Genetics
Abstract
Developmental and epileptic encephalopathies (DEE) are characterized by pharmacoresistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant; only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared to mice treated with a control ASO (non-hybridizing sequence). ASO administration at neonatal age was also well-tolerated and effective in controlling seizures and extending the lifespan of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.
Authors
Lisseth E. Burbano, Melody Li, Nikola Jancovski, Paymaan Jafar-nejad, Kay Richards, Alicia Sedo, Armand Soriano, Ben Rollo, Linghan Jia, Elena V. Gazina, Sandra Piltz, Fatwa Adikusuma, Paul Q. Thomas, Helen Kopsidas, Frank Rigo, Christopher A. Reid, Snezana Maljevic, Steven Petrou
Abstract
The LAMA5 gene encodes laminin α5, an indispensable component of glomerular basement membrane and other types of basement membrane. A homozygous pathological variant in LAMA5 is known to cause systemic developmental syndrome, including glomerulopathy. However, the roles of heterozygous LAMA5 gene variants in human renal and systemic disease have remained unclear. We performed whole-exome sequence analyses of a family with slowly progressive nephropathy associated with hereditary focal segmental glomerulosclerosis; we identified a probable pathogenic novel variant of LAMA5, NP_005551.3:p.Val3687Met. In vitro analyses revealed cell type-dependent changes in secretion of variant laminin α5 LG4-5 domain. Heterozygous and homozygous knock-in mice with a corresponding variant of human LAMA5, p.Val3687Met, developed focal segmental glomerulosclerosis-like pathology with reduced laminin α5 and increased glomerular vinculin levels; this suggested that impaired cell adhesion may underlie this glomerulopathy. We also identified pulmonary defects such as bronchial deformity and alveolar dilation. Re-examinations of the family revealed phenotypes compatible with reduced laminin α5 and increased vinculin levels in affected tissues. Thus, the heterozygous p.Val3687Met variant may cause a new syndromic nephropathy with focal segmental glomerulosclerosis through possibly defective secretion of laminin α5. Enhanced vinculin may be a useful disease marker.
Authors
Jun-Ya Kaimori, Yamato Kikkawa, Daisuke Motooka, Tomoko Namba-Hamano, Ayako Takuwa, Atsuko Imai-Okazaki, Kaori Kobayashi, Arisa Tanigawa, Yuko Kotani, Yoshihiro Uno, Kazuto Yoshimi, Koki Hattori, Yuta Asahina, Sachio Kajimoto, Yohei Doi, Tatsufumi Oka, Yusuke Sakaguchi, Tomoji Mashimo, Kiyotoshi Sekiguchi, Akihiro Nakaya, Motoyoshi Nomizu, Yoshitaka Isaka
Abstract
Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the Southwestern United States and Mexico. Illness occurs in approximately 30% of those infected, <1% of whom develop disseminated disease. To address why some individuals allow dissemination, we enrolled DCM patients and performed whole-exome sequencing. In an exploratory set of 67 DCM patients, two had haploinsufficient STAT3 mutations, while defects in β-glucan sensing and response were seen in 34/67 (50.7%) cases. Damaging CLEC7A (n=14) and PLCG2 (n=11) variants were associated with impaired production of β-glucan-stimulated TNF-α from peripheral blood mononuclear cells compared to healthy controls (P<0.005). Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were over-represented in DCM (P=0.0206, P=0.015, respectively) including CLEC7A Y238* (P=0.0105) and PLCG2 R268W (P=0.0025). A validation cohort of 111 DCM patients confirmed PLCG2 R268W (P=0.0276), CLEC7A I223S (P=0.044), and CLEC7A Y238* (P=0.0656). Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived H2O2 in transfected cells. Heterozygous DUOX1 or DUOXA1 variants which impaired H2O2 production were overrepresented in discovery and validation cohorts. Patients with DCM have impaired β-glucan sensing or response affecting TNF-α and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.
Authors
Amy P. Hsu, Agnieszka Korzeniowska, Cynthia C. Aguilar, Jingwen Gu, Eric Karlins, Andrew J. Oler, Gang Chen, Glennys V. Reynoso, Joie Davis, Alexandria Chaput, Tao Peng, Ling Sun, Justin B. Lack, Derek J. Bays, Ethan R. Stewart, Sarah E. Waldman, Daniel A. Powell, Fariba M. Donovan, Jigar V. Desai, Nima Pouladi, Debra A. Long Priel, Daisuke Yamanaka, Sergio D. Rosenzweig, Julie E. Niemela, Jennifer Stoddard, Alexandra F. Freeman, Christa S. Zerbe, Douglas B. Kuhns, Yves A. Lussier, Kenneth N. Olivier, Richard C. Boucher, Heather D. Hickman, Jeffrey Frelinger, Joshua Fierer, Lisa F. Shubitz, Thomas L. Leto, George R. Thompson III, John N. Galgiani, Michail S. Lionakis, Steven M. Holland
Abstract
Wolfram syndrome is a rare genetic disorder largely caused by pathogenic variants in the WFS1 gene and manifested by diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration. Recent genetic and clinical findings have revealed Wolfram syndrome as a spectrum disorder. Therefore, a genotype-phenotype correlation analysis is needed for diagnosis and therapeutic development. Here, we focus on the WFS1 c.1672C>T, p.R558C variant, which is highly prevalent in the Ashkenazi Jewish population. Clinical investigation indicated that patients carrying the homozygous WFS1 c.1672C>T, p.R558C variant showed mild forms of Wolfram syndrome phenotypes. Expression of WFS1 p.R558C was more stable compared with the other known recessive pathogenic variants associated with Wolfram syndrome. Human induced pluripotent stem cell–derived (iPSC-derived) islets (SC-islets) homozygous for WFS1 c.1672C>T variant recapitulated genotype-related Wolfram syndrome phenotypes. Enhancing residual WFS1 function through a combination treatment of chemical chaperones mitigated detrimental effects caused by the WFS1 c.1672C>T, p.R558C variant and increased insulin secretion in SC-islets. Thus, the WFS1 c.1672C>T, p.R558C variant causes a mild form of Wolfram syndrome phenotypes, which can be remitted with a combination treatment of chemical chaperones. We demonstrate that our patient iPSC–derived disease model provides a valuable platform for further genotype-phenotype analysis and therapeutic development for Wolfram syndrome.
Authors
Rie Asada Kitamura, Kristina G. Maxwell, Wenjuan Ye, Kelly Kries, Cris M. Brown, Punn Augsornworawat, Yoel Hirsch, Martin M. Johansson, Tzvi Weiden, Joseph Ekstein, Joshua Cohen, Justin Klee, Kent Leslie, Anton Simeonov, Mark J. Henderson, Jeffrey R. Millman, Fumihiko Urano
Allele-specific silencing of the gain-of-function mutation in Huntington's disease using CRISPR-Cas9
Abstract
Dominant gain-of-function mechanisms in Huntington's disease (HD) suggest selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing on exonic PAM-Altering SNP (PAS), we developed an allele-specific CRISPR-Cas9 strategy to permanently inactivate mutant HTT through nonsense- mediated decay (NMD). Comprehensive sequence/haplotype analysis identified SNP-generated NGG PAM sites on exons of common HTT haplotypes in HD subjects, revealing a clinically relevant PAS-based mutant- specific CRISPR-Cas9 strategy. Alternative allele of rs363099 (29th exon) eliminates the NGG PAM site on the most frequent normal HTT haplotype in HD, permitting mutant-specific CRISPR-Cas9 therapeutics in a predicted ~20% of HD subjects with European ancestry. Our rs363099-based CRISPR-Cas9 showed perfect allele specificity and good targeting efficiencies in patient-derived cells. Dramatically reduced mutant HTT mRNA and complete loss of mutant protein suggest that our allele-specific CRISPR-Cas9 strategy inactivate mutant HTT through NMD. In addition, GUIDE-seq analysis and subsequent validation experiments supported high levels of on-target gene specificity. Together, our data demonstrated a significant target population, complete mutant specificity, decent targeting efficiency in patient-derived cells, and minimal off-target effects on protein-coding genes, proving the concept of PAS-based allele-specific NMD-CRISPR-Cas9 and supporting its therapeutic potential in HD.
Authors
Jun Wan Shin, Eun Pyo Hong, Seri S. Park, Doo Eun Choi, Ihn Sik Seong, Madelynn N. Whittaker, Benjamin P. Kleinstiver, Richard Z. Chen, Jong-Min Lee
Abstract
The complex genomic landscape of prostate cancer evolves across disease states under therapeutic pressure directed toward inhibiting androgen receptor (AR) signaling. While significantly altered genes in prostate cancer have been extensively defined, there have been fewer systematic analyses of how structural variation shapes the genomic landscape of this disease across disease states. We uniformly characterized structural alterations across 531 localized and 143 metastatic prostate cancers profiled by whole genome sequencing, 125 metastatic samples of which were also profiled via whole transcriptome sequencing. We observed distinct significantly recurrent breakpoints in localized and metastatic castration-resistant prostate cancers (mCRPC), with pervasive alterations in noncoding regions flanking the AR, MYC, FOXA1, and LSAMP genes enriched in mCRPC and TMPRSS2-ERG rearrangements enriched in localized prostate cancer. We defined nine subclasses of mCRPC based on signatures of structural variation, each associated with distinct genetic features and clinical outcomes. Our results comprehensively define patterns of structural variation in prostate cancer and identify clinically actionable subgroups based on whole genome profiling.
Authors
Meng Zhou, Minjeong Ko, Anna C.H. Hoge, Kelsey Luu, Yuzhen Liu, Magdalena L. Russell, William W. Hannon, Zhenwei Zhang, Jian Carrot-Zhang, Rameen Beroukhim, Eliezer M. Van Allen, Atish D. Choudhury, Peter S. Nelson, Matthew Freedman, Mary-Ellen Taplin, Matthew Meyerson, Srinivas R. Viswanathan, Gavin Ha
Abstract
Pathogenic variants in the human F Box and Leucine Rich Repeat Protein 4 (FBXL4) gene result in an autosomal recessive, multi-systemic, mitochondrial disorder involving variable mitochondrial depletion and respiratory chain (RC) complex deficiencies with lactic acidemia. As no FDA-approved effective therapies exist, we sought to characterize translational C. elegans and zebrafish animal models, as well as human fibroblasts, to study FBXL4-/- disease mechanisms and identify preclinical therapeutic leads. Developmental delay, impaired fecundity and neurologic and/or muscular activity, mitochondrial dysfunction, and altered lactate metabolism were identified in fbxl-1(ok3741) C. elegans. Detailed studies of a pyruvate dehydrogenase complex activator, dichloroacetate (DCA) in fbxl-1(ok3741) C. elegans demonstrated its beneficial effects on fecundity, neuromotor activity, and mitochondrial function. Validation studies were performed in fbxl4sa12470 zebrafish larvae and in FBXL4-/- human fibroblasts, which showed DCA efficacy in preventing brain damage, impairment of neurologic and/or muscular function, mitochondrial biochemical dysfunction, and stress-induced morphologic and ultrastructural mitochondrial defects. These data demonstrate that fbxl-1 (ok3741) C. elegans and fbxl4sa12470 zebrafish provide robust translational models to study mechanisms and identify preclinical therapeutic candidates for FBXL4-/- disease. Further, DCA is a lead therapeutic candidate with therapeutic benefit on diverse aspects of survival, neurologic and/or muscular function, and mitochondrial physiology that warrants rigorous clinical trial study in human subjects with FBXL4-/- disease.
Authors
Manuela Lavorato, Eiko Nakamaru-Ogiso, Neal D. Mathew, Elizabeth Herman, Nina K. Shah, Suraiya Haroon, Rui Xiao, Christoph Seiler, Marni J. Falk
Abstract
Polyamine dysregulation plays key roles in a broad range of human diseases from cancer to neurodegeneration. Snyder-Robinson syndrome (SRS) is the first known genetic disorder of the polyamine pathway, caused by X-linked recessive loss-of-function mutations in spermine synthase. In the Drosophila SRS model, altered spermidine/spermine balance has been associated with increased generation of ROS and aldehydes, consistent with elevated spermidine catabolism. These toxic byproducts cause mitochondrial and lysosomal dysfunction, which are also observed in cells from SRS patients. No efficient therapy is available. We explored the biochemical mechanism and discovered acetyl-CoA reduction and altered protein acetylation as potentially novel pathomechanisms of SRS. We repurposed the FDA-approved drug phenylbutyrate (PBA) to treat SRS using an in vivo Drosophila model and patient fibroblast cell models. PBA treatment significantly restored the function of mitochondria and autolysosomes and extended life span in vivo in the Drosophila SRS model. Treating fibroblasts of patients with SRS with PBA ameliorated autolysosome dysfunction. We further explored the mechanism of drug action and found that PBA downregulates the first and rate-limiting spermidine catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), reduces the production of toxic metabolites, and inhibits the reduction of the substrate acetyl-CoA. Taken together, we revealed PBA as a potential modulator of SAT1 and acetyl-CoA levels and propose PBA as a therapy for SRS and potentially other polyamine dysregulation–related diseases.
Authors
Xianzun Tao, Yi Zhu, Zoraida Diaz-Perez, Seok-Ho Yu, Jackson R. Foley, Tracy Murray Stewart, Robert A. Casero Jr., Richard Steet, R. Grace Zhai
Abstract
Ciliopathies are a class of genetic diseases resulting in cilia dysfunction in multiple organ systems, including the olfactory system. Currently, there are no available curative treatments for olfactory dysfunction and other symptoms in ciliopathies. The loss or shortening of olfactory cilia, as seen in multiple mouse models of the ciliopathy Bardet-Biedl syndrome (BBS), results in olfactory dysfunction. However, the underlying mechanism of the olfactory cilia reduction is unknown, thus limiting the development of therapeutic approaches for BBS and other ciliopathies. Here, we demonstrated that PI(4,5)P2, a phosphoinositide typically excluded from olfactory cilia, aberrantly redistributed into the residual cilia of BBS mouse models, which caused F-actin ciliary infiltration. Importantly, PI(4,5)P2 and F-actin were necessary for olfactory cilia shortening. Using a gene therapeutic approach, the hydrolyzation of PI(4,5)P2 by overexpression of INPP5E restored cilia length, and rescued odor detection and odor perception in BBS. Together, our data indicate that PI(4,5)P2 and F-actin-dependent cilia disassembly is a common mechanism contributing to the loss of olfactory cilia in BBS and provide valuable pan therapeutic intervention targets for the treatment of ciliopathies.
Authors
Chao Xie, Julien C. Habif, Kirill Ukhanov, Cedric R. Uytingco, Lian Zhang, Robert J. Campbell, Jeffrey R. Martens
Abstract
Familial exudative vitreoretinopathy (FEVR) is a hereditary disorder that can cause vision loss. The CTNND1 gene encodes a cellular adhesion protein p120-catenin (p120), which is essential for vascularization, yet the function of p120 in postnatal physiological angiogenesis remains unclear. Here, we applied whole-exome sequencing (WES) on 140 probands of FEVR families and identified three candidate variants in the human CTNND1 gene. We performed inducible deletion of Ctnnd1 in the postnatal mouse endothelial cells (ECs) and observed typical phenotypes of FEVR. Immunofluorescence of retina flat mounts also revealed immune responses, including reactive astrogliosis and microgliosis accompanied by abnormal Vegfa expression. Using an unbiased proteomics analysis in combination with in vivo or in vitro approaches, we propose that p120 is critical for the integrity of cadherin/catenin complex, and that p120 activates Wnt signaling activity by protecting β-catenin from Gsk3β-ubiquitin-guided degradation. Treatment of CTNND1-depleted HRECs with Gsk3β inhibitors LiCl or CHIR-99021 successfully enhanced cell proliferation by preventing β-catenin from degradation. Moreover, LiCl treatment increased vessel density in Ctnnd1-deficient mouse retinas. Functional analysis also revealed that variants in CTNND1 cause FEVR by compromising the expression of adherens junctions (AJs) and Wnt signaling activity. Additionally, genetic interactions between p120 and β-catenin or α-catenin revealed by double heterozygous deletion in mice further confirmed that p120 regulates vascular development through the Wnt/Cadherin axis. Together, we propose that CTNND1 is a novel candidate gene associated with FEVR, and that variants in CTNND1 can cause FEVR through the Wnt/Cadherin axis.
Authors
Mu Yang, Shujin Li, Li Huang, Rulian Zhao, Erkuan Dai, Xiaoyan Jiang, Yunqi He, Jinglin Lu, Li Peng, Wenjing Liu, Zhaotian Zhang, Dan Jiang, Yi Zhang, Zhilin Jiang, Yeming Yang, Peiquan Zhao, Xianjun Zhu, Xiaoyan Ding, Zhenglin Yang
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