Congenital iRHOM2 deficiency causes ADAM17 dysfunction and environmentally directed immunodysregulatory disease.

We report a pleiotropic disease due to loss-of-function mutations in RHBDF2, the gene encoding iRHOM2, in two kindreds with recurrent infections in different organs. One patient had recurrent pneumonia but no colon involvement, another had recurrent infectious hemorrhagic colitis but no lung involvement and the other two experienced recurrent respiratory infections. Loss of iRHOM2, a rhomboid superfamily member that regulates the ADAM17 metalloproteinase, caused defective ADAM17-dependent cleavage and release of cytokines, including tumor-necrosis factor and amphiregulin. To understand the diverse clinical phenotypes, we challenged Rhbdf2-/- mice with Pseudomonas aeruginosa by nasal gavage and observed more severe pneumonia, whereas infection with Citrobacter rodentium caused worse inflammatory colitis than in wild-type mice. The fecal microbiota in the colitis patient had characteristic oral species that can predispose to colitis. Thus, a human immunodeficiency arising from iRHOM2 deficiency causes divergent disease phenotypes that can involve the local microbial environment.

Perturbations of the T-cell receptor repertoire in response to SARS-CoV-2 in immunocompetent and immunocompromised individuals.

BACKGROUND: Functional T-cell responses are essential for virus clearance and long-term protection after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, whereas certain clinical factors, such as older age and immunocompromise, are associated with worse outcome. OBJECTIVE: We sought to study the breadth and magnitude of T-cell responses in patients with coronavirus disease 2019 (COVID-19) and in individuals with inborn errors of immunity (IEIs) who had received COVID-19 mRNA vaccine. METHODS: Using high-throughput sequencing and bioinformatics tools to characterize the T-cell receptor ß repertoire signatures in 540 individuals after SARS-CoV-2 infection, 31 IEI recipients of COVID-19 mRNA vaccine, and healthy controls, we quantified HLA class I- and class II-restricted SARS-CoV-2-specific responses and also identified several HLA allele-clonotype motif associations in patients with COVID-19, including a subcohort of anti-type 1 interferon (IFN-1)-positive patients. RESULTS: Our analysis revealed that elderly patients with COVID-19 with critical disease manifested lower SARS-CoV-2 T-cell clonotype diversity as well as T-cell responses with reduced magnitude, whereas the SARS-CoV-2-specific clonotypes targeted a broad range of HLA class I- and class II-restricted epitopes across the viral proteome. The presence of anti-IFN-I antibodies was associated with certain HLA alleles. Finally, COVID-19 mRNA immunization induced an increase in the breadth of SARS-CoV-2-specific clonotypes in patients with IEIs, including those who had failed to seroconvert. CONCLUSIONS: Elderly individuals have impaired capacity to develop broad and sustained T-cell responses after SARS-CoV-2 infection. Genetic factors may play a role in the production of anti-IFN-1 antibodies. COVID-19 mRNA vaccines are effective in inducing T-cell responses in patients with IEIs.

Clinical exome sequencing of 1000 families with complex immune phenotypes: Toward comprehensive genomic evaluations.

BACKGROUND: Prospective genetic evaluation of patients at this referral research hospital presents clinical research challenges. OBJECTIVES: This study sought not only a single-gene explanation for participants' immune-related presentations, but viewed each participant holistically, with the potential to have multiple genetic contributions to their immune phenotype and other heritable comorbidities relevant to their presentation and health. METHODS: This study developed a program integrating exome sequencing, chromosomal microarray, phenotyping, results return with genetic counseling, and reanalysis in 1505 individuals from 1000 families with suspected or known inborn errors of immunity. RESULTS: Probands were 50.8% female, 71.5% were ≥18 years, and had diverse immune presentations. Overall, 327 of 1000 probands (32.7%) received 361 molecular diagnoses. These included 17 probands with diagnostic copy number variants, 32 probands with secondary findings, and 31 probands with multiple molecular diagnoses. Reanalysis added 22 molecular diagnoses, predominantly due to new disease-gene associations (9 of 22, 40.9%). One-quarter of the molecular diagnoses (92 of 361) did not involve immune-associated genes. Molecular diagnosis was correlated with younger age, male sex, and a higher number of organ systems involved. This program also facilitated the discovery of new gene-disease associations such as SASH3-related immunodeficiency. A review of treatment options and ClinGen actionability curations suggest that at least 251 of 361 of these molecular diagnoses (69.5%) could translate into ≥1 management option. CONCLUSIONS: This program contributes to our understanding of the diagnostic and clinical utility whole exome analysis on a large scale.

Susceptibility to kidney fibrosis in mice is associated with early growth response-2 protein and tissue inhibitor of metalloproteinase-1 expression.

Patients with chronic kidney disease and experimental animal models of kidney fibrosis manifest diverse progression rates. Genetic susceptibility may contribute to this diversity, but the causes remain largely unknown. We have previously described kidney fibrosis with a mild or severe phenotype in mice expressing transforming growth factor-beta1 (TGF-ß1) under the control of a mouse albumin promoter (Alb/TGF-ß1), on a mixed genetic background with CBAxC57Bl6 mice. Here, we aimed to examine how genetic background may influence kidney fibrosis in TGF-ß1 transgenic mice, and in the unilateral ureteral obstruction (UUO) and subtotal nephrectomy (SNX) mouse models. Congenic C57Bl6(B6)-TGFß and CBAxB6-TGFß (F1) transgenic mice were generated and survival, proteinuria, kidney histology, transcriptome and protein expressions were analyzed. We investigated the kidneys of B6 and CBA mice subjected to UUO and SNX, and the effects of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) neutralization on the fibrotic process. CBAxB6-TGFß mice developed severe kidney fibrosis and premature death, while B6-TGF-ß mice had mild fibrosis and prolonged survival. Kidney early growth response factor-2 (EGR2) and TIMP-1 expression were induced only in CBAxB6-TGFß mice. Similar strain-dependent early changes in EGR2 and TIMP-1 of mice subjected to UUO or SNX were observed. TIMP-1 neutralization in vivo hindered fibrosis both in transgenic mice and the SNX model. EGR2 over-expression in cultured HEK293 cells induced TIMP-1 while EGR2 silencing hindered TGF-ß induced TIMP-1 production in HK-2 cells and ureteral obstructed kidneys. Finally, EGR2 and TIMP1 was increased in human kidneys manifesting focal segmental glomerulosclerosis suggesting a correlation between animal studies and patient clinical settings. Thus, our observations demonstrate a strong relationship between genetic background and the progression of kidney fibrosis, which might involve early altered EGR2 and TIMP-1 response, but the relationship to patient genetics remains to be explored.

Extended clinical and immunological phenotype and transplant outcome in CD27 and CD70 deficiency.

Biallelic mutations in the genes encoding CD27 or its ligand CD70 underlie inborn errors of immunity (IEIs) characterized predominantly by Epstein-Barr virus (EBV)-associated immune dysregulation, such as chronic viremia, severe infectious mononucleosis, hemophagocytic lymphohistiocytosis (HLH), lymphoproliferation, and malignancy. A comprehensive understanding of the natural history, immune characteristics, and transplant outcomes has remained elusive. Here, in a multi-institutional global collaboration, we collected the clinical information of 49 patients from 29 families (CD27, n = 33; CD70, n = 16), including 24 previously unreported individuals and identified a total of 16 distinct mutations in CD27, and 8 in CD70, respectively. The majority of patients (90%) were EBV+ at diagnosis, but only ∼30% presented with infectious mononucleosis. Lymphoproliferation and lymphoma were the main clinical manifestations (70% and 43%, respectively), and 9 of the CD27-deficient patients developed HLH. Twenty-one patients (43%) developed autoinflammatory features including uveitis, arthritis, and periodic fever. Detailed immunological characterization revealed aberrant generation of memory B and T cells, including a paucity of EBV-specific T cells, and impaired effector function of CD8+ T cells, thereby providing mechanistic insight into cellular defects underpinning the clinical features of disrupted CD27/CD70 signaling. Nineteen patients underwent allogeneic hematopoietic stem cell transplantation (HSCT) prior to adulthood predominantly because of lymphoma, with 95% survival without disease recurrence. Our data highlight the marked predisposition to lymphoma of both CD27- and CD70-deficient patients. The excellent outcome after HSCT supports the timely implementation of this treatment modality particularly in patients presenting with malignant transformation to lymphoma.

PhenoRerank: A re-ranking model for phenotypic concept recognition pre-trained on human phenotype ontology.

The study aims at developing a neural network model to improve the performance of Human Phenotype Ontology (HPO) concept recognition tools. We used the terms, definitions, and comments about the phenotypic concepts in the HPO database to train our model. The document to be analyzed is first split into sentences and annotated with a base method to generate candidate concepts. The sentences, along with the candidate concepts, are then fed into the pre-trained model for re-ranking. Our model comprises the pre-trained BlueBERT and a feature selection module, followed by a contrastive loss. We re-ranked the results generated by three robust HPO annotation tools and compared the performance against most of the existing approaches. The experimental results show that our model can improve the performance of the existing methods. Significantly, it boosted 3.0% and 5.6% in F1 score on the two evaluated datasets compared with the base methods. It removed more than 80% of the false positives predicted by the base methods, resulting in up to 18% improvement in precision. Our model utilizes the descriptive data in the ontology and the contextual information in the sentences for re-ranking. The results indicate that the additional information and the re-ranking model can significantly enhance the precision of HPO concept recognition compared with the base method.

Global selective sweep of a highly inbred genome of the cattle parasite Neospora caninum.

Neospora caninum, a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular diseases in dogs as well as fetal abortion in cattle worldwide. The importance of the domestic and sylvatic life cycles of Neospora, and the role of vertical transmission in the expansion and transmission of infection in cattle, is not sufficiently understood. To elucidate the population genomics of Neospora, we genotyped 50 isolates collected worldwide from a wide range of hosts using 19 linked and unlinked genetic markers. Phylogenetic analysis and genetic distance indices resolved a single genotype of N. caninum Whole-genome sequencing of 7 isolates from 2 different continents identified high linkage disequilibrium, significant structural variation, but only limited polymorphism genome-wide, with only 5,766 biallelic single nucleotide polymorphisms (SNPs) total. Greater than half of these SNPs (∼3,000) clustered into 6 distinct haploblocks and each block possessed limited allelic diversity (with only 4 to 6 haplotypes resolved at each cluster). Importantly, the alleles at each haploblock had independently segregated across the strains sequenced, supporting a unisexual expansion model that is mosaic at 6 genomic blocks. Integrating seroprevalence data from African cattle, our data support a global selective sweep of a highly inbred livestock pathogen that originated within European dairy stock and expanded transcontinentally via unisexual mating and vertical transmission very recently, likely the result of human activities, including recurrent migration, domestication, and breed development of bovid and canid hosts within similar proximities.

Genome-wide association study in patients with pulmonary Mycobacterium avium complex disease.

RATIONALE: Nontuberculous mycobacteria (NTM) are environmental mycobacteria that can cause a chronic progressive lung disease. Although epidemiological data indicate potential genetic predisposition, its nature remains unclear. OBJECTIVES: We aimed to identify host susceptibility loci for Mycobacterium avium complex (MAC), the most common NTM pathogen. METHODS: This genome-wide association study (GWAS) was conducted in Japanese patients with pulmonary MAC and healthy controls, followed by genotyping of candidate single-nucleotide polymorphisms (SNPs) in another Japanese cohort. For verification by Korean and European ancestry, we performed SNP genotyping. RESULTS: The GWAS discovery set included 475 pulmonary MAC cases and 417 controls. Both GWAS and replication analysis of 591 pulmonary MAC cases and 718 controls revealed the strongest association with chromosome 16p21, particularly with rs109592 (p=1.64×10-13, OR 0.54), which is in an intronic region of the calcineurin-like EF-hand protein 2 (CHP2). Expression quantitative trait loci analysis demonstrated an association with lung CHP2 expression. CHP2 was expressed in the lung tissue in pulmonary MAC disease. This SNP was associated with the nodular bronchiectasis subtype. Additionally, this SNP was significantly associated with the disease in patients of Korean (p=2.18×10-12, OR 0.54) and European (p=5.12×10-03, OR 0.63) ancestry. CONCLUSIONS: We identified rs109592 in the CHP2 locus as a susceptibility marker for pulmonary MAC disease.

The transcription factor T-bet is induced by multiple pathways and prevents an endogenous Th2 cell program during Th1 cell responses.

T-bet is a critical transcription factor for T helper 1 (Th1) cell differentiation. To study the regulation and functions of T-bet, we developed a T-bet-ZsGreen reporter mouse strain. We determined that interleukin-12 (IL-12) and interferon-γ (IFN-γ) were redundant in inducing T-bet in mice infected with Toxoplasma gondii and that T-bet did not contribute to its own expression when induced by IL-12 and IFN-γ. By contrast, T-bet and the transcription factor Stat4 were critical for IFN-γ production whereas IFN-γ signaling was dispensable for inducing IFN-γ. Loss of T-bet resulted in activation of an endogenous program driving Th2 cell differentiation in cells expressing T-bet-ZsGreen. Genome-wide analyses indicated that T-bet directly induced many Th1 cell-related genes but indirectly suppressed Th2 cell-related genes. Our study revealed redundancy and synergy among several Th1 cell-inducing pathways in regulating the expression of T-bet and IFN-γ, and a critical role of T-bet in suppressing an endogenous Th2 cell-associated program.

Magnesium transporter 1 (MAGT1) deficiency causes selective defects in N-linked glycosylation and expression of immune-response genes.

Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches. In humans, loss-of-function mutations in the MAGT1 gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasia (XMEN), a disease that has a broad range of clinical and immunological consequences. We have previously shown that EBV susceptibility in XMEN is associated with defective expression of the antiviral natural-killer group 2 member D (NKG2D) protein and abnormal Mg2+ transport. New evidence suggests that MAGT1 is the human homolog of the yeast OST3/OST6 proteins that form an integral part of the N-linked glycosylation complex, although the exact contributions of these perturbations in the glycosylation pathway to disease pathogenesis are still unknown. Using MS-based glycoproteomics, along with CRISPR/Cas9-KO cell lines, natural killer cell-killing assays, and RNA-Seq experiments, we now demonstrate that humans lacking functional MAGT1 have a selective deficiency in both immune and nonimmune glycoproteins, and we identified several critical glycosylation defects in important immune-response proteins and in the expression of genes involved in immunity, particularly CD28. We show that MAGT1 function is partly interchangeable with that of the paralog protein tumor-suppressor candidate 3 (TUSC3) but that each protein has a different tissue distribution in humans. We observed that MAGT1-dependent glycosylation is sensitive to Mg2+ levels and that reduced Mg2+ impairs immune-cell function via the loss of specific glycoproteins. Our findings reveal that defects in protein glycosylation and gene expression underlie immune defects in an inherited disease due to MAGT1 deficiency.

Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs.

Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin (HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies. Antibody-free propagation following antigenic escape rapidly selected viruses with mutations that modulated receptor binding avidity through the addition of N-linked glycans to the HA globular domain. These findings expand our understanding of the viral mechanisms that maintain fitness during antigenic evolution to include glycan addition, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms.

Correction: Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs.

[This corrects the article DOI: 10.1371/journal.ppat.1006796.].

Hypomorphic caspase activation and recruitment domain 11 (CARD11) mutations associated with diverse immunologic phenotypes with or without atopic disease.

BACKGROUND: Caspase activation and recruitment domain 11 (CARD11) encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nuclear factor κB, c-Jun N-terminal kinase, and mechanistic target of rapamycin complex 1. Germline CARD11 mutations cause several distinct primary immune disorders in human subjects, including severe combined immune deficiency (biallelic null mutations), B-cell expansion with nuclear factor κB and T-cell anergy (heterozygous, gain-of-function mutations), and severe atopic disease (loss-of-function, heterozygous, dominant interfering mutations), which has focused attention on CARD11 mutations discovered by using whole-exome sequencing. OBJECTIVES: We sought to determine the molecular actions of an extended allelic series of CARD11 and to characterize the expanding range of clinical phenotypes associated with heterozygous CARD11 loss-of-function alleles. METHODS: Cell transfections and primary T-cell assays were used to evaluate signaling and function of CARD11 variants. RESULTS: Here we report on an expanded cohort of patients harboring novel heterozygous CARD11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with CARD11 mutations. In addition to (and sometimes excluding) severe atopy, heterozygous missense and indel mutations in CARD11 presented with immunologic phenotypes similar to those observed in signal transducer and activator of transcription 3 loss of function, dedicator of cytokinesis 8 deficiency, common variable immunodeficiency, neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome. Pathogenic variants exhibited dominant negative activity and were largely confined to the CARD or coiled-coil domains of the CARD11 protein. CONCLUSION: These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in human subjects and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity.

Strand-Specific Dual RNA Sequencing of Bronchial Epithelial Cells Infected with Influenza A/H3N2 Viruses Reveals Splicing of Gene Segment 6 and Novel Host-Virus Interactions.

Host-influenza virus interplay at the transcript level has been extensively characterized in epithelial cells. Yet, there are no studies that simultaneously characterize human host and influenza A virus (IAV) genomes. We infected human bronchial epithelial BEAS-2B cells with two seasonal IAV/H3N2 strains, Brisbane/10/07 and Perth/16/09 (reference strains for past vaccine seasons) and the well-characterized laboratory strain Udorn/307/72. Strand-specific RNA sequencing (RNA-seq) of the infected BEAS-2B cells allowed for simultaneous analysis of host and viral transcriptomes, in addition to pathogen genomes, to reveal changes in mRNA expression and alternative splicing (AS). In general, patterns of global and immune gene expression induced by the three IAVs were mostly shared. However, AS of host transcripts and small nuclear RNAs differed between the seasonal and laboratory strains. Analysis of viral transcriptomes showed deletions of the polymerase components (defective interfering-like RNAs) within the genome. Surprisingly, we found that the neuraminidase gene undergoes AS and that the splicing event differs between seasonal and laboratory strains. Our findings reveal novel elements of the host-virus interaction and highlight the importance of RNA-seq in identifying molecular changes at the genome level that may contribute to shaping RNA-based innate immunity.IMPORTANCE The use of massively parallel RNA sequencing (RNA-seq) has revealed insights into human and pathogen genomes and their evolution. Dual RNA-seq allows simultaneous dissection of host and pathogen genomes and strand-specific RNA-seq provides information about the polarity of the RNA. This is important in the case of negative-strand RNA viruses like influenza virus, which generate positive (complementary and mRNA) and negative-strand RNAs (genome) that differ in their potential to trigger innate immunity. Here, we characterize interactions between human bronchial epithelial cells and three influenza A/H3N2 strains using strand-specific dual RNA-seq. We focused on this subtype because of its epidemiological importance in causing significant morbidity and mortality during influenza epidemics. We report novel elements that differ between seasonal and laboratory strains highlighting the complexity of the host-virus interplay at the RNA level.

Nephele: a cloud platform for simplified, standardized and reproducible microbiome data analysis.

Motivation: Widespread interest in the study of the microbiome has resulted in data proliferation and the development of powerful computational tools. However, many scientific researchers lack the time, training, or infrastructure to work with large datasets or to install and use command line tools. Results: The National Institute of Allergy and Infectious Diseases (NIAID) has created Nephele, a cloud-based microbiome data analysis platform with standardized pipelines and a simple web interface for transforming raw data into biological insights. Nephele integrates common microbiome analysis tools as well as valuable reference datasets like the healthy human subjects cohort of the Human Microbiome Project (HMP). Nephele is built on the Amazon Web Services cloud, which provides centralized and automated storage and compute capacity, thereby reducing the burden on researchers and their institutions. Availability and implementation: https://nephele.niaid.nih.gov and https://github.com/niaid/Nephele. Contact: darrell.hurt@nih.gov.

Recombinant Origins of Pathogenic and Nonpathogenic Mouse Gammaretroviruses with Polytropic Host Range.

Ecotropic, xenotropic, and polytropic mouse leukemia viruses (E-, X-, and P-MLVs) exist in mice as infectious viruses and endogenous retroviruses (ERVs) inserted into mouse chromosomes. All three MLV subgroups are linked to leukemogenesis, which involves generation of recombinants with polytropic host range. Although P-MLVs are deemed to be the proximal agents of disease induction, few biologically characterized infectious P-MLVs have been sequenced for comparative analysis. We analyzed the complete genomes of 16 naturally occurring infectious P-MLVs, 12 of which were typed for pathogenic potential. We sought to identify ERV progenitors, recombinational hot spots, and segments that are always replaced, never replaced, or linked to pathogenesis or host range. Each P-MLV has an E-MLV backbone with P- or X-ERV replacements that together cover 100% of the recombinant genomes, with different substitution patterns for X- and P-ERVs. Two segments are always replaced, both coding for envelope (Env) protein segments: the N terminus of the surface subunit and the cytoplasmic tail R peptide. Viral gag gene replacements are influenced by host restriction genes Fv1 and Apobec3 Pathogenic potential maps to the env transmembrane subunit segment encoding the N-heptad repeat (HR1). Molecular dynamics simulations identified three novel interdomain salt bridges in the lymphomagenic virus HR1 that could affect structural stability, entry or sensitivity to host immune responses. The long terminal repeats of lymphomagenic P-MLVs are differentially altered by recombinations, duplications, or mutations. This analysis of the naturally occurring, sometimes pathogenic P-MLV recombinants defines the limits and extent of intersubgroup recombination and identifies specific sequence changes linked to pathogenesis and host interactions.IMPORTANCE During virus-induced leukemogenesis, ecotropic mouse leukemia viruses (MLVs) recombine with nonecotropic endogenous retroviruses (ERVs) to produce polytropic MLVs (P-MLVs). Analysis of 16 P-MLV genomes identified two segments consistently replaced: one at the envelope N terminus that alters receptor choice and one in the R peptide at the envelope C terminus, which is removed during virus assembly. Genome-wide analysis shows that nonecotropic replacements in the progenitor ecotropic MLV genome are more extensive than previously appreciated, covering 100% of the genome; contributions from xenotropic and polytropic ERVs differentially alter the regions responsible for receptor determination or subject to APOBEC3 and Fv1 restriction. All pathogenic viruses had modifications in the regulatory elements in their long terminal repeats and differed in a helical segment of envelope involved in entry and targeted by the host immune system. Virus-induced leukemogenesis thus involves generation of complex recombinants, and specific replacements are linked to pathogenesis and host restrictions.

Prospective study of DNA methylation at chromosome 8q24 in peripheral blood and prostate cancer risk.

BACKGROUND: Chromosome 8q24 has emerged as an important genetic susceptibility region for several cancers, including prostate cancer; however, little is known about the contribution of DNA methylation in this region to risk. METHODS: We prospectively evaluated DNA methylation at 8q24 in relation to prostate cancer using pre-diagnostic blood samples from 694 prostate cancer cases (including 172 aggressive cases) and 703 controls in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. We used logistic regression to estimate odds ratios and 95% confidence intervals. RESULTS: Although none remained significant after adjustment for multiple testing (q>0.05), of the 50 CpG sites meeting quality control, we identified 8 sites that were nominally associated with prostate cancer (Ptrend<0.05), including 6 correlated (Spearman ρ: 0.20-0.52) sites in POU5F1B and 2 intergenic sites (most significant site: Chr8:128428897 in POU5F1B, Ptrend=0.01). We also identified two correlated (ρ=0.39) sites in MYC (Chr8:128753187 and Chr8:128753154) that were associated with aggressive (Ptrend=0.02 and 0.03), but not non-aggressive disease (Ptrend=0.70 and 0.20; Pheterogeneity=0.01 and 4.6 × 10-3). These findings persisted after adjustment for the top 8q24 prostate cancer variants in our study. CONCLUSIONS: Although requiring replication, our findings provide some evidence that 8q24 DNA methylation levels may be associated with prostate cancer risk.

Whole-Genome Sequencing of Mycobacterium tuberculosis Provides Insight into the Evolution and Genetic Composition of Drug-Resistant Tuberculosis in Belarus.

The emergence and spread of drug-resistant Mycobacterium tuberculosis (DR-TB) are critical global health issues. Eastern Europe has some of the highest incidences of DR-TB, particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. To better understand the genetic composition and evolution of MDR- and XDR-TB in the region, we sequenced and analyzed the genomes of 138 M. tuberculosis isolates from 97 patients sampled between 2010 and 2013 in Minsk, Belarus. MDR and XDR-TB isolates were significantly more likely to belong to the Beijing lineage than to the Euro-American lineage, and known resistance-conferring loci accounted for the majority of phenotypic resistance to first- and second-line drugs in MDR and XDR-TB. Using a phylogenomic approach, we estimated that the majority of MDR-TB was due to the recent transmission of already-resistant M. tuberculosis strains rather than repeated de novo evolution of resistance within patients, while XDR-TB was acquired through both routes. Longitudinal sampling of M. tuberculosis from 34 patients with treatment failure showed that most strains persisted genetically unchanged during treatment or acquired resistance to fluoroquinolones. HIV+ patients were significantly more likely to have multiple infections over time than HIV- patients, highlighting a specific need for careful infection control in these patients. These data provide a better understanding of the genomic composition, transmission, and evolution of MDR- and XDR-TB in Belarus and will enable improved diagnostics, treatment protocols, and prognostic decision-making.

