Notch signaling is a highly conserved intercellular pathway with tightly regulated and pleiotropic roles in normal tissue development and homeostasis. Dysregulated Notch signaling has also been implicated in human disease, including multiple forms of cancer, and represents an emerging therapeutic target. Successful development of such therapeutics requires a detailed understanding of potential on-target toxicities. Here, we identify autosomal dominant mutations of the canonical Notch ligand Jagged1 (or JAG1) as a cause of peripheral nerve disease in 2 unrelated families with the hereditary axonal neuropathy Charcot-Marie-Tooth disease type 2 (CMT2). Affected individuals in both families exhibited severe vocal fold paresis, a rare feature of peripheral nerve disease that can be life-threatening. Our studies of mutant protein posttranslational modification and localization indicated that the mutations (p.Ser577Arg, p.Ser650Pro) impair protein glycosylation and reduce JAG1 cell surface expression. Mice harboring heterozygous CMT2-associated mutations exhibited mild peripheral neuropathy, and homozygous expression resulted in embryonic lethality by midgestation. Together, our findings highlight a critical role for JAG1 in maintaining peripheral nerve integrity, particularly in the recurrent laryngeal nerve, and provide a basis for the evaluation of peripheral neuropathy as part of the clinical development of Notch pathway–modulating therapeutics.
Jeremy M. Sullivan, William W. Motley, Janel O. Johnson, William H. Aisenberg, Katherine L. Marshall, Katy E.S. Barwick, Lingling Kong, Jennifer S. Huh, Pamela C. Saavedra-Rivera, Meriel M. McEntagart, Marie-Helene Marion, Lucy A. Hicklin, Hamid Modarres, Emma L. Baple, Mohamed H. Farah, Aamir R. Zuberi, Cathleen M. Lutz, Rachelle Gaudet, Bryan J. Traynor, Andrew H. Crosby, Charlotte J. Sumner
Background: Neurofibroma/schwannoma hybrid nerve sheath tumors (N/S HNSTs) are neoplasms associated with larger nerves that occur sporadically and in the context of schwannomatosis or neurofibromatosis type 2 or 1. Clinical management of N/S HNST is challenging, especially for large tumors, and established systemic treatments are lacking. Methods: We used next-generation sequencing and array-based DNA methylation profiling to determine the clinically actionable genomic and epigenomic landscapes of N/S HNST. Results: Whole-exome sequencing within a precision oncology program identified an activating mutation (p.Asp769Tyr) in the catalytic domain of the ERBB2 receptor tyrosine kinase in a patient with schwannomatosis-associated N/S HNST, and targeted treatment with the small-molecule ERBB inhibitor lapatinib led to prolonged clinical benefit and a lasting radiographic and metabolic response. Analysis of a multicenter validation cohort revealed recurrent ERBB2 mutations (p.Leu755Ser, p.Asp769Tyr, p.Val777Leu) in N/S HNSTs occurring in patients who met diagnostic criteria for sporadic schwannomatosis (3 of 7 patients), but not in N/S HNSTs arising in the context of neurofibromatosis (6 patients) or outside a tumor syndrome (1 patient), and showed that ERBB2-mutant N/S HNSTs cluster in a distinct subgroup of peripheral nerve sheath tumors based on genome-wide DNA methylation patterns. Conclusion: These findings uncover a key biological feature of N/S HNST that may have important diagnostic and therapeutic implications. Funding: This work was supported by grant H021 from DKFZ-HIPO. MWR and PNH have received fellowships from UCT Frankfurt, and MWR has received funding from the Frankfurt Research Funding Clinician Scientist Program.
Michael W. Ronellenfitsch, Patrick N. Harter, Martina Kirchner, Christoph Heining, Barbara Hutter, Laura Gieldon, Jens Schittenhelm, Martin U. Schuhmann, Marcos Tatagiba, Gerhard Marquardt, Marlies Wagner, Volker Endris, Christian H. Brandts, Victor-Felix Mautner, Evelin Schröck, Wilko Weichert, Benedikt Brors, Andreas von Deimling, Michel Mittelbronn, Joachim P. Steinbach, David E. Reuss, Hanno Glimm, Albrecht Stenzinger, Stefan Fröhling
Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon–mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies.
