61 results on '"Bowles KR"'
Search Results
2. Development of MAPT S305 mutation models exhibiting elevated 4R tau expression, resulting in altered neuronal and astrocytic function
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Bowles, KR, primary, Pugh, DA, additional, Pedicone, C, additional, Oja, L, additional, Weitzman, SA, additional, Liu, Y, additional, Chen, JL, additional, Disney, MD, additional, and Goate, AM, additional
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- 2023
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3. 17q21.31 sub-haplotypes underlying H1-associated risk for Parkinson’s disease and progressive supranuclear palsy converge on altered glial regulation
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Bowles, KR, Pugh, DA, Farrell, K, Han, N, TCW, J, Liu, Y, Liang, SA, Qian, L, Bendl, J, Fullard, JF, Renton, AE, Casella, A, Iida, MA, Bandres-Ciga, S, Gan-Or, Z, Heutink, P, Siitonen, A, Bertelsen, S, Karch, CM, Frucht, SJ, Kopell, BH, Peter, I, Park, YJ, Crane, PK, Kauwe, JSK, Boehme, KL, Höglinger, GU, Charney, A, Roussos, P, Wang, JC, Poon, WW, Raj, T, Crary, JF, and Goate, AM
- Abstract
Parkinson’s disease (PD) and progressive supranuclear palsy (PSP) are clinically similar neurodegenerative movement disorders that display unique neuropathological features (i.e. Lewy body pathology and Tau pathology, respectively). While each disorder has distinct clinical and genetic risk factors, both are associated with the MAPT 17q.21.31 locus H1 haplotype. This suggests a pleiotropic effect of this genomic region. To better understand the genetic contribution of this region to these diseases, we fine-mapped the apparent pleiotropy of this locus. Our study indicates that PD and PSP are associated with different sub-haplotypes of the H1 clade. PD-associated sub-haplotypes were associated with altered LRRC37A copy number and expression, which, like other PD risk-associated genes, we hypothesize to be most relevant to astroglial function. In contrast, PSP was associated with grossly altered LD structure across the 17q21.31 locus, and risk-associated variants were found to impact chromatin structure in both neurons and microglia. We conclude that the contribution of the 17q21.31 locus to multiple disorders is a result of its structural and haplotypic complexity, which in turn impacts the regulation of multiple genes and neural cell types. This raises the possibility of novel disease-specific pathogenic mechanisms driven by 17q21.31 structural variation and altered epigenetic regulation that appear to converge on glial function and gene expression. By fine-mapping the association of H1 with PD and PSP, we have begun to untangle the apparent pleiotropy of this locus, and gain better insight into the mechanism of each disease, which will guide future functional analyses and disease models for PD and PSP.
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- 2019
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4. Danon disease as an underrecognized cause of hypertrophic cardiomyopathy in children.
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Yang Z, McMahon CJ, Smith LR, Bersola J, Adesina AM, Breinholt JP, Kearney DL, Dreyer WJ, Denfield SW, Price JF, Grenier M, Kertesz NJ, Clunie SK, Fernbach SD, Southern JF, Berger S, Towbin JA, Bowles KR, and Bowles NE
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- 2005
5. Detection of viruses in myocardial tissues by polymerase chain reaction. evidence of adenovirus as a common cause of myocarditis in children and adults.
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Bowles NE, Ni J, Kearney DL, Pauschinger M, Schultheiss H, McCarthy R, Hare J, Bricker JT, Bowles KR, Towbin JA, Bowles, Neil E, Ni, Jiyuan, Kearney, Debra L, Pauschinger, Matthias, Schultheiss, Heinz-Peter, McCarthy, Robert, Hare, Joshua, Bricker, J Timothy, Bowles, Karla R, and Towbin, Jeffrey A
- Abstract
Objectives: The purpose of this study was to analyze cardiac tissue and blood for viral genomes using polymerase chain reaction (PCR) to define the common viral etiologies of myocarditis by age group.Background: Enteroviruses are considered the most common cause of myocarditis at all ages. Diagnosis relies on viral cultures, serology, and cardiac histology, which lack sensitivity, as well as PCR. However, in many cases enteroviruses are not detected.Methods: Cardiac samples were obtained for PCR analysis from patients with myocarditis (n = 624) and dilated cardiomyopathy (DCM) (n = 149). Patients were analyzed by age group, including neonates (n = 116), infants (n = 191), toddlers (n = 87), children (n = 110), adolescents (n = 92), and adults (n = 177). After nucleic acids had been extracted from an endomyocardial biopsy, an explant, or autopsy samples, PCR and reverse transcription PCR were performed to detect the genomic sequences of enterovirus, adenovirus, cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), parvovirus, respiratory syncytial virus (RSV), and influenza A virus.Results: Viral genome was amplified (adenovirus = 142, enterovirus = 85, CMV = 18, parvovirus = 6, influenza A = 5, HSV = 5, EBV = 3, RSV = 1) from 239 (38%) of the 624 samples from myocarditis patients, including 26 patient samples in which dual infection was found. Virus was detected in 30 (20%) of 149 DCM patient samples; only adenovirus (n = 18) and enterovirus (n = 12) were detected.Conclusions: Polymerase chain reaction identified adenovirus as the most common virus in the myocardium of children and adults with myocarditis and DCM. Although enteroviruses are also found in these patients, they appear to be a less common cause of myocarditis than adenovirus. [ABSTRACT FROM AUTHOR]- Published
- 2003
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6. Mutations in Cypher/ZASP in patients with dilated cardiomyopathy and left ventricular non-compaction
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Jiuann Huey Lin, William J. McKenna, Andrea Di Lenarda, Matteo Vatta, Michelle A. Chrisco, Lisa A. Schimmenti, Karla R. Bowles, Neil E. Bowles, Michelle Fox, Ju Chen, Georgine Faulkner, Thuy M. Vu, Jeffrey A. Towbin, Qiang Zhou, Ross T. Murphy, Gianfranco Sinagra, Bhagyalaxmi Mohapatra, Giorgio Valle, Perry M. Elliott, Zeev Perles, Ximena Sanchez, Shinawe Jimenez, Vatta, M, Mohapatra, B, Jimenez, S, Sanchez, X, Faulkner, G, Perles, Z, Sinagra, Gianfranco, Lin, Jh, Vu, Tm, Zhou, Q, Bowles, Kr, DI LENARDA, A, Schimmenti, L, Fox, M, Chrisco, Ma, Murphy, Rt, Mckenna, W, Elliott, P, Bowles, Ne, Chen, J, Valle, G, and Towbin, Ja
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Cardiomyopathy, Dilated ,medicine.medical_specialty ,Heart Ventricles ,Blotting, Western ,Cardiomyopathy ,Muscle Proteins ,030204 cardiovascular system & hematology ,Transfection ,Denaturing high performance liquid chromatography ,03 medical and health sciences ,Ventricular Dysfunction, Left ,0302 clinical medicine ,Internal medicine ,medicine ,Humans ,cardiovascular diseases ,Chromatography, High Pressure Liquid ,030304 developmental biology ,Adaptor Proteins, Signal Transducing ,Homeodomain Proteins ,0303 health sciences ,business.industry ,Barth syndrome ,Dilated cardiomyopathy ,LDB3 ,LIM Domain Proteins ,medicine.disease ,Left ventricular noncompaction cardiomyopathy ,Blotting, Northern ,Immunohistochemistry ,3. Good health ,Phospholamban ,Echocardiography ,Mutagenesis ,Heart failure ,Mutation ,Cardiology ,cardiovascular system ,Cardiology and Cardiovascular Medicine ,business ,Carrier Proteins - Abstract
OBJECTIVES We evaluated the role of Cypher/ZASP in the pathogenesis of dilated cardiomyopathy (DCM) with or without isolated non-compaction of the left ventricular myocardium (INLVM). BACKGROUND Dilated cardiomyopathy, characterized by left ventricular dilation and systolic dysfunction with signs of heart failure, is genetically transmitted in 30% to 40% of cases. Genetic heterogeneity has been identified with mutations in multiple cytoskeletal and sarcomeric genes causing the phenotype. In addition, INLVM with a hypertrophic dilated left ventricle, ventricular dysfunction, and deep trabeculations, is also inherited, and the genes identified to date differ from those causing DCM. Cypher/ZASP is a newly identified gene encoding a protein that is a component of the Z-line in both skeletal and cardiac muscle. METHODS Diagnosis of DCM was performed by echocardiogram, electrocardiogram, and physical examination. In addition, levels of the muscular isoform of creatine kinase were measured to evaluate for skeletal muscle involvement. Cypher/ZASP was screened by denaturing high performance liquid chromatography (DHPLC) and direct deoxyribonucleic acid sequencing. RESULTS We identified and screened 100 probands with left ventricular dysfunction. Five mutations in six probands (6% of cases) were identified in patients with familial or sporadic DCM or INLVM. In vitro studies showed cytoskeleton disarray in cells transfected with mutated Cypher/ZASP. CONCLUSIONS These data suggest that mutated Cypher/ZASP can cause DCM and INLVM and identify a mechanistic basis.
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- 2003
7. Development of MAPT S305 mutation human iPSC lines exhibiting elevated 4R tau expression and functional alterations in neurons and astrocytes.
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Bowles KR, Pedicone C, Pugh DA, Oja LM, Sousa FH, Keavey LK, Fulton-Howard B, Weitzman SA, Liu Y, Chen JL, Disney MD, and Goate AM
- Abstract
Due to the importance of 4R tau (with four microtubule-binding-repeat domains) in the pathogenicity of primary tauopathies, it has been challenging to model these diseases in induced pluripotent stem cell (iPSC)-derived neurons, which express very low levels of 4R tau. To address this, we have developed a panel of isogenic iPSC lines carrying MAPT splice-site mutations, S305S, S305I, or S305N, derived from four different donors. All mutations significantly increase 4R tau expression in iPSC neurons and astrocytes. Functional analyses of S305 mutant neurons reveal shared disruption in synaptic signaling and maturity but divergent effects on mitochondrial bioenergetics. In iPSC astrocytes, S305 mutations promote internalization of exogenous tau that may be a precursor to glial pathology. These lines recapitulate previously characterized tauopathy-relevant phenotypes and highlight functional differences between the wild-type 4R and the mutant 4R proteins in both neurons and astrocytes. As such, these lines enable a more complete understanding of pathogenic mechanisms underlying 4R tauopathies across different cell types., Competing Interests: Declaration of interests A.M.G. is a member of the science advisory boards of Genentech and Muna Therapeutics and is a consultant for Biogen., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2024
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8. Novel avenues of tau research.
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Sexton CE, Bitan G, Bowles KR, Brys M, Buée L, Maina MB, Clelland CD, Cohen AD, Crary JF, Dage JL, Diaz K, Frost B, Gan L, Goate AM, Golbe LI, Hansson O, Karch CM, Kolb HC, La Joie R, Lee SE, Matallana D, Miller BL, Onyike CU, Quiroz YT, Rexach JE, Rohrer JD, Rommel A, Sadri-Vakili G, Schindler SE, Schneider JA, Sperling RA, Teunissen CE, Weninger SC, Worley SL, Zheng H, and Carrillo MC
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- Humans, tau Proteins, Alzheimer Disease, Tauopathies
- Abstract
Introduction: The pace of innovation has accelerated in virtually every area of tau research in just the past few years., Methods: In February 2022, leading international tau experts convened to share selected highlights of this work during Tau 2022, the second international tau conference co-organized and co-sponsored by the Alzheimer's Association, CurePSP, and the Rainwater Charitable Foundation., Results: Representing academia, industry, and the philanthropic sector, presenters joined more than 1700 registered attendees from 59 countries, spanning six continents, to share recent advances and exciting new directions in tau research., Discussion: The virtual meeting provided an opportunity to foster cross-sector collaboration and partnerships as well as a forum for updating colleagues on research-advancing tools and programs that are steadily moving the field forward., (© 2024 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)
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- 2024
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9. Improved Protocol for Reproducible Human Cortical Organoids Reveals Early Alterations in Metabolism with MAPT Mutations.
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Bertucci T, Bowles KR, Lotz S, Qi L, Stevens K, Goderie SK, Borden S, Oja LM, Lane K, Lotz R, Lotz H, Chowdhury R, Joy S, Arduini BL, Butler DC, Miller M, Baron H, Sandhof CA, Silva MC, Haggarty SJ, Karch CM, Geschwind DH, Goate AM, and Temple S
- Abstract
Cerebral cortical-enriched organoids derived from human pluripotent stem cells (hPSCs) are valuable models for studying neurodevelopment, disease mechanisms, and therapeutic development. However, recognized limitations include the high variability of organoids across hPSC donor lines and experimental replicates. We report a 96-slitwell method for efficient, scalable, reproducible cortical organoid production. When hPSCs were cultured with controlled-release FGF2 and an SB431542 concentration appropriate for their TGFBR1 / ALK5 expression level, organoid cortical patterning and reproducibility were significantly improved. Well-patterned organoids included 16 neuronal and glial subtypes by single cell RNA sequencing (scRNA-seq), frequent neural progenitor rosettes and robust BCL11B+ and TBR1+ deep layer cortical neurons at 2 months by immunohistochemistry. In contrast, poorly-patterned organoids contain mesendoderm-related cells, identifiable by negative QC markers including COL1A2 . Using this improved protocol, we demonstrate increased sensitivity to study the impact of different MAPT mutations from patients with frontotemporal dementia (FTD), revealing early changes in key metabolic pathways.
- Published
- 2023
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10. Development of MAPT S305 mutation models exhibiting elevated 4R tau expression, resulting in altered neuronal and astrocytic function.
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Bowles KR, Pugh DA, Pedicone C, Oja L, Weitzman SA, Liu Y, Chen JL, Disney MD, and Goate AM
- Abstract
Due to the importance of 4R tau in the pathogenicity of primary tauopathies, it has been challenging to model these diseases in iPSC-derived neurons, which express very low levels of 4R tau. To address this problem we have developed a panel of isogenic iPSC lines carrying the MAPT splice-site mutations S305S, S305I or S305N, derived from four different donors. All three mutations significantly increased the proportion of 4R tau expression in iPSC-neurons and astrocytes, with up to 80% 4R transcripts in S305N neurons from as early as 4 weeks of differentiation. Transcriptomic and functional analyses of S305 mutant neurons revealed shared disruption in glutamate signaling and synaptic maturity, but divergent effects on mitochondrial bioenergetics. In iPSC-astrocytes, S305 mutations induced lysosomal disruption and inflammation and exacerbated internalization of exogenous tau that may be a precursor to the glial pathologies observed in many tauopathies. In conclusion, we present a novel panel of human iPSC lines that express unprecedented levels of 4R tau in neurons and astrocytes. These lines recapitulate previously characterized tauopathy-relevant phenotypes, but also highlight functional differences between the wild type 4R and mutant 4R proteins. We also highlight the functional importance of MAPT expression in astrocytes. These lines will be highly beneficial to tauopathy researchers enabling a more complete understanding of the pathogenic mechanisms underlying 4R tauopathies across different cell types.
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- 2023
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11. A Mozambican marine protected area provides important habitat for vulnerable pelagic sharks.
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Murie CJG, Lebrato M, Lawrence A, Brown J, Gavard L, Bowles KR, Jije MG, Dicken M, and Oliver SP
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- Animals, Humans, Mozambique, Oceans and Seas, Seasons, Ecosystem, Sharks
- Abstract
Pelagic sharks play key roles in marine ecosystems, but are increasingly threatened by human extraction, habitat degradation and mismanagement. We investigated the use of protected and unprotected coastal habitats by bull (Carcharhinus leucas) and oceanic blacktip (Carcharhinus limbatus) sharks in southern Mozambique. Five INNOVASEA VR2W-69 kHz acoustic receivers were positioned in the Bazaruto Archipelago National Park (BANP) as well as one to the south of the park's boundaries. Seven receivers were also deployed 250 km south in the Inhambane estuary and on reef sites off Praia de Tofo. Twelve bull, and six oceanic blacktip sharks, were fitted with INNOVASEA V16 acoustic tags, which generated 933 detections of bull and 12,381 detections of oceanic blacktip sharks over a period of 1391 days. A generalised additive model was used to estimate the effects of seven spatiotemporal and environmental parameters on the frequency of each species' detections. In general, calculated residency indices were highest around the locations monitored in the BANP and one unprotected location off Tofo. Both species were more abundant across the monitored sites, during the summer when water temperatures were ~ 27 °C, when the moon was < 50% illuminated, and when the tide was rising. Detections coincided with each species' reproductive season indicating that both species may be reproductively active in the BANP region. Oceanic blacktip sharks were largely resident and so fisheries management may significantly benefit their population(s) around certain reef habitats in the BANP. The low residency and seasonal detections of bull sharks indicates that they may be transient and so effective conservation may require coordination between regional fisheries managers., (© 2023. The Author(s).)
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- 2023
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12. Discordance between germline genetic findings and abnormal tumor immunohistochemistry staining of mismatch repair proteins in individuals with suspected Lynch syndrome.
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Pan S, Cox H, Willmott J, Mundt E, Gorringe H, Landon M, Bowles KR, Coffee B, Roa BB, and Mancini-DiNardo D
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Background and Aims: Tumor immunohistochemical staining (IHC) of DNA mismatch repair (MMR) proteins is often used to guide germline genetic testing and variant classification for patients with suspected Lynch syndrome. This analysis examined the spectrum of germline findings in a cohort of individuals showing abnormal tumor IHC., Methods: We assessed individuals with reported abnormal IHC findings and referred for testing with a six-gene syndrome-specific panel (n=703). Pathogenic variants (PVs) and variants of uncertain significance (VUS) in MMR genes were designated expected/unexpected relative to IHC results., Results: The PV positive rate was 23.2% (163/703; 95% confidence interval [CI], 20.1%-26.5%); 8.0% (13/163; 95% CI, 4.3%-13.3%) of PV carriers had a PV in an unexpected MMR gene. Overall, 121 individuals carried VUS in MMR genes expected to be mutated based on IHC results. Based on independent evidence, in 47.1% (57/121; 95% CI, 38.0%-56.4%) of these individuals the VUSs were later reclassified as benign and in 14.0% (17/121; 95% CI, 8.4%-21.5%) of these individuals the VUSs were reclassified as pathogenic., Conclusions: Among patients with abnormal IHC findings, IHC-guided single-gene genetic testing may miss 8% of individuals with Lynch syndrome. In addition, in patients with VUS identified in MMR genes predicted to be mutated by IHC, extreme caution must be taken when the IHC results are considered in variant classification., Competing Interests: All authors were employees of Myriad Genetic Laboratories, Inc., at the time of this work and received salary and stock options as compensation., (Copyright © 2023 Pan, Cox, Willmott, Mundt, Gorringe, Landon, Bowles, Coffee, Roa and Mancini-DiNardo.)
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- 2023
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13. The influence of 17q21.31 and APOE genetic ancestry on neurodegenerative disease risk.
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Harerimana NV, Goate AM, and Bowles KR
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Advances in genomic research over the last two decades have greatly enhanced our knowledge concerning the genetic landscape and pathophysiological processes involved in multiple neurodegenerative diseases. However, current insights arise almost exclusively from studies on individuals of European ancestry. Despite this, studies have revealed that genetic variation differentially impacts risk for, and clinical presentation of neurodegenerative disease in non-European populations, conveying the importance of ancestry in predicting disease risk and understanding the biological mechanisms contributing to neurodegeneration. We review the genetic influence of two important disease-associated loci, 17q21.31 (the " MAPT locus") and APOE , to neurodegenerative disease risk in non-European populations, touching on global population differences and evolutionary genetics by ancestry that may underlie some of these differences. We conclude there is a need to increase representation of non-European ancestry individuals in genome-wide association studies (GWAS) and biomarker analyses in order to help resolve existing disparities in understanding risk for, diagnosis of, and treatment for neurodegenerative diseases in diverse populations., Competing Interests: Author AG is a member of the scientific advisory boards of Genentech and Muna Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Harerimana, Goate and Bowles.)
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- 2022
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14. 17q21.31 sub-haplotypes underlying H1-associated risk for Parkinson's disease are associated with LRRC37A/2 expression in astrocytes.
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Bowles KR, Pugh DA, Liu Y, Patel T, Renton AE, Bandres-Ciga S, Gan-Or Z, Heutink P, Siitonen A, Bertelsen S, Cherry JD, Karch CM, Frucht SJ, Kopell BH, Peter I, Park YJ, Charney A, Raj T, Crary JF, and Goate AM
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- Astrocytes pathology, DNA Copy Number Variations genetics, Genetic Predisposition to Disease, Haplotypes, Humans, Polymorphism, Single Nucleotide, alpha-Synuclein genetics, tau Proteins genetics, Parkinson Disease genetics, Parkinson Disease pathology
- Abstract
Background: Parkinson's disease (PD) is genetically associated with the H1 haplotype of the MAPT 17q.21.31 locus, although the causal gene and variants underlying this association have not been identified., Methods: To better understand the genetic contribution of this region to PD and to identify novel mechanisms conferring risk for the disease, we fine-mapped the 17q21.31 locus by constructing discrete haplotype blocks from genetic data. We used digital PCR to assess copy number variation associated with PD-associated blocks, and used human brain postmortem RNA-seq data to identify candidate genes that were then further investigated using in vitro models and human brain tissue., Results: We identified three novel H1 sub-haplotype blocks across the 17q21.31 locus associated with PD risk. Protective sub-haplotypes were associated with increased LRRC37A/2 copy number and expression in human brain tissue. We found that LRRC37A/2 is a membrane-associated protein that plays a role in cellular migration, chemotaxis and astroglial inflammation. In human substantia nigra, LRRC37A/2 was primarily expressed in astrocytes, interacted directly with soluble α-synuclein, and co-localized with Lewy bodies in PD brain tissue., Conclusion: These data indicate that a novel candidate gene, LRRC37A/2, contributes to the association between the 17q21.31 locus and PD via its interaction with α-synuclein and its effects on astrocytic function and inflammatory response. These data are the first to associate the genetic association at the 17q21.31 locus with PD pathology, and highlight the importance of variation at the 17q21.31 locus in the regulation of multiple genes other than MAPT and KANSL1, as well as its relevance to non-neuronal cell types., (© 2022. The Author(s).)
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- 2022
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15. Dysregulated coordination of MAPT exon 2 and exon 10 splicing underlies different tau pathologies in PSP and AD.
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Bowles KR, Pugh DA, Oja LM, Jadow BM, Farrell K, Whitney K, Sharma A, Cherry JD, Raj T, Pereira AC, Crary JF, and Goate AM
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- Alternative Splicing genetics, Brain pathology, Exons genetics, Humans, Neurons pathology, Protein Isoforms, Tauopathies genetics, Tauopathies pathology, Alzheimer Disease genetics, Alzheimer Disease pathology, Supranuclear Palsy, Progressive genetics, Supranuclear Palsy, Progressive pathology, tau Proteins genetics
- Abstract
Understanding regulation of MAPT splicing is important to the etiology of many nerurodegenerative diseases, including Alzheimer disease (AD) and progressive supranuclear palsy (PSP), in which different tau isoforms accumulate in pathologic inclusions. MAPT, the gene encoding the tau protein, undergoes complex alternative pre-mRNA splicing to generate six isoforms. Tauopathies can be categorized by the presence of tau aggregates containing either 3 (3R) or 4 (4R) microtubule-binding domain repeats (determined by inclusion/exclusion of exon 10), but the role of the N-terminal domain of the protein, determined by inclusion/exclusion of exons 2 and 3 has been less well studied. Using a correlational screen in human brain tissue, we observed coordination of MAPT exons 2 and 10 splicing. Expressions of exon 2 splicing regulators and subsequently exon 2 inclusion are differentially disrupted in PSP and AD brain, resulting in the accumulation of 1N4R isoforms in PSP and 0N isoforms in AD temporal cortex. Furthermore, we identified different N-terminal isoforms of tau present in neurofibrillary tangles, dystrophic neurites and tufted astrocytes, indicating a role for differential N-terminal splicing in the development of disparate tau neuropathologies. We conclude that N-terminal splicing and combinatorial regulation with exon 10 inclusion/exclusion is likely to be important to our understanding of tauopathies., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
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- 2022
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16. Genome-wide association study and functional validation implicates JADE1 in tauopathy.
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Farrell K, Kim S, Han N, Iida MA, Gonzalez EM, Otero-Garcia M, Walker JM, Richardson TE, Renton AE, Andrews SJ, Fulton-Howard B, Humphrey J, Vialle RA, Bowles KR, de Paiva Lopes K, Whitney K, Dangoor DK, Walsh H, Marcora E, Hefti MM, Casella A, Sissoko CT, Kapoor M, Novikova G, Udine E, Wong G, Tang W, Bhangale T, Hunkapiller J, Ayalon G, Graham RR, Cherry JD, Cortes EP, Borukov VY, McKee AC, Stein TD, Vonsattel JP, Teich AF, Gearing M, Glass J, Troncoso JC, Frosch MP, Hyman BT, Dickson DW, Murray ME, Attems J, Flanagan ME, Mao Q, Mesulam MM, Weintraub S, Woltjer RL, Pham T, Kofler J, Schneider JA, Yu L, Purohit DP, Haroutunian V, Hof PR, Gandy S, Sano M, Beach TG, Poon W, Kawas CH, Corrada MM, Rissman RA, Metcalf J, Shuldberg S, Salehi B, Nelson PT, Trojanowski JQ, Lee EB, Wolk DA, McMillan CT, Keene CD, Latimer CS, Montine TJ, Kovacs GG, Lutz MI, Fischer P, Perrin RJ, Cairns NJ, Franklin EE, Cohen HT, Raj T, Cobos I, Frost B, Goate A, White Iii CL, and Crary JF
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- Aged, Aged, 80 and over, Aging pathology, Animals, Cohort Studies, Drosophila, Female, Genome-Wide Association Study, Humans, Male, Middle Aged, Polymorphism, Single Nucleotide, Homeodomain Proteins genetics, Tauopathies genetics, Tauopathies pathology, Tumor Suppressor Proteins genetics
- Abstract
Primary age-related tauopathy (PART) is a neurodegenerative pathology with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-β (Aβ) plaques. The pathogenesis of PART is not known, but evidence suggests an association with genes that promote tau pathology and others that protect from Aβ toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (n = 647) using Braak neurofibrillary tangle stage as a quantitative trait. We found some significant associations with candidate loci associated with AD (SLC24A4, MS4A6A, HS3ST1) and progressive supranuclear palsy (MAPT and EIF2AK3). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including JADE1 which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brains with four microtubule-binding domain repeats (4R) isoforms and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation in post-mortem human PART brain tissue revealed a specific binding of JADE1 protein to four repeat tau lacking N-terminal inserts (0N4R). Finally, knockdown of the Drosophila JADE1 homolog rhinoceros (rno) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model, as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a modifier of neurofibrillary degeneration., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
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- 2022
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17. ELAVL4, splicing, and glutamatergic dysfunction precede neuron loss in MAPT mutation cerebral organoids.
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Bowles KR, Silva MC, Whitney K, Bertucci T, Berlind JE, Lai JD, Garza JC, Boles NC, Mahali S, Strang KH, Marsh JA, Chen C, Pugh DA, Liu Y, Gordon RE, Goderie SK, Chowdhury R, Lotz S, Lane K, Crary JF, Haggarty SJ, Karch CM, Ichida JK, Goate AM, and Temple S
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- Autophagy drug effects, Autophagy genetics, Biomarkers metabolism, Body Patterning drug effects, Body Patterning genetics, Cell Death drug effects, Cell Line, Humans, Hydrazones pharmacology, Lysosomes drug effects, Lysosomes metabolism, Morpholines pharmacology, Neurons drug effects, Neurons metabolism, Organoids drug effects, Organoids ultrastructure, Phosphorylation drug effects, Pyrimidines pharmacology, RNA Splicing drug effects, Signal Transduction drug effects, Stress Granules drug effects, Stress Granules metabolism, Synapses metabolism, Up-Regulation drug effects, Up-Regulation genetics, Cerebrum pathology, ELAV-Like Protein 4 genetics, Glutamic Acid metabolism, Mutation genetics, Neurons pathology, Organoids metabolism, RNA Splicing genetics, tau Proteins genetics
- Abstract
Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD., Competing Interests: Declaration of interests J.D.L. employee, Amgen. A.M.G.: Scientific Advisory Board (SAB), Denali Therapeutics (2015–2018); Genetic SAB, Pfizer (2019); SAB, Genentech; consultant, GSK, AbbVie, Biogen, and Eisai. S.J.H.: SAB, Rodin Therapeutics, Frequency Therapeutics, Psy Therapeutics, Vesigen Therapeutics, and Souvien Therapeutics; inventor, patent 6,475,723. S.T.: president, StemCultures; cofounder, LUXA Biotech; SAB, Sana Biotechnology, Blue Rock Therapeutics, and Vita Therapeutics; inventor, patent 16/331,063. J.K.I.: cofounder, AcuraStem and Modulo Bio; SAB, Spinogenix. Named companies were not involved in this project., (Copyright © 2021. Published by Elsevier Inc.)
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- 2021
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18. Alzheimer's-associated PU.1 expression levels regulate microglial inflammatory response.
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Pimenova AA, Herbinet M, Gupta I, Machlovi SI, Bowles KR, Marcora E, and Goate AM
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- Alzheimer Disease metabolism, Amyloid beta-Peptides pharmacology, Animals, Apoptosis drug effects, Cell Line, Cytokines drug effects, Cytokines metabolism, Gene Expression Profiling, Gene Knockdown Techniques, Inflammation metabolism, Lipopolysaccharides pharmacology, Mice, Microglia drug effects, Nitric Oxide metabolism, Peptide Fragments pharmacology, Rotenone pharmacology, Staurosporine, Uncoupling Agents pharmacology, Alzheimer Disease genetics, Apoptosis genetics, Inflammation genetics, Microglia metabolism, Proto-Oncogene Proteins genetics, Trans-Activators genetics
- Abstract
More than forty loci contribute to genetic risk for Alzheimer's disease (AD). These risk alleles are enriched in myeloid cell enhancers suggesting that microglia, the brain-resident macrophages, contribute to AD risk. We have previously identified SPI1/PU.1, a master regulator of myeloid cell development in the brain and periphery, as a genetic risk factor for AD. Higher expression of SPI1 is associated with increased risk for AD, while lower expression is protective. To investigate the molecular and cellular phenotypes associated with higher and lower expression of PU.1 in microglia, we used stable overexpression and knock-down of PU.1 in BV2, an immortalized mouse microglial cell line. Transcriptome analysis suggests that reduced PU.1 expression suppresses expression of homeostatic genes similar to the disease-associated microglia response to amyloid plaques in mouse models of AD. Moreover, PU.1 knock-down resulted in activation of protein translation, antioxidant action and cholesterol/lipid metabolism pathways with a concomitant decrease of pro-inflammatory gene expression. PU.1 overexpression upregulated and knock-down downregulated phagocytic uptake in BV2 cells independent of the nature of the engulfed material. However, cells with reduced PU.1 expression retained their ability to internalize myelin similar to control albeit with a delay, which aligns with their anti-inflammatory profile. Here we identified several microglial responses that are modulated by PU.1 expression levels and propose that risk association of PU.1 to AD is driven by increased pro-inflammatory response due to increased viability of cells under cytotoxic conditions. In contrast, low expression of PU.1 leads to increased cell death under cytotoxic conditions accompanied by reduced pro-inflammatory signaling that decreased A1 reactive astrocytes signature supporting the protective effect of SPI1 genotype in AD. These findings inform future in vivo validation studies and design of small molecule screens for therapeutic discovery in AD., (Copyright © 2020. Published by Elsevier Inc.)
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- 2021
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19. Impact of a Cancer Gene Variant Reclassification Program Over a 20-Year Period.
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Esterling L, Wijayatunge R, Brown K, Morris B, Hughes E, Pruss D, Manley S, Bowles KR, and Ross TS
- Abstract
Purpose: Hereditary cancer genetic testing can inform personalized medical management for individuals at increased cancer risk. However, many variants in cancer predisposition genes are individually rare, and traditional tools may be insufficient to evaluate pathogenicity. This analysis presents data on variant classification and reclassification over a 20-year period., Patients and Methods: This is a retrospective analysis of > 1.9 million individuals who received hereditary cancer genetic testing from a single clinical laboratory (March 1997 to December 2017). Variant classification included review of evidence from traditional tools (eg, population frequency databases, literature) and laboratory-developed tools (eg, novel statistical methods, in-house RNA analysis) by a multidisciplinary expert committee. Variants may have been reclassified more than once and with more than one line of evidence., Results: In this time period, 62,842 unique variants were observed across 25 cancer predisposition genes, and 2,976 variants were reclassified. Overall, 82.1% of reclassification events were downgrades (eg, variant of uncertain significance [VUS] to benign), and 17.9% were upgrades (eg, VUS to pathogenic). Among reclassified variants, 82.8% were initially classified as VUS, and 47.5% were identified in ≤ 20 individuals (allele frequency ≤ 0.001%). Laboratory-developed tools were used in 72.3% of variant reclassification events, which affected > 600,000 individuals. More than 1.3 million patients were identified as carrying a variant that was reclassified within this 20-year time period., Conclusion: The variant classification program used by the laboratory evaluated here enabled the reclassification of variants that were individually rare. Laboratory-developed tools were a key component of this program and were used in the majority of reclassifications. This demonstrates the importance of using robust and novel tools to reclassify rare variants to appropriately inform personalized medical management., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Lisa EsterlingEmployment: Myriad Genetics Stock and Other Ownership Interests: Myriad GeneticsKrystal BrownEmployment: Myriad Genetics Stock and Other Ownership Interests: Myriad GeneticsBrian MorrisEmployment: Myriad Genetics Stock and Other Ownership Interests: Myriad GeneticsElisha HughesEmployment: Myriad Genetics Stock and Other Ownership Interests: Myriad GeneticsDmitry PrussEmployment: Myriad Genetics Stock and Other Ownership Interests: Myriad GeneticsSusan ManleyEmployment: Myriad Genetics Stock and Other Ownership Interests: Myriad GeneticsKarla R. BowlesEmployment: Myriad Genetic Laboratories Leadership: Myriad Genetic Laboratories Stock and Other Ownership Interests: Myriad GeneticsTheodora S. RossEmployment: Merck & Co. No other potential conflicts of interest were reported., (© 2020 by American Society of Clinical Oncology.)
- Published
- 2020
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20. A Comprehensive Resource for Induced Pluripotent Stem Cells from Patients with Primary Tauopathies.
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Karch CM, Kao AW, Karydas A, Onanuga K, Martinez R, Argouarch A, Wang C, Huang C, Sohn PD, Bowles KR, Spina S, Silva MC, Marsh JA, Hsu S, Pugh DA, Ghoshal N, Norton J, Huang Y, Lee SE, Seeley WW, Theofilas P, Grinberg LT, Moreno F, McIlroy K, Boeve BF, Cairns NJ, Crary JF, Haggarty SJ, Ichida JK, Kosik KS, Miller BL, Gan L, Goate AM, and Temple S
- Subjects
- Cell Line, Fibroblasts metabolism, Fibroblasts pathology, Gene Editing, Humans, Induced Pluripotent Stem Cells metabolism, Mutation, Neural Stem Cells metabolism, Neural Stem Cells pathology, Neurogenesis, Neurons metabolism, Neurons pathology, Tauopathies genetics, tau Proteins genetics, Induced Pluripotent Stem Cells pathology, Tauopathies pathology
- Abstract
Primary tauopathies are characterized neuropathologically by inclusions containing abnormal forms of the microtubule-associated protein tau (MAPT) and clinically by diverse neuropsychiatric, cognitive, and motor impairments. Autosomal dominant mutations in the MAPT gene cause heterogeneous forms of frontotemporal lobar degeneration with tauopathy (FTLD-Tau). Common and rare variants in the MAPT gene increase the risk for sporadic FTLD-Tau, including progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). We generated a collection of fibroblasts from 140 MAPT mutation/risk variant carriers, PSP, CBD, and cognitively normal controls; 31 induced pluripotent stem cell (iPSC) lines from MAPT mutation carriers, non-carrier family members, and autopsy-confirmed PSP patients; 33 genome engineered iPSCs that were corrected or mutagenized; and forebrain neural progenitor cells (NPCs). Here, we present a resource of fibroblasts, iPSCs, and NPCs with comprehensive clinical histories that can be accessed by the scientific community for disease modeling and development of novel therapeutics for tauopathies., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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21. Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting.
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Bowles KR, Tcw J, Qian L, Jadow BM, and Goate AM
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- AC133 Antigen metabolism, Cell Differentiation, Cell Line, Cellular Reprogramming Techniques, Flow Cytometry methods, Humans, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells metabolism, Magnetics, Nerve Tissue Proteins metabolism, Nestin metabolism, Neural Stem Cells metabolism, Neurons metabolism, Receptors, Nerve Growth Factor metabolism, SOXB1 Transcription Factors metabolism, Cell Separation methods, Neural Stem Cells cytology, Neurons cytology
- Abstract
Genetic and epigenetic variability between iPSC-derived neural progenitor cells (NPCs) combined with differences in investigator technique and selection protocols contributes to variability between NPC lines, which subsequently impacts the quality of differentiated neuronal cultures. We therefore sought to develop an efficient method to reduce this variability in order to improve the purity of NPC and neuronal cultures. Here, we describe a magnetic activated cell sorting (MACS) method for enriching NPC cultures for CD271-/CD133+ cells at both early (<2-3) and late (>10) passage. MACS results in a similar sorting efficiency to fluorescence activated cell sorting (FACS), while achieving an increased yield of live cells and reduced cellular stress. Furthermore, neurons derived from MACS NPCs showed greater homogeneity between cell lines compared to those derived from unsorted NPCs. We conclude that MACS is a cheap technique for incorporation into standard NPC differentiation and maintenance protocols in order to improve culture homogeneity and consistency., Competing Interests: The authors have declared that no competing interests exist.
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- 2019
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22. Hereditary cancer testing challenges: assembling the analytical pieces to solve the patient clinical puzzle.
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Bowles KR, Mancini-DiNardo D, Coffee B, Cox HC, Qian Y, Elias M, Singh N, Judkins T, Leclair B, and Roa BB
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- Gene Rearrangement, Genetic Predisposition to Disease, Genetic Testing standards, High-Throughput Nucleotide Sequencing standards, Humans, Mismatch Repair Endonuclease PMS2 genetics, Mosaicism, Pseudogenes, Quality Control, Reproducibility of Results, Genetic Testing methods, High-Throughput Nucleotide Sequencing methods, Neoplastic Syndromes, Hereditary genetics
- Abstract
Expanded genetic test utilization to guide cancer management has driven the development of larger gene panels and greater diversity in the patient population pursuing testing, resulting in increased identification of atypical or technically challenging genetic findings. To ensure appropriate patient care, it is critical that genetic tests adequately identify and characterize these findings. We describe genetic testing challenges frequently encountered by our laboratory and the methodologies we employ to improve test accuracy for the identification and characterization of atypical genetic findings. While these findings may be individually rare, 15,745 (9%) individuals tested by our laboratory for hereditary cancer risk had an atypical genetic finding, highlighting the importance of employing highly accurate and comprehensive methods in clinical genetic testing.
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- 2019
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23. High-resolution temporal and regional mapping of MAPT expression and splicing in human brain development.
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Hefti MM, Farrell K, Kim S, Bowles KR, Fowkes ME, Raj T, and Crary JF
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- Adult, Aged, Brain metabolism, Case-Control Studies, Exons, Humans, Middle Aged, Neurodegenerative Diseases etiology, Protein Isoforms, Transcriptome, Alternative Splicing, Brain growth & development, Computational Biology methods, Neurodegenerative Diseases pathology, RNA Precursors genetics, tau Proteins genetics
- Abstract
The microtubule associated protein tau plays a critical role in the pathogenesis of neurodegenerative disease. Recent studies suggest that tau also plays a role in disorders of neuronal connectivity, including epilepsy and post-traumatic stress disorder. Animal studies have shown that the MAPT gene, which codes for the tau protein, undergoes complex pre-mRNA alternative splicing to produce multiple isoforms during brain development. Human data, particularly on temporal and regional variation in tau splicing during development are however lacking. In this study, we present the first detailed examination of the temporal and regional sequence of MAPT alternative splicing in the developing human brain. We used a novel computational analysis of large transcriptomic datasets (total n = 502 patients), quantitative polymerase chain reaction (qPCR) and western blotting to examine tau expression and splicing in post-mortem human fetal, pediatric and adult brains. We found that MAPT exons 2 and 10 undergo abrupt shifts in expression during the perinatal period that are unique in the canonical human microtubule-associated protein family, while exon 3 showed small but significant temporal variation. Tau isoform expression may be a marker of neuronal maturation, temporally correlated with the onset of axonal growth. Immature brain regions such as the ganglionic eminence and rhombic lip had very low tau expression, but within more mature regions, there was little variation in tau expression or splicing. We thus demonstrate an abrupt, evolutionarily conserved shift in tau isoform expression during the human perinatal period that may be due to tau expression in maturing neurons. Alternative splicing of the MAPT pre-mRNA may play a vital role in normal brain development across multiple species and provides a basis for future investigations into the developmental and pathological functions of the tau protein.
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- 2018
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24. Complexities of Variant Classification in Clinical Hereditary Cancer Genetic Testing.
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Mundt E, Nix P, Brown K, Bowles KR, and Manley S
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- Clinical Decision-Making, Genetic Predisposition to Disease, Heredity, Humans, Neoplasms classification, Neoplasms pathology, Neoplasms therapy, Observer Variation, Pedigree, Phenotype, Practice Guidelines as Topic, Predictive Value of Tests, Reproducibility of Results, Biomarkers, Tumor genetics, Genetic Variation, Molecular Diagnostic Techniques standards, Neoplasms genetics
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- 2017
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25. Identification of pathogenic retrotransposon insertions in cancer predisposition genes.
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Qian Y, Mancini-DiNardo D, Judkins T, Cox HC, Brown K, Elias M, Singh N, Daniels C, Holladay J, Coffee B, Bowles KR, and Roa BB
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- Alu Elements genetics, Base Sequence, Founder Effect, Haplotypes genetics, Humans, Mutation genetics, Risk Factors, Genes, Neoplasm, Genetic Predisposition to Disease, Mutagenesis, Insertional genetics, Neoplasms genetics, Retroelements genetics
- Abstract
Cancer risks have been previously reported for some retrotransposon element (RE) insertions; however, detection of these insertions is technically challenging and very few oncogenic RE insertions have been reported. Here we evaluate RE insertions identified during hereditary cancer genetic testing using a comprehensive testing strategy. Individuals who had single-syndrome or pan-cancer hereditary cancer genetic testing from February 2004 to March 2017 were included. RE insertions were identified using Sanger sequencing, Next Generation Sequencing, or multiplex quantitative PCR, and further characterized using targeted PCR and sequencing analysis. Personal cancer history, ancestry, and haplotype were evaluated. A total of 37 unique RE insertions were identified in 10 genes, affecting 211 individuals. BRCA2 accounted for 45.9% (17/37) of all unique RE insertions. Several RE insertions were detected with high frequency in populations of conserved ancestry wherein up to 100% of carriers shared a high degree of haplotype conservation, suggesting founder effects. Our comprehensive testing strategy resulted in a substantial increase in the number of reported oncogenic RE insertions, several of which may have possible founder effects. Collectively, these data show that the detection of RE insertions is an important component of hereditary cancer genetic testing and may be more prevalent than previously reported., (Copyright © 2017 Myriad Genetics, Inc. Published by Elsevier Inc. All rights reserved.)
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- 2017
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26. An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells.
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Tcw J, Wang M, Pimenova AA, Bowles KR, Hartley BJ, Lacin E, Machlovi SI, Abdelaal R, Karch CM, Phatnani H, Slesinger PA, Zhang B, Goate AM, and Brennand KJ
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- Astrocytes metabolism, Calcium metabolism, Cell Culture Techniques, Cells, Cultured, Computational Biology methods, Cytokines metabolism, Gene Expression Profiling, Gene Expression Regulation, Developmental, Humans, Induced Pluripotent Stem Cells metabolism, Microglia immunology, Microglia metabolism, Molecular Sequence Annotation, Neural Stem Cells cytology, Neural Stem Cells metabolism, Phagocytosis, Transcriptome, Astrocytes cytology, Cell Differentiation genetics, Induced Pluripotent Stem Cells cytology
- Abstract
Growing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of replicative astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. We evaluated this protocol across 42 NPC lines (derived from 30 individuals). Transcriptomic analysis demonstrated that hiPSC-astrocytes from four individuals are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity, and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our protocol is a reproducible, straightforward (single medium), and rapid (<30 days) method to generate populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2017
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27. Correction: Huntingtin Subcellular Localisation Is Regulated by Kinase Signalling Activity in the StHdhQ111 Model of HD.
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Bowles KR, Brooks SP, Hughes AC, Dunnett SB, and Jones L
- Abstract
[This corrects the article DOI: 10.1371/journal.pone.0144864.].
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- 2016
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28. Classification of genetic variants in genes associated with Lynch syndrome using a clinical history weighting algorithm.
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Morris B, Hughes E, Rosenthal E, Gutin A, and Bowles KR
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- Algorithms, Colorectal Neoplasms, Hereditary Nonpolyposis genetics, DNA Mismatch Repair, Female, Genetic Association Studies, Genetic Predisposition to Disease, Genetic Testing, Humans, Colorectal Neoplasms, Hereditary Nonpolyposis classification, DNA-Binding Proteins genetics, MutL Protein Homolog 1 genetics, MutS Homolog 2 Protein genetics, Mutation
- Abstract
Background: Lynch syndrome is a hereditary cancer syndrome associated with high risks of colorectal and endometrial cancer that is caused by pathogenic variants in the mismatch repair genes (MLH1, MSH2, MSH6, PMS2, EPCAM). Accurate classification of variants identified in these genes as pathogenic or benign enables informed medical management decisions. Previously, we developed a clinical History Weighting Algorithm (HWA) for the classification of variants of uncertain significance (VUSs) in BRCA1 and BRCA2. The BRCA1/2 HWA is based on the premise that pathogenic variants in these genes will be identified more often in individuals with strong personal and/or family histories of breast and/or ovarian cancer, while the identification of benign variants should be independent of cancer history. Here we report the development of a similar HWA to allow for classification of VUSs in genes associated with Lynch syndrome using data collected through both syndrome-specific and pan-cancer panel testing., Methods: Upon completion of algorithm development, the HWA was tested using simulated variants constructed from 79,214 probands, as well as 379 true variants. Positive (PPV) and negative predictive values (NPV) were calculated on a per gene basis., Results: 25,500 pathogenic and 50,500 benign simulated variants were analyzed using the HWA and the PPVs and NPVs for each gene were greater than 0.997 and 0.999, respectively. The HWA was also evaluated using 100 trials for each of the 379 true variants. PPVs of >0.998 and NPVs of >0.999 were obtained for all genes., Conclusions: We have developed and implemented a HWA to aid in the classification of VUSs in genes associated with Lynch syndrome. The work presented here demonstrates that this HWA is able to classify MLH1, MSH2, and MSH6 VUSs as either benign or pathogenic with high accuracy.
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- 2016
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29. Huntingtin Subcellular Localisation Is Regulated by Kinase Signalling Activity in the StHdhQ111 Model of HD.
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Bowles KR, Brooks SP, Dunnett SB, and Jones L
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- Animals, Cell Line, Transformed, Cell Nucleus metabolism, Corpus Striatum pathology, Cytosol metabolism, Disease Models, Animal, Gene Expression Regulation, Gene Knock-In Techniques, Humans, Huntington Disease metabolism, Huntington Disease pathology, MAP Kinase Kinase 1 antagonists & inhibitors, MAP Kinase Kinase 1 metabolism, MAP Kinase Kinase 2 antagonists & inhibitors, MAP Kinase Kinase 2 metabolism, Mice, Mice, Transgenic, Neural Stem Cells pathology, Phosphorylation, Primary Cell Culture, Protein Kinase Inhibitors pharmacology, Protein Transport, Proto-Oncogene Proteins c-akt antagonists & inhibitors, Proto-Oncogene Proteins c-akt metabolism, Serotonin Plasma Membrane Transport Proteins metabolism, Signal Transduction, Corpus Striatum metabolism, Huntington Disease genetics, MAP Kinase Kinase 1 genetics, MAP Kinase Kinase 2 genetics, Neural Stem Cells metabolism, Proto-Oncogene Proteins c-akt genetics, Serotonin Plasma Membrane Transport Proteins genetics
- Abstract
Huntington's disease is a neurodegenerative disorder characterised primarily by motor abnormalities, and is caused by an expanded polyglutamine repeat in the huntingtin protein. Huntingtin dynamically shuttles between subcellular compartments, and the mutant huntingtin protein is mislocalised to cell nuclei, where it may interfere with nuclear functions, such as transcription. However, the mechanism by which mislocalisation of mutant huntingtin occurs is currently unknown. An immortalised embryonic striatal cell model of HD (StHdhQ111) was stimulated with epidermal growth factor in order to determine whether the subcellular localisation of huntingtin is dependent on kinase signalling pathway activation. Aberrant phosphorylation of AKT and MEK signalling pathways was identified in cells carrying mutant huntingtin. Activity within these pathways was found to contribute to the regulation of huntingtin and mutant huntingtin localisation, as well as to the expression of immediate-early genes. We propose that altered kinase signalling is a phenotype of Huntington's disease that occurs prior to cell death; specifically, that altered kinase signalling may influence huntingtin localisation, which in turn may impact upon nuclear processes such as transcriptional regulation. Aiming to restore the balance of activity between kinase signalling networks may therefore prove to be an effective approach to delaying Huntington's disease symptom development and progression.
- Published
- 2015
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30. Exceptions to the rule: case studies in the prediction of pathogenicity for genetic variants in hereditary cancer genes.
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Rosenthal ET, Bowles KR, Pruss D, van Kan A, Vail PJ, McElroy H, and Wenstrup RJ
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- Genetic Testing methods, Genetic Testing standards, Genetic Testing statistics & numerical data, Humans, Neoplasms diagnosis, Practice Guidelines as Topic standards, Predictive Value of Tests, Prognosis, Reproducibility of Results, Sensitivity and Specificity, BRCA1 Protein genetics, BRCA2 Protein genetics, Genetic Predisposition to Disease genetics, Genetic Variation, MutS Homolog 2 Protein genetics, Neoplasms genetics
- Abstract
Based on current consensus guidelines and standard practice, many genetic variants detected in clinical testing are classified as disease causing based on their predicted impact on the normal expression or function of the gene in the absence of additional data. However, our laboratory has identified a subset of such variants in hereditary cancer genes for which compelling contradictory evidence emerged after the initial evaluation following the first observation of the variant. Three representative examples of variants in BRCA1, BRCA2 and MSH2 that are predicted to disrupt splicing, prematurely truncate the protein, or remove the start codon were evaluated for pathogenicity by analyzing clinical data with multiple classification algorithms. Available clinical data for all three variants contradicts the expected pathogenic classification. These variants illustrate potential pitfalls associated with standard approaches to variant classification as well as the challenges associated with monitoring data, updating classifications, and reporting potentially contradictory interpretations to the clinicians responsible for translating test outcomes to appropriate clinical action. It is important to address these challenges now as the model for clinical testing moves toward the use of large multi-gene panels and whole exome/genome analysis, which will dramatically increase the number of genetic variants identified., (© 2015 The Authors. Clinical Genetics published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)
- Published
- 2015
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31. Identification of a Variety of Mutations in Cancer Predisposition Genes in Patients With Suspected Lynch Syndrome.
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Yurgelun MB, Allen B, Kaldate RR, Bowles KR, Judkins T, Kaushik P, Roa BB, Wenstrup RJ, Hartman AR, and Syngal S
- Subjects
- Adult, Colorectal Neoplasms, Hereditary Nonpolyposis diagnosis, DNA Mutational Analysis, Female, Gene Expression Profiling, Gene Frequency, Genetic Predisposition to Disease, Heredity, High-Throughput Nucleotide Sequencing, Humans, Male, Middle Aged, Pedigree, Phenotype, Predictive Value of Tests, Risk Assessment, Risk Factors, Biomarkers, Tumor genetics, Colorectal Neoplasms, Hereditary Nonpolyposis genetics, Genetic Testing methods, Germ-Line Mutation
- Abstract
Background & Aims: Multigene panels are commercially available tools for hereditary cancer risk assessment that allow for next-generation sequencing of numerous genes in parallel. However, it is not clear if these panels offer advantages over traditional genetic testing. We investigated the number of cancer predisposition gene mutations identified by parallel sequencing in individuals with suspected Lynch syndrome., Methods: We performed germline analysis with a 25-gene, next-generation sequencing panel using DNA from 1260 individuals who underwent clinical genetic testing for Lynch syndrome from 2012 through 2013. All patients had a history of Lynch syndrome-associated cancer and/or polyps. We classified all identified germline alterations for pathogenicity and calculated the frequencies of pathogenic mutations and variants of uncertain clinical significance (VUS). We also analyzed data on patients' personal and family history of cancer, including fulfillment of clinical guidelines for genetic testing., Results: Of the 1260 patients, 1112 met National Comprehensive Cancer Network (NCCN) criteria for Lynch syndrome testing (88%; 95% confidence interval [CI], 86%-90%). Multigene panel testing identified 114 probands with Lynch syndrome mutations (9.0%; 95% CI, 7.6%-10.8%) and 71 with mutations in other cancer predisposition genes (5.6%; 95% CI, 4.4%-7.1%). Fifteen individuals had mutations in BRCA1 or BRCA2; 93% of these met the NCCN criteria for Lynch syndrome testing and 33% met NCCN criteria for BRCA1 and BRCA2 analysis (P = .0017). An additional 9 individuals carried mutations in other genes linked to high lifetime risks of cancer (5 had mutations in APC, 3 had bi-allelic mutations in MUTYH, and 1 had a mutation in STK11); all of these patients met NCCN criteria for Lynch syndrome testing. A total of 479 individuals had 1 or more VUS (38%; 95% CI, 35%-41%)., Conclusions: In individuals with suspected Lynch syndrome, multigene panel testing identified high-penetrance mutations in cancer predisposition genes, many of which were unexpected based on patients' histories. Parallel sequencing also detected a high number of potentially uninformative germline findings, including VUS., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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32. A comprehensive laboratory-based program for classification of variants of uncertain significance in hereditary cancer genes.
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Eggington JM, Bowles KR, Moyes K, Manley S, Esterling L, Sizemore S, Rosenthal E, Theisen A, Saam J, Arnell C, Pruss D, Bennett J, Burbidge LA, Roa B, and Wenstrup RJ
- Subjects
- Genes, BRCA1, Genes, BRCA2, Humans, Algorithms, Classification methods, Databases, Genetic, Genes, Neoplasm genetics, Genetic Variation
- Abstract
Genetic testing has the potential to guide the prevention and treatment of disease in a variety of settings, and recent technical advances have greatly increased our ability to acquire large amounts of genetic data. The interpretation of this data remains challenging, as the clinical significance of genetic variation detected in the laboratory is not always clear. Although regulatory agencies and professional societies provide some guidance regarding the classification, reporting, and long-term follow-up of variants, few protocols for the implementation of these guidelines have been described. Because the primary aim of clinical testing is to provide results to inform medical management, a variant classification program that offers timely, accurate, confident and cost-effective interpretation of variants should be an integral component of the laboratory process. Here we describe the components of our laboratory's current variant classification program (VCP), based on 20 years of experience and over one million samples tested, using the BRCA1/2 genes as a model. Our VCP has lowered the percentage of tests in which one or more BRCA1/2 variants of uncertain significance (VUSs) are detected to 2.1% in the absence of a pathogenic mutation, demonstrating how the coordinated application of resources toward classification and reclassification significantly impacts the clinical utility of testing., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)
- Published
- 2014
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33. Development and validation of a new algorithm for the reclassification of genetic variants identified in the BRCA1 and BRCA2 genes.
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Pruss D, Morris B, Hughes E, Eggington JM, Esterling L, Robinson BS, van Kan A, Fernandes PH, Roa BB, Gutin A, Wenstrup RJ, and Bowles KR
- Subjects
- Case-Control Studies, Female, Humans, Neoplasm Staging, Prognosis, Algorithms, BRCA1 Protein genetics, BRCA2 Protein genetics, Breast Neoplasms classification, Breast Neoplasms genetics, Genetic Predisposition to Disease, Genetic Testing, Genetic Variation genetics
- Abstract
BRCA1 and BRCA2 sequencing analysis detects variants of uncertain clinical significance in approximately 2 % of patients undergoing clinical diagnostic testing in our laboratory. The reclassification of these variants into either a pathogenic or benign clinical interpretation is critical for improved patient management. We developed a statistical variant reclassification tool based on the premise that probands with disease-causing mutations are expected to have more severe personal and family histories than those having benign variants. The algorithm was validated using simulated variants based on approximately 145,000 probands, as well as 286 BRCA1 and 303 BRCA2 true variants. Positive and negative predictive values of ≥99 % were obtained for each gene. Although the history weighting algorithm was not designed to detect alleles of lower penetrance, analysis of the hypomorphic mutations c.5096G>A (p.Arg1699Gln; BRCA1) and c.7878G>C (p.Trp2626Cys; BRCA2) indicated that the history weighting algorithm is able to identify some lower penetrance alleles. The history weighting algorithm is a powerful tool that accurately assigns actionable clinical classifications to variants of uncertain clinical significance. While being developed for reclassification of BRCA1 and BRCA2 variants, the history weighting algorithm is expected to be applicable to other cancer- and non-cancer-related genes.
- Published
- 2014
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34. Kinase signalling in Huntington's disease.
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Bowles KR and Jones L
- Subjects
- Animals, Disease Models, Animal, Humans, Apoptosis physiology, Huntington Disease metabolism, Huntington Disease pathology, MAP Kinase Signaling System physiology
- Abstract
Alterations in numerous signal transduction pathways and aberrant activity of specific kinases have been identified in multiple cell and mouse models of Huntington's disease (HD), as well as in human HD brain. The balance and integration of a network of kinase signalling pathways is paramount for the regulation of a wide range of cellular and physiological processes, such as proliferation, differentiation, inflammation, neuronal plasticity and apoptosis. Unbalanced activity within these pathways provides a potential mechanism for many of the pathological phenotypes associated with HD, such as transcriptional dysregulation, inflammation and ultimately neurodegeneration. The characterisation of aberrant kinase signalling regulation in HD has been inconsistent and may be a result of failure to consider integration between multiple signalling pathways, as well as alterations that may occur over time with both age and disease progression. Collating the information about the effect of mHTT on signalling pathways demonstrates that it has wide ranging effects on multiple pro- and anti-apoptotic kinases, resulting in the dysregulation of numerous complex interactions within a dynamic network.
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- 2014
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35. Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay.
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Biswas K, Das R, Eggington JM, Qiao H, North SL, Stauffer S, Burkett SS, Martin BK, Southon E, Sizemore SC, Pruss D, Bowles KR, Roa BB, Hunter N, Tessarollo L, Wenstrup RJ, Byrd RA, and Sharan SK
- Subjects
- Amino Acid Sequence, Animals, BRCA2 Protein chemistry, Cell Cycle Proteins, Cell Survival, Cells, Cultured, Chromosome Mapping, Conserved Sequence, DNA Breaks, Double-Stranded, DNA Repair, DNA-Binding Proteins, Embryonic Stem Cells drug effects, Embryonic Stem Cells physiology, Fanconi Anemia Complementation Group N Protein, Female, Genetic Association Studies, Humans, Likelihood Functions, Male, Mice, Mice, Transgenic, Mitomycin pharmacology, Models, Molecular, Mutagens pharmacology, Protein Binding, Protein Interaction Domains and Motifs genetics, Protein Structure, Quaternary, Structural Homology, Protein, BRCA2 Protein genetics, Breast Neoplasms genetics, Embryonic Stem Cells metabolism, Mutation, Nuclear Proteins metabolism, Tumor Suppressor Proteins metabolism
- Abstract
Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.
- Published
- 2012
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36. Gene expression and behaviour in mouse models of HD.
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Bowles KR, Brooks SP, Dunnett SB, and Jones L
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- Animals, Humans, Mice, Mice, Transgenic, Serotonin Plasma Membrane Transport Proteins genetics, Behavior, Animal physiology, Disease Models, Animal, Gene Expression Regulation, Huntington Disease genetics
- Abstract
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease, resulting in expansion of the CAG repeat in exon 1 of the HTT gene. The resulting mutant huntingtin protein has been implicated in the disruption of a variety of cellular functions, including transcription. Mouse models of HD have been central to the development of our understanding of gene expression changes in this disease, and are now beginning to elucidate the relationship between gene expression and behaviour. Here, we review current mouse models of HD and their characterisation in terms of gene expression. In addition, we look at how this can inform behaviours observed in mouse models of disease. The relationship between gene expression and behaviour in mouse models of HD is important, as this will further our knowledge of disease progression and its underlying molecular events, highlight new treatment targets, and potentially provide new biomarkers for therapeutic trials., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2012
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37. Viral endomyocardial infection is an independent predictor and potentially treatable risk factor for graft loss and coronary vasculopathy in pediatric cardiac transplant recipients.
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Moulik M, Breinholt JP, Dreyer WJ, Kearney DL, Price JF, Clunie SK, Moffett BS, Kim JJ, Rossano JW, Jefferies JL, Bowles KR, O'Brian Smith E, Bowles NE, Denfield SW, and Towbin JA
- Subjects
- Adolescent, Child, Child, Preschool, Coronary Disease virology, Female, Graft Survival, Humans, Infant, Infant, Newborn, Male, Polymerase Chain Reaction, Prevalence, Risk Factors, Genome, Viral, Graft Rejection virology, Heart Transplantation, Myocarditis epidemiology, Myocarditis virology
- Abstract
Objectives: This study sought to evaluate the outcome and prevalence of viral endomyocardial infection after cardiac transplantation., Background: Viral myocardial infection causes heart failure, but its role after cardiac transplantation is unclear. We hypothesized that viral infection of the cardiac allograft reduces graft survival., Methods: Between June 1999 and November 2004, 94 pediatric cardiac transplant patients were screened for the presence of viral genome in serial endomyocardial biopsies (EMBs) using polymerase chain reaction (PCR) assays. Graft loss, advanced transplant coronary artery disease (TCAD), and acute rejection (AR) were compared in the PCR-positive (n = 37) and PCR-negative (n = 57) groups, using time-dependent Kaplan-Meier and Cox regression analyses. From November 2002 to November 2004, intravenous immunoglobulin therapy (IVIG) was administered to patients with PCR-positive EMBs. The outcomes of the IVIG-treated, PCR-positive patients (n = 20) were compared with IVIG-untreated, PCR-positive patients (n = 17)., Results: Viral genomes were detected in EMBs from 37 (39%) patients; parvovirus B19, adenovirus, and Epstein-Barr virus (EBV) were the most common. The PCR-positive group (n = 37, 25% graft loss at 2.4 years) had decreased graft survival (p < 0.001) compared with the PCR-negative group (n = 57, 25% graft loss at 8.7 years) and developed advanced TCAD prematurely (p = 0.001). The number of AR episodes was similar in both groups. On multivariate analysis, presence of viral genome was an independent risk factor for graft loss (relative risk: 4.2, p = 0.015). The time to advanced TCAD after becoming PCR-positive was longer in the IVIG-treated patients (p = 0.03) with a trend toward improved graft survival (p = 0.06)., Conclusions: Viral endomyocardial infection is an independent predictor of graft loss in pediatric cardiac transplant recipients. This effect appears to be mediated through premature development of advanced TCAD. IVIG therapy in this subgroup may improve survival and merits further investigation., (Copyright (c) 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)
- Published
- 2010
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38. Viral epidemiologic shift in inflammatory heart disease: the increasing involvement of parvovirus B19 in the myocardium of pediatric cardiac transplant patients.
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Breinholt JP, Moulik M, Dreyer WJ, Denfield SW, Kim JJ, Jefferies JL, Rossano JW, Gates CM, Clunie SK, Bowles KR, Kearney DL, Bowles NE, and Towbin JA
- Subjects
- Adenoviridae, Adenoviridae Infections complications, Adenoviridae Infections epidemiology, Adolescent, Biopsy, Child, Child, Preschool, Coronary Artery Disease epidemiology, Coxsackievirus Infections complications, Coxsackievirus Infections epidemiology, DNA, Viral blood, Enterovirus, Heart Diseases blood, Humans, Incidence, Infant, Infant, Newborn, Kaplan-Meier Estimate, Myocardium pathology, Retrospective Studies, Risk Factors, Heart virology, Heart Diseases virology, Heart Transplantation trends, Parvoviridae Infections complications, Parvoviridae Infections epidemiology, Parvovirus B19, Human isolation & purification
- Abstract
Background: Detection of viral genome in rejecting cardiac transplant patients has been reported, with coxsackievirus and adenovirus causing premature graft failure. Recently, parvovirus B19 (PVB19) genome in myocardial samples has been increasingly reported, but its role in cardiac pathology and effect on transplant graft survival are unknown. The objectives of this study were to determine if changes in the viruses identified in the myocardium represent an epidemiologic shift in viral myocardial disease and whether PVB19 adversely affects transplant graft survival., Methods: From September 2002 to December 2005, nested polymerase chain reaction was used to evaluate endomyocardial biopsy specimens for 99 children (aged 3 weeks-18 years) with heart transplants for the presence of viral genome. Cellular rejection was assessed by histology of specimens. Transplant coronary artery disease (TCAD) was diagnosed by coronary angiography or histopathology., Results: Specimens from 700 biopsies were evaluated from 99 patients; 121 specimens had viral genome, with 100 (82.6%) positive for PVB19, 24 for Epstein-Barr virus (EBV; 7 positive for PVB19 and EBV), 3 for CMV, and 1 for adenovirus. Presence of PVB19 genome did not correlate with rejection score, nor did a higher viral copy number. Early development of advanced TCAD (p < 0.001) occurred in 20 children with persistent PVB19 infection (> 6 months)., Conclusions: PVB19 is currently the predominant virus detected in heart transplant surveillance biopsy specimens, possibly representing an epidemiologic shift. Cellular rejection does not correlate with the presence or quantity of PVB19 genome in the myocardium, but children with chronic PVB19 infection have increased risk for earlier TCAD, supporting the hypothesis that PVB19 negatively affects graft survival.
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- 2010
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39. Genetic analysis in patients with left ventricular noncompaction and evidence for genetic heterogeneity.