Sequence Diversity, Intersubgroup Relationships, and Origins of the Mouse Leukemia Gammaretroviruses of Laboratory and Wild Mice.

UNLABELLED: Mouse leukemia viruses (MLVs) are found in the common inbred strains of laboratory mice and in the house mouse subspecies ofMus musculus Receptor usage and envelope (env) sequence variation define three MLV host range subgroups in laboratory mice: ecotropic, polytropic, and xenotropic MLVs (E-, P-, and X-MLVs, respectively). These exogenous MLVs derive from endogenous retroviruses (ERVs) that were acquired by the wild mouse progenitors of laboratory mice about 1 million years ago. We analyzed the genomes of seven MLVs isolated from Eurasian and American wild mice and three previously sequenced MLVs to describe their relationships and identify their possible ERV progenitors. The phylogenetic tree based on the receptor-determining regions ofenvproduced expected host range clusters, but these clusters are not maintained in trees generated from other virus regions. Colinear alignments of the viral genomes identified segmental homologies to ERVs of different host range subgroups. Six MLVs show close relationships to a small xenotropic ERV subgroup largely confined to the inbred mouse Y chromosome.envvariations define three E-MLV subtypes, one of which carries duplications of various sizes, sequences, and locations in the proline-rich region ofenv Outside theenvregion, all E-MLVs are related to different nonecotropic MLVs. These results document the diversity in gammaretroviruses isolated from globally distributedMussubspecies, provide insight into their origins and relationships, and indicate that recombination has had an important role in the evolution of these mutagenic and pathogenic agents. IMPORTANCE: Laboratory mice carry mouse leukemia viruses (MLVs) of three host range groups which were acquired from their wild mouse progenitors. We sequenced the complete genomes of seven infectious MLVs isolated from geographically separated Eurasian and American wild mice and compared them with endogenous germ line retroviruses (ERVs) acquired early in house mouse evolution. We did this because the laboratory mouse viruses derive directly from specific ERVs or arise by recombination between different ERVs. The six distinctively different wild mouse viruses appear to be recombinants, often involving different host range subgroups, and most are related to a distinctive, largely Y-chromosome-linked MLV ERV subtype. MLVs with ecotropic host ranges show the greatest variability with extensive inter- and intrasubtype envelope differences and with homologies to other host range subgroups outside the envelope. The sequence diversity among these wild mouse isolates helps define their relationships and origins and emphasizes the importance of recombination in their evolution.

Endogenous retrovirus induces leukemia in a xenograft mouse model for primary myelofibrosis.

The compound immunodeficiencies in nonobese diabetic (NOD) inbred mice homozygous for the Prkdc(scid) and Il2rg(null) alleles (NSG mice) permit engraftment of a wide-range of primary human cells, enabling sophisticated modeling of human disease. In studies designed to define neoplastic stem cells of primary myelofibrosis (PMF), a myeloproliferative neoplasm characterized by profound disruption of the hematopoietic microenvironment, we observed a high frequency of acute myeloid leukemia (AML) in NSG mice. AML was of mouse origin, confined to PMF-xenografted mice, and contained multiple clonal integrations of ecotropic murine leukemia virus (E-MuLV). Significantly, MuLV replication was not only observed in diseased mice, but also in nontreated NSG controls. Furthermore, in addition to the single ecotropic endogenous retrovirus (eERV) located on chromosome 11 (Emv30) in the NOD genome, multiple de novo germ-line eERV integrations were observed in mice from each of four independent NSG mouse colonies. Analysis confirmed that E-MuLV originated from the Emv30 provirus and that recombination events were not necessary for virus replication or AML induction. Pathogenicity is thus likely attributable to PMF-mediated paracrine stimulation of mouse myeloid cells, which serve as targets for retroviral infection and transformation, as evidenced by integration into the Evi1 locus, a hotspot for retroviral-induced myeloid leukemia. This study thus corroborates a role of paracrine stimulation in PMF disease progression, underlines the importance of target cell type and numbers in MuLV-induced disease, and mandates awareness of replicating MuLV in NOD immunodeficient mice, which can significantly influence experimental results and their interpretation.