Publications by authors named "Ian Blair"

339 Publications

Polygenic risk score analysis for amyotrophic lateral sclerosis leveraging cognitive performance, educational attainment and schizophrenia.

Eur J Hum Genet 2021 Apr 27. Epub 2021 Apr 27.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia.

Amyotrophic Lateral Sclerosis (ALS) is recognised to be a complex neurodegenerative disease involving both genetic and non-genetic risk factors. The underlying causes and risk factors for the majority of cases remain unknown; however, ever-larger genetic data studies and methodologies promise an enhanced understanding. Recent analyses using published summary statistics from the largest ALS genome-wide association study (GWAS) (20,806 ALS cases and 59,804 healthy controls) identified that schizophrenia (SCZ), cognitive performance (CP) and educational attainment (EA) related traits were genetically correlated with ALS. To provide additional evidence for these correlations, we built single and multi-trait genetic predictors using GWAS summary statistics for ALS and these traits, (SCZ, CP, EA) in an independent Australian cohort (846 ALS cases and 665 healthy controls). We compared methods for generating the risk predictors and found that the combination of traits improved the prediction (Nagelkerke-R) of the case-control logistic regression. The combination of ALS, SCZ, CP, and EA, using the SBayesR predictor method gave the highest prediction (Nagelkerke-R) of 0.027 (P value = 4.6 × 10), with the odds-ratio for estimated disease risk between the highest and lowest deciles of individuals being 3.15 (95% CI 1.96-5.05). These results support the genetic correlation between ALS, SCZ, CP and EA providing a better understanding of the complexity of ALS.
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http://dx.doi.org/10.1038/s41431-021-00885-yDOI Listing
April 2021

Meta-analysis of genome-wide DNA methylation identifies shared associations across neurodegenerative disorders.

Genome Biol 2021 Mar 26;22(1):90. Epub 2021 Mar 26.

Centre for Clinical Research, The University of Queensland, Brisbane, QLD, 4019, Australia.

Background: People with neurodegenerative disorders show diverse clinical syndromes, genetic heterogeneity, and distinct brain pathological changes, but studies report overlap between these features. DNA methylation (DNAm) provides a way to explore this overlap and heterogeneity as it is determined by the combined effects of genetic variation and the environment. In this study, we aim to identify shared blood DNAm differences between controls and people with Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease.

Results: We use a mixed-linear model method (MOMENT) that accounts for the effect of (un)known confounders, to test for the association of each DNAm site with each disorder. While only three probes are found to be genome-wide significant in each MOMENT association analysis of amyotrophic lateral sclerosis and Parkinson's disease (and none with Alzheimer's disease), a fixed-effects meta-analysis of the three disorders results in 12 genome-wide significant differentially methylated positions. Predicted immune cell-type proportions are disrupted across all neurodegenerative disorders. Protein inflammatory markers are correlated with profile sum-scores derived from disease-associated immune cell-type proportions in a healthy aging cohort. In contrast, they are not correlated with MOMENT DNAm-derived profile sum-scores, calculated using effect sizes of the 12 differentially methylated positions as weights.

Conclusions: We identify shared differentially methylated positions in whole blood between neurodegenerative disorders that point to shared pathogenic mechanisms. These shared differentially methylated positions may reflect causes or consequences of disease, but they are unlikely to reflect cell-type proportion differences.
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http://dx.doi.org/10.1186/s13059-021-02275-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004462PMC
March 2021

ALS/FTD-causing mutation in cyclin F causes the dysregulation of SFPQ.

Hum Mol Genet 2021 Mar 17. Epub 2021 Mar 17.

Department of Biomedical Sciences, Centre for Motor Neuron Disease Research, Faculty of Medicine and Health Sciences, Macquarie University, 2 Technology Place, North Ryde, NSW 2109, Australia.

Previously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Hallmark features of these diseases include the build-up of insoluble protein aggregates as well as the mislocalisation of proteins such as TDP-43. In recent years, the dysregulation of SFPQ has also emerged as a pathological hallmark of ALS/FTD. CCNF encodes for the protein cyclin F, a substrate recognition component of an E3-ubiquitin ligase. We have previously shown that ALS/FTD-linked mutations in CCNF cause disruptions to overall protein homeostasis that leads to a build-up of K48-linked ubiquitylated proteins as well as defects in autophagic machinery. To investigate further processes that may be affected by cyclin F, we used a protein-proximity ligation method, known as BioID, standard immunoprecipitations and mass spectrometry (MS) to identify novel interaction partners of cyclin F and infer further process that may be affected by the ALS/FTD-causing mutation. Results demonstrate that cyclin F closely associates with proteins involved with RNA metabolism as well as a number of RNA binding proteins previously linked to ALS/FTD, including splicing factor proline and glutamine rich (SFPQ). Notably, the overexpression of cyclin F(S621G) led to the aggregation and altered subcellular distribution of SFPQ in HEK293 cells, whilst leading to altered degradation in primary neurons. Overall, our data links ALS/FTD-causing mutations in CCNF to converging pathological features of ALS/FTD and provides a link between defective protein degradation systems and the pathological accumulation of a protein involved in RNA processing and metabolism.
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http://dx.doi.org/10.1093/hmg/ddab073DOI Listing
March 2021

Genetic analysis of GLT8D1 and ARPP21 in Australian familial and sporadic amyotrophic lateral sclerosis.

Neurobiol Aging 2021 Jan 16. Epub 2021 Jan 16.

Macquarie University Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, New South Wales, Australia.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the progressive degeneration of motor neurons. Recently, genetic variants in GLT8D1 and ARPP21 were associated with ALS in a cohort of European descent. A synergistic relationship was proposed between ALS associated variants in GLT8D1 and ARPP21. We aimed to determine the prevalence of genetic variation in GLT8D1 and ARPP21 in an Australian cohort of familial (n = 81) and sporadic ALS (n = 618) cases using whole-exome and whole-genome sequencing data. No novel mutations were identified in either gene, nor was there significant enrichment of protein-altering sequence variation among ALS cases. GLT8D1 and ARPP21 mutations are not a common cause of ALS in Australian familial and sporadic cohorts.
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http://dx.doi.org/10.1016/j.neurobiolaging.2021.01.005DOI Listing
January 2021

Effects of systemic inflammation on relapse in early breast cancer.

NPJ Breast Cancer 2021 Jan 22;7(1). Epub 2021 Jan 22.

Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA.

Chronic inflammation has been a proposed mechanism of resistance to aromatase inhibitors in breast cancer. Stratifying by HER2 status, a matched case-control study from the Wellness After Breast Cancer-II cohort was performed to assess whether or not elevated serum inflammatory biomarkers (C-Reactive protein [CRP], interleukin-6 [IL-6], and serum amyloid A [SAA]) and/or the presence of a high-risk IL-6 promoter genotype were associated with recurrence of hormone receptor positive (HR+) early breast cancer. Estrogen levels were also measured and correlated with biomarkers and disease outcomes. CRP and SAA were significantly associated with an increased risk of recurrence in the HR+/HER2- group, but not the HR+/HER2+ group. Mean serum estrogen levels were non-significantly elevated in patients who relapsed vs. non-relapsed patients. Surprisingly, high-risk IL-6 promoter polymorphisms were strongly associated with HER2+ breast cancer relapse, which has potential therapeutic implications, as elevated intracellular IL-6 has been associated with trastuzumab resistance in pre-clinical models.
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http://dx.doi.org/10.1038/s41523-020-00212-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822844PMC
January 2021

Using biochemistry and biophysics to extinguish androgen receptor signaling in prostate cancer.

J Biol Chem 2021 Jan 9;296:100240. Epub 2021 Jan 9.

Department Systems Pharmacology & Translational Therapeutics, Perelman School of Medicine University of Pennsylvania, Philadelphia, Pennsylvania, USA. Electronic address:

Castration resistant prostate cancer (CRPC) continues to be androgen receptor (AR) driven. Inhibition of AR signaling in CRPC could be advanced using state-of-the-art biophysical and biochemical techniques. Structural characterization of AR and its complexes by cryo-electron microscopy would advance the development of N-terminal domain (NTD) and ligand-binding domain (LBD) antagonists. The structural basis of AR function is unlikely to be determined by any single structure due to the intrinsic disorder of its NTD, which not only interacts with coregulators but likely accounts for the constitutive activity of AR-splice variants (SV), which lack the LBD and emerge in CRPC. Using different AR constructs lacking the LBD, their effects on protein folding, DNA binding, and transcriptional activity could reveal how interdomain coupling explains the activity of AR-SVs. The AR also interacts with coregulators that promote chromatin looping. Elucidating the mechanisms involved can identify vulnerabilities to treat CRPC, which do not involve targeting the AR. Phosphorylation of the AR coactivator MED-1 by CDK7 is one mechanism that can be blocked by the use of CDK7 inhibitors. CRPC gains resistance to AR signaling inhibitors (ARSI). Drug resistance may involve AR-SVs, but their role requires their reliable quantification by SILAC-mass spectrometry during disease progression. ARSI drug resistance also occurs by intratumoral androgen biosynthesis catalyzed by AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase), which is unique in that its acts as a coactivator of AR. Novel bifunctional inhibitors that competitively inhibit AKR1C3 and block its coactivator function could be developed using reverse-micelle NMR and fragment-based drug discovery.
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http://dx.doi.org/10.1074/jbc.REV120.012411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949100PMC
January 2021

Lactate Limits T Cell Proliferation via the NAD(H) Redox State.

