Genetics
Abstract
Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disorder caused by depletion of DLD from α-ketoacid dehydrogenase complexes. Caenorhabditis elegans animal models of DLD deficiency generated by graded feeding of dld-1(RNAi) revealed that full or partial reduction of DLD-1 expression recapitulated increased pyruvate levels typical of pyruvate dehydrogenase complex deficiency and significantly altered animal survival and health, with reductions in brood size, adult length, and neuromuscular function. DLD-1 deficiency dramatically increased mitochondrial unfolded protein stress response induction and adaptive mitochondrial proliferation. While ATP levels were reduced, respiratory chain enzyme activities and in vivo mitochondrial membrane potential were not significantly altered. DLD-1 depletion directly correlated with the induction of mitochondrial stress and impairment of worm growth and neuromuscular function. The safety and efficacy of dichloroacetate, thiamine, riboflavin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), l-carnitine, and lipoic acid supplemental therapies empirically used for human DLD disease were objectively evaluated by life span and mitochondrial stress response studies. Only dichloroacetate and thiamine showed individual and synergistic therapeutic benefits. Collectively, these C. elegans dld-1(RNAi) animal model studies demonstrate the translational relevance of preclinical modeling of disease mechanisms and therapeutic candidates. Results suggest that clinical trials are warranted to evaluate the safety and efficacy of dichloroacetate and thiamine in human DLD disease.
Authors
Chynna N. Broxton, Prabhjot Kaur, Manuela Lavorato, Smruthi Ganesh, Rui Xiao, Neal D. Mathew, Eiko Nakamaru-Ogiso, Vernon E. Anderson, Marni J. Falk
Abstract
We describe affected members of a two-generation family segregating a Stargardt disease-like phenotype caused by a two base pair deletion-insertion, c.1014_1015delGAinsCT;p(Trp338_Asn339delinsCysTyr), in BEST1. The variant was identified by whole exome sequencing and its pathogenicity was verified through chloride channel recording using wild-type (WT) and transfected mutant HEK293 cells. Clinical examination of both patients revealed a similar phenotype at two different disease stages that were attributable to difference in their age at presentation. Hyperautofluorescent flecks along the arcades were observed in the proband, while the affected mother exhibited more advanced retinal pigment epithelium (RPE) loss in the central macula. Full-field electroretinogram testing was unremarkable in the daughter, however, moderate attenuation of generalized cone function was detected in the mother. Electro-oculogram testing in the daughter was consistent with widespread dysfunction of the RPE characteristic of Best disease. Whole-cell patch clamp recordings revealed statistically significant decrease in chloride conductance of the mutant compared to WT cells. This report broadens the clinical spectrum of BEST1-associated retinopathy in the form of a mother and daughter with BEST1 genotype phenocopying Stargardt disease.
Authors
Masha Kolesnikova, Jin Kyun Oh, Jiali Wang, Winston Lee, Jana Zernant, Pei-Yin Su, Angela H. Kim, Laura A. Jenny, Tingting Yang, Rando Allikmets, Stephen H. Tsang
Abstract
Deficiency of glycogen debranching enzyme in glycogen storage disease type III (GSD III) results in excessive glycogen accumulation in multiple tissues, primarily the liver, heart, and skeletal muscle. We recently reported that an adeno-associated virus (AAV) vector expressing a bacterial debranching enzyme (Pullulanase) driven by the ubiquitous CMV enhancer/chicken β-actin (CB) promoter cleared glycogen in major affected tissues of infant GSD IIIa mice. In this study, we developed a novel dual promoter consisting of a liver-specific promoter (LSP) and the CB promoter for gene therapy in adult GSD IIIa mice. Ten-week treatment with an AAV vector containing the LSP-CB dual promoter in adult GSD IIIa mice significantly increased Pullulanase expression and reduced glycogen contents in the liver (-60%), heart (-76%), and skeletal muscle (-63%), accompanied by the reversal of liver fibrosis, improved muscle function, and significant decrease in plasma biomarkers alanine aminotransferase, aspartate aminotransferase, and creatine kinase. Compared to the CB promoter, the dual promoter effectively decreased Pullulanase-induced cytotoxic T lymphocyte responses and enabled persistent therapeutic gene expression in adult GSD IIIa mice. Future studies are needed to determine the long-term durability of the dual promoter mediated expression of Pullulanase in adult GSD IIIa mice and in large animal models.
Authors
Jeong-A Lim, Priya S. Kishnani, Baodong Sun
Abstract
TP53 mutation (TP53mut) is one of the most important driver events facilitating tumorigenesis, which could induce a series of chain reactions to promote tumor malignant transformation. However, the malignancy progression patterns under TP53 mutation still remain less known. Clarifying the molecular landscapes of TP53mut tumors will help us understand the process of tumor development and aid precise treatment. Here, we distilled genetic and epigenetic features altered in TP53mut cancers for cluster-of-cluster analysis. Using integrated classification, we derived five different subtypes of TP53mut patients. These subtypes have distinct features in genomic alteration, clinical relevance, microenvironment dysregulation and potential therapeutics. Among the five subtypes, COCA3 was identified as the subtype with worst prognosis, causing an immunosuppressive microenvironment and immunotherapeutic resistantance. Further drug efficacy research highlighted olaparib as the most promising therapeutic agents for COCA3 tumors. Importantly, the therapeutic efficacy of olaparib in COCA3 and immunotherapy in non-COCA3 tumors was validated in vivo experiment. Summarily, our study first explored the important molecular events and developed a subtype classification system with distinct targeted therapy strategies for different subtypes of TP53mut tumors. These multi-omics classification systems provided a valuable resource that significantly expands the knowledge of TP53mut tumors and might eventually benefit in clinical practice.
Authors
Xin Chen, Tianqi Liu, Wu Jianqi, Chen Zhu, Gefei Guan, Cunyi Zou, Qing Guo, Xiaolin Ren, Chen Li, Peng Cheng, Wen Cheng, Anhua Wu
Abstract
Antithrombin, a major endogenous anticoagulant, is a serine protease inhibitor (serpin). We characterized the biological and clinical impact of variants involving C-terminal antithrombin. We performed comprehensive molecular, cellular, and clinical characterization of patients with C-terminal antithrombin variants from a cohort of 444 unrelated individuals with confirmed antithrombin deficiency. We identified 17 patients carrying 12 C-terminal variants, 5 of whom had the p.Arg445Serfs*17 deletion. Five missense variants caused qualitative deficiency, and 7, including 4 insertion-deletion variants, induced severe quantitative deficiency, particularly p.Arg445Serfs*17 (antithrombin <40%). This +1 frameshift variant had a molecular size similar to that of WT antithrombin but possessed a different C-terminus. Morphologic and cotransfection experiments showed that recombinant p.Arg445Serfs*17 was retained at the endoplasmic reticulum and had a dominant-negative effect on WT antithrombin. Characterization of different 1+ frameshift, aberrant C-terminal variants revealed that protein secretion was determined by frameshift site. The introduction of Pro441 in the aberrant C-terminus, shared by 5 efficiently secreted variants, partially rescued p.Arg445Serfs*17 secretion. C-terminal antithrombin mutants have notable heterogeneity, related to variant type and localization. Aberrant C-terminal variants caused by 1+ frameshift, with similar size as WT antithrombin, may be secreted or not, depending on frameshift site. The severe clinical phenotypes of these genetic changes are consistent with their dominant-negative effects.
Authors
Carlos Bravo-Pérez, Mara Toderici, Joseph E. Chambers, José A. Martínez-Menárguez, Pedro Garrido-Rodriguez, Horacio Pérez-Sanchez, Belén de la Morena-Barrio, José Padilla, Antonia Miñano, Rosa Cifuentes-Riquelme, Vicente Vicente, Maria L. Lozano, Stefan J. Marciniak, Maria Eugenia de la Morena-Barrio, Javier Corral
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
No posts were found with this tag.
