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Background Prostate cancer is a clinically and molecularly heterogeneous disease, with highest incidence and mortality among men of African ancestry. To date, prostate cancer patient-derived xenograft (PCPDX) models to study this disease have been difficult to establish because of limited specimen availability and poor uptake rates in immunodeficient mice. Ancestrally diverse PCPDXs are even more rare, and only six PCPDXs from self-identified African American patients from one institution were recently made available. Methods In the present study, we established a PCPDX from prostate cancer tissue from a patient of estimated 90% West African ancestry with metastatic castration resistant disease, and characterized this model's pathology, karyotype, hotspot mutations, copy number, gene fusions, gene expression, growth rate in normal and castrated mice, therapeutic response, and experimental metastasis. Results This PCPDX has a mutation in TP53 and loss of PTEN and RB1. We have documented a 100% take rate in mice after thawing the PCPDX tumor from frozen stock. The PCPDX is castrate- and docetaxel-resistant and cisplatin-sensitive, and has gene expression patterns associated with such drug responses. After tail vein injection, the PCPDX tumor cells can colonize the lungs of mice. Conclusion This PCPDX, along with others that are established and characterized, will be useful pre-clinically for studying the heterogeneity of prostate cancer biology and testing new therapeutics in models expected to be reflective of the clinical setting.

Interleukin 7 receptor α-chain is crucial for the development and maintenance of T cells and is genetically associated with autoimmune disorders including multiple sclerosis (MS), a demyelinating disease of the CNS. Exon 6 of IL7R encodes for the transmembrane domain of the receptor and is regulated by alternative splicing: inclusion or skipping of IL7R exon 6 results in membrane-bound or soluble IL7R isoforms, respectively. We previously identified a SNP (rs6897932) in IL7R exon 6, strongly associated with MS risk and showed that the risk allele (C) increases skipping of the exon, resulting in elevated levels of sIL7R. This has important pathological consequences as elevated levels of sIL7R has been shown to exacerbate the disease in the experimental autoimmune encephalomyelitis mouse model of MS. Understanding the regulation of exon 6 splicing provides important mechanistic insights into the pathogenesis of MS. Here we report two mechanisms by which IL7R exon 6 is controlled. First, a competition between PTBP1 and U2AF2 at the polypyrimidine tract (PPT) of intron 5, and second, an unexpected U2AF2-mediated assembly of spicing factors in the exon. We noted the presence of a branchpoint sequence (BPS) (TACTAAT or TACTAAC) within exon 6, which is stronger with the C allele. We also noted that the BPS is followed by a PPT and conjectured that silencing could be mediated by the binding of U2AF2 to that tract. In support of this model, we show that evolutionary conservation of the exonic PPT correlates well with the degree of alternative splicing of exon 6 in two non-human primate species and that U2AF2 binding to this PPT recruits U2 snRNP components to the exon. These observations provide the first explanation for the stronger silencing of IL7R exon 6 with the disease associated C allele at rs6897932.

The genomic regulatory networks underlying the pathogenesis of non-ST-segment elevation acute coronary syndrome (NSTE-ACS) are incompletely understood. As intermediate traits, protein biomarkers report on underlying disease severity and prognosis in NSTE-ACS. We hypothesized that integration of dense microRNA (miRNA) profiling with biomarker measurements would highlight potential regulatory pathways that underlie the relationships between prognostic biomarkers, miRNAs, and cardiovascular phenotypes. We performed miRNA sequencing using whole blood from 186 patients from the TRILOGY-ACS trial. Seven circulating prognostic biomarkers were measured: NH2-terminal pro-B-type natriuretic peptide (NT-proBNP), high-sensitivity C-reactive protein, osteopontin (OPN), myeloperoxidase, growth differentiation factor 15, monocyte chemoattractant protein, and neopterin. We tested miRNAs for association with each biomarker with generalized linear models and controlled the false discovery rate at 0.05. Ten miRNAs, including known cardiac-related miRNAs 25-3p and 423-3p, were associated with NT-proBNP levels (min. P = 7.5 × 10−4) and 48 miRNAs, including cardiac-related miRNAs 378a-3p, 20b-5p and 320a, -b, and -d, were associated with OPN levels (min. P = 1.6 × 10−6). NT-proBNP and OPN were also associated with time to cardiovascular death, myocardial infarction (MI), or stroke in the sample. By integrating large-scale miRNA profiling with circulating biomarkers as intermediate traits, we identified associations of known cardiac-related and novel miRNAs with two prognostic biomarkers and identified potential genomic networks regulating these biomarkers. These results, highlighting plausible biological pathways connecting miRNAs with biomarkers and outcomes, may inform future studies seeking to delineate genomic pathways underlying NSTE-ACS outcomes.

Mutations in IRF6, TFAP2A and GRHL3 cause orofacial clefting syndromes in humans. However, Tfap2a and Grhl3 are also required for neurulation in mice. Here, we found that homeostasis of Irf6 is also required for development of the neural tube and associated structures. Over-expression of Irf6 caused exencephaly, a rostral neural tube defect, through suppression of Tfap2a and Grhl3 expression. Conversely, loss of Irf6 function caused a curly tail and coincided with a reduction of Tfap2a and Grhl3 expression in tail tissues. To test whether Irf6 function in neurulation was conserved, we sequenced samples obtained from human cases of spina bifida and anencephaly. We found two likely disease-causing variants in two samples from patients with spina bifida. Overall, these data suggest that the Tfap2a-Irf6-Grhl3 genetic pathway is shared by two embryologically distinct morphogenetic events that previously were considered independent during mammalian development. In addition, these data suggest new candidates to delineate the genetic architecture of neural tube defects and new therapeutic targets to prevent this common birth defect.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurrent abscesses, nodules, and sinus tracts in areas of high hair follicle and sweat gland density. These sinus tracts can present with purulent drainage and scar formation. Dysregulation of multiple immune pathways drives the complexity of HS pathogenesis and may account for the heterogeneity of treatment response in HS patients. Using transcriptomic approaches, including single-cell sequencing and protein analysis, we here characterize the innate inflammatory landscape of HS lesions. We identified a shared upregulation of genes involved in interferon (IFN) and antimicrobial defense signaling through transcriptomic overlap analysis of differentially expressed genes (DEGs) in datasets from HS skin, diabetic foot ulcers (DFUs), and the inflammatory stage of normal healing wounds. Overlap analysis between HS- and DFU-specific DEGs revealed an enrichment of gene signatures associated with monocyte/macrophage functions. Single-cell RNA sequencing further revealed monocytes/macrophages with polarization toward a pro-inflammatory M1-like phenotype and increased effector function, including antiviral immunity, phagocytosis, respiratory burst, and antibody-dependent cellular cytotoxicity. Specifically, we identified the STAT1/IFN-signaling axis and the associated IFN-stimulated genes as central players in monocyte/macrophage dysregulation. Our data indicate that monocytes/macrophages are a potential pivotal player in HS pathogenesis and their pathways may serve as therapeutic targets and biomarkers in HS treatment.

Background The neuropeptide oxytocin regulates mammalian social behavior. Disruptions in oxytocin signaling are a feature of many psychopathologies. One commonly studied biomarker for oxytocin involvement in psychiatric diseases is DNA methylation at the oxytocin receptor gene (OXTR). Such studies focus on DNA methylation in two regions of OXTR, exon 3 and a region termed MT2 which overlaps exon 1 and intron 1. However, the relative contribution of exon 3 and MT2 in regulating OXTR gene expression in the brain is currently unknown. Results Here, we use the prairie vole as a translational animal model to investigate genetic, epigenetic, and environmental factors affecting Oxtr gene expression in a region of the brain that has been shown to drive Oxtr related behavior in the vole, the nucleus accumbens. We show that the genetic structure of Oxtr in prairie voles resembles human OXTR. We then studied the effects of early life experience on DNA methylation in two regions of a CpG island surrounding the Oxtr promoter: MT2 and exon 3. We show that early nurture in the form of parental care results in DNA hypomethylation of Oxtr in both MT2 and exon 3, but only DNA methylation in MT2 is associated with Oxtr gene expression. Network analyses indicate that CpG sites in the 3′ portion of MT2 are most highly associated with Oxtr gene expression. We also identify two novel SNPs in exon 3 of Oxtr in prairie voles and a novel alternative transcript originating from the third intron of the gene. Expression of the novel alternative transcript is associated with genotype at SNP KLW2. Conclusions These results identify putative regulatory features of Oxtr in prairie voles which inform future studies examining OXTR in human social behaviors and disorders. These studies indicate that in prairie voles, DNA methylation in MT2, particularly in the 3′ portion, is more predictive of Oxtr gene expression than DNA methylation in exon 3. Similarly, in human temporal cortex, we find that DNA methylation in the 3′ portion of MT2 is associated with OXTR expression. Together, these results suggest that among the CpG sites studied, DNA methylation of MT2 may be the most reliable indicator of OXTR gene expression. We also identify novel features of prairie vole Oxtr, including SNPs and an alternative transcript, which further develop the prairie vole as a translational model for studies of OXTR.

Lymphoblastoid cell lines (LCLs) are generated by transforming primary B cells with Epstein–Barr virus (EBV) and are used extensively as model systems in viral oncology, immunology, and human genetics research. In this study, we characterized single-cell transcriptomic profiles of five LCLs and present a simple discrete-time simulation to explore the influence of stochasticity on LCL clonal evolution. Single-cell RNA sequencing (scRNA-seq) revealed substantial phenotypic heterogeneity within and across LCLs with respect to immunoglobulin isotype; virus-modulated host pathways involved in survival, activation, and differentiation; viral replication state; and oxidative stress. This heterogeneity is likely attributable to intrinsic variance in primary B cells and host–pathogen dynamics. Stochastic simulations demonstrate that initial primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness can contribute substantially to dynamic diversity in populations of nominally clonal cells.

Men with circulating tumor cell (CTC) AR-V7–positive metastatic castration-resistant prostate cancer (mCRPC) have worse outcomes when treated with enzalutamide/abiraterone. However, most men lack CTC AR-V7 detection, and additional predictive biomarkers are needed. We conducted a retrospective secondary analysis of the prospective PROPHECY trial (NCT02269982) of men with mCRPC undergoing treatment with enzalutamide/abiraterone, analyzing pooled CTC and germline DNA for whole-genome copy-number alterations (CNA) in 73 samples from 48 men over time along with pooled CTC and germline whole-exome sequencing on 22 paired samples before and following progression on androgen receptor (AR) inhibitor therapy to identify somatic genomic alterations associated with acquired resistance. We observed broad interpatient and longitudinal CTC genomic heterogeneity from AR-V7–negative men with mCRPC, including common gains of KDM6A, MYCN, and AR, and loss of ZFHX3, BRCA1, and PTEN. Men who had progression-free survival of ≤3 months despite enzalutamide/abiraterone treatment were more likely to have baseline CTC genomic loss of CHD1, PTEN, PHLPP1, and ZFHX3 and gains of BRCA2, KDM5D, MYCN, and SPARC. After progression on abiraterone/enzalutamide, we observed clonal evolution of CTCs harboring TP53 mutations and gain of ATM, KDM6A, and MYC, and loss of NCOR1, PTEN, RB1, and RUNX2. CTC genomic findings were independently confirmed in a separate cohort of mCRPC men who progressed despite prior treatment with abiraterone/enzalutamide (NCT02204943). Implications: We identified common and reproducible genomic alterations in CTCs from AR-V7–negative mCRPC men associated with poor outcomes during enzalutamide/abiraterone treatment, including CNAs in genes linked to lineage plasticity and epigenetic signaling, DNA repair, AR, TP53/RB1, PTEN, and WNT pathways.

Lymphoblastoid Cell Lines (LCLs) are generated by transforming primary B cells with Epstein-Barr Virus (EBV) and are used extensively as model systems in viral oncology, immunology, and human genetics research. In this study, we characterized single-cell transcriptomic profiles of five LCLs and present a simple discrete-time simulation to explore the influence of stochasticity on LCL clonal evolution. Single-cell RNA sequencing revealed substantial phenotypic heterogeneity within and across LCLs with respect to immunoglobulin isotype; virus-modulated host pathways involved in survival, proliferation, and differentiation; viral replication state; and oxidative stress. This heterogeneity is likely attributable to intrinsic variance in primary B cells and host-pathogen dynamics. Stochastic simulations demonstrate that initial primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness can contribute substantially to dynamic diversity in populations of nominally clonal cells.

Adult liver has enormous regenerative capacity; it can regenerate after losing two-thirds of its mass while sustaining essential metabolic functions. How the liver balances dual demands for increased proliferative activity with maintenance of organ function is unknown but essential to prevent liver failure. Using partial hepatectomy (PHx) in mice to model liver regeneration, we integrated single-cell RNA- and ATAC-Seq to map state transitions in approximately 13,000 hepatocytes at single-cell resolution as livers regenerated, and validated key findings with IHC, to uncover how the organ regenerates hepatocytes while simultaneously fulfilling its vital tissue-specific functions. After PHx, hepatocytes rapidly and transiently diversified into multiple distinct populations with distinct functional bifurcation: some retained the chromatin landscapes and transcriptomes of hepatocytes in undamaged adult livers, whereas others transitioned to acquire chromatin landscapes and transcriptomes of fetal hepatocytes. Injury-related signaling pathways known to be critical for regeneration were activated in transitioning hepatocytes, and the most fetal-like hepatocytes exhibited chromatin landscapes that were enriched with transcription factors regulated by those pathways., Mapping state transitions in hepatocytes at single-cell resolution as livers regenerated revealed bifurcation into adult and fetal-like hepatocyte populations.

Cancer therapies targeting metabolism have been limited due to a lack of understanding of the controlling properties of vulnerable pathways. The Na+/K+ATPase is responsible for a large portion of cellular energy demands but how these demands influence metabolism and create metabolic liabilities are not known. Using metabolomic approaches, we first show that digoxin, a cardiac glycoside widely used in humans, acts through disruption to central carbon metabolism via on target inhibition of the Na+/K+ATPase that was fully recovered by expression of an allele resistant to digoxin. We further show in vivo that administration of digoxin inhibits glycolysis in both malignant and healthy cells, particularly within clinically relevant cardiac tissue, while exhibiting tumor-specific cytotoxic activity in an allografted soft tissue sarcoma. Single-cell expression analysis of over 31,000 cells within the sarcoma shows that acute Na+/K+ATPase inhibition shifts the immune composition of the tumor microenvironment, leading to selective alterations to metabolic programs in specific immune cells thus acting both through tumor cell and microenvironmental (e.g. macrophage) cells. These results provide evidence that altering energy demands can be used to regulate glycolysis with cell-type specific consequences in a multicellular environment of biomedical interest.

The immune system plays a multifunctional role throughout the regenerative process, regulating both pro-/anti-inflammatory phases and progenitor cell function. In the present study, we identify the myokine/cytokine Meteorin-like (Metrnl) as a critical regulator of muscle regeneration. Mice genetically lacking Metrnl have impaired muscle regeneration associated with a reduction in immune cell infiltration and an inability to transition towards an anti-inflammatory phenotype. Isochronic parabiosis, joining wild-type and whole-body Metrnl knock-out (KO) mice, returns Metrnl expression in the injured muscle and improves muscle repair, providing supportive evidence for Metrnl secretion from infiltrating immune cells. Macrophage-specific Metrnl KO mice are also deficient in muscle repair. During muscle regeneration, Metrnl works, in part, through Stat3 activation in macrophages, resulting in differentiation to an anti-inflammatory phenotype. With regard to myogenesis, Metrnl induces macrophage-dependent insulin-like growth factor 1 production, which has a direct effect on primary muscle satellite cell proliferation. Perturbations in this pathway inhibit efficacy of Metrnl in the regenerative process. Together, these studies identify Metrnl as an important regulator of muscle regeneration and a potential therapeutic target to enhance tissue repair.

We elucidated the molecular cross-talk between cartilage and synovium in osteoarthritis, the most widespread arthritis in the world, using the powerful tool of single-cell RNA-sequencing. Multiple cell types were identified based on profiling of 10,640 synoviocytes and 26,192 chondrocytes: 12 distinct synovial cell types and 7 distinct articular chondrocyte phenotypes from matched tissues. Intact cartilage was enriched for homeostatic and hypertrophic chondrocytes, while damaged cartilage was enriched for prefibro- and fibro-, regulatory, reparative and prehypertrophic chondrocytes. A total of 61 cytokines and growth factors were predicted to regulate the 7 chondrocyte cell phenotypes. Based on production by > 1% of cells, 55% of the cytokines were produced by synovial cells (39% exclusive to synoviocytes and not expressed by chondrocytes) and their presence in osteoarthritic synovial fluid confirmed. The synoviocytes producing IL-1beta (a classic pathogenic cytokine in osteoarthritis), mainly inflammatory macrophages and dendritic cells, were characterized by co-expression of surface proteins corresponding to HLA-DQA1, HLA-DQA2, OLR1 or TLR2. Strategies to deplete these pathogenic intra-articular cell subpopulations could be a therapeutic option for human osteoarthritis.

Chiari malformations are considered to have a multifactorial etiology, likely influenced by environmental and genetic factors. This chapter will detail the evidence that supports a genetic contribution to the disorder, including discussions of twin studies, familial aggregation, co-occurrence with known genetic syndromes, and previous genetic studies. While no susceptibility genes have been identified to date, gene identification efforts are continuing. It is expected that researchers will have a more complete understanding of the specific genes and biological pathways that contribute to disease development in the coming years. The future benefits from genetic research of Chiari I and II may include the development of genetic tests that result in more accurate and faster diagnoses as well as new targeted treatment options for patients.

This study examined the utility of serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) as biomarkers in primary progressive multiple sclerosis in context with clinical severity, progression, and treatment. Using a single-molecule array (Quanterix), serum protein concentrations were measured from twenty-five participants semiannually for five years. There was no association between levels of either biomarker and disease severity, disease duration, or treatment group. Enrollment sNfL level was not associated with future clinical worsening. Precedent clinical worsening was not associated with last sGFAP measurement. These results suggest a limited role for these biomarkers in primary progressive disease management.

The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-seq analysis of zebrafish granulomas as well as analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell-autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.

The balance of myeloid populations and lymphoid populations must be well controlled. Here we found that osteopontin (OPN) skewed this balance during pathogenic conditions such as infection and autoimmunity. Notably, two isoforms of OPN exerted distinct effects in shifting this balance through cell-type-specific regulation of apoptosis. Intracellular OPN (iOPN) diminished the population size of myeloid progenitor cells and myeloid cells, and secreted OPN (sOPN) increase the population size of lymphoid cells. The total effect of OPN on skewing the leukocyte population balance was observed as host sensitivity to early systemic infection with Candida albicans and T cell-mediated colitis. Our study suggests previously unknown detrimental roles for two OPN isoforms in causing the imbalance of leukocyte populations.

Although circulating microRNA (miRNAs) have emerged as biomarkers predicting mortality in acute coronary syndrome (ACS), more data are needed to understand these mechanisms. Mapping miRNAs to high-risk traits may identify miRNAs involved in pathways conferring risk for poor outcome in ACS. We aim to investigate the relationship between circulating miRNAs and high-risk traits in non-ST-segment elevation acute coronary syndrome (NSTE-ACS).Whole-genome miRNA sequencing was performed on RNA extracted from whole blood of 199 patients with NSTE-ACS. Generalized linear models were used to test associations of miRNAs and 13 high-risk clinical traits, including the Global Registry of Acute Coronary Events (GRACE) score, a widely validated risk score for mortality in NSTE-ACS.There were 205 nominally significant miRNA-risk factor associations (p 0.05) observed. Significant associations occurred most frequently with chronic heart failure (HF) (43 miRs), GRACE risk score (30 miRs), and renal function (32 miRs). In hierarchical cluster analysis, chronic HF and GRACE risk score clustered most tightly together, sharing 14 miRNAs with matching fold-change direction. Controlling for a false discovery rate of 5%, chronic HF was significantly associated with lower circulating levels of miR-3135b (p 0.0006), miR-126-5p (p 0.0001), miR-142-5p (p = 0.0004) and miR-144-5p (p = 0.0007), while increasing GRACE risk score inversely correlated with levels of miR-3135b (p 0.0001) and positively correlated with levels of miR-28-3p (p = 0.0002).Circulating miRs clustered around two powerful traits for mortality risk in NSTE-ACS. MiR-3135b, which was under-expressed in chronic HF and increasing GRACE risk score, and miR-28-3p, which has no known association with cardiovascular disease, warrant further investigation.

The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal organization are highly heritable. To understand the genetic aetiology of Fuchs endothelial corneal dystrophy (FECD), the most prevalent corneal disorder requiring transplantation, we conducted a genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry, followed by replication and meta-analysis, for a total of 2,075 cases and 3,342 controls. We identify three novel loci meeting genome-wide significance (P, Fuchs endothelial corneal dystrophy (FECD) is one of the most common reasons for corneal transplantation, and is known to cluster in families. Here, the authors discover new genetic loci associated with FECD with sex-specific effects and implications for disease mechanism.

Purpose: Beyond enumeration, circulating tumor cells (CTCs) can provide genetic information from metastatic cancer that may facilitate a greater understanding of tumor biology and enable a precision medicine approach. Experimental Design: CTCs and paired leukocytes from men with metastatic castration-resistant prostate cancer (mCRPC) were isolated from blood through red cell lysis, CD45 depletion, and flow sorting based on EpCAM/CD45 expression. We next performed whole genomic copy number analysis of CTCs and matched patient leukocytes (germline) using array-based comparative genomic hybridization (aCGH) from 16 men with mCRPC, including longitudinal and sequential aCGH analyses of CTCs in the context of enzalutamide therapy. Results: All patients had mCRPC and primary or acquired resistance to abiraterone acetate or enzalutamide. We compiled copy gains and losses, with a particular focus on those genes highly implicated in mCRPC progression and previously validated as being aberrant in metastatic tissue samples and genomic studies of reference mCRPC datasets. Genomic gains in >25% of CTCs were observed in AR, FOXA1, ABL1, MET, ERG, CDK12, BRD4, and ZFHX3, while common genomic losses involved PTEN, ZFHX3, PDE4DIP, RAF1, and GATA2. Analysis of aCGH in a sample with sequential enzalutamide-resistant visceral progression showed acquired loss of AR amplification concurrent with gain of MYCN, consistent with evolution toward a neuroendocrine-like, AR-independent clone. Conclusions: Genomic analysis of pooled CTCs in men with mCRPC suggests a reproducible, but highly complex molecular profile that includes common aberrations in AR, ERG, c-MET, and PI3K signaling during mCRPC progression, which may be useful for predictive biomarker development. Clin Cancer Res; 23(5); 1346–57. ©2016 AACR.

Inflammation induced by innate immunity influences the development of T cell-mediated autoimmunity in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). We found that strong activation of innate immunity induced Nod-like receptor protein 3 (NLRP3) inflammasome-independent and interferon-β (IFNβ)-resistant EAE (termed type B EAE), whereas EAE induced by weak activation of innate immunity requires the NLRP3 inflammasome and is sensitive to IFNβ treatment. Instead, an alternative inflammatory mechanism, including membrane-bound lymphotoxin-β receptor (LTβR) and CXC chemokine receptor 2 (CXCR2), is involved in type B EAE development, and type B EAE is ameliorated by antagonizing these receptors. Relative expression of Ltbr and Cxcr2 genes was indeed enhanced in patients with IFNβ-resistant multiple sclerosis. Remission was minimal in type B EAE due to neuronal damages induced by semaphorin 6B upregulation on CD4+ T cells. Our data reveal a new inflammatory mechanism by which an IFNβ-resistant EAE subtype develops.

Circulating tumor cell (CTC) and cell-free (cf) DNA-based genomic alterations are increasingly being used for clinical decision-making in oncology. However, the concordance and discordance between paired CTC and cfDNA genomic profiles remain largely unknown. We performed comparative genomic hybridization (CGH) on CTCs and cfDNA, and low-pass whole genome sequencing (lpWGS) on cfDNA to characterize genomic alterations (CNA) and tumor content in two independent prospective studies of 93 men with mCRPC treated with enzalutamide/abiraterone, or radium-223. Comprehensive analysis of 69 patient CTCs and 72 cfDNA samples from 93 men with mCRPC, including 64 paired samples, identified common concordant gains in FOXA1, AR, and MYC, and losses in BRCA1, PTEN, and RB1 between CTCs and cfDNA. Concordant PTEN loss and discordant BRCA2 gain were associated with significantly worse outcomes in Epic AR-V7 negative men with mCRPC treated with abiraterone/enzalutamide. We identified and externally validated CTC-specific genomic alternations that were discordant in paired cfDNA, even in samples with high tumor content. These CTC/cfDNA-discordant regions included key genomic regulators of lineage plasticity, osteomimicry, and cellular differentiation, including MYCN gain in CTCs (31%) that was rarely detected in cfDNA. CTC MYCN gain was associated with poor clinical outcomes in AR-V7 negative men and small cell transformation. In conclusion, we demonstrated concordance of multiple genomic alterations across CTC and cfDNA platforms; however, some genomic alterations displayed substantial discordance between CTC DNA and cfDNA despite the use of identical copy number analysis methods, suggesting tumor heterogeneity and divergent evolution associated with poor clinical outcomes.

Osteoclasts are multinucleated cells of the monocyte/macrophage lineage that degrade bone. Here, we used lineage tracing studies—labelling cells expressing Cx3cr1, Csf1r or Flt3—to identify the precursors of osteoclasts in mice. We identified an erythromyeloid progenitor (EMP)-derived osteoclast precursor population. Yolk-sac macrophages of EMP origin produced neonatal osteoclasts that can create a space for postnatal bone marrow haematopoiesis. Furthermore, EMPs gave rise to long-lasting osteoclast precursors that contributed to postnatal bone remodelling in both physiological and pathological settings. Our single-cell RNA-sequencing data showed that EMP-derived osteoclast precursors arose independently of the haematopoietic stem cell (HSC) lineage and the data from fate tracking of EMP and HSC lineages indicated the possibility of cell–cell fusion between these two lineages. Cx3cr1+ yolk-sac macrophage descendants resided in the adult spleen, and parabiosis experiments showed that these cells migrated through the bloodstream to the remodelled bone after injury. Yahara et al. show that yolk-sac macrophages, which are derived from erythromyeloid progenitors, produce neonatal osteoclasts that support bone homeostasis and repair and also provide long-lasting osteoclasts in adult bone.

Centromeres are defined by a specialized chromatin organization that includes nucleosomes that contain the centromeric histone variant centromere protein A (CENP-A) instead of canonical histone H3. Studies in various organisms have shown that centromeric chromatin (i.e., CENP-A chromatin or centrochromatin) exhibits plasticity, in that it can assemble on different types of DNA sequences. However, once established on a chromosome, the centromere is maintained at the same position. In humans, this location is the highly homogeneous repetitive DNA alpha satellite. Mislocalization of centromeric chromatin to atypical locations can lead to genome instability, indicating that restriction of centromeres to a distinct genomic position is important for cell and organism viability. Here, we describe a rearrangement of Homo sapiens chromosome 17 (HSA17) that has placed alpha satellite DNA next to euchromatin. We show that on this mutant chromosome, CENP-A chromatin has spread from the alpha satellite into the short arm of HSA17, establishing a ∼700 kb hybrid centromeric domain that spans both repetitive and unique sequences and changes the expression of at least one gene over which it spreads. Our results illustrate the plasticity of human centromeric chromatin and suggest that heterochromatin normally constrains CENP-A chromatin onto alpha satellite DNA. This work highlights that chromosome rearrangements, particularly those that remove the pericentromere, create opportunities for centromeric nucleosomes to move into non-traditional genomic locations, potentially changing the surrounding chromatin environment and altering gene expression.

OXTR modulates a variety of behaviors in mammals, including social memory and recognition. Genetic and epigenetic dysregulation of OXTR has been suggested to be implicated in neuropsychiatric disorders, including autism spectrum disorder (ASD). While the involvement of DNA methylation is suggested, the mechanism underlying epigenetic regulation of OXTR is largely unknown. This has hampered the experimental design and interpretation of the results of epigenetic studies of OXTR in neuropsychiatric disorders. From the generation and characterization of a new line of Tet1 mutant mice - by deleting the largest coding exon 4 (Tet1Δe4) - we discovered for the first time to our knowledge that Oxtr has an array of mRNA isoforms and a complex transcriptional regulation. Select isoforms of Oxtr are significantly reduced in the brain of Tet1Δe4-/- mice. Accordingly, CpG islands of Oxtr are hypermethylated during early development and persist into adulthood. Consistent with the reduced express of OXTR, Tet1Δe4-/- mice display impaired maternal care, social behavior, and synaptic responses to oxytocin stimulation. Our findings elucidate a mechanism mediated by TET1 protein in regulating Oxtr expression by preventing DNA hypermethylation of Oxtr. The discovery of epigenetic dysregulation of Oxtr in TET1-deficient mouse brain supports the necessity of a reassessment of existing findings and a value of future studies of OXTR in neuropsychiatric disorders.

Scope Fumonisin (FB) occurs in maize and is an inhibitor of ceramide synthase (CerS). We determined the urinary FB1 (UFB1) and sphingoid base 1-phosphate levels in blood from women consuming maize in high and low FB exposure communities in Guatemala. Methods and results FB1 intake was estimated using the UFB1. Sphinganine 1-phosphate (Sa 1-P), sphingosine 1-phosphate (So 1-P), and the Sa 1-P/So 1-P ratio were determined in blood spots collected on absorbent paper at the same time as urine collection. In the first study, blood spots and urine were collected every 3 months (March 2011 to February 2012) from women living in low (Chimaltenango and Escuintla) and high (Jutiapa) FB exposure communities (1240 total recruits). The UFB1, Sa 1-P/So 1-P ratio, and Sa 1-P/mL in blood spots were significantly higher in the high FB1 intake community compared to the low FB1 intake communities. The results were confirmed in a follow-up study (February 2013) involving 299 women living in low (Sacatepequez) and high (Santa Rosa and Chiquimula) FB exposure communities. Conclusions High levels of FB1 intake are correlated with changes in Sa 1-P and the Sa 1-P/So 1-P ratio in human blood in a manner consistent with FB1 inhibition of CerS.

Co-occurrence of fumonisin B1 (FB1) and aflatoxin B1 (AFB1) in maize has been demonstrated in many surveys. Combined-exposure to FB1 and AFB1 was of concern to the Joint FAO/WHO Expert Committee on Food Additives because of the known genotoxicity of AFB1 and the ability of FB1 to induce regenerative proliferation in target tissues. Humans living where maize is a dietary staple are at high risk for exposure to both mycotoxins. Our work has focused on Guatemala, a country in Central America where maize is consumed in large amounts every day and where intake of FB1 has been shown to be potentially quite high using biomarker-based studies. In 2012 a survey was conducted which analysed maize samples for FB1 and AFB1 from all 22 departments of Guatemala. The results show that the levels of AFB1 exposure are also potentially quite high in Guatemala, and likely throughout Central America and Mexico. The implications of co-exposure for human health are numerous, but one area of particular concern is the potential of FB1 to modulate AFB1 hepatoxicity and/or hepatocarcinogenicity. Both the mechanism of action of FB1 and its ability to promote liver carcinogenicity in rats and rainbow trout is consistent with this concern. In farm and laboratory animals FB1 inhibits ceramide synthases, key enzymes in de novo ceramide biosynthesis. The inhibition of sphingolipid signalling pathways mediating programmed cell death and activation of pathways stimulating cell proliferation in livers of individuals exposed to AFB1 could contribute to the tumorigenicity of AFB1. Studies investigating the health effects of either toxin should consider the potential for co-exposure to both toxins. Also, in countries where maize-based food are prepared by alkaline treatment of the maize kernels, the effect of traditional processing on AFB1 levels and toxicity needs to be determined, especially for maize highly contaminated with AFB1.

Summary Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5′ UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.

Background Neural tube defects (NTD) have a strong genetic component, with up to 70% of variance in human prevalence determined by heritable factors. Although the identification of causal DNA variants by sequencing candidate genes from functionally relevant pathways and model organisms has provided some success, alternative approaches are demanded.

DNA methylation age (DNAm age) has become a widely utilized epigenetic biomarker for the aging process. The Horvath method for determining DNAm age is perhaps the most widely utilized and validated DNA methylation age assessment measure. Horvath DNAm age is calculated based on methylation measurements at 353 loci, present on Illumina's 450k and 27k DNA methylation microarrays. With increasing use of the more recently developed Illumina MethylationEPIC (850k) microarray, it is worth revisiting this aging measure to evaluate estimation differences due to array design. Of the requisite 353 loci, 17 are missing from the 850k microarray. Similarly, an alternate, 71 loci DNA methylation age assessment measure created by Hannum et al. is missing 6 requisite loci. Using 17 datasets with 27k, 450k, and/or 850k methylation data, we compared each sample's epigenetic age estimated from all 353 loci required by the Horvath DNAm age calculator, and using only the 336 loci available on the 850k array. In 450k/27k data, removing loci not on the 850k array resulted in underestimation of Horvath's DNAm age. Underestimation of Horvath DNAm age increased from ages 0 to ~20, remaining stable thereafter (mean deviation = -3.46 y, SD = 1.13 for individuals ≥20 years). Underestimation of Horvath's DNAm age by the reduced 450k/27k data was similar to the underestimation observed in the 850k data indicating it is driven by missing probes. In analogous examination of Hannum's DNAm age, the magnitude and direction of epigenetic age misestimation varied with chronological age. In conclusion, inter-array deviations in DNAm age estimations may be largely driven by missing probes between arrays, despite default probe imputation procedures. Though correlations and associations based on Horvath's DNAm age may be unaffected, researchers should exercise caution when interpreting results based on absolute differences in DNAm age or when mixing samples assayed on different arrays.

Scope Fumonisin (FB) intake can be high when maize is a dietary staple. We determined (i) urinary FB (UFB) in women consuming maize in high- and low-exposure communities in Guatemala, (ii) the FB levels in maize, (iii) the relationship between UFB and FB intake, and (iv) the relative excretion of UFB1, UFB2, and UFB3. Methods and results Urine and maize were analyzed for FB for 1 year in three departments. Maize consumption was estimated by an interview questionnaire. Fumonisin B1, B2, and B3 (FB1, FB2 and FB3), were detected in 100% of maize samples. FB1 in maize and urine was significantly higher in Jutiapa compared to Chimaltenango or Escuintla. The FB intake paralleled UFB1 in a dose-dependent manner but UFB1 was present in much higher levels than UFB2 or UFB3 compared to maize. Conclusion In Jutiapa, agroecological conditions favored FB production. UFB1 mirrored the estimated FB intake. UFB1 > 0.1 ng/mL resulted in a dose-dependent increase in the risk of exceeding FB intake of 2 μg/kg b.w./day compared to women with no detectable UFB1. More than 50% exceeded 2 μg/kg b.w./day when UFB1 was >0.5 ng/mL. UFB2 and UFB3 were rarely detected confirming that FB1 is either absorbed better or preferentially excreted in urine.

Neural tube defects (NTDs) are caused by improper neural tube closure during the early stages of embryonic development. NTDs are hypothesized to have a complex genetic origin and numerous candidate genes have been proposed. The nitric oxide synthase 3 (NOS3) G594T polymorphism has been implicated in risk for spina bifida, and interactions between that single nucleotide polymorphism (SNP) and the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism have also been observed. To evaluate other genetic variation in the NO pathway in the development of NTDs, we examined all three NOS genes: NOS1, NOS2, and NOS3. Using 3109 Caucasian samples in 745 families, we evaluated association in the overall dataset and within specific phenotypic subsets. Haplotype tagging SNPs in the NOS genes were tested for genetic association with NTD subtypes, both for main effects as well as for the presence of interactions with the MTHFR C677T polymorphism. Nominal main effect associations were found with all subtypes, across all three NOS genes, and interactions were observed between SNPs in all three NOS genes and MTHFR C677T. Unlike the previous report, the most significant associations in our dataset were with cranial subtypes and the AG genotype of rs4795067 in NOS2 (p = 0.0014) and the interaction between the rs9658490 G allele in NOS1 and MTHFR 677TT genotype (p = 0.0014). Our data extend the previous findings by implicating a role for all three NOS genes, independently and through interactions with MTHFR, in risk not only for spina bifida, but all NTD subtypes.

We performed a gene–smoking interaction analysis using families from an early-onset coronary artery disease cohort (GENECARD). This analysis was focused on validating and expanding results from previous studies implicating single nucleotide polymorphisms (SNPs) on chromosome 3 in smoking-mediated coronary artery disease. We analyzed 430 SNPs on chromosome 3 and identified 16 SNPs that showed a gene–smoking interaction at P < 0.05 using association in the presence of linkage—ordered subset analysis, a method that uses permutations of the data to empirically estimate the strength of the association signal. Seven of the 16 SNPs were in the Rho-GTPase pathway indicating a 1.87-fold enrichment for this pathway. A meta-analysis of gene–smoking interactions in three independent studies revealed that rs9289231 in KALRN had a Fisher’s combined P value of 0.0017 for the interaction with smoking. In a gene-based meta-analysis KALRN had a P value of 0.026. Finally, a pathway-based analysis of the association results using WebGestalt revealed several enriched pathways including the regulation of the actin cytoskeleton pathway as defined by the Kyoto Encyclopedia of Genes and Genomes.

Only two genome-wide significant loci associated with longevity have been identified so far, probably because of insufficient sample sizes of centenarians, whose genomes may harbor genetic variants associated with health and longevity. Here we report a genome-wide association study (GWAS) of Han Chinese with a sample size 2.7 times the largest previously published GWAS on centenarians. We identified 11 independent loci associated with longevity replicated in Southern-Northern regions of China, including two novel loci (rs2069837-IL6; rs2440012-ANKRD20A9P) with genome-wide significance and the rest with suggestive significance (P Only two genome-wide significant loci associated with longevity have been identified so far, probably because of insufficient sample sizes of centenarians, whose genomes may harbor genetic variants associated with health and longevity. Here we report a genome-wide association study (GWAS) of Han Chinese with a sample size 2.7 times the largest previously published GWAS on centenarians. We identified 11 independent loci associated with longevity replicated in Southern-Northern regions of China, including two novel loci (rs2069837-IL6; rs2440012-ANKRD20A9P) with genome-wide significance and the rest with suggestive significance (P

There is a growing literature indicating that genetic variants modify many of the associations between environmental exposures and clinical outcomes, potentially by increasing susceptibility to these exposures. However, genome-scale investigations of these interactions have been rarely performed particularly in the case of air pollution exposures. We performed race-stratified genome-wide gene-environment interaction association studies on European-American (EA, N = 1623) and African-American (AA, N = 554) cohorts to investigate the joint influence of common single nucleotide polymorphisms (SNPs) and residential exposure to traffic ("traffic exposure")-a recognized vascular disease risk factor-on peripheral arterial disease (BMP8A eQTLs in tissue types highlight relevant for PAD such as rs755249 (tibial nerve, eQTL P = 3.6x10(-6)) and rs1180341 (tibial artery, eQTL P = 5.3x10(-6)). Together these results reveal a novel gene, and possibly gene family, associated with PAD via an interaction with traffic air pollution exposure. These results also highlight the potential for interactions studies, particularly at the genome scale, to reveal novel biology linking environmental exposures to clinical outcomes.PAD). Traffic exposure was estimated via the distance from the primary residence to the nearest major roadway, defined as the nearest limited access highways or major arterial. The rs755249-traffic exposure interaction was associated with PAD at a genome-wide significant level (P = 2.29x10(-8)) in European-Americans. Rs755249 is located in the 3' untranslated region of BMP8A, a member of the bone morphogenic protein (BMP) gene family. Further investigation revealed several variants in BMP genes associated with PAD via an interaction with traffic exposure in both the EA and AA cohorts; this included interactions with non-synonymous variants in BMP2, which is regulated by air pollution exposure. The BMP family of genes is linked to vascular growth and calcification and is a novel gene family for the study of PAD pathophysiology. Further investigation of BMP8A using the Genotype Tissue Expression Database revealed multiple variants with nominally significant (P < 0.05) interaction P-values in our EA cohort were significant BMP8A eQTLs in tissue types highlight relevant for PAD such as rs755249 (tibial nerve, eQTL P = 3.6x10(-6)) and rs1180341 (tibial artery, eQTL P = 5.3x10(-6)). Together these results reveal a novel gene, and possibly gene family, associated with PAD via an interaction with traffic air pollution exposure. These results also highlight the potential for interactions studies, particularly at the genome scale, to reveal novel biology linking environmental exposures to clinical outcomes.

On the basis of the genotypic/phenotypic data from Chinese Longitudinal Healthy Longevity Survey (CLHLS) and Cox proportional hazard model, the present study demonstrates that interactions between carrying FOXO1A-209 genotypes and tea drinking are significantly associated with lower risk of mortality at advanced ages. Such a significant association is replicated in two independent Han Chinese CLHLS cohorts (p = 0.028-0.048 in the discovery and replication cohorts, and p = 0.003-0.016 in the combined dataset). We found the associations between tea drinking and reduced mortality are much stronger among carriers of the FOXO1A-209 genotype compared to non-carriers, and drinking tea is associated with a reversal of the negative effects of carrying FOXO1A-209 minor alleles, that is, from a substantially increased mortality risk to substantially reduced mortality risk at advanced ages. The impacts are considerably stronger among those who carry two copies of the FOXO1A minor allele than those who carry one copy. On the basis of previously reported experiments on human cell models concerning FOXO1A-by-tea-compounds interactions, we speculate that results in the present study indicate that tea drinking may inhibit FOXO1A-209 gene expression and its biological functions, which reduces the negative impacts of FOXO1A-209 gene on longevity (as reported in the literature) and offers protection against mortality risk at oldest-old ages. Our empirical findings imply that the health outcomes of particular nutritional interventions, including tea drinking, may, in part, depend upon individual genetic profiles, and the research on the effects of nutrigenomics interactions could potentially be useful for rejuvenation therapies in the clinic or associated healthy aging intervention programs.

Glioblastoma is universally fatal because of its propensity for rapid recurrence due to highly migratory tumor cells. Unraveling the genomic complexity that underlies this migratory characteristic could provide therapeutic targets that would greatly complement current surgical therapy. Using multiple high-resolution genomic screening methods, we identified a single locus, adherens junctional associated protein 1 (AJAP1) on chromosome 1p36 that is lost or epigenetically silenced in many glioblastomas. We found AJAP1 expression absent or reduced in 86% and 100% of primary glioblastoma tumors and cell lines, respectively, and the loss of expression correlates with AJAP1 methylation. Restoration of AJAP1 gene expression by transfection or demethylation agents results in decreased tumor cell migration in glioblastoma cell lines. This work shows the significant loss of expression of AJAP1 in glioblastoma and provides evidence of its role in the highly migratory characteristic of these tumors. Mol Cancer Res; 10(2); 208–17. ©2012 AACR.

Received: June 10, 2010 , Accepted: July 29, 2010 , Published: August 8, 2010 // The glioblastoma genome displays remarkable chromosomal aberrations, which harbor critical glioblastoma-specific genes contributing to several oncogenetic pathways. To identify glioblastoma-targeted genes, we completed a multifaceted genome-wide analysis to characterize the most significant aberrations of DNA content occurring in glioblastomas. We performed copy number analysis of 111 glioblastomas by Digital Karyotyping and Illumina BeadChip assays and validated our findings using data from the TCGA (The Cancer Genome Atlas) glioblastoma project. From this study, we identified recurrent focal copy number alterations in 1p36.23 and 4p16.3. Expression analyses of genes located in the two regions revealed genes which are dysregulated in glioblastomas. Specifically, we identify EGFR negative regulator, ERRFI1, within the minimal region of deletion in 1p36.23. In glioblastoma cells with a focal deletion of the ERRFI1 locus, restoration of ERRFI1 expression slowed cell migration. Furthermore, we demonstrate that TACC3, an Aurora-A kinase substrate, on 4p16.3, displays gain of copy number, is overexpressed in a glioma-grade-specific pattern, and correlates with Aurora kinase overexpression in glioblastomas. Our multifaceted genomic evaluation of glioblastoma establishes ERRFI1 as a potential candidate tumor suppressor gene and TACC3 as a potential oncogene, and provides insight on targets for oncogenic pathway-based therapy.

Alternative splicing is a general mechanism for regulating gene expression that affects the RNA products of more than 90% of human genes. Not surprisingly, alternative splicing is observed among gene products of metazoan immune systems, which have evolved to efficiently recognize pathogens and discriminate between “self” and “non-self”, and thus need to be both diverse and flexible. In this review we focus on the specific interface between alternative splicing and autoimmune diseases, which result from a malfunctioning of the immune system and are characterized by the inappropriate reaction to self-antigens. Despite the widespread recognition of alternative splicing as one of the major regulators of gene expression, the connections between alternative splicing and autoimmunity have not been apparent. We summarize recent findings connecting splicing and autoimmune disease, and attempt to find common patterns of splicing regulation that may advance our understanding of autoimmune diseases and open new avenues for therapy.

Objective The genetic contributions to the multifactorial disorder osteoarthritis (OA) have been increasingly recognized. The goal of the current study was to use OA-related biomarkers of severity and disease burden as quantitative traits to identify genetic susceptibility loci for OA. Methods In a large multigenerational extended family (n = 350), we measured 5 OA-related biomarkers: hyaluronan (HA), cartilage oligomeric matrix protein (COMP), N-propeptide of type IIA collagen (PIIANP), C-propeptide of type II procollagen (CPII), and type II collagen neoepitope (C2C). Single-nucleotide polymorphism markers (n = 6,090) covering the whole genome were genotyped using the Illumina HumanLinkage-12 BeadChip. Variance components analysis, as implemented in the Sequential Oligogenic Linkage Analysis Routines, was used to estimate heritabilities of the quantitative traits and to calculate 2-point and multipoint logarithm of odds (LOD) scores using a polygenic model. Results After adjusting for age and sex, we found that 4 of the 5 biomarkers exhibited significant heritability (PIIANP 0.57, HA 0.49, COMP 0.43, C2C 0.30; P ≤ 0.01 for all). Fourteen of the 19 loci that had multipoint LOD scores of >1.5 were near to or overlapped with previously reported OA susceptibility loci. Four of these loci were identified by more than 1 biomarker. The maximum multipoint LOD scores for the heritable quantitative biomarker traits were 4.3 for PIIANP (chromosome 8p23.2), 3.2 for COMP (chromosome 8q11.1), 2.0 for HA (chromosome 6q16.3), and 2.0 for C2C (chromosome 5q31.2). Conclusion Herein, we report the first evidence of genetic susceptibility loci identified by OA-related biomarkers in an extended family. Our results demonstrate that serum concentrations of PIIANP, HA, COMP, and C2C have substantial heritable components, and using these biomarkers, several genetic loci potentially contributing to the genetic diversity of OA were identified.

We studied the status of chromosomes 1 and 19 in 363 astrocytic and oligodendroglial tumors. Whereas the predominant pattern of copy number abnormality was a concurrent loss of the entire 1p and 19q regions (total 1p/19q loss) among oligodendroglial tumors and partial deletions of 1p and/or 19q in astrocytic tumors, a subset of apparently astrocytic tumors also had total 1p/19q loss. The presence of total 1p/19q loss was associated with longer survival of patients with all types of adult gliomas independent of age and diagnosis (P = .041). The most commonly deleted region on 19q in astrocytic tumors spans 885 kb in 19q13.33–q13.41, which is telomeric to the previously proposed region. Novel regions of homozygous deletion, including a part of DPYD (1p21.3) or the KLK cluster (19q13.33), were observed in anaplastic oligodendrogliomas. Amplifications encompassing AKT2 (19q13.2) or CCNE1 (19q12) were identified in some glioblastomas. Deletion mapping of the centromeric regions of 1p and 19q in the tumors that had total 1p/19q loss, indicating that the breakpoints lie centromeric to NOTCH2 within the pericentromeric regions of 1p and 19q. Thus, we show that the copy number abnormalities of 1p and 19q in human gliomas are complex and have distinct patterns that are prognostically predictive independent of age and pathological diagnosis. An accurate identification of total 1p/19q loss and discriminating this from other 1p/19q changes is, however, critical when the 1p/19q copy number status is used to stratify patients in clinical trials.

Leukotrienes are arachidonic acid derivatives long known for their inflammatory properties and their involvement with a number of human diseases, most particularly asthma. Recently, leukotriene-based inflammation has also been shown to play an important role in atherosclerosis: ALOX5AP and LTA4H, both genes in the leukotriene biosynthesis pathway, have individually been shown to be associated with various cardiovascular disease (CVD) phenotypes. To assess the role of the leukotriene pathway in CVD pathogenesis, we performed genetic association studies of ALOX5AP and LTA4H in a family based study of early onset coronary artery disease (EOCAD) (GENECARD, 1,101 families) and in a non-familial dataset of EOCAD (CATHGEN, 656 cases and 405 controls). We found weak to moderate association between single nucleotide polymorphisms (SNPs) in ALOX5AP and LTA4H with EOCAD. The previously reported four-SNP haplotype (HapA) in ALOX5AP showed association with EOCAD in CATHGEN (P = 0.02), while controlling for age, race and CVD risk factors. HapK, the previously reported ten-SNP haplotype in LTA4H was associated with EOCAD in CATHGEN (P = 0.04). Another previously reported four-SNP haplotype in ALOX5AP (HapB) was not significant in our sample (P = 0.39). The overall lack of (or weak) association of single SNPs as compared with the haplotype results demonstrates the need for analyzing multiple SNPs within each gene in such studies. Interestingly, we detected an association of SNPs in ALOX5 (P < 0.05), the target of ALOX5AP, with CVD. Using a pathway-based approach, we also detected statistical evidence for interactions among ALOX5, ALOX5AP and LTA4H using RNA expression data from a collection of freshly harvested human aortas with varying degrees of atherosclerosis. The GENECARD families did not demonstrate evidence for linkage or association with ALOX5, ALOX5AP or LTA4H. Our results support a modest role for the leukotriene pathway in atherosclerosis pathogenesis, reveal important genomic interactions within the pathway, and suggest the importance of using pathway-based modeling for evaluating the genomics of atherosclerosis susceptibility.

Multiple sclerosis is a demyelinating neurodegenerative disease with a strong genetic component. Previous genetic risk studies have failed to identify consistently linked regions or genes outside of the major histocompatibility complex on chromosome 6p. We describe allelic association of a polymorphism in the gene encoding the interleukin 7 receptor α chain ( IL7R ) as a significant risk factor for multiple sclerosis in four independent family-based or case-control data sets (overall P = 2.9 × 10−7). Further, the likely causal SNP, rs6897932, located within the alternatively spliced exon 6 of IL7R, has a functional effect on gene expression. The SNP influences the amount of soluble and membrane-bound isoforms of the protein by putatively disrupting an exonic splicing silencer.

A susceptibility locus for coronary artery disease (CAD) has been mapped to chromosome 3q13-21 in a linkage study of early-onset CAD. We completed an association-mapping study across the 1-LOD–unit-down supporting interval, using two independent white case-control data sets (CATHGEN, initial and validation) to evaluate association under the peak. Single-nucleotide polymorphisms (SNPs) evenly spaced at 100-kb intervals were screened in the initial data set (N=468). Promising SNPs (P

Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately two-fold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we demonstrate that engineered haplodeficiency of IL-2 gene expression not only reduces T cell IL-2 production by two-fold but also mimics the autoimmune dysregulatory effects of the naturally-occurring susceptibility alleles of IL-2. Reduced IL-2 production achieved by both genetic mechanisms correlates with fewer and less functional CD4+CD25+ regulatory T cells, which are critical for maintaining immune homeostasis.

It is well established that the major histocompatibility complex (MHC) on chromosome 6p21 contains at least one gene that influences susceptibility to multiple sclerosis.1–6 Although this association was first identified more than 30 years ago1 through the study of class I human leukocyte antigens (HLAs), it was quickly realized that this signal was predominantly, if not exclusively, the result of linkage disequilibrium (LD) with class II HLA genes, and that these exert the primary effect on susceptibility.7, 8 The complex nature of the MHC, especially its high gene content, extreme polymorphism, and extensive LD,9 has confounded efforts to resolve the nature of the MHC association in multiple sclerosis, although progress and useful clarifications have been made, especially in recent years. In virtually every population studied, multiple sclerosis is found to be associated with the DRB1*1501 allele.10 The only exceptions are those populations where this allele has a low frequency, and analysis is therefore underpowered; but even in these situations, DRB1*1501 is generally overrepresented in cases.11 The DRB1*1501 allele is carried on a particularly extensive haplotype,12 the most common DR15 haplotype found in white Europeans. As a result, many variants from flanking genes, even some located quite a distance from DRB1, have sufficient LD with DRB1*1501 that they invariably also show evidence for association with the disease in any population where association with DRB1*1501 can be demonstrated.11, 13–17 This extensive LD has made it difficult to establish which of the variants making up this haplotype is primarily responsible for the association. The distinction between DRB1*1501 and DQB1*0602 has been particularly taxing because the LD between these closely mapped genes is especially tight in those populations where the disease is frequent, that is, white Europeans and their migrant descendants. However, recent studies in the admixed African American population indicate the supremacy of the DRB1*1501 allele.18 In the presence of one susceptibility allele it is difficult to identify effects attributable to a second allele,19 especially if the second allele exerts a more modest effect or has a low frequency, or both. However, by analyzing populations where DR15 haplotypes are less common, and by using large cohorts, it has been possible to demonstrate that the DRB1*0301 allele also confers susceptibility to multiple sclerosis, thereby confirming allelic heterogeneity at the DRB1 locus.4, 6, 18, 20 Furthermore available evidence suggests that the susceptibility effects of the DRB1*1501 allele may be modulated by other DRB1 alleles.6, 20 The relation between the MHC and multiple sclerosis is further complicated by the accumulating evidence suggesting that MHC loci mapping outside DRB1 also influence susceptibility to the disease.11, 13–16 Work in animal models suggests that clustering of susceptibility loci is a common phenomenon in complex disease,21 and it therefore appears reasonable to expect that other genes from the MHC region may influence susceptibility to multiple sclerosis. The observation of positive logarithm of odds scores in the MHC region in linkage studies stratified for the effects of DRB1 supports the existence of secondary loci,22–24 although none of these data reaches a level providing statistical confidence. In considering these linkage data, it is important to remember that early linkage studies in multiple sclerosis25–27 were significantly underpowered,28 to the point that they could not even convincingly demonstrate evidence for linkage resulting from the effects of DRB1. Confirmation of linkage in this region has been established only in more recent studies involving many hundreds of families.23, 24, 29 Given the inherently limited resolution of linkage-based studies,28 the absence of statistically significant linkage in the MHC region after exclusion of primary effects attributable to DRB1 does not exclude the presence of secondary loci. Several authors have attempted to identify secondary loci using more powerful association-based methods. Two groups have typed dense microsatellite maps of the region and both found evidence for a secondary locus maximal in a region close to HLA-A: one group identifying the marker D6S1683 just telomeric of HLA-A,11 and the second group implicating a region including HLA-A extending from MOGCA to D6S265 marker.14 Follow-up studies in Norway also found evidence implicating the D6S265 marker.16 In another smaller study, a microsatellite marker close to HLA-C (marker C1_3_2) also showed evidence for an independent effect.15 In contrast, a systematic effort to screen the MHC and flanking regions using single nucleotide polymorphisms (SNPs) found no evidence for association beyond that attributable to DRB1*1501, although this study was limited by a high genotyping failure rate (40%) and, more importantly, a distribution of markers leaving regions close to the classical loci essentially unexplored.17 In all of these studies, statistical power has inevitably been reduced by the processes required to filter out the primary effect attributable to DRB1 and the large correction required for multiple testing. Unfortunately, none of the published studies has used sufficient samples to compensate for these statistical penalties, and thus none is able to provide unequivocal evidence supporting any particular secondary locus.

Background: Bicuspid aortic valves predispose to ascending aortic aneurysms, but the mechanisms underlying this aortopathy remain incompletely characterized. We sought to identify epigenetic pathways predisposing to aneurysm formation in bicuspid patients.Methods: Ascending aortic aneurysm tissue samples were collected at the time of aortic replacement in subjects with bicuspid and trileaflet aortic valves. Genome-wide DNA methylation status was determined on DNA from tissue using the Illumina 450K methylation chip, and gene expression was profiled on the same samples using Illumina Whole-Genome DASL arrays. Gene methylation and expression were compared between bicuspid and trileaflet individuals using an unadjusted Wilcoxon rank sum test. Results: Twenty-seven probes in 9 genes showed significant differential methylation and expression (P-4). The top gene was protein tyrosine phosphatase, non-receptor type 22 (PTPN22), which was hypermethylated (delta beta range: +15.4 to +16.0%) and underexpressed (log 2 gene expression intensity: bicuspid 5.1 vs. trileaflet 7.9, P=2x10-5) in bicuspid patients, as compared to tricuspid patients. Numerous genes involved in cardiovascular development were also differentially methylated, but not differentially expressed, including ACTA2 (4 probes, delta beta range: -10.0 to -22.9%), which when mutated causes the syndrome of familial thoracic aortic aneurysms and dissectionsConclusions: Using an integrated, unbiased genomic approach, we have identified novel genes associated with ascending aortic aneurysms in patients with bicuspid aortic valves, modulated through epigenetic mechanisms. The top gene was PTPN22, which is involved in T-cell receptor signaling and associated with various immune disorders. These differences highlight novel potential mechanisms of aneurysm development in the bicuspid population.

TMEM231, a functional component of the MKS complex at the ciliary transition zone, is mutated in orofaciodigital syndrome type 3 and Meckel syndrome., The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS.