Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy, characterized by a pathognomonic hindbrain malformation. All known JBTS-genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we use the recently identified JBTS-associated protein ARMC9 in tandem-affinity purification and yeast two-hybrid screens to identify a novel ciliary module whose dysfunction underlies JBTS. In addition to known JBTS-associated proteins CEP104 and CSPP1, we identify CCDC66 and TOGARAM1 as ARMC9 interaction partners. We show that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses and characterization of patient-derived fibroblasts, CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrate that dysfunction of ARMC9 or TOGARAM1 results in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant cold- and serum-induced ciliary loss in both ARMC9 and TOGARAM1 patient cell lines suggests a role for this new JBTS-associated protein module in ciliary stability.
Brooke L. Latour, Julie C. Van De Weghe, Tamara D.S. Rusterholz, Stef J.F. Letteboer, Arianna Gomez, Ranad Shaheen, Matthias Gesemann, Arezou Karamzade, Mostafa Asadollahi, Miguel Barroso-Gil, Manali Chitre, Megan E. Grout, Jeroen van Reeuwijk, Sylvia E.C. van Beersum, Caitlin V. Miller, Jennifer C. Dempsey, Heba Morsy, Michael J. Bamshad, Deborah A. Nickerson, Stephan C.F. Neuhauss, Karsten Boldt, Marius Ueffing, Mohammad Keramatipour, John A. Sayer, Fowzan S. Alkuraya, Ruxandra Bachmann-Gagescu, Ronald Roepman, Dan Doherty
The transcription factor ISL1 is expressed in pituitary gland stem cells and the thyrotrope and gonadotrope lineages. Pituitary-specific Isl1 deletion causes hypopituitarism with increased stem cell apoptosis, reduced differentiation of thyrotropes and gonadotropes, and reduced body size. Conditional Isl1 deletion causes development of multiple Rathke’s cleft-like cysts, with 100% penetrance. Foxa1 and Foxj1 are abnormally expressed in the pituitary gland and associated with a ciliogenic gene expression program in the cysts. We confirmed expression of FOXA1, FOXJ1 and stem cell markers in human Rathke's cleft cyst tissue, but not craniopharyngiomas, which suggests these transcription factors are useful, pathological markers for diagnosis of Rathke's cleft cysts. These studies support a model whereby expression of ISL1 in pituitary progenitors drives differentiation into thyrotropes and gonadotropes, and without it, activation of FOXA1 and FOXJ1 permits development of an oral epithelial cell fate with mucinous cysts. This pituitary specific Isl1 mouse knockout sheds light on the etiology of Rathke's cleft cysts and the role of ISL1 in normal pituitary development.
Michelle L. Brinkmeier, Hironori Bando, Adriana C. Camarano, Shingo Fujio, Koji Yoshimoto, Flávio S. J. de Souza, Sally A. Camper
Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by a selective predisposition to clinical disease caused by the Bacille Calmette-Guérin (BCG) vaccine and environmental mycobacteria. The known genetic etiologies of MSMD are inborn errors of IFN-γ immunity due to mutations of 15 genes controlling the production of or response to IFN-γ. Since the first MSMD-causing mutations were reported in 1996, biallelic mutations in the genes encoding IFN-γ receptor 1 (IFN-γR1) and IFN-γR2 have been reported in many patients of diverse ancestries. Surprisingly, mutations of the gene encoding the IFN-γ cytokine itself have not been reported, raising the remote possibility that there might be other agonists of the IFN-γ receptor. We describe 2 Lebanese cousins with MSMD, living in Kuwait, who are both homozygous for a small deletion within the IFNG gene (c.354_357del), causing a frameshift that generates a premature stop codon (p.T119Ifs4*). The mutant allele is loss of expression and loss of function. We also show that the patients’ herpesvirus Saimiri–immortalized T lymphocytes did not produce IFN-γ, a phenotype that can be rescued by retrotransduction with WT IFNG cDNA. The blood T and NK lymphocytes from these patients also failed to produce and secrete detectable amounts of IFN-γ. Finally, we show that human IFNG has evolved under stronger negative selection than IFNGR1 or IFNGR2, suggesting that it is less tolerant to heterozygous deleterious mutations than IFNGR1 or IFNGR2. This may account for the rarity of patients with autosomal-recessive, complete IFN-γ deficiency relative to patients with complete IFN-γR1 and IFN-γR2 deficiencies.
Gaspard Kerner, Jérémie Rosain, Antoine Guérin, Ahmad Al-Khabaz, Carmen Oleaga-Quintas, Franck Rapaport, Michel J. Massaad, Jing-Ya Ding, Taushif Khan, Fatima Al Ali, Mahbuba Rahman, Caroline Deswarte, Rubén Martinez-Barricarte, Raif S. Geha, Valentine Jeanne-Julien, Diane Garcia, Chih-Yu Chi, Rui Yang, Manon Roynard, Bernhard Fleckenstein, Flore Rozenberg, Stéphanie Boisson-Dupuis, Cheng-Lung Ku, Yoann Seeleuthner, Vivien Béziat, Nico Marr, Laurent Abel, Waleed Al-Herz, Jean-Laurent Casanova, Jacinta Bustamante
Haploinsufficiency of factors governing genome stability underlies hereditary breast and ovarian cancer. Homologous recombination (HR) repair is a major pathway disabled in these cancers. With the aim of identifying new candidate genes, we examined early onset breast cancer patients negative for BRCA1 and BRCA2 pathogenic variants. Here, we focused on CtIP (RBBP8 gene) that mediates HR repair through the end-resection of DNA double-strand breaks (DSB). Notably, the patients exhibited a number of rare germline RBBP8 variants, and functional analysis revealed that these variants did not affect DNA DSB end-resection efficiency. However, expression of a subset of variants led to deleterious nucleolytic degradation of stalled DNA replication forks in a manner similar to cells lacking BRCA1 or BRCA2. In contrast to BRCA1 and BRCA2, CtIP deficiency promoted the helicase-driven destabilization of RAD51 nucleofilaments at damaged DNA replication forks. Taken together, our work identifies CtIP as a critical regulator of DNA replication fork integrity, which when compromised, may predispose to the development of early onset breast cancer.
