To improve our limited understanding of the pathogenesis of thoracic aortic aneurysm (TAA) leading to acute aortic dissection, single-cell RNA sequencing (scRNA-seq) was employed to profile disease-relevant transcriptomic changes of aortic cell populations in a well-characterized mouse model of the most commonly diagnosed form of Marfan syndrome (MFS). As result, two discrete sub-populations of aortic cells (SMC3 and EC4) were identified only in the aorta of Fbn1mgR/mgR mice. SMC3 highly express genes related to extracellular matrix formation and nitric oxide signaling, whereas EC4 transcriptional profile is enriched in SMC, fibroblast, and immune cell-related genes. Trajectory analysis predicted close phenotypic modulation between SMC3 and EC4, which were therefore analyzed together as a discrete MFS-modulated (MFSmod) sub-population. In situ hybridizations of diagnostic transcripts located MFSmod cells to the intima of Fbn1mgR/mgR aortas. Reference-based dataset integration revealed transcriptomic similarity between MFSmod and an SMC-derived cell cluster modulated in human TAA. Consistent with angiotensin II type I receptor (At1r) contribution to TAA development, MFSmod cells were absent in the aorta of Fbn1mgR/mgR mice treated with the At1r antagonist losartan. Altogether, our findings indicate that a discrete dynamic alteration of aortic cell identity is associated with dissecting TAA in MFS mice and increased risk of aortic dissection in MFS patients.
Yifei Sun, Keiichi Asano, Lauriane Sedes, Anna Cantalupo, Jens Hansen, Ravi Iyengar, Martin J. Walsh, Francesco Ramirez
Overgrowth syndromes can be caused by pathogenic genetic variants in epigenetic writers, such as DNA and histone methyltransferases. However, no overgrowth disorder has previously been ascribed to variants in a gene that acts primarily as an epigenetic reader. Here, we studied a male individual with generalized overgrowth of prenatal onset. Exome sequencing identified a hemizygous frameshift variant in Spindlin 4 (SPIN4), with X-linked inheritance. We found evidence that SPIN4 binds specific histone modifications, promotes canonical WNT signaling, and inhibits cell proliferation in vitro and that the identified frameshift variant had lost all of these functions. Ablation of Spin4 in mice recapitulated the human phenotype with generalized overgrowth, including increased longitudinal bone growth. Growth plate analysis revealed increased cell proliferation in the proliferative zone and an increased number of progenitor chondrocytes in the resting zone. We also found evidence of decreased canonical Wnt signaling in growth plate chondrocytes, providing a potential explanation for the increased number of resting zone chondrocytes. Taken together, our findings provide strong evidence that SPIN4 is an epigenetic reader that negatively regulates mammalian body growth, and that loss of SPIN4 causes an overgrowth syndrome in humans, expanding our knowledge of the epigenetic regulation of human growth.
Julian C. Lui, Jacob Wagner, Elaine Zhou, Lijin Dong, Kevin M. Barnes, Youn Hee Jee, Jeffrey Baron
BACKGROUND. Cellular stressors influence the development of clonal hematopoiesis (CH). We hypothesized that aging, environmental, inflammatory, and genotoxic stresses drive the emergence of CH in patients with severe lung disease undergoing lung transplantation. METHODS. We performed a cross-sectional cohort study of 85 patients with severe lung disease undergoing transplantation to characterize CH prevalence. We evaluated somatic variants using duplex error-corrected sequencing and germline variants using whole exome sequencing. We evaluated CH frequency and burden using chi-square and Poisson regression, associations with clinical and demographic variables using logistic regression, and associations with clinical outcomes using chi-square, logistic, and Cox regression. RESULTS. CH in DNA damage response (DDR) genes TP53, PPM1D, and ATM was observed at high frequency in transplant recipients compared to a control group of older adults [28% vs. 0%, aOR 12.9 (1.7-100.3), p=0.0002]. Age [OR 1.13 (1.03-1.25), p=0.014] and smoking history [OR 4.25 (1.02-17.82), p=0.048] were associated with CH in DDR genes. Germline variants causing predisposition to idiopathic pulmonary fibrosis, including telomere biology disorders and surfactant-related lung disease were identified but not associated with CH. DDR CH was associated with increased cytomegalovirus viremia compared to patients with no CH [OR 7.23 (1.95-26.8), p=0.009]] or non-DDR CH [OR 7.64 (1.77-32.89, p=0.012)], decreased lymphopenia (aHR 0.49 (0.27 – 0.90), p=0.021) and mycophenolate discontinuation [aOR 3.8 (1.3-12.9), p=0.031]. CONCLUSION. In patients with severe lung disease requiring lung transplantation, CH due to somatic variants in PPM1D, TP53 or ATM is highly prevalent and associated with post-transplant outcomes including cytomegalovirus activation and mycophenolate intolerance. FUNDING. NIH/NHLBI K01HL155231 (LKT), R25HL105400 (LKT), Foundation for Barnes-Jewish Hospital (LKT), Evans MDS Center at Washington University (KAO, MJW), ASH Scholar Award (KAO), NIH K12CA167540 (KAO), NIH P01AI116501 (AEG, DK), NIH R01HL094601 (AEG), and NIH P01CA101937 (DCL).
Laneshia K. Tague, Karolyn A. Oetjen, Anirudh Mahadev, Matthew J. Walter, Hephzibah Anthony, Daniel Kreisel, Daniel C. Link, Andrew E. Gelman
Targeted biologic therapies can elicit an undesirable host immune response characterized by the development of antidrug antibodies (ADA), an important cause of treatment failure. The most widely used biologic across immune-mediated diseases is adalimumab, a tumor necrosis factor inhibitor. This study aimed to identify genetic variants that contribute to the development of ADA against adalimumab, thereby influencing treatment failure. In patients with psoriasis on their first course of adalimumab, in whom serum ADA had been evaluated 6–36 months after starting treatment, we observed a genome-wide association with ADA against adalimumab within the major histocompatibility complex (MHC). The association signal mapped to the presence of tryptophan at position 9 and lysine at position 71 of the HLA-DR peptide-binding groove, with both residues conferring protection against ADA. Underscoring their clinical relevance, these residues were also protective against treatment failure. Our findings highlight antigenic peptide presentation via MHC class II as a critical mechanism in the development of ADA against biologic therapies and downstream treatment response.
Teresa Tsakok, Jake Saklatvala, Theo Rispens, Floris C. Loeff, Annick de Vries, Michael H. Allen, Ines A. Barbosa, David Baudry, Tejus Dasandi, Michael Duckworth, Freya Meynell, Alice Russell, Anna Chapman, Sandy McBride, Kevin McKenna, Gayathri Perera, Helen Ramsay, Raakhee Ramesh, Kathleen Sands, Alexa Shipman, the Biomarkers of Systemic Treatment Outcomes in Psoriasis (BSTOP) Study Group, A. David Burden, Christopher E.M. Griffiths, Nick J. Reynolds, Richard B. Warren, Satveer Mahil, Jonathan Barker, Nick Dand, Catherine Smith, Michael A. Simpson
The genomic and immune landscapes of prostate cancer differ by self-identified race. However, few studies have examined the genome-wide copy number landscape and immune content of matched cohorts with genetic ancestry data and clinical outcomes. Here, we assessed prostate cancer somatic copy number alterations (sCNA) and tumor immune content of a grade-matched, surgically treated cohort of 145 self-identified Black (BL) and 145 self-identified White (WH) patients with genetic ancestry estimation. A generalized linear model adjusted with age, preoperative prostate-specific antigen (PSA), and Gleason Grade Group and filtered for germline copy number variations (gCNV) identified 143 loci where copy number varied significantly by percent African ancestry, clustering on chromosomes 6p, 10q, 11p, 12p, and 17p. Multivariable Cox regression models adjusted for age, preoperative PSA levels, and Gleason Grade Group revealed that chromosome 8q gains (including MYC) were significantly associated with biochemical recurrence and metastasis, independent of genetic ancestry. Finally, Treg density in BL and WH patients was significantly correlated with percent genome altered, and these findings were validated in the TCGA cohort. Taken together, our findings identify specific sCNA linked to genetic ancestry and outcome in primary prostate cancer and demonstrate that Treg infiltration varies by global sCNA burden in primary disease.
Thiago Vidotto, Eddie L. Imada, Farzana Faisal, Sanjana Murali, Adrianna A. Mendes, Harsimar Kaur, Siqun Zheng, Jianfeng Xu, Edward M. Schaeffer, William B. Isaacs, Karen S. Sfanos, Luigi Marchionni, Tamara L. Lotan
In vertebrate species, fertility is controlled by gonadotropin-releasing hormone (GnRH) neurons. GnRH cells arise outside the central nervous system, in the developing olfactory pit, and migrate along olfactory/vomeronasal/terminal nerve axons into the forebrain during embryonic development. Congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS) are rare genetic disorders characterized by infertility and they are associated with defects in GnRH neuron migration and/or altered GnRH secretion and signaling. Here, we documented the expression of Jagged 1/Notch signaling pathway in GnRH neurons and along the GnRH neuron migratory route both in zebrafish embryos and in human fetuses. Genetic knock-down of the zebrafish ortholog of JAG1 (jag1b) resulted in altered GnRH migration and olfactory axonal projections to the olfactory bulbs. Next-generation sequencing was performed in 467 CHH unrelated probands leading to the identification of heterozygous rare variants in JAG1. Functional in vitro validation of JAG1 mutants revealed that 7 out of the 9 studied variants exhibit reduced protein levels and altered subcellular localization. Altogether our data provide compelling evidence that Jag1/Notch signaling plays a prominent role in the development of GnRH neurons and we propose that JAG1 insufficiency may contribute to the pathogenesis of CHH in humans.
Ludovica Cotellessa, Federica Marelli, Paolo Duminuco, Michela Adamo, Georgios E. Papadakis, Lucia Bartoloni, Naoko Sato, Mariarosaria Lang-Muritano, Amineh Troendle, Waljit S. Dhillo, Annamaria Morelli, Giulia Guarnieri, Nelly Pitteloud, Luca Persani, Marco Bonomi, Paolo Giacobini, Valeria Vezzoli
Antisense oligonucleotide (AO)-mediated therapy is a promising strategy to treat several neurological diseases including spinal muscular atrophy (SMA). However, limited delivery to the central nervous system (CNS) with AOs administered intravenously or subcutaneously is a major challenge. Here we demonstrate a single subcutaneous administration of cell-penetrating peptide DG9 conjugated to an AO called phosphorodiamidate morpholino oligomers (PMOs) reaches the CNS and significantly prolonged the median survival compared to unconjugated PMO and R6G-PMO in a severe SMA mouse model. Treated mice exhibited significantly higher expression of full-length SMN2 expression (FL-SMN2) in both the CNS and systemic tissues compared to non-treated and unmodified AO-treated mice. The treatment ameliorated the atrophic musculature and improved breathing function accompanied by improved muscle strength and innervation at the neuromuscular junction with no signs of apparent toxicity. We also demonstrated DG9-conjugated PMO localizes in nuclei in the spinal cord and brain after subcutaneous injections. Our data identify DG9 peptide conjugation as a powerful way to improve the efficacy of AO-mediated splice modulation. Finally, DG9-PMO is a promising therapeutic option to treat SMA and other neurological diseases, overcoming the necessity for intrathecal injections and treating body-wide tissues without apparent toxicity.
Tejal Aslesh, Esra Erkut, Jun Ren, Kenji Rowel Q. Lim, Stanley Woo, Susan Hatlevig, Hong M. Moulton, Simon Gosgnach, John Greer, Rika Maruyama, Toshifumi Yokota
Human papillomaviruses (HPVs), are DNA viruses, including ~450 types, classified into five genera (α-, β-, γ-, µ-, and 𝜈-HPV). The γ- and β-HPVs are present in low-copy numbers in healthy individuals, however, in patients with an inborn error of immunity, certain species of β-HPVs can cause epidermodysplasia verruciformis (EV), manifesting as recalcitrant cutaneous warts and skin cancer. EV presents as either “typical” or “atypical”. Manifestations in typical EV are limited to the skin and are caused by abnormal keratinocyte-intrinsic immunity to β-HPVs due to pathogenic sequence variants in TMC6, TMC8, or CIB1. We applied a transcriptome-based computational pipeline, VirPy, on RNA extracted from normal-appearing skin and wart samples of patients with typical EV, to explore the viral and human genetic determinants. In 26 patients, nine distinct biallelic mutations in TMC6 (5), TMC8 (1), and CIB1 (3), seven being previously unreported, were detected. Additionally, 20 different HPV species, including three α-, 16 β-, and one γ-HPVs, were detected, eight of which are being reported for the first time in EV patients (β-HPV-37, -47, -80, -151, -159, α-HPV-2, -57, and γ-HPV-128). This study expands the TMC6, TMC8, and CIB1 sequence variant spectrum and implicates new HPV subtypes in the pathogenesis of typical EV.
Amir Hossein Saeidian, Leila Youssefian, Mahtab Naji, Hamidreza Mahmoudi, Samantha M. Barnada, Charles Y. Huang, Karim Naghipoor, Amir Hozhabrpour, Jason S. Park, Flavia Manzo Margiotta, Fatemeh Vahidnezhad, Zahra Saffarian, Kambiz Kamyab-Hesari, Mohammad Tolouei, Niloofar Faraji, Seyyede Zeinab Azimi, Ghazal Namdari, Parvin Mansouri, Jean-Laurent Casanova, Vivien Béziat, Emmanuelle Jouanguy, Jouni Uitto, Hassan Vahidnezhad
SMA with respiratory distress type 1 (SMARD1) and Charcot Marie Tooth type 2S (CMT2S) are a result of mutations in immunoglobulin mu DNA binding protein 2 (IGHMBP2). IGHMBP2 is an UPF1-like helicase with proposed roles in several cellular processes including translation. This study examines activator of basal transcription (ABT1), a modifier of the FVB-Ighmbp2nmd/nmd phenotype. Microscale thermophoresis and dynamic light scattering demonstrate IGHMBP2 and ABT1 proteins directly interact with high affinity. The association of ABT1 with IGHMBP2 significantly increases the ATPase and helicase activity as well as the processivity of IGHMBP2. The IGHMBP2-ABT1 complex interacts with the 5' external transcribed spacer and U3 snoRNA suggesting that the IGHMBP2-ABT1 complex is important for pre-rRNA processing. Intracerebroventricular injection of scAAV9-Abt1 decreases FVB-Ighmbp2nmd/nmd disease pathology, significantly increases lifespan and substantially decreases neuromuscular junction denervation. ABT1 is the first disease modifying gene identified for SMARD1. We provide a mechanism that proposes ABT1 decreases disease pathology in FVB-Ighmbp2nmd/nmd mutants by optimizing IGHMBP2 biochemical activity (ATPase and helicase activity). Our studies provide important insight into SMARD1 pathogenesis suggesting ABT1 modifies IGHMBP2 activity as a means to regulate pre-rRNA processing.
Gangadhar P. Vadla, Sara M. Ricardez Hernandez, Jiude Mao, Mona O. Garro-Kacher, Zachary C. Lorson, Ronin P. Rice, Sarah A. Hansen, Christian L. Lorson, Kamal Singh, Monique A. Lorson
Oncogenic FOXO1 gene fusions drive a subset of rhabdomyosarcoma (RMS) with poor survival and to date these cancer drivers are therapeutically intractable. To identify new therapies for this disease, we undertook an isogenic CRISPR-interference screen to define PAX3-FOXO1 specific genetic dependencies and identified genes in the GATOR2 complex. GATOR2 loss in RMS abrogated amino acid-induced lysosomal localization of mTORC1 and consequent downstream signaling, slowing G1-S cell cycle transition. In vivo suppression of GATOR2 impaired the growth of tumor xenografts and favored the outgrowth of cells lacking PAX3-FOXO1. Loss of a subset of GATOR2 members can be compensated by direct genetic activation of mTORC1. RAS mutations are also sufficient to decouple mTORC1 activation from GATOR2, and indeed fusion negative RMS harboring such mutations exhibit amino acid-independent mTORC1 activity. A bi-steric, mTORC1-selective small molecule induced tumor regressions in fusion positive patient-derived tumor xenografts. These findings highlight a vulnerability in FOXO1 fusion positive RMS and provide rationale for the clinical evaluation of bi-steric mTORC1 inhibitors, currently in phase 1 testing, to treat this disease. Isogenic genetic screens can thus identify potentially exploitable vulnerabilities in fusion driven pediatric cancers which otherwise remain mostly undruggable.
Jacqueline Morales, David V. Allegakoen, José A. Garcia, Kristen Kwong, Pushpendra K. Sahu, Drew A. Fajardo, Yue Pan, Max A. Horlbeck, Jonathan S. Weissman, W. Clay Gustafson, Trever G. Bivona, Amit J. Sabnis
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