patients. Methods: This article describes the genetic characterization of a cohort of patients. Telomere length was determined by Southern blot and quantitative PCR. Nucleotide variants were analyzed either by high-resolution melting analysis and Sanger sequencing of selected exons or by massive sequencing of a panel of genes. Results: Forty-seven patients with telomere length below the 10% of normal population, affected with three telomeropathies: dyskeratosis congenita (4), aplastic anemia (22) or pulmonary fibrosis (21) were analyzed. Eighteen of these patients presented known pathogenic or novel possibly pathogenic variants in the telomere-related genes TERT, TERC, RTEL1, CTC1 and ACD. In addition, the analyses of a panel of 188 genes related to haematological disorders indicated that a relevant proportion of the patients (up to 35%) presented rare variants in genes related to DNA repair or in genes coding for proteins involved in the resolution of complex DNA structures, that participate in telomere replication. Mutations in some of these genes are causative of several syndromes previously associated to telomere shortening

Prospective individual patient data meta-analysis of two randomized trials on convalescent plasma for COVID-19 outpatients

Data on convalescent plasma (CP) treatment in COVID-19 outpatients are scarce. We aimed to assess whether CP administered during the first week of symptoms reduced the disease progression or risk of hospitalization of outpatients. Two multicenter, double-blind randomized trials (NCT04621123, NCT04589949) were merged with data pooling starting when = 50 years and symptomatic for <= 7days were included. The intervention consisted of 200-300mL of CP with a predefined minimum level of antibodies. Primary endpoints were a 5-point disease severity scale and a composite of hospitalization or death by 28 days. Amongst the 797 patients included, 390 received CP and 392 placebo; they had a median age of 58 years, 1 comorbidity, 5 days symptoms and 93% had negative IgG antibody-test. Seventy-four patients were hospitalized, 6 required mechanical ventilation and 3 died. The odds ratio (OR) of CP for improved disease severity scale was 0.936 (credible interval (CI) 0.667-1.311); OR for hospitalization or death was 0.919 (CI 0.592-1.416). CP effect on hospital admission or death was largest in patients with <= 5 days of symptoms (OR 0.658, 95%CI 0.394-1.085). CP did not decrease the time to full symptom resolution

Novel genes and sex differences in COVID-19 severity

[EN] Here, we describe the results of a genome-wide study conducted in 11 939 coronavirus disease 2019 (COVID-19) positive cases with an extensive clinical information that were recruited from 34 hospitals across Spain (SCOURGE consortium). In sex-disaggregated genome-wide association studies for COVID-19 hospitalization, genome-wide significance (P < 5 × 10−8) was crossed for variants in 3p21.31 and 21q22.11 loci only among males (P = 1.3 × 10−22 and P = 8.1 × 10−12, respectively), and for variants in 9q21.32 near TLE1 only among females (P = 4.4 × 10−8). In a second phase, results were combined with an independent Spanish cohort (1598 COVID-19 cases and 1068 population controls), revealing in the overall analysis two novel risk loci in 9p13.3 and 19q13.12, with fine-mapping prioritized variants functionally associated with AQP3 (P = 2.7 × 10−8) and ARHGAP33 (P = 1.3 × 10−8), respectively. The meta-analysis of both phases with four European studies stratified by sex from the Host Genetics Initiative (HGI) confirmed the association of the 3p21.31 and 21q22.11 loci predominantly in males and replicated a recently reported variant in 11p13 (ELF5, P = 4.1 × 10−8). Six of the COVID-19 HGI discovered loci were replicated and an HGI-based genetic risk score predicted the severity strata in SCOURGE. We also found more SNP-heritability and larger heritability differences by age (<60 or ≥60 years) among males than among females. Parallel genome-wide screening of inbreeding depression in SCOURGE also showed an effect of homozygosity in COVID-19 hospitalization and severity and this effect was stronger among older males. In summary, new candidate genes for COVID-19 severity and evidence supporting genetic disparities among sexes are provided.S