Reihaneh Zarrizi, Martin R. Higgs, Karolin Voßgröne, Maria Rossing, Birgitte Bertelsen, Muthiah Bose, Arne N. Kousholt, Heike I. Rösner, Bent Ejlertsen, Grant S. Stewart, Finn Cilius Nielsen, Claus Sørensen
Molecular mechanisms governing the development of mammalian cochlea, the hearing organ, remain largely unknown. Through genome sequencing in three subjects from two families with non-syndromic cochlear aplasia, we identified homozygous 221 KB and 338 KB deletions in a non-coding region on chromosome 8 with an ~200 KB overlapping section. Genomic location of the overlapping deleted region was starting from ~350 KB downstream of GDF6. Otic lineage cells differentiated from induced pluripotent stem cells derived from an affected individual show reduced expression of GDF6 compared to control cells. A mouse knock-out of Gdf6 reveals cochlear aplasia closely resembling the human phenotype. We conclude that GDF6 plays a necessary role in early cochlear development controlled by cis-regulatory elements located within ~500 KB region of the genome in humans and that its disruption leads to deafness due to cochlear aplasia.
Guney Bademci, Clemer Abad, Filiz Basak Cengiz, Serhat Seyhan, Armagan Incesulu, Shengru Guo, Suat Fitoz, Emine Ikbal Atli, Nicholas C. Gosstola, Selma Demir, Brett M. Colbert, Gozde Cosar Seyhan, Claire J. Sineni, Duygu Duman, Hakan Gurkan, Cynthia Casson Morton, Derek M. Dykxhoorn, Katherina Walz, Mustafa Tekin
Transcriptional dysregulation is a hallmark of prostate cancer (PCa). We mapped the RNA Polymerase II (RNA Pol II) associated chromatin interactions in normal prostate cells and PCa cells. We discovered thousands of enhancer-promoter, enhancer-enhancer, as well as promoter-promoter chromatin interactions. These transcriptional hubs operate within the framework set by structural proteins—CTCF and cohesins, and are regulated by the cooperative action of master transcription factors, such as the Androgen Receptor (AR) and FOXA1. By combining analyses from metastatic castration resistant PCa (mCRPC) specimens, we show that AR locus amplification contributes to the transcriptional up-regulation of AR gene by increasing the total number of chromatin interaction modules comprising of the AR gene and its distal enhancer. We deconvoluted the transcription control modules of several PCa genes, notably, the biomarker KLK3, lineage-restricted genes (KRT8, KRT18, HOXB13, FOXA1, ZBTB16), the drug target EZH2, and the oncogene MYC. By integrating clinical PCa data, we defined a novel germline-somatic interplay between the PCa risk allele rs684232 and the somatically acquired TMPRSS2-ERG gene fusion in the transcriptional regulation of multiple target genes—VPS53, FAM57A and GEMIN4. Our studies implicate changes in genome organization as a critical determinant of aberrant transcriptional regulation in PCa.
Susmita G. Ramanand, Yong Chen, Jiapei Yuan, Kelly Daescu, Maryou Lambros, Kathleen E. Houlahan, Suzanne Carreira, Wei Yuan, GuemHee Baek, Adam Sharp, Alec Paschalis, Mohammed Kanchwala, Yunpeng Gao, Adam Aslam, Nida Safdar, Xiaowei Zhan, Ganesh V. Raj, Chao Xing, Paul C. Boutros, Johann de Bono, Michael Q. Zhang, Ram S. Mani
Tumor DNA circulates in the plasma of cancer patients admixed with DNA from noncancerous cells. The genomic landscape of plasma DNA has been characterized in metastatic castration-resistant prostate cancer (mCRPC) but the plasma methylome has not been extensively explored. Here, we performed next-generation sequencing (NGS) on plasma DNA with and without bisulfite treatment from mCRPC patients receiving either abiraterone or enzalutamide in the pre- or post-chemotherapy setting. Principal component analysis on the mCRPC plasma methylome indicated that the main contributor to methylation variance (principal component one, or PC1) was strongly correlated with genomically determined tumor fraction (r = –0.96; P < 10–8) and characterized by hypermethylation of targets of the polycomb repressor complex 2 components. Further deconvolution of the PC1 top-correlated segments revealed that these segments are comprised of methylation patterns specific to either prostate cancer or prostate normal epithelium. To extract information specific to an individual’s cancer, we then focused on an orthogonal methylation signature, which revealed enrichment for androgen receptor binding sequences and hypomethylation of these segments associated with AR copy number gain. Individuals harboring this methylation pattern had a more aggressive clinical course. Plasma methylome analysis can accurately quantitate tumor fraction and identify distinct biologically relevant mCRPC phenotypes.
Anjui Wu, Paolo Cremaschi, Daniel Wetterskog, Vincenza Conteduca, Gian Marco Franceschini, Dimitrios Kleftogiannis, Anuradha Jayaram, Shahneen Sandhu, Stephen Q. Wong, Matteo Benelli, Samanta Salvi, Giorgia Gurioli, Andrew Feber, Mariana Buongermino Pereira, Anna Maria Wingate, Enrique Gonzalez-Billalebeita, Ugo De Giorgi, Francesca Demichelis, Stefano Lise, Gerhardt Attard
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