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Xing Y, Ichida F, Matsuoka T, Isobe T, Ikemoto Y, Higaki T, Tsuji T, Haneda N, Kuwabara A, Chen R, Futatani T, Tsubata S, Watanabe S, Watanabe K, Hirono K, Uese K, Miyawaki T, Bowles KR, Bowles NE, and Towbin JA
- Subjects
- Acyltransferases, Adaptor Proteins, Signal Transducing genetics, Aged, Asian People genetics, Dystrophin-Associated Proteins genetics, Female, Humans, Infant, Newborn, LIM Domain Proteins, Male, Neuropeptides genetics, Pedigree, Point Mutation, Proteins genetics, Sequence Analysis, DNA, Transcription Factors genetics, Cardiomyopathies genetics, Genetic Heterogeneity, Hypertrophy, Left Ventricular genetics
- Abstract
Left ventricular noncompaction (LVNC) is a cardiomyopathy characterized by numerous excessively trabeculations and deep intertrabecular recesses. This study was performed to investigate Japanese LVNC patients for disease-causing mutations in a series of selected candidate genes. DNA was isolated from the peripheral blood of 79 cases including 20 familial cases and 59 sporadic cases. DNA samples were screened for mutations in the genes encoding G4.5 (TAZ), alpha-dystrobrevin (DTNA), alpha1-syntrophin (SNTA1), FK506 Binding protein 1A (FKBP1A or FKPB12: FKBP1A), and LIM Domain Binding protein 3 (Cypher/ZASP: LDB3), using single-strand conformational polymorphism analysis and DNA sequencing. DNA variants were identified in 6 of the 79 cases, including four familial cases and two sporadic cases. A splice acceptor mutation of intron 8 in TAZ (IVS8-1G>C) was identified in one family with isolated LVNC, resulting in deletion of exon 9 from mRNA. In a sporadic case of isolated LVNC and Barth syndrome (BTHS), a 158insC in exon 2 of TAZ resulting in a frame-shift mutation was identified. A 1876G>A substitution changing an aspartic acid to asparagine (D626N) was identified in LDB3 in four members of two families with LVNC. A 163G>A polymorphism was identified in LDB3, which changed a valine to isoleucine (V55I) in one patient with isolated LVNC. In addition, in a family with nonisolated LVNC, a 362C>T mutation was identified in DTNA. LVNC, like other forms of inherited cardiomyopathy, is a genetically heterogeneous disease, associated with variable clinical symptoms and can be inherited as an autosomal or X-linked recessive disorder.
- Published
- 2006
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40. Intrafamilial variability of noncompaction of the ventricular myocardium.
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Johnson MT, Zhang S, Gilkeson R, Ameduri R, Siwik E, Patel CR, Chebotarev O, Kenton AB, Bowles KR, Towbin JA, Robin NH, Brozovich F, and Hoit BD
- Subjects
- Adolescent, Cardiomyopathies complications, Cardiomyopathies embryology, Electrocardiography, Female, Heart Ventricles, Humans, Infant, Infant, Newborn, Male, Middle Aged, Pedigree, Phenotype, Ultrasonography, Prenatal, Ventricular Dysfunction etiology, Ventricular Dysfunction physiopathology, Cardiomyopathies diagnosis, Cardiomyopathies genetics, Echocardiography, Genetic Variation
- Abstract
Background: Noncompaction of the ventricular myocardium (NVM) is a relatively uncommon form of cardiomyopathy characterized by a highly trabeculated myocardium. This report describes the clinical and genetic evaluation of a 3-generation kindred., Methods: Family members were initially evaluated by 2-dimensional echocardiography. Most family members with signs of NVM were further evaluated by magnetic resonance imaging. Genetic analyses included mutational screening of the taffazin (TAZ) and alpha-dystrobrevin (DTNA) genes., Results: Eight family members had signs of NVM. Considerable interindividual variation was noted in terms of spatial distribution and severity of affected regions and ventricular dysfunction. Depending on which of 2 previously proposed quantitative diagnostic criteria were used and where ventricular myocardial measurements were taken, between 4 and 7 of these individuals had findings that were considered diagnostic. Magnetic resonance imaging served as a useful adjunct for confirming or establishing diagnoses in all 8 individuals. No mutation was found in TAZ or DTNA., Conclusions: This kindred demonstrates the remarkably wide phenotypic spectrum that can be seen in familial cases of NVM, ranging from prenatal/neonatal lethality to a complete lack of symptoms. The fact that all 8 affected individuals either have shown improvement in ventricular function or symptoms during childhood or have been asymptomatic indicates that NVM can have a relatively benign course. The degree and nature of cardiac involvement are also quite varied, and there is a weak correlation with ventricular function and symptoms. Evaluation of families with NVM requires careful assessment that uses a combination of imaging techniques and diagnostic criteria.
- Published
- 2006
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41. Viral genomic detection and outcome in myocarditis.
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Bowles NE, Bowles KR, and Towbin JA
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- Animals, Endocardial Fibroelastosis etiology, Humans, Myocarditis diagnosis, Myocarditis therapy, Prognosis, Virus Diseases genetics, Genome, Viral, Myocarditis virology, Virus Diseases diagnosis
- Published
- 2005
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42. Genetics of inherited cardiomyopathies.
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Bowles KR and Bowles NE
- Subjects
- Cardiomyopathies diagnosis, Cardiomyopathies epidemiology, Genetic Testing, Humans, Mutation, Cardiomyopathies genetics
- Abstract
Cardiomyopathies are the most common disorders resulting in heart failure, with dilated cardiomyopathy being responsible for the majority of cases. Other forms of cardiomyopathy, especially hypertrophic forms, are also important causes of heart failure. The mortality rate due to cardiomyopathy in the USA is over 10,000 deaths per year, and the costs associated with heart failure are approximately 200 million US dollars per year in the USA alone. Over the past few years, breakthroughs have occurred in understanding the basic mechanisms of these disorders, potentially enabling clinicians to devise improved diagnostic strategies and therapies. As at least 30 to 40% of cases are inherited, it is now imperative that the genetic basis for these disorders is clearly recognized by caregivers and scientists. However, it has also become clear that these diseases are genetically highly heterogeneous, with multiple genes identified for each of the major forms of cardiomyopathy, and most patients having private mutations. These data suggest that the genetic diagnosis of most patients with cardiomyopathy will be impractical with current technologies. However, there are a few exceptions, such as patients with X-linked cardiomyopathies, with or without the concomitant abnormalities of cyclic neutropenia and 3-methylglutaconic aciduria, or patients with cardiomyopathy associated with conduction disease: these appear to be associated with mutations in a small subset of genes, and can be investigated by certified diagnostic laboratories. This review will summarize current knowledge of the genetics of inherited cardiomyopathies and how findings from research laboratories may be translated into the diagnostic laboratory.
- Published
- 2004
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43. Isolated left ventricular noncompaction is rarely caused by mutations in G4.5, alpha-dystrobrevin and FK Binding Protein-12.
- Author
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Kenton AB, Sanchez X, Coveler KJ, Makar KA, Jimenez S, Ichida F, Murphy RT, Elliott PM, McKenna W, Bowles NE, Towbin JA, and Bowles KR
- Subjects
- Acyltransferases, Base Sequence, Humans, RNA Splice Sites genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Dystrophin-Associated Proteins genetics, Hypertrophy, Left Ventricular genetics, Mutation genetics, Proteins genetics, Tacrolimus Binding Protein 1A genetics, Transcription Factors genetics
- Abstract
Isolated left ventricular noncompaction (LVNC) is a form of cardiomyopathy that most commonly presents in infancy with a hypertrophic and dilated left ventricle characterized by deep trabeculations and intertrabecular recesses. Our goal was to determine the frequency of mutations in G4.5, alpha-dystrobrevin, and FK Binding protein-12 in isolated LVNC patients. No mutations were identified in 47 of the 48 patients studied, while a splice site acceptor site mutation of intron 10 of G4.5 was identified in one patient, resulting in the deletion of exon 10 from the mRNA.
- Published
- 2004
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44. Mutations in Cypher/ZASP in patients with dilated cardiomyopathy and left ventricular non-compaction.
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Vatta M, Mohapatra B, Jimenez S, Sanchez X, Faulkner G, Perles Z, Sinagra G, Lin JH, Vu TM, Zhou Q, Bowles KR, Di Lenarda A, Schimmenti L, Fox M, Chrisco MA, Murphy RT, McKenna W, Elliott P, Bowles NE, Chen J, Valle G, and Towbin JA
- Subjects
- Adaptor Proteins, Signal Transducing, Blotting, Northern, Blotting, Western, Cardiomyopathy, Dilated diagnosis, Chromatography, High Pressure Liquid, Echocardiography, Humans, Immunohistochemistry, LIM Domain Proteins, Mutagenesis, Transfection, Cardiomyopathy, Dilated genetics, Carrier Proteins genetics, Heart Ventricles pathology, Homeodomain Proteins genetics, Muscle Proteins genetics, Mutation, Ventricular Dysfunction, Left genetics
- Abstract
Objectives: We evaluated the role of Cypher/ZASP in the pathogenesis of dilated cardiomyopathy (DCM) with or without isolated non-compaction of the left ventricular myocardium (INLVM)., Background: Dilated cardiomyopathy, characterized by left ventricular dilation and systolic dysfunction with signs of heart failure, is genetically transmitted in 30% to 40% of cases. Genetic heterogeneity has been identified with mutations in multiple cytoskeletal and sarcomeric genes causing the phenotype. In addition, INLVM with a hypertrophic dilated left ventricle, ventricular dysfunction, and deep trabeculations, is also inherited, and the genes identified to date differ from those causing DCM. Cypher/ZASP is a newly identified gene encoding a protein that is a component of the Z-line in both skeletal and cardiac muscle., Methods: Diagnosis of DCM was performed by echocardiogram, electrocardiogram, and physical examination. In addition, levels of the muscular isoform of creatine kinase were measured to evaluate for skeletal muscle involvement. Cypher/ZASP was screened by denaturing high performance liquid chromatography (DHPLC) and direct deoxyribonucleic acid sequencing., Results: We identified and screened 100 probands with left ventricular dysfunction. Five mutations in six probands (6% of cases) were identified in patients with familial or sporadic DCM or INLVM. In vitro studies showed cytoskeleton disarray in cells transfected with mutated Cypher/ZASP., Conclusions: These data suggest that mutated Cypher/ZASP can cause DCM and INLVM and identify a mechanistic basis.
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- 2003
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45. Mutations in the muscle LIM protein and alpha-actinin-2 genes in dilated cardiomyopathy and endocardial fibroelastosis.
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Mohapatra B, Jimenez S, Lin JH, Bowles KR, Coveler KJ, Marx JG, Chrisco MA, Murphy RT, Lurie PR, Schwartz RJ, Elliott PM, Vatta M, McKenna W, Towbin JA, and Bowles NE
- Subjects
- Actinin metabolism, Actins metabolism, Animals, Base Sequence, Cell Nucleus genetics, Cell Nucleus metabolism, Cells, Cultured, Dystrophin metabolism, Humans, LIM Domain Proteins, Mice, Molecular Sequence Data, Muscle Proteins metabolism, Mutation, Myoblasts cytology, Myocardium metabolism, Protein Binding, Sarcomeres genetics, Sarcomeres metabolism, Actinin genetics, Cardiomyopathy, Dilated genetics, Endocardial Fibroelastosis genetics, Muscle Proteins genetics, Myoblasts metabolism, Myocardium pathology
- Abstract
Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two genes have been identified for the X-linked forms (dystrophin and tafazzin), while mutations in multiple genes cause autosomal dominant DCM. Muscle LIM protein (MLP) is a member of the cysteine-rich protein (CRP) family and has been implicated in both myogenesis and sarcomere assembly. In the latter role, it binds zyxin and alpha-actinin, both of which are involved in actin organization. An MLP-deficient mouse has been described; these mice develop dilated cardiomyopathy and heart failure. Based upon these data, and the recent descriptions of mutations in MLP in patients with DCM or hypertrophic cardiomyopathy, we screened patients for mutations in the MLP and alpha-actinin-2 genes. We identified a patient with DCM and EFE, having a mutation in MLP with the residue lysine 69 substituted by arginine (K69R). This is within a highly conserved region adjacent to the first LIM domain involved in alpha-actinin binding. Analysis in cell culture systems demonstrated that the mutation abolishes the interaction between MLP and alpha-actinin-2 and the cellular localization of MLP was altered. In another individual with DCM, a W4R mutation was identified. However, this mutation did not segregate with disease in this family. In another patient with DCM, a Q9R mutation was identified in alpha-actinin-2. This mutation also disrupted the interaction with MLP and appeared to inhibit alpha-actinin function in cultured cells, in respect to the nuclear localization of actinin and the initiation of cellular differentiation.
- Published
- 2003
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46. Mutation analysis of the G4.5 gene in patients with isolated left ventricular noncompaction.
- Author
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Chen R, Tsuji T, Ichida F, Bowles KR, Yu X, Watanabe S, Hirono K, Tsubata S, Hamamichi Y, Ohta J, Imai Y, Bowles NE, Miyawaki T, and Towbin JA
- Subjects
- Acyltransferases, Cardiomyopathies genetics, DNA Mutational Analysis, Female, Genetic Diseases, X-Linked, Genotype, Humans, Hypertrophy, Left Ventricular physiopathology, Male, Pedigree, Phenotype, Polymorphism, Single-Stranded Conformational, RNA Processing, Post-Transcriptional genetics, Hypertrophy, Left Ventricular genetics, Proteins genetics, Transcription Factors
- Abstract
Mutations in the gene G4.5, originally associated with Barth syndrome, have been reported to result in a wide spectrum of severe infantile X-linked cardiomyopathies. The purpose of this study was to investigate patients with isolated left ventricular noncompaction (LVNC) for disease-causing mutations in G4.5. In 27 patients including 10 families with isolated LVNC, mutation analysis of G4.5 was performed using single-strand DNA conformation polymorphism (SSCP) analysis and DNA sequencing. A novel splice acceptor site mutation of intron 8 of G4.5 was identified in a family with severe infantile X-linked LVNC without the usual findings of Barth syndrome. This mutation results in deletion of exon 9 from the mRNA, and is predicted to significantly disrupt the protein product. Genotype-phenotype correlation of G4.5 mutations in all 38 cases reported in the literature to date revealed that there was no correlation between location or type of mutation and either cardiac phenotype or disease severity. We suggest that males presenting with cardiomyopathy, particularly during infancy, even in the absence of the typical signs of Barth syndrome, should be evaluated for mutations in G4.5.
- Published
- 2002
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47. Novel gene mutations in patients with left ventricular noncompaction or Barth syndrome.
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Ichida F, Tsubata S, Bowles KR, Haneda N, Uese K, Miyawaki T, Dreyer WJ, Messina J, Li H, Bowles NE, and Towbin JA
- Subjects
- Acyltransferases, Base Sequence, Cardiomyopathies pathology, Cardiomyopathy, Dilated pathology, DNA chemistry, DNA genetics, DNA Mutational Analysis, Family Health, Female, Humans, Hypertrophy, Left Ventricular pathology, Male, Mutation, Pedigree, Polymorphism, Single-Stranded Conformational, Syndrome, Cardiomyopathies genetics, Cardiomyopathy, Dilated genetics, Cytoskeletal Proteins genetics, Dystrophin-Associated Proteins, Hypertrophy, Left Ventricular genetics, Membrane Proteins genetics, Proteins genetics, Transcription Factors
- Abstract
Background: Mutations in the gene G4.5 result in a wide spectrum of severe infantile cardiomyopathic phenotypes, including isolated left ventricular noncompaction (LVNC), as well as Barth syndrome (BTHS) with dilated cardiomyopathy (DCM). The purpose of this study was to investigate patients with LVNC or BTHS for mutations in G4.5 or other novel genes., Methods and Results: DNA was isolated from 2 families and 3 individuals with isolated LVNC or LVNC with congenital heart disease (CHD), as well as 4 families with BTHS associated with LVNC or DCM, and screened for mutations by single-strand DNA conformation polymorphism analysis and DNA sequencing. In 1 family with LVNC and CHD, a C-->T mutation was identified at nucleotide 362 of alpha-dystrobrevin, changing a proline to leucine (P121L). Mutations in G4.5 were identified in 2 families with isolated LVNC: a missense mutation in exon 4 (C118R) in 1 and a splice donor mutation (IVS10+2T-->A) in intron 10 in the other. In a family with cardiomyopathies ranging from BTHS or fatal infantile cardiomyopathy to asymptomatic DCM, a splice acceptor mutation in exon 2 of G4.5 (398-2 A-->G) was identified, and a 1-bp deletion in exon 2 of G4.5, resulting in a stop codon after amino acid 41, was identified in a sporadic case of BTHS., Conclusions: These data demonstrate genetic heterogeneity in LVNC, with mutation of a novel gene, alpha-dystrobrevin, identified in LVNC associated with CHD. In addition, these results confirm that mutations in G4.5 result in a wide phenotypic spectrum of cardiomyopathies.
- Published
- 2001
- Full Text
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48. Mutations in the human delta-sarcoglycan gene in familial and sporadic dilated cardiomyopathy.
- Author
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Tsubata S, Bowles KR, Vatta M, Zintz C, Titus J, Muhonen L, Bowles NE, and Towbin JA
- Subjects
- Adolescent, Cardiomyopathy, Dilated etiology, Child, Child, Preschool, Cloning, Molecular, Female, Humans, Infant, Infant, Newborn, Male, Myocardium pathology, Pedigree, Phenotype, Polymorphism, Single-Stranded Conformational, Sarcoglycans, Sequence Analysis, DNA, Cardiomyopathy, Dilated genetics, Cytoskeletal Proteins genetics, Cytoskeleton pathology, Membrane Glycoproteins genetics, Mutation
- Abstract
Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two genes have been identified for the X-linked forms (dystrophin and tafazzin), whereas three other genes (actin, lamin A/C, and desmin) cause autosomal dominant DCM; seven other loci for autosomal dominant DCM have been mapped but the genes have not been identified. Hypothesizing that DCM is a disease of the cytoskeleton and sarcolemma, we have focused on candidate genes whose products are found in these structures. Here we report the screening of the human delta-sarcoglycan gene, a member of the dystrophin-associated protein complex, by single-stranded DNA conformation polymorphism analysis and by DNA sequencing in patients with DCM. Mutations affecting the secondary structure were identified in one family and two sporadic cases, whereas immunofluorescence analysis of myocardium from one of these patients demonstrated significant reduction in delta-sarcoglycan staining. No skeletal muscle disease occurred in any of these patients. These data suggest that delta-sarcoglycan is a disease-causing gene responsible for familial and idiopathic DCM and lend support to our "final common pathway" hypothesis that DCM is a cytoskeletalopathy.
- Published
- 2000
- Full Text
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49. Construction of a high-resolution physical map of the chromosome 10q22-q23 dilated cardiomyopathy locus and analysis of candidate genes.
- Author
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Bowles KR, Abraham SE, Brugada R, Zintz C, Comeaux J, Sorajja D, Tsubata S, Li H, Brandon L, Gibbs RA, Scherer SE, Bowles NE, and Towbin JA
- Subjects
- Chromosomes, Bacterial, DNA Mutational Analysis, Expressed Sequence Tags, Family Health, Female, Genetic Predisposition to Disease genetics, Genomic Library, Humans, Male, Pedigree, Phenotype, Sequence Analysis, DNA, Cardiomyopathy, Dilated genetics, Chromosomes, Human, Pair 10 genetics, Physical Chromosome Mapping
- Abstract
Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality and a leading cause of cardiac transplantation worldwide. Multiple loci and three genes encoding cardiac actin, desmin, and lamin A/C have been described for autosomal dominant DCM. Using recombination analysis, we have narrowed the 10q21-q23 locus to a region of approximately 4.1 cM. In addition, we have constructed a BAC contig, composed of 199 clones, which was used to develop a high-resolution physical map that contains the DCM critical region (approximately 3.9 Mb long). Seven genes, including ANX11, PPIF, DLG5, RPC155, RPS24, SFTPA1, and KCNMA1, have been mapped to the region of interest. RPC155, RPS24, SFTPA1, and KCNMA1 were excluded from further analysis based on their known functions and tissue-specific expression patterns. Mutational analysis of ANX11, DLG5, and PPIF revealed no disease-associated mutations. Multiple ESTs have also been mapped to the critical region., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
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50. The "final common pathway" hypothesis and inherited cardiovascular disease. The role of cytoskeletal proteins in dilated cardiomyopathy.
- Author
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Bowles NE, Bowles KR, and Towbin JA
- Subjects
- Cardiomyopathy, Hypertrophic genetics, DNA Mutational Analysis, Genetic Predisposition to Disease genetics, Genotype, Humans, Long QT Syndrome genetics, Cardiomyopathy, Dilated genetics, Cytoskeletal Proteins genetics
- Abstract
The genetic basis of a number of inherited cardiovascular diseases has been elucidated over the last few years, including the long QT syndromes, hypertrophic cardiomyopathy and dilated cardiomyopathy. While genetic heterogeneity has been demonstrated in most of these diseases, a pattern has emerged, specifically that genes encoding proteins with similar functions or involved in the same pathway are responsible for a particular disease or syndrome. Based on this observation we proposed the "final common pathway" hypothesis. In the case of the arrhythmogenic disorders, the long QT syndromes and Brugada syndrome, mutations have been described in a number of ion channel proteins, including cardiac potassium (KVLQT1, HERG and minK) and sodium (SCN5A) channels. Thus, using the "final common pathway" hypothesis we have proposed these diseases to be "ion channelopathies". Hypertrophic cardiomyopathy appears to be a disease of the sarcomere ("sarcomyopathy") since all the disease-causing mutations have been identified in the gene encoding many of the sarcomeric proteins, including beta-myosin heavy chain, alpha-tropomyosin, troponin I and troponin T, as well as in actin, close to the beta-myosin heavy chain binding site. The genes responsible for familial dilated cardiomyopathy have been less well characterized. For X-linked dilated cardiomyopathy, mutations in the dystrophin and G4.5 genes have been reported. In addition, mutations in actin (close to the dystrophin binding domain) and desmin, a component of the intermediate filaments, have been reported. However, the genes at a further 6 loci associated with autosomal dominant dilated cardiomyopathy (associated with conduction disease in 2 cases) remain unidentified. Due to the mutations in dystrophin, actin and desmin, we have proposed that dilated cardiomyopathy is a "cytoskeletalopathy", and we are currently investigating the involvement of these genes in patients.
- Published
- 2000
- Full Text
- View/download PDF
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