Cell Rep 2020 Dec;33(11):108500

Division of Nephrology and Department of Pediatrics, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Immune cell function is influenced by metabolic conditions. Low-glucose, high-lactate environments, such as the placenta, gastrointestinal tract, and the tumor microenvironment, are immunosuppressive, especially for glycolysis-dependent effector T cells. We report that nicotinamide adenine dinucleotide (NAD), which is reduced to NADH by lactate dehydrogenase in lactate-rich conditions, is a key point of metabolic control in T cells. Reduced NADH is not available for NAD-dependent enzymatic reactions involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate dehydrogenase (PGDH). We show that increased lactate leads to a block at GAPDH and PGDH, leading to the depletion of post-GAPDH glycolytic intermediates, as well as the 3-phosphoglycerate derivative serine that is known to be important for T cell proliferation. Supplementing serine rescues the ability of T cells to proliferate in the presence of lactate-induced reductive stress. Directly targeting the redox state may be a useful approach for developing novel immunotherapies in cancer and therapeutic immunosuppression.
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http://dx.doi.org/10.1016/j.celrep.2020.108500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830708PMC
December 2020

Ubiquitin Homeostasis Is Disrupted in TDP-43 and FUS Cell Models of ALS.

iScience 2020 Nov 20;23(11):101700. Epub 2020 Oct 20.

Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia.

A major feature of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitin (Ub) into intracellular inclusions. This sequestration of Ub may reduce the availability of free Ub, disrupting Ub homeostasis and ultimately compromising cellular function and survival. We previously reported significant disturbance of Ub homeostasis in neuronal-like cells expressing mutant SOD1. Here, we show that Ub homeostasis is also perturbed in neuronal-like cells expressing either TDP-43 or FUS. The expression of mutant TDP-43 and mutant FUS led to UPS dysfunction, which was associated with a redistribution of Ub and depletion of the free Ub pool. Redistribution of Ub is also a feature of sporadic ALS, with an increase in Ub signal associated with inclusions and no compensatory increase in Ub expression. Together, these findings suggest that alterations to Ub homeostasis caused by the misfolding and aggregation of ALS-associated proteins play an important role in the pathogenesis of ALS.
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http://dx.doi.org/10.1016/j.isci.2020.101700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644588PMC
November 2020

Upregulation of Antioxidant Capacity and Nucleotide Precursor Availability Suffices for Oncogenic Transformation.

Cell Metab 2021 Jan 6;33(1):94-109.e8. Epub 2020 Nov 6.

Department of Cancer Biology and Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The emergence of cancer from diverse normal tissues has long been rationalized to represent a common set of fundamental processes. However, these processes are not fully defined. Here, we show that forced expression of glucose-6-phosphate dehydrogenase (G6PD) affords immortalized mouse and human cells anchorage-independent growth in vitro and tumorigenicity in animals. Mechanistically, G6PD augments the NADPH pool by stimulating NAD kinase-mediated NADP biosynthesis in addition to converting NADP to NADPH, bolstering antioxidant defense. G6PD also increases nucleotide precursor levels through the production of ribose and NADPH, promoting cell proliferation. Supplementation of antioxidants or nucleosides suffices to convert immortalized mouse and human cells into a tumorigenic state, and supplementation of both is required when their overlapping metabolic consequences are minimized. These results suggest that normal cells have a limited capacity for redox balance and nucleotide synthesis, and overcoming this limit might represent a key aspect of oncogenic transformation.
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http://dx.doi.org/10.1016/j.cmet.2020.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846267PMC
January 2021

Genome-wide Meta-analysis Finds the ACSL5-ZDHHC6 Locus Is Associated with ALS and Links Weight Loss to the Disease Genetics.

Cell Rep 2020 10;33(4):108323

Centre for Clinical Research, The University of Queensland, Brisbane QLD, Australia; Department of Neurology, Royal Brisbane and Women's Hospital, Brisbane QLD, Australia; School of Biomedical Sciences, The University of Queensland, Brisbane QLD, Australia.

We meta-analyze amyotrophic lateral sclerosis (ALS) genome-wide association study (GWAS) data of European and Chinese populations (84,694 individuals). We find an additional significant association between rs58854276 spanning ACSL5-ZDHHC6 with ALS (p = 8.3 × 10), with replication in an independent Australian cohort (1,502 individuals; p = 0.037). Moreover, B4GALNT1, G2E3-SCFD1, and TRIP11-ATXN3 are identified using a gene-based analysis. ACSL5 has been associated with rapid weight loss, as has another ALS-associated gene, GPX3. Weight loss is frequent in ALS patients and is associated with shorter survival. We investigate the effect of the ACSL5 and GPX3 single-nucleotide polymorphisms (SNPs), using longitudinal body composition and weight data of 77 patients and 77 controls. In patients' fat-free mass, although not significant, we observe an effect in the expected direction (rs58854276: -2.1 ± 1.3 kg/A allele, p = 0.053; rs3828599: -1.0 ± 1.3 kg/A allele, p = 0.22). No effect was observed in controls. Our findings support the increasing interest in lipid metabolism in ALS and link the disease genetics to weight loss in patients.
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http://dx.doi.org/10.1016/j.celrep.2020.108323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610013PMC
October 2020

Extra-mitochondrial mouse frataxin and its implications for mouse models of Friedreich's ataxia.

Sci Rep 2020 09 25;10(1):15788. Epub 2020 Sep 25.

Penn Medicine/CHOP Center of Excellence in Friedreich's Ataxia, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Mature frataxin is essential for the assembly of iron-sulfur cluster proteins including a number of mitochondrial enzymes. Reduced levels of mature frataxin (81-20) in human subjects caused by the genetic disease Friedreich's ataxia results in decreased mitochondrial function, neurodegeneration, and cardiomyopathy. Numerous studies of mitochondrial dysfunction have been conducted using mouse models of frataxin deficiency. However, mouse frataxin that is reduced in these models, is assumed to be mature frataxin (78-207) by analogy with human mature frataxin (81-210). Using immunoaffinity purification coupled with liquid chromatography-high resolution tandem mass spectrometry, we have discovered that mature frataxin in mouse heart (77%), brain (86%), and liver (47%) is predominantly a 129-amino acid truncated mature frataxin (79-207) in which the N-terminal lysine residue has been lost. Mature mouse frataxin (78-207) only contributes 7-15% to the total frataxin protein present in mouse tissues. We have also found that truncated mature frataxin (79-207) is present primarily in the cytosol of mouse liver; whereas, frataxin (78-207) is primarily present in the mitochondria. These findings, which provide support for the role of extra-mitochondrial frataxin in the etiology of Friedreich's ataxia, also have important implications for studies of mitochondrial dysfunction conducted in mouse models of frataxin deficiency.
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http://dx.doi.org/10.1038/s41598-020-72884-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519113PMC
September 2020

Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes.

PLoS One 2020 23;15(9):e0239625. Epub 2020 Sep 23.

Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York, United States of America.

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239625PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510980PMC
November 2020

Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations.

Mol Neurodegener 2020 09 9;15(1):51. Epub 2020 Sep 9.

Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, 75 Talavera Road NSW, North Ryde, NSW, 2109, Australia.

Background: Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response.

Methods: We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or HO treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage.

Results: We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm.

Conclusions: This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.
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http://dx.doi.org/10.1186/s13024-020-00386-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7488163PMC
September 2020

A Simple Differentiation Protocol for Generation of Induced Pluripotent Stem Cell-Derived Basal Forebrain-Like Cholinergic Neurons for Alzheimer's Disease and Frontotemporal Dementia Disease Modeling.

Cells 2020 09 2;9(9). Epub 2020 Sep 2.

Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia.

The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer's disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.
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http://dx.doi.org/10.3390/cells9092018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564334PMC
September 2020

Identity by descent analysis identifies founder events and links familial and sporadic ALS cases.

NPJ Genom Med 2020 7;5:32. Epub 2020 Aug 7.

Macquarie University Centre for Motor Neuron Disease Research, Department of Biological Sciences, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW Australia.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterised by the loss of upper and lower motor neurons resulting in paralysis and eventual death. Approximately 10% of ALS cases have a family history of disease, while the remainder present as apparently sporadic cases. Heritability studies suggest a significant genetic component to sporadic ALS, and although most sporadic cases have an unknown genetic aetiology, some familial ALS mutations have also been found in sporadic cases. This suggests that some sporadic cases may be unrecognised familial cases with reduced disease penetrance in their ancestors. A powerful strategy to uncover a familial link is identity-by-descent (IBD) analysis, which detects genomic regions that have been inherited from a common ancestor. IBD analysis was performed on 83 Australian familial ALS cases from 25 families and three sporadic ALS cases, each of whom carried one of three mutations (p.I114T, p.V149G and p.E101G). We defined five unique 350-SNP haplotypes that carry these mutations in our cohort, indicative of five founder events. This included two founder haplotypes that carry p.I114T; linking familial and sporadic cases. We found that p.E101G arose independently in each family that carries this mutation and linked two families that carry p.V149G. The age of disease onset varied between cases that carried each p.I114T haplotype. Linking families with identical ALS mutations allows for larger sample sizes and increased statistical power to identify putative phenotypic modifiers.
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http://dx.doi.org/10.1038/s41525-020-00139-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414871PMC
August 2020

Neuronal intranuclear inclusion disease is genetically heterogeneous.

Ann Clin Transl Neurol 2020 09 10;7(9):1716-1725. Epub 2020 Aug 10.

Department of Pathology, Faculty of Medicine, University of Helsinki, Helsinki, Finland.

Neuronal intranuclear inclusion disease (NIID) is a clinically heterogeneous neurodegenerative condition characterized by pathological intranuclear eosinophilic inclusions. A CGG repeat expansion in NOTCH2NLC was recently identified to be associated with NIID in patients of Japanese descent. We screened pathologically confirmed European NIID, cases of neurodegenerative disease with intranuclear inclusions and applied in silico-based screening using whole-genome sequencing data from 20 536 participants in the 100 000 Genomes Project. We identified a single European case harbouring the pathogenic repeat expansion with a distinct haplotype structure. Thus, we propose new diagnostic criteria as European NIID represents a distinct disease entity from East Asian cases.
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http://dx.doi.org/10.1002/acn3.51151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480908PMC
September 2020

Chaperone-mediated autophagy regulates the pluripotency of embryonic stem cells.

Science 2020 07;369(6502):397-403

Department of Cancer Biology and Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Embryonic stem cells can propagate indefinitely in a pluripotent state, able to differentiate into all types of specialized cells when restored to the embryo. What sustains their pluripotency during propagation remains unclear. Here, we show that core pluripotency factors OCT4 and SOX2 suppress chaperone-mediated autophagy (CMA), a selective form of autophagy, until the initiation of differentiation. Low CMA activity promotes embryonic stem cell self-renewal, whereas its up-regulation enhances differentiation. CMA degrades isocitrate dehydrogenases IDH1 and IDH2 and reduces levels of intracellular α-ketoglutarate, an obligatory cofactor for various histone and DNA demethylases involved in pluripotency. These findings suggest that CMA mediates the effect of core pluripotency factors on metabolism, shaping the epigenetic landscape of stem cells and governing the balance between self-renewal and differentiation.
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http://dx.doi.org/10.1126/science.abb4467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939502PMC
July 2020

Impaired Redox and Protein Homeostasis as Risk Factors and Therapeutic Targets in Toxin-Induced Biliary Atresia.

Gastroenterology 2020 09 4;159(3):1068-1084.e2. Epub 2020 Jun 4.

Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

Background & Aims: Extrahepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA.

Methods: We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screening of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model, with subsequent validation.

Results: Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors and other cyclic guanosine monophosphate signaling activators worked synergistically with the glutathione precursor N-acetylcysteine in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. Phosphodiesterase-5 inhibitors enhanced proteasomal degradation and required intact HSP90 chaperone.

Conclusion: Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone and may account for the reported association between BA transplant-free survival and glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with N-acetylcysteine and cyclic guanosine monophosphate signaling enhancers warrants further investigation as therapy for BA.
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http://dx.doi.org/10.1053/j.gastro.2020.05.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856536PMC
September 2020

Changes in Aged Fibroblast Lipid Metabolism Induce Age-Dependent Melanoma Cell Resistance to Targeted Therapy via the Fatty Acid Transporter FATP2.

Cancer Discov 2020 Sep 4;10(9):1282-1295. Epub 2020 Jun 4.

The Wistar Institute, Philadelphia, Pennsylvania.

Older patients with melanoma (>50 years old) have poorer prognoses and response rates to targeted therapy compared with young patients (<50 years old), which can be driven, in part, by the aged microenvironment. Here, we show that aged dermal fibroblasts increase the secretion of neutral lipids, especially ceramides. When melanoma cells are exposed to the aged fibroblast lipid secretome, or cocultured with aged fibroblasts, they increase the uptake of lipids via the fatty acid transporter FATP2, which is upregulated in melanoma cells in the aged microenvironment and known to play roles in lipid synthesis and accumulation. We show that blocking FATP2 in melanoma cells in an aged microenvironment inhibits their accumulation of lipids and disrupts their mitochondrial metabolism. Inhibiting FATP2 overcomes age-related resistance to BRAF/MEK inhibition in animal models, ablates tumor relapse, and significantly extends survival time in older animals. SIGNIFICANCE: These data show that melanoma cells take up lipids from aged fibroblasts, via FATP2, and use them to resist targeted therapy. The response to targeted therapy is altered in aged individuals because of the influences of the aged microenvironment, and these data suggest FATP2 as a target to overcome resistance...
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http://dx.doi.org/10.1158/2159-8290.CD-20-0329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483379PMC
September 2020

Evidence for polygenic and oligogenic basis of Australian sporadic amyotrophic lateral sclerosis.

J Med Genet 2020 May 14. Epub 2020 May 14.

Macquarie University Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with phenotypic and genetic heterogeneity. Approximately 10% of cases are familial, while remaining cases are classified as sporadic. To date, >30 genes and several hundred genetic variants have been implicated in ALS.

Methods: Seven hundred and fifty-seven sporadic ALS cases were recruited from Australian neurology clinics. Detailed clinical data and whole genome sequencing (WGS) data were available from 567 and 616 cases, respectively, of which 426 cases had both datasets available. As part of a comprehensive genetic analysis, 853 genetic variants previously reported as ALS-linked mutations or disease-associated alleles were interrogated in sporadic ALS WGS data. Statistical analyses were performed to identify correlation between clinical variables, and between phenotype and the number of ALS-implicated variants carried by an individual. Relatedness between individuals carrying identical variants was assessed using identity-by-descent analysis.

Results: Forty-three ALS-implicated variants from 18 genes, including , , and were identified in Australian sporadic ALS cases. One-third of cases carried at least one variant and 6.82% carried two or more variants, implicating a potential oligogenic or polygenic basis of ALS. Relatedness was detected between two sporadic ALS cases carrying a p.I114T mutation, and among three cases carrying a p.K238E mutation. Oligogenic/polygenic sporadic ALS cases showed earlier age of onset than those with no reported variant.

Conclusion: We confirm phenotypic associations among ALS cases, and highlight the contribution of genetic variation to all forms of ALS.
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http://dx.doi.org/10.1136/jmedgenet-2020-106866DOI Listing
May 2020

FBP1 loss disrupts liver metabolism and promotes tumorigenesis through a hepatic stellate cell senescence secretome.

Nat Cell Biol 2020 06 4;22(6):728-739. Epub 2020 May 4.

Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The crosstalk between deregulated hepatocyte metabolism and cells within the tumour microenvironment, as well as the consequent effects on liver tumorigenesis, are not completely understood. We show here that hepatocyte-specific loss of the gluconeogenic enzyme fructose 1,6-bisphosphatase 1 (FBP1) disrupts liver metabolic homeostasis and promotes tumour progression. FBP1 is universally silenced in both human and murine liver tumours. Hepatocyte-specific Fbp1 deletion results in steatosis, concomitant with activation and senescence of hepatic stellate cells (HSCs), exhibiting a senescence-associated secretory phenotype. Depleting senescent HSCs by 'senolytic' treatment with dasatinib/quercetin or ABT-263 inhibits tumour progression. We further demonstrate that FBP1-deficient hepatocytes promote HSC activation by releasing HMGB1; blocking its release with the small molecule inflachromene limits FBP1-dependent HSC activation, the subsequent development of the senescence-associated secretory phenotype and tumour progression. Collectively, these findings provide genetic evidence for FBP1 as a metabolic tumour suppressor in liver cancer and establish a critical crosstalk between hepatocyte metabolism and HSC senescence that promotes tumour growth.
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http://dx.doi.org/10.1038/s41556-020-0511-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286794PMC
June 2020

CYLD is a causative gene for frontotemporal dementia - amyotrophic lateral sclerosis.

Brain 2020 03;143(3):783-799

UK Dementia Research Institute, Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Maurice Wohl Clinical Neuroscience Institute, King's College London, London SE5 9RX, UK.

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.
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http://dx.doi.org/10.1093/brain/awaa039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089666PMC
March 2020

Significant out-of-sample classification from methylation profile scoring for amyotrophic lateral sclerosis.

NPJ Genom Med 2020 27;5:10. Epub 2020 Feb 27.

4Centre for Motor Neuron Disease Research, Macquarie University, Sydney, NSW 2109 Australia.

We conducted DNA methylation association analyses using Illumina 450K data from whole blood for an Australian amyotrophic lateral sclerosis (ALS) case-control cohort (782 cases and 613 controls). Analyses used mixed linear models as implemented in the OSCA software. We found a significantly higher proportion of neutrophils in cases compared to controls which replicated in an independent cohort from the Netherlands (1159 cases and 637 controls). The OSCA MOMENT linear mixed model has been shown in simulations to best account for confounders. When combined in a methylation profile score, the 25 most-associated probes identified by MOMENT significantly classified case-control status in the Netherlands sample (area under the curve, AUC = 0.65, CI = [0.62-0.68],  = 8.3 × 10). The maximum AUC achieved was 0.69 (CI = [0.66-0.71],  = 4.3 × 10) when cell-type proportion was included in the predictor.
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http://dx.doi.org/10.1038/s41525-020-0118-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046630PMC
February 2020

The mRNA-based reprogramming of fibroblasts from a SOD1 familial amyotrophic lateral sclerosis patient to induced pluripotent stem cell line UOWi007.

Stem Cell Res 2020 01 16;42:101701. Epub 2020 Jan 16.

Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia; School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia. Electronic address:

Dermal fibroblasts were donated by a 43 year old male patient with clinically diagnosed familial amyotrophic lateral sclerosis (ALS), carrying the SOD1 mutation. The induced pluripotent stem cell (iPSC) line UOWi007-A was generated using repeated mRNA transfections for pluripotency transcription factors Oct4, Klf4, Sox2, c-Myc, Lin28 and Nanog. The iPSCs carried the SOD1 genotype and had a normal karyotype, expressed expected pluripotency markers and were capable of in vitro differentiation into endodermal, mesodermal and ectodermal lineages. This iPSC line may be useful for investigating familial ALS resulting from a SOD1 mutation.
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http://dx.doi.org/10.1016/j.scr.2020.101701DOI Listing
January 2020

Gut microbiota modulate dendritic cell antigen presentation and radiotherapy-induced antitumor immune response.

J Clin Invest 2020 01;130(1):466-479

Department of Radiation Oncology and.

Alterations in gut microbiota impact the pathophysiology of several diseases, including cancer. Radiotherapy (RT), an established curative and palliative cancer treatment, exerts potent immune modulatory effects, inducing tumor-associated antigen (TAA) cross-priming with antitumor CD8+ T cell elicitation and abscopal effects. We tested whether the gut microbiota modulates antitumor immune response following RT distal to the gut. Vancomycin, an antibiotic that acts mainly on gram-positive bacteria and is restricted to the gut, potentiated the RT-induced antitumor immune response and tumor growth inhibition. This synergy was dependent on TAA cross presentation to cytolytic CD8+ T cells and on IFN-γ. Notably, butyrate, a metabolite produced by the vancomycin-depleted gut bacteria, abrogated the vancomycin effect. In conclusion, depletion of vancomycin-sensitive bacteria enhances the antitumor activity of RT, which has important clinical ramifications.
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http://dx.doi.org/10.1172/JCI124332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934221PMC
January 2020

Amyotrophic lateral sclerosis-linked UBQLN2 mutants inhibit endoplasmic reticulum to Golgi transport, leading to Golgi fragmentation and ER stress.

Cell Mol Life Sci 2020 Oct 4;77(19):3859-3873. Epub 2019 Dec 4.

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, Australia.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases that are related genetically and pathologically. Mutations in the UBQLN2 gene, encoding the ubiquitin-like protein ubiquilin2, are associated with familial ALS/FTD, but the pathophysiological mechanisms remain unclear. Here, we demonstrate that ALS/FTD UBQLN2 mutants P497H and P506T inhibit protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in neuronal cells. In addition, we observed that Sec31-positive ER exit sites are clustered in UBQLN2 patient spinal cord tissues. Both the ER-Golgi intermediate (ERGIC) compartment and the Golgi become disorganised and fragmented. This activates ER stress and inhibits ER-associated degradation. Hence, this study highlights perturbations in secretory protein trafficking and ER homeostasis as pathogenic mechanisms associated with ALS/FTD-associated forms of UBQLN2.
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http://dx.doi.org/10.1007/s00018-019-03394-wDOI Listing
October 2020

Cervicovaginal fluid proteomic analysis to identify potential biomarkers for preterm birth.

Am J Obstet Gynecol 2020 05 20;222(5):493.e1-493.e13. Epub 2019 Nov 20.

Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, AL.

Background: Spontaneous preterm birth is a leading cause of neonatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could reduce the spontaneous preterm birth rate, but conventional methods such as obstetrical history and maternal cervical length screening identify only a minority of spontaneous preterm birth cases. Cervicovaginal fluid might prove to be a useful, readily available biological fluid for identifying spontaneous preterm birth biomarkers.

Objective: The objective of the study was to identify cervicovaginal fluid biomarkers of early spontaneous preterm birth in a high-risk cohort of pregnant women with a history of spontaneous preterm birth using targeted and shotgun proteomic analyses.

Study Design: A nested case control study (cases were spontaneous preterm birth <34 weeks in the current pregnancy; controls were spontaneous labor and delivery at 39-41 weeks) was performed using cervicovaginal fluid samples collected at 3 study visits (10 to 18 weeks, 19 to 23 weeks, and 28 to 31 weeks). All participants had a history of at least 1 prior spontaneous preterm birth. Targeted proteomic analysis was performed using a stable isotope-labeled proteome derived from endocervical and vaginal mucosal cells. This served as a standard to quantitate candidate protein levels in individual cervicovaginal fluid samples from the second and third study visits using liquid chromatography-multiple reaction monitoring mass spectrometry. The ratio of endogenous peptide area/stable isotope-labeled proteome-derived peptide area was used to measure levels of 42 peptides in 22 proteins. To maximize biomarker discovery in the cervicovaginal fluid samples, shotgun proteomic analysis also was performed utilizing liquid chromatography and ion trap mass spectrometry. A validation study was performed in second-trimester cervicovaginal fluid samples from an independent study group (12 spontaneous preterm birth cases, 19 term delivery controls) using enzyme-linked immunosorbent assay for 5 proteins expressed at higher levels in spontaneous preterm birth cases compared with controls in targeted or shotgun proteomic analyses.

Results: For targeted proteomics, cervicovaginal fluid samples from 33 cases and 32 controls at 19 to 23 weeks and 16 cases and 14 controls at 28 to 31 weeks from the same pregnancies were analyzed. When samples were compared between cases and controls, the relative abundance of 5 proteins was greater (P = .02-.05) in cases at both visits, while the relative abundance of 1 protein was lower (P = .03) in cases at both visits. For shotgun proteomics analyses, cervicovaginal fluid samples were pooled for 9 spontaneous preterm birth cases and 9 term delivery controls at each study visit. Shotgun proteomics yielded 28 proteins that were detected at levels >2 times higher and 1 protein that was detected at a level <0.5 times lower in spontaneous preterm birth cases compared with controls at all 3 study visits. Validation enzyme-linked immunosorbent assay for 5 proteins that were detected at higher levels in cervicovaginal fluid samples from spontaneous preterm birth cases compared with term delivery controls in proteomics analyses did not demonstrate statistically significant differences between spontaneous preterm birth cases and controls.

