An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS ...in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC‐derived motor neurons, decreased cell survival is correlated with dysfunction in Ca2+ homeostasis, reduced levels of the antiapoptotic protein Bcl‐2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC‐derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063–2078
This study presents a comprehensive characterization of cellular pathways that are contributing to cell death in iPSC‐derived motor and cortical neurons from ALS/FTD patients carrying pathogenic hexanucleotide repeats in the C9orf72 gene. The apoptotic pathway that we identified in the C9orf72 neurons involves ER calcium elevation and stress, followed by mitochondrial alterations, the release of cytochrome c from the mitochondria and cleavage of caspase‐3. Aggregates positive for p62 were detected in these neurons along with high frequency of stress granules, indicating altered proteostatis in the C9orf72 motor and cortical neurons.
Amyotrophic lateral sclerosis overlaps aetiologically and genetically with frontotemporal dementia and occurs in both familial and apparently sporadic forms. The most commonly implicated genes are
,
...,
and
. Penetrance of disease-causing variants in these genes is known to be incomplete, but has not been well studied at population level.
We sought to determine the population-level penetrance of pathogenic and likely pathogenic variants in genes commonly causing amyotrophic lateral sclerosis.
Published epidemiological data for amyotrophic lateral sclerosis and frontotemporal dementia were used to calculate expected frequencies of disease-causing variants per gene at population level. Variant data from gnomAD and ClinVar databases were used to ascertain observed numbers of disease-causing variants and to estimate population-level penetrance per gene. Data for
were obtained from the published literature.
Maximum population penetrance for either amyotrophic lateral sclerosis or frontotemporal dementia was found to be 33% for
(95% CI (20.9 to 53.2)), 54% for
(95% CI (32.7 to 88.6)), 38% for
(95% CI (21.1 to 69.8)) and 19% for
(95% CI (13.0 to 28.4)).
Population-level penetrance of amyotrophic lateral sclerosis disease genes is reduced. This finding has implications for the genetic testing and counselling of affected individuals and their unaffected relatives.
RNA splicing analysis in genomic medicine Wai, Htoo; Douglas, Andrew G.L.; Baralle, Diana
International journal of biochemistry & cell biology,
March 2019, 2019-03-00, 20190301, Volume:
108
Journal Article
Peer reviewed
Open access
High-throughput next-generation sequencing technologies have led to a rapid increase in the number of sequence variants identified in clinical practice via diagnostic genetic tests. Current ...bioinformatic analysis pipelines fail to take adequate account of the possible splicing effects of such variants, particularly where variants fall outwith canonical splice site sequences, and consequently the pathogenicity of such variants may often be missed. The regulation of splicing is highly complex and as a result, in silico prediction tools lack sufficient sensitivity and specificity for reliable use. Variants of all kinds can be linked to aberrant splicing in disease and the need for correct identification and diagnosis grows ever more crucial as novel splice-switching antisense oligonucleotide therapies start to enter clinical usage. RT-PCR provides a useful targeted assay of the splicing effects of identified variants, while minigene assays, massive parallel reporter assays and animal models can also be used for more detailed study of a particular splicing system, given enough time and resources. However, RNA-sequencing (RNA-seq) has the potential to be used as a rapid diagnostic tool in genomic medicine. By utilising data science approaches and machine learning, it may prove possible to finally understand and interpret the ‘splicing code’ and apply this knowledge in human disease diagnostics.
A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise ...molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.
Apnea (breath-holding) elicits co-activation of sympathetic and parasympathetic nervous systems, affecting cardiac control. In situations of autonomic co-activation (e.g., cold water immersion), ...cardiac arrhythmias are observed during apnea. Chronic endurance training reduces resting heart rate in part via elevation in parasympathetic tone, and has been identified as a risk factor for development of arrhythmias. However, few studies have investigated autonomic control of the heart in trained athletes during stress. Therefore, we determined whether heightened vagal tone resulting from endurance training promotes a higher incidence of arrhythmia during apnea. We assessed the heart rate, rhythm (ECG lead II), and cardiac inotropic (speckle-tracking echocardiography) response to apnea in 10 endurance trained and 7 untrained participants. Participants performed an apnea at rest and following sympathetic activation using post-exercise circulatory occlusion (PECO). All apneas were performed prior to control (CON) and following vagal block using glycopyrrolate (GLY). Trained participants had lower heart rates at rest (
= 0.03) and during apneas (
= 0.009) under CON. At rest, 3 trained participants exhibited instances of junctional rhythm and 4 trained participants developed ectopy during CON apneas, whereas 3 untrained participants developed ectopic beats only with concurrent sympathetic activation (PECO). Following GLY, no arrhythmias were noted in either group. Vagal block also revealed increased cardiac chronotropy (heart rate) and inotropy (strain rate) during apnea, demonstrating a greater sympathetic influence in the absence of parasympathetic drive. Our results highlight that endurance athletes may be more susceptible to ectopy via elevated vagal tone, whereas untrained participants may only develop ectopy through autonomic conflict.
Barriers remain in the hepatitis C virus (HCV) cascade of care (CoC), limiting the overall impact of direct acting antivirals. This study examines movement between the stages of the HCV CoC and ...identifies reasons why patients and specific patient populations fail to advance through care in a real world population. We performed a single-center, ambispective cohort study of patients receiving care in an outpatient infectious diseases clinic between October 2015 and September 2016. Patients were followed from treatment referral through sustained virologic response. Univariate and multivariate analyses were performed to identify factors related to completion of each step of the CoC. Of 187 patients meeting inclusion criteria, 120 (64%) completed an evaluation for HCV treatment, 119 (64%) were prescribed treatment, 114 (61%) were approved for treatment, 113 (60%) initiated treatment, 107 (57%) completed treatment, and 100 (53%) achieved a sustained virologic response. In univariate and multivariate analyses, patients with Medicaid insurance were less likely to complete an evaluation and were less likely to be approved for treatment. Treatment completion and SVR rates are much improved from historical CoC reports. However, linkage to care following referral continues to be a formidable challenge for the HCV CoC in the DAA era. Ongoing efforts should focus on linkage to care to capitalize on DAA treatment advances and improving access for patients with Medicaid insurance.
The structure of proline prevents it from adopting an optimal position for rapid protein synthesis. Poly-proline-tract (PPT) associated ribosomal stalling is resolved by highly conserved eIF5A, the ...only protein to contain the amino acid hypusine. We show that de novo heterozygous EIF5A variants cause a disorder characterized by variable combinations of developmental delay, microcephaly, micrognathia and dysmorphism. Yeast growth assays, polysome profiling, total/hypusinated eIF5A levels and PPT-reporters studies reveal that the variants impair eIF5A function, reduce eIF5A-ribosome interactions and impair the synthesis of PPT-containing proteins. Supplementation with 1 mM spermidine partially corrects the yeast growth defects, improves the polysome profiles and restores expression of PPT reporters. In zebrafish, knockdown eif5a partly recapitulates the human phenotype that can be rescued with 1 µM spermidine supplementation. In summary, we uncover the role of eIF5A in human development and disease, demonstrate the mechanistic complexity of EIF5A-related disorder and raise possibilities for its treatment.
Competition and competitiveness are roundly celebrated as public values and key indicators of a dynamic and forward-thinking society. But the headlong embrace of competitive market principles, ...increasingly prevalent in our neoliberal age, often obscures the enduring divisiveness of a society set up to produce winners and losers. In this inspired and thoughtfully argued book, Andrew J. Douglas turns to the later writings of W. E. B. Du Bois to reevaluate the very terms of the competitive society.
Situating Du Bois in relation to the Depression-era roots of contemporary neoliberal thinking, Douglas shows that into the 1930s Du Bois ratcheted up a race-conscious indictment of capitalism and liberal democracy and posed unsettling questions about how the compulsory pull of market relations breeds unequal outcomes and underwrites the perpetuation of racial animosities. Blending historical analysis with ethical and political theory, and casting new light on several aspects of Du Bois's thinking, this book makes a compelling case that Du Bois's sweeping disillusionment with Western liberalism is as timely now as ever.
This book charts the progress of integration in basketball, from the first black professional basketball player in 1902 to the modern game. These crucial steps in the history of basketball are placed ...within the larger context of American history, making this book an essential addition to the literature on sports and race in America.
Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA ...splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories.
Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software.
Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity.
Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics.
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