Genetics
Abstract
Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of IRD patients and other inherited diseases.
Authors
Valentin J. Weber, Alice Reschigna, Maximilian J. Gerhardt, Thomas Heigl, Klara S. Hinrichsmeyer, Sander van den Engel, Dina Y. Otify, Zoran Gavrilov, Frank Blaser, Isabelle Meneau, Christian Betz, Hanno J. Bolz, Martin Biel, Stylianos Michalakis, Elvir Becirovic
Abstract
Ischemic cardiomyopathy (ICM) is a leading cause of heart failure characterized by extensive remodeling of the cardiac extracellular matrix (ECM). While initially adaptive, ECM deposition following ischemic injury eventually turns maladaptive, promoting adverse cardiac remodeling. The strong link between the extent of fibrosis and adverse clinical outcomes has led to growing interest in ECM targeted therapies to prevent or reverse maladaptive cardiac remodeling in ICM; yet, the precise composition of the ECM in ICM remains poorly defined. In this study, we employed a sequential protein extraction enabled by the photocleavable surfactant Azo to enrich ECM proteins from left ventricular tissues of patients with end-stage ICM (n=16) and nonfailing donor hearts (n=16). High-resolution mass spectrometry-based quantitative proteomics identified and quantified over 6,000 unique protein groups, including 315 ECM proteins. We discovered significant upregulation of key ECM components, particularly glycoproteins, proteoglycans, collagens, and ECM regulators. Notably, LOXL1, FBLN1, and VCAN were among the most differentially expressed. Functional enrichment analyses revealed enhanced TGFβ signaling, integrin-mediated adhesion, and complement activation in ICM tissues, suggesting a feedback loop driving continued ECM deposition in the end-stage failing heart. Together, our findings provide a comprehensive proteomic landscape of ECM alterations in the end-stage ICM myocardium and identify promising molecular targets for therapeutic intervention.
Authors
Kevin M. Buck, Holden T. Rogers, Zachery R. Gregorich, Morgan W. Mann, Timothy J. Aballo, Zhan Gao, Emily A. Chapman, Andrew J. Perciaccante, Scott J. Price, Ienglam Lei, Paul C. Tang, Ying Ge
Abstract
Genetic diseases such as ion-channelopathies substantially burden human health. Existing treatments are limited and not genotype specific. Here, we report a two-step high-throughput approach to rapidly identify drug candidates for repurposing as genotype-specific therapy. We first screened 1,680 medicines using a new thallium-flux trafficking assay against KV11.1 gene variants causing Long QT Syndrome (LQTS), an ion-channelopathy associated with fatal cardiac arrhythmias. We identify evacetrapib as a suitable drug candidate that improves membrane trafficking and activates channels. We then use deep mutational scanning to prospectively identify all KV11.1 missense variants in a LQTS hotspot region responsive to treatment with evacetrapib. Combining high-throughput drug screens with deep mutational scanning establishes a new paradigm for mutation-specific drug discovery translatable to personalized treatment of patients with rare genetic disorders.
Authors
Christian L. Egly, Alex Shen, Tri Q. Do, Carlos Tellet Cabiya, Paxton A. Ritschel, Suah Woo, Matthew J. Ku, Brian P. Delisle, Brett Kroncke, Bjorn C. Knollmann
Abstract
Focal cortical dysplasia (FCD) is a major cause of refractory epilepsy and is associated with pathogenic variants in mTOR pathway genes, including DEPDC5, the most common cause of familial focal epilepsy. The mechanisms of epileptogenesis associated with FCD and hyperactive mTOR signaling remain unclear in DEPDC5-related epilepsy. To test whether DEPDC5 loss leading to seizures require in utero cortical developmental defects or if postnatal neuronal dysfunction of mTORC1 is sufficient to drive seizures, we developed a postnatal focal cortical Depdc5 knockout mouse model. Postnatal day 0-1 Depdc5-floxed mice received unilateral motor cortex injections of either AAV-Cre-GFP or control AAV-GFP. The AAV-Cre-GFP injected hemisphere had decreased DEPDC5 levels with hyperactivation of mTOR that increased with age compared to both the contralateral hemisphere and the AAV-GFP injected mice. Cortical lamination was not disrupted by postnatal DEPDC5 loss. Pathologic hallmarks of FCDs were identified in the Depdc5 knockout hemisphere, including increased SMI-311 neurofilament staining, hypomyelination, astrogliosis, and microglial activation. Mice with postnatal cortical DEPDC5 loss exhibited lower seizure thresholds, increased focal seizures, and increased rates of seizure-induced death compared to control mice. This study demonstrates that postnatal DEPDC5 loss and subsequent mTOR hyperactivation without disruption of cortical migration is sufficient to cause epilepsy.
Authors
Karenna J. Groff, Yini Liang, Christopher Morici, Jinita Modasia, Leena Mehendale, Nishtha Gupta, Angelica D'Amore, Yongho Choe, Mustafa Q. Hameed, Alexander Rotenberg, Mustafa Sahin, Christopher J. Yuskaitis
Abstract
Tumor suppressor NF1 is recurrently mutated in glioblastoma, leading to aberrant activation of Ras/rapidly accelerated fibrosarcoma (RAF)/MEK signaling. However, how tumor heterogeneity shapes the molecular landscape and efficacy of targeted therapies remains unclear. Here, we combined bulk and single-cell genomics of human somatic NF1-mutant, isocitrate dehydrogenase (IDH) wild-type glioblastomas with functional studies in cell lines and mouse intracranial tumor models to identify mechanisms of tumor heterogeneity underlying clinical outcome and MEK inhibitor response. Targeted DNA sequencing identified CDKN2A/B homozygous deletion as a poor prognostic marker in somatic NF1-mutant, but not NF1 wild-type, glioblastoma. Single-nucleus RNA sequencing of human patient NF1-mutant glioblastomas demonstrated that mesenchymal-like (MES-like) tumor cells were enriched for MEK activation signatures. Single-cell RNA-sequencing of mouse intracranial glioblastomas treated with the MEK inhibitor selumetinib identified distinct responses among tumor subpopulations. MEK inhibition selectively depleted MES-like cells, and selumetinib-resistant MES-like cells upregulated Ras signaling while resistant non-MES cells expressed markers of glial differentiation. Finally, genome-wide CRISPR interference screens validated Ras/RAF/MEK signaling as a key mediator of selumetinib response. Repression of the RAF regulator SHOC2 sensitized glioblastomas to selumetinib in vitro and in vivo, suggesting a synergistic treatment strategy. Taken together, these results highlighted the heterogeneity of NF1-mutant glioblastomas and informed future combination therapies.
Authors
Sixuan Pan, Kanish Mirchia, Emily Payne, S. John Liu, Nadeem Al-Adli, Zain Peeran, Poojan Shukla, Jacob S. Young, Rohit Gupta, Jasper Wu, Joanna Pak, Tomoko Ozawa, Brian Na, Alyssa T. Reddy, Steve E. Braunstein, Joanna J. Phillips, Susan Chang, David A. Solomon, Arie Perry, David R. Raleigh, Mitchel S. Berger, Adam R. Abate, Harish N. Vasudevan
Abstract
Wiedemann-Steiner syndrome (WDSTS) is a rare genetic cause of intellectual disability that is primarily caused by heterozygous loss of function variants in the gene encoding the histone lysine methyltransferase 2A (KMT2A). Prior studies have shown successful postnatal amelioration of disease phenotypes for Rett, Rubinstein-Taybi and Kabuki syndromes, which are related Mendelian disorders of the epigenetic machinery. To explore whether the neurological phenotype in WDSTS is treatable in-utero, we created a mouse model carrying a loss of function variant placed between two loxP sites. Kmt2a+/LSL mice demonstrated core features of WDSTS including growth retardation, craniofacial abnormalities, and hypertrichosis as well as hippocampal memory defects. The neurological phenotypes were rescued upon restoration of KMT2A in-utero following breeding to a nestin-Cre. Together, our data provided a mouse model to explore the potential therapeutic window in WDSTS. Our work suggested that WDSTS has a window of opportunity extending at least until the mid-point of in-utero development, making WDSTS an ideal candidate for future therapeutic strategies.
Authors
Tinna Reynisdottir, Kimberley J. Anderson, Katrin Möller, Stefán Pétursson, Andrew Brinn, Katheryn P. Franklin, Juan Ouyang, Asbjorg O. Snorradottir, Cathleen M. Lutz, Aamir R. Zuberi, Valerie B. DeLeon, Hans T. Bjornsson
Abstract
Methylmalonic acidemia (MMA) is a severe metabolic disorder affecting multiple organs because of a distal block in branched-chain amino acid (BCAA) catabolism. Standard of care is limited to protein restriction and supportive care during metabolic decompensation. Severe cases require liver/kidney transplantation, and there is a clear need for better therapy. Here, we investigated the effects of a small molecule branched-chain amino acid transaminase (BCAT) inhibitor in human MMA hepatocytes and an MMA mouse model. Mitochondrial BCAT is the first step in BCAA catabolism, and reduction of flux through an early enzymatic step is successfully used in other amino acid metabolic disorders. Metabolic flux analyses confirmed robust BCAT inhibition, with reduction of labeling of proximal and distal BCAA-derived metabolites in MMA hepatocytes. In vivo experiments verified the BCAT inhibition, but total levels of distal BCAA catabolite disease markers and clinical symptoms were not normalized, indicating contributions of substrates other than BCAA to these distal metabolite pools. Our study demonstrates the importance of understanding the underlying pathology of metabolic disorders for identification of therapeutic targets and the use of multiple, complementary models to evaluate them.
Authors
Madeline G. Hemmingsen, Guo-Fang Zhang, Yunhan Ma, Hannah Marchuk, Kalyani R. Patel, Tong Chen, Xinning Li, Mark Chapman, Sabrina Collias, Dolores H. Lopez-Terrada, James Beasley, Ashlee R. Stiles, Randy J. Chandler, Charles P. Venditti, Sarah P. Young, Mercedes Barzi, Beatrice Bissig-Choisat, Doug Krafte, Christopher B. Newgard, Karl-Dimiter Bissig
Abstract
Patients with Dravet syndrome (DS) present with severe, spontaneous seizures and ataxia. While most patients with DS have variants in the sodium channel Nav1.1 α subunit gene, SCN1A, variants in the sodium channel β1 subunit gene, SCN1B, are also linked to DS. Scn1b null mice model DS, with spontaneous generalized seizures that start in the second week of life. In Scn1b null cerebellum, neuronal pathfinding is severely altered, and Purkinje cells (PCs) and granule neurons have altered excitability. Here, we show that Scn1b null mice are ataxic. Expression of β1 protein in WT cerebellum, assessed using a CRISPR transgenic mouse model containing an in-frame V5 epitope tag at the β1 C-terminus, is widespread. Scn1b null PCs and interneurons in cerebellar slices have increased thresholds for action potential initiation and decreased repetitive firing frequency compared with WT. Scn1b null PCs have reduced transient and resurgent sodium current densities. We propose that reduced PC excitability underlies the ataxic phenotype of Scn1b mice. In addition, because cerebellar output to other areas of the brain can result in termination of seizures, we propose that PC hypoexcitability exacerbates the severe phenotype of this mouse model.
Authors
Yukun Yuan, Heather A. O’Malley, Jesse J. Winters, Alfonso Lavado, Nicholas S. Denomme, Shreeya Bakshi, Samantha L. Hodges, Luis Lopez-Santiago, Chunling Chen, Lori L. Isom
Abstract
Authors
Yorihiro Iwasaki, Monica Reyes, Arnaud Molin, Mari Muurinen, Marie-Laure Kottler, Murat Bastepe, Harald Jüppner
Abstract
Ehlers-Danlos syndromes (EDS) comprise a genetically and clinically heterogenous group of rare diseases that cause severe, often fatal, damage to connective tissue. The molecular basis of EDS implicates defects in extracellular matrix components, including various fibrillar collagens and glycosaminoglycans (GAGs). However, the precise pathogenic mechanisms behind EDS remain elusive. Here, we have implemented a multi-tiered approach to demonstrate the functional impact of B3GALT6 mutations on biochemical and developmental processes, ultimately leading to the spondylodysplastic subtype of EDS (spEDS), characterized by severe musculoskeletal symptoms. We show that the loss of function of β1,3-galactosyltransferase 6 (β3GalT6) is partially compensated by β1,3-glucuronosyltransferase 3 (GlcAT-I), the next enzyme in the GAG biosynthetic pathway. In addition, results from transcriptomics, collagen analysis, and biophysical experiments revealed that impaired collagen maturation, including defective glycosylation of collagen XII, contributes to altered tissue structure and biomechanics, the hallmarks of spEDS. Our findings unravel a new pathogenic mechanism of spEDS and bring us one step closer to therapeutic strategies, including cell and tissue engineering.
Authors
Roméo Milan Diana, Benjamin Jolivet, Jean-Baptiste Vincourt, Sébastien Hergalant, Grégory Francius, Yasaman Karami, Hamed Khakzad, Rebekka Wild, Marie Bourgeais, Anne Robert, Alison Wurtz, Guillermo Barreto, Nick Ramalanjaona, Déborah Helle, Rachel Onifarasoaniaina, Sophie Front, Chrystel Lopin-Bon, Delfien Syx, Fransiska Malfait, Sylvie Fournel-Gigleux, Sandrine Gulberti, Catherine Bui
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