Conclusions: Potential biomarkers of spontaneous preterm birth were identified by targeted and shotgun proteomics analyses in cervicovaginal fluid samples from high-risk, asymptomatic women. Many of the proteins detected at higher levels in cervicovaginal fluid samples from spontaneous preterm birth cases are extracellular matrix proteins and/or regulate cell membrane physiology. These proteins have substantial biological interest, but validation enzyme-linked immunosorbent assay for 5 of these proteins did not yield clinically useful biomarkers for spontaneous preterm birth.
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http://dx.doi.org/10.1016/j.ajog.2019.11.1252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196033PMC
May 2020

CMS-01 Genetic testing for familial amyotrophic lateral sclerosis (ALS): insights and challenges.

Amyotroph Lateral Scler Frontotemporal Degener 2019 Nov;20(sup1):327-347

Department of Clinical Medicine.

Pathogenic variants in ALS genes are known to be present in up to 70% of familial and 10% of apparently sporadic ALS cases, and can be associated with risks for ALS only, or risks for other neurodegenerative diseases (eg. frontotemporal dementia). While there are no changes to medical management for patients confirmed as pathogenic variant carriers, genetic testing may be important for future drug trials. Confirmation of a pathogenic variant also provides relatives with the opportunity to consider predictive and/or reproductive genetic testing. Genetic counselling is an important aspect of testing decision-making as it enables individuals to make informed decisions about genetic testing while minimising adverse psychological, ethical and legal outcomes. Few studies have explored how individuals decide whether to pursue testing, nor the needs and experiences of familial ALS families. To identify factors that influence patient and family member decision-making about genetic testing for ALS genes, assess the impact of familial disease on the patient and their family, and identify information and support needs. In-depth, semi-structured interviews with individuals from Australian ALS families with known pathogenic gene variants explored experiences of familial ALS, and factors that influenced genetic testing decision-making. Interviews were analysed using an inductive approach. Thirty-four individuals from 24 families were interviewed and included patients ( = 4), spouses ( = 4), and asymptomatic at-risk relatives ( = 26). Life stage, experience of disease, costs, research opportunities, and attitudes to familial ALS and/or reproductive options influenced decision-making. Some patients and relatives experienced difficulty gaining accurate information from their health professionals about the costs and implications of genetic counselling or testing, resulting in a reluctance to proceed. This study provides new insight into the Australian experience of genetic testing and counselling for familial ALS. It highlights the need to work together with other health professionals to ensure the complexities of genetic testing decision-making, and referral pathways are better understood.
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http://dx.doi.org/10.1080/21678421.2019.1647002DOI Listing
November 2019

Theme 3 In vitro experimental models.

Amyotroph Lateral Scler Frontotemporal Degener 2019 Nov;20(sup1):135-159

Centre for MND Research, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, Australia.

Ongoing disease gene discoveries continue to drive our understanding of the molecular and cellular mechanisms underlying ALS. Causative genes from 60% of ALS families have been identified using modern genetic techniques, but the causal gene defect is yet to be identified in the remaining 40% of families. These remaining families often do not follow true Mendelian inheritance patterns and are challenging to solve using traditional genetic analysis alone. and studies have become critical in assessing and validating these ALS candidate genes. In this study, we aim to develop and validate the utility of an functional pipeline for the discovery and validation of novel ALS candidate genes. A panel of cell based-assays were applied to candidate genes to examine the presence/absence of known ALS pathologies in cell lines as well as human autopsy tissues. These include immunofluorescence, flow cytometry and western blotting to study toxicity, neuronal inclusion formation, interaction with TDP-43, aberrant protein degradation and accumulation in detergent-insoluble cellular fractions. Immunohistochemistry and immunofluorescence were also used to examine if candidates were present in neuronal inclusions from ALS patient spinal cord tissues. The pipeline was applied to five candidate genes from an ALS family that is negative for known ALS gene mutations. Two candidates were prioritized as top candidates based on their capacity to induce known ALS cellular pathologies. In transfected cells, the variants in these two genes caused a significantly higher toxicity than wild type, formed detergent insoluble inclusions and was able to co-aggregate with TDP-43 in neuronal cells. The variants have also led to protein degradation defects. One of the candidates also co-localised with TDP-43-positive neuronal inclusions in sporadic ALS patient post-mortem tissues, a signature pathology of ALS. We have demonstrated the utility of a functional prioritization pipeline and successfully prioritized two novel candidate ALS genes. These genes, and its associated pathways, will be further investigated through the development of animal models to establish if there is support for its role in ALS. New ALS genes offer fresh diagnostic and therapeutic targets and tools for the generation of novel animal models to better understand disease biology and offer preclinical testing of candidate treatments for ALS in the future.
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http://dx.doi.org/10.1080/21678421.2019.1646991DOI Listing
November 2019