Andreas Marg, Helena Escobar, Sina Gloy, Markus Kufeld, Joseph Zacher, Andreas Spuler, Carmen Birchmeier, Zsuzsanna Izsvák, Simone Spuler
Multipass membrane proteins have a myriad of functions, including transduction of cell-cell signals, ion transport, and photoreception. Insertion of these proteins into the membrane depends on the endoplasmic reticulum (ER) membrane protein complex (EMC). Recently, birth defects have been observed in patients with variants in the gene encoding a member of this complex, EMC1. Patient phenotypes include congenital heart disease, craniofacial malformations, and neurodevelopmental disease. However, a molecular connection between EMC1 and these birth defects is lacking. Using Xenopus, we identified defects in neural crest cells (NCCs) upon emc1 depletion. We then used unbiased proteomics and discovered a critical role for emc1 in WNT signaling. Consistent with this, readouts of WNT signaling and Frizzled (Fzd) levels were reduced in emc1-depleted embryos, while NCC defects could be rescued with β-catenin. Interestingly, other transmembrane proteins were mislocalized upon emc1 depletion, providing insight into additional patient phenotypes. To translate our findings back to humans, we found that EMC1 was necessary for human NCC development in vitro. Finally, we tested patient variants in our Xenopus model and found the majority to be loss-of-function alleles. Our findings define molecular mechanisms whereby EMC1 dysfunction causes disease phenotypes through dysfunctional multipass membrane protein topogenesis.
Jonathan Marquez, June Criscione, Rebekah M. Charney, Maneeshi S. Prasad, Woong Y. Hwang, Emily K. Mis, Martín I. García-Castro, Mustafa K. Khokha
Background: DICER1 is the only miRNA biogenesis component associated with an inherited tumor syndrome, featuring multinodular goiter (MNG) and rare pediatric-onset lesions. Other susceptibility genes for familial forms of MNG likely exist. Methods: Whole exome sequencing of a kindred with early-onset MNG and schwannomatosis was followed by investigation of germline pathogenic variants that fully segregated with the disease. Genome wide analyses were performed on 13 tissue samples from familial and non-familial DGCR8-E518K positive tumors, including MNG, schwannomas, papillary thyroid cancers (PTC) and Wilms Tumors. MiRNA profiles of four tissue types were compared, and sequencing of miRNA, pre-miRNA and mRNA was performed in a subset of 9 schwannomas, four of which harbor DGCR8-E518K. Results: We identified c.1552G>A;p.E518K in DGCR8, a microprocessor located in 22q, in the kindred. The variant identified is a somatic hotspot in Wilms Tumors and has been identified in two PTCs. Copy number loss of chromosome 22q, leading to loss of heterozygosity at the DGCR8 locus, was found in all 13 samples harboring c.1552G>A;p.E518K. miRNA profiling of PTC, MNG, schwannomas and Wilms Tumors revealed a common profile among E518K hemizygous tumors. In vitro cleavage demonstrated improper processing of pre-miRNA by DGCR8-E518K. MicroRNA and RNA profiling show that this variant disrupts precursor microRNA production, impacting populations of canonical microRNAs and mirtrons. Conclusions: We identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis. Funded by CIHR, Compute Canada, Alex’s Lemonade Stand Foundation, and the Mia Neri Foundation for Childhood Cancer.
Barbara Rivera, Javad Nadaf, Somayyeh Fahiminiya, Maria Apellaniz-Ruiz, Avi Saskin, Anne-Sophie Chong, Sahil Sharma, Rabea Wagener, Timothée Revil, Vincenzo Condello, Zineb Harra, Nancy Hamel, Nelly Sabbaghian, Karl Muchantef, Christian Thomas, Leanne de Kock, Marie-Noëlle Hébert-Blouin, Angelia V. Bassenden, Hannah Rabenstein, Ozgur Mete, Ralf Paschke, Marc P. Pusztaszeri, Werner Paulus, Albert Berghuis, Jiannis Ragoussis, Yuri E. Nikiforov, Reiner Siebert, Steffen Albrecht, Robert Turcotte, Martin Hasselblatt, Marc R. Fabian, William D. Foulkes
Immune response to therapeutic enzymes poses a detriment to patient safety and treatment outcome. Enzyme replacement therapy (ERT) is a standard therapeutic option for some types of Mucopolysaccharidoses including Morquio A syndrome caused by GALNS deficiency. Current protocols tolerize patients using cytotoxic immunosuppressives which can cause adverse effects. Here we show development of tolerance in Morquio A mice via oral delivery of peptide or GALNS during ten days prior to ERT. Our results show that using an immunodominant peptide (I10) or the complete enzyme (GALNS) to orally induce tolerance to GALNS prior to ERT, resulted in several improvements to ERT in mice: i) decreased splenocyte proliferation after in-vitro GALNS stimulation; ii) modulation of cytokine secretion profile; iii) decline in GALNS-specific IgG or IgE plasma; iv) decreased GAG storage in liver; and v) fewer circulating immune-complexes in plasma. This model could be extrapolated to other lysosomal storage disorders where immune response hinders ERT.
Angela C. Sosa, Barbara Kariuki, Qi Gan, Alan P. Knutsen, Clifford J. Bellone, Miguel A. Guzmán, Luis A. Barrera, Shunji Tomatsu, Anil K. Chauhan, Eric Armbrecht, Adriana M. Montaño
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.
Kathryn E. Oliver, Robert Rauscher, Marjolein Mijnders, Wei Wang, Matthew J. Wolpert, Jessica Maya, Carleen M. Sabusap, Robert A. Kesterson, Kevin L. Kirk, Andras Rab, Ineke Braakman, Jeong S. Hong, John L. Hartman IV, Zoya Ignatova, Eric J. Sorscher
Gout is caused by deposition of monosodium urate crystals in joints when plasma uric acid levels are chronically elevated beyond the saturation threshold, mostly due to renal underexcretion of uric acid. Although molecular pathways of this underexcretion have been elucidated, its etiology remains mostly unknown. We demonstrate that gout can be caused by a mutation in LDHD within the putative catalytic site of the encoded d-lactate dehydrogenase, resulting in augmented blood levels of d-lactate, a stereoisomer of l-lactate, which is normally present in human blood in miniscule amounts. Consequent excessive renal secretion of d-lactate in exchange for uric acid reabsorption culminated in hyperuricemia and gout. We showed that LDHD expression is enriched in tissues with a high metabolic rate and abundant mitochondria and that d-lactate dehydrogenase resides in the mitochondria of cells overexpressing the human LDHD gene. Notably, the p.R370W mutation had no effect on protein localization. In line with the human phenotype, injection of d-lactate into naive mice resulted in hyperuricemia. Thus, hyperuricemia and gout can result from the accumulation of metabolites whose renal excretion is coupled to uric acid reabsorption.
Max Drabkin, Yuval Yogev, Lior Zeller, Raz Zarivach, Ran Zalk, Daniel Halperin, Ohad Wormser, Evgenia Gurevich, Daniel Landau, Rotem Kadir, Yonatan Perez, Ohad S. Birk
Background: Proteinuria is considered as an unfavorable clinical condition that accelerates renal and cardiovascular disease. However, it is not clear if all forms of proteinuria are damaging. Mutations in CUBN cause Imerslund-Gräsbeck syndrome (IGS) featured by intestinal malabsorption of vitamin B12 and in some cases proteinuria. CUBN encodes for cubilin, an intestinal and proximal tubular uptake receptor containing 27 CUB domains for ligand binding. Methods: We used next-generation sequencing for renal disease genes to genotype cohorts of patients with suspected hereditary renal disease and chronic proteinuria. CUBN variants were analyzed using bioinformatics, structural modeling and epidemiological methods. Results: We identified 39 patients, in whom biallelic pathogenic variants in the CUBN gene are associated with chronic isolated proteinuria with childhood onset. Since the proteinuria displayed a high proportion of albuminuria, glomerular diseases such as steroid-resistant nephrotic syndrome or Alport syndrome were often the primary clinical diagnosis, motivating renal biopsies and proteinuria-lowering treatments. Yet, renal function was normal in all cases. By contrast, we did not find any biallelic pathogenic CUBN variants in patients with reduced renal function or focal segmental glomerulosclerosis. Unlike the more N-terminal IGS mutations, 37 out of the 41 proteinuria-associated CUBN variants led to modifications or truncations after the vitamin B12-binding domain. Finally, we show that four C-terminal CUBN variants are associated with albuminuria and moderately increased GFR in meta-analyses of large population-based cohorts. Conclusions: Collectively, our data suggest an important role for the C-terminal half of cubilin in renal albumin reabsorption. Albuminuria due to reduced cubilin function could be an unexpectedly common benign condition in humans that may not require any proteinuria-lowering treatment or renal biopsies.
Mathilda Bedin, Olivia Boyer, Aude Servais, Yong Li, Laure Villoing-Gaudé, Marie-Josephe Tête, Alexandra Cambier, Julien Hogan, Veronique Baudouin, Saoussen Krid, Albert Bensman, Florie Lammens, Ferielle Louillet, Bruno Ranchin, Cecile Vigneau, Iseline Bouteau, Corinne Isnard-Bagnis, Christoph J. Mache, Tobias Schäfer, Lars Pape, Markus Gödel, Tobias B. Huber, Marcus Benz, Günter Klaus, Matthias Hansen, Kay Latta, Olivier Gribouval, Vincent Morinière, Carole Tournant, Maik Grohmann, Elisa Kuhn, Timo Wagner, Christine Bole-Feysot, Fabienne Jabot-Hanin, Patrick Nitschké, Tarunveer S. Ahluwalia, Anna Köttgen, Christian Brix Folsted Andersen, Carsten Bergmann, Corinne Antignac, Matias Simons
Atrial fibrillation (AF), defined by disorganized atrial cardiac rhythm, is the most prevalent cardiac arrhythmia worldwide. Recent genetic studies have highlighted a major heritable component and identified numerous loci associated with AF risk, including the cardiogenic transcription factor genes TBX5, GATA4, and NKX2-5. We report that Tbx5 and Gata4 interact with opposite signs for atrial rhythm controls compared with cardiac development. Using mouse genetics, we found that AF pathophysiology caused by Tbx5 haploinsufficiency, including atrial arrhythmia susceptibility, prolonged action potential duration, and ectopic cardiomyocyte depolarizations, were all rescued by Gata4 haploinsufficiency. In contrast, Nkx2-5 haploinsufficiency showed no combinatorial effect. The molecular basis of the TBX5/GATA4 interaction included normalization of intra-cardiomyocyte calcium flux and expression of calcium channel genes Atp2a2 and Ryr2. Furthermore, GATA4 and TBX5 showed antagonistic interactions on an Ryr2 enhancer. Atrial rhythm instability caused by Tbx5 haploinsufficiency was rescued by a decreased dose of phospholamban, a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, consistent with a role for decreased sarcoplasmic reticulum calcium flux in Tbx5-dependent AF susceptibility. This work defines a link between Tbx5 dose, sarcoplasmic reticulum calcium flux, and AF propensity. The unexpected interactions between Tbx5 and Gata4 in atrial rhythm control suggest that evaluating specific interactions between genetic risk loci will be necessary for ascertaining personalized risk from genetic association data.
Brigitte Laforest, Wenli Dai, Leonid Tyan, Sonja Lazarevic, Kaitlyn M. Shen, Margaret Gadek, Michael T. Broman, Christopher R. Weber, Ivan P. Moskowitz