Characterization of C9orf72 haplotypes to evaluate the effects of normal and pathological variations on its expression and splicing

Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. All C9orf72-ALS/FTD patients share a common risk (R) haplotype. To study C9orf72 expression and splicing from the mutant R allele compared to the complementary normal allele in ALS/FTD patients, we initially created a detailed molecular map of the single nucleotide polymorphism (SNP) signature and the HR length of the various C9orf72 haplotypes in Caucasians. We leveraged this map to determine the allelic origin of transcripts per patient, and decipher the effects of pathological and normal HR lengths on C9orf72 expression and splicing. In C9orf72 ALS patients’ cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of a downstream initiated transcript variant and increased levels of transcripts initiated upstream of the HR. HR expanded R alleles correlated with high levels of unspliced intron 1 and activation of cryptic donor splice sites along intron 1. Retention of intron 1 was associated with sequential intron 2 retention. The SNP signature of C9orf72 haplotypes described here enables allele-specific analysis of transcriptional products and may pave the way to allele-specific therapeutic strategies.

Download Full-text

Carriership of two copies of C9orf72 hexanucleotide repeat intermediate-length alleles is a risk factor for ALS in the Finnish population

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01059-5 ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Karri Kaivola ◽

Samuli J. Salmi ◽

Lilja Jansson ◽

Jyrki Launes ◽

Laura Hokkanen ◽

...

Keyword(s):

Risk Factor ◽

Age Groups ◽

Corticobasal Degeneration ◽

Control Group ◽

Hexanucleotide Repeat ◽

Intron 1 ◽

Hexanucleotide Repeat Expansion ◽

Als Patients ◽

Lateral Sclerosis

Abstract The hexanucleotide repeat expansion in intron 1 of the C9orf72 gene causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. In addition to the effects of the pathogenic expansion, a role of intermediate-length alleles has been suggested in ALS, corticobasal degeneration and Parkinson’s disease. Due to the rarity of intermediate-length alleles with over 20 repeats and the geographical variability in their frequency, large studies that account for population stratification are needed to elucidate their effects. To this aim, we used repeat-primed PCR and confirmatory PCR assays to determine the C9orf72 repeat allele lengths in 705 ALS patients and 3958 controls from Finland. After exclusion of expansion carriers (25.5% of the ALS patients and 0.2% of the controls), we compared the frequency of intermediate-length allele carriers of 525 ALS cases and 3950 controls using several intermediate-length allele thresholds (7–45, 17–45, 21–45, 24–45 and 24–30). The carriership of an intermediate-length allele did not associate with ALS (Fisher’s test, all p ≥ 0.15) nor was there any association with survival (p ≥ 0.33), when we divided our control group into three age groups (18–65, 66–84 and 85–105 years). Carriership of two intermediate-length alleles was associated with ALS, when the longer allele was ≥ 17 repeats (p = 0.002, OR 5.32 95% CI 2.02–14.05) or ≥ 21 repeats (p = 0.00016, OR 15.21 95% CI 3.79–61.0). Our results show that intermediate-length alleles are a risk factor of ALS when present in both alleles, whereas carrying just one intermediate-length allele was not associated with ALS or survival.

Download Full-text

Involvement of cortico-efferent tracts in flail arm syndrome: a tract-of-interest-based DTI study

Journal of Neurology ◽

10.1007/s00415-021-10854-6 ◽

2021 ◽

Author(s):

Angela Rosenbohm ◽

Kelly Del Tredici ◽

Heiko Braak ◽

Hans-Jürgen Huppertz ◽

Albert C. Ludolph ◽

...

Keyword(s):

White Matter ◽

Diffusion Tensor ◽

Statistical Comparison ◽

Specific Analysis ◽

Cerebral Pathology ◽

Propagation Pattern ◽

Als Patients ◽

Lateral Sclerosis ◽

Diffusion Tensor Imaging Dti

Abstract Background Flail arm syndrome is a restricted phenotype of motor neuron disease that is characterized by progressive, predominantly proximal weakness and atrophy of the upper limbs. Objective The study was designed to investigate specific white matter alterations in diffusion tensor imaging (DTI) data from flail arm syndrome patients using a hypothesis-guided tract-of-interest-based approach to identify in vivo microstructural changes according to a neuropathologically defined amyotrophic lateral sclerosis (ALS)-related pathology of the cortico-efferent tracts. Methods DTI-based white matter mapping was performed both by an unbiased voxel-wise statistical comparison and by a hypothesis-guided tract-wise analysis of fractional anisotropy (FA) maps according to the neuropathological ALS-propagation pattern for 43 flail arm syndrome patients vs 43 ‘classical’ ALS patients vs 40 matched controls. Results The analysis of white matter integrity demonstrated regional FA reductions for the flail arm syndrome group predominantly along the CST. In the tract-specific analysis according to the proposed sequential cerebral pathology pattern of ALS, the flail arm syndrome patients showed significant alterations of the specific tract systems that were identical to ‘classical’ ALS if compared to controls. Conclusions The DTI study including the tract-of-interest-based analysis showed a microstructural involvement pattern in the brains of flail arm syndrome patients, supporting the hypothesis that flail arm syndrome is a phenotypical variant of ALS.

Download Full-text

Genetic Polymorphism of CYP1A2 in Ethiopians Affecting Induction and Expression: Characterization of Novel Haplotypes with Single-Nucleotide Polymorphisms in Intron 1

Molecular Pharmacology ◽

10.1124/mol.64.3.659 ◽

2003 ◽

Vol 64 (3) ◽

pp. 659-669 ◽

Cited By ~ 112

Author(s):

Eleni Aklillu ◽

Juan Antonio Carrillo ◽

Eyasu Makonnen ◽

Karin Hellman ◽

Marià Pitarque ◽

...

Keyword(s):

Genetic Polymorphism ◽

Single Nucleotide Polymorphisms ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Intron 1 ◽

Expression Characterization

Download Full-text

Genetics and Sex in the Pathogenesis of Amyotrophic Lateral Sclerosis (ALS): Is There a Link?

International Journal of Molecular Sciences ◽

10.3390/ijms21103647 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3647 ◽

Cited By ~ 1

Author(s):

Francesca Trojsi ◽

Giulia D’Alvano ◽

Simona Bonavita ◽

Gioacchino Tedeschi

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Disease Risk ◽

Genetic Mutation ◽

Chromosome 9 ◽

Patient Specific ◽

Reading Frame ◽

Hexanucleotide Repeat ◽

Sporadic Als ◽

Als Patients ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no known cure. Approximately 90% of ALS cases are sporadic, although multiple genetic risk factors have been recently revealed also in sporadic ALS (SALS). The pathological expansion of a hexanucleotide repeat in chromosome 9 open reading frame 72 (C9orf72) is the most common genetic mutation identified in familial ALS, detected also in 5–10% of SALS patients. C9orf72-related ALS phenotype appears to be dependent on several modifiers, including demographic factors. Sex has been reported as an independent factor influencing ALS development, with men found to be more susceptible than women. Exposure to both female and male sex hormones have been shown to influence disease risk or progression. Moreover, interplay between genetics and sex has been widely investigated in ALS preclinical models and in large populations of ALS patients carrying C9orf72 repeat expansion. In light of the current need for reclassifying ALS patients into pathologically homogenous subgroups potentially responsive to targeted personalized therapies, we aimed to review the recent literature on the role of genetics and sex as both independent and synergic factors, in the pathophysiology, clinical presentation, and prognosis of ALS. Sex-dependent outcomes may lead to optimizing clinical trials for developing patient-specific therapies for ALS.

Download Full-text

Targeting S100A4 with niclosamide attenuates inflammatory and profibrotic pathways in models of amyotrophic lateral sclerosis

Journal of Neuroinflammation ◽

10.1186/s12974-021-02184-1 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Martina Milani ◽

Eleonora Mammarella ◽

Simona Rossi ◽

Chiara Miele ◽

Serena Lattante ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Cell Types ◽

Specific Protein ◽

Beneficial Effects ◽

Hexanucleotide Repeat ◽

Hexanucleotide Repeat Expansion ◽

Als Patients ◽

Different Cell Types ◽

Lateral Sclerosis

Abstract Background An increasing number of studies evidences that amyotrophic lateral sclerosis (ALS) is characterized by extensive alterations in different cell types and in different regions besides the CNS. We previously reported the upregulation in ALS models of a gene called fibroblast-specific protein-1 or S100A4, recognized as a pro-inflammatory and profibrotic factor. Since inflammation and fibrosis are often mutual-sustaining events that contribute to establish a hostile environment for organ functions, the comprehension of the elements responsible for these interconnected pathways is crucial to disclose novel aspects involved in ALS pathology. Methods Here, we employed fibroblasts derived from ALS patients harboring the C9orf72 hexanucleotide repeat expansion and ALS patients with no mutations in known ALS-associated genes and we downregulated S100A4 using siRNA or the S100A4 transcriptional inhibitor niclosamide. Mice overexpressing human FUS were adopted to assess the effects of niclosamide in vivo on ALS pathology. Results We demonstrated that S100A4 underlies impaired autophagy and a profibrotic phenotype, which characterize ALS fibroblasts. Indeed, its inhibition reduces inflammatory, autophagic, and profibrotic pathways in ALS fibroblasts, and interferes with different markers known as pathogenic in the disease, such as mTOR, SQSTM1/p62, STAT3, α-SMA, and NF-κB. Importantly, niclosamide in vivo treatment of ALS-FUS mice reduces the expression of S100A4, α-SMA, and PDGFRβ in the spinal cord, as well as gliosis in central and peripheral nervous tissues, together with axonal impairment and displays beneficial effects on muscle atrophy, by promoting muscle regeneration and reducing fibrosis. Conclusion Our findings show that S100A4 has a role in ALS-related mechanisms, and that drugs such as niclosamide which are able to target inflammatory and fibrotic pathways could represent promising pharmacological tools for ALS.

Download Full-text

Niclosamide Targets Inflammatory and Profibrotic Pathways in Amyotrophic Lateral Sclerosis

10.21203/rs.3.rs-138253/v1 ◽

2021 ◽

Author(s):

Martina Milani ◽

Eleonora Mammarella ◽

Simona Rossi ◽

Serena Lattante ◽

Mario Sabatelli ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Cell Types ◽

Specific Protein ◽

Beneficial Effects ◽

Hexanucleotide Repeat ◽

Sporadic Als ◽

Als Patients ◽

Different Cell Types ◽

Lateral Sclerosis

Abstract BackgroundAn increasing number of studies evidence that amyotrophic lateral sclerosis (ALS) is characterized by extensive alterations in different cell types and in different regions besides the CNS. We previously reported the up-regulation in ALS models of a gene called fibroblast-specific protein (FSP)-1 or S100A4, generally recognized as a pro-inflammatory and profibrotic factor. Since inflammation and fibrosis are often mutual-sustaining events that contribute to establish a hostile environment for organ functioning, the comprehension of the elements responsible for these interconnected pathways is crucial to disclose novel aspects involved in ALS pathology.MethodsHere we employed fibroblasts derived from ALS patients harboring the C9orf72 hexanucleotide repeat expansion and sporadic ALS patients with no mutations in known ALS-associated genes and we downregulated S100A4 using siRNA or the S100A4 transcriptional inhibitor niclosamide. Mice overexpressing human FUS were adopted to assess the effects of niclosamide in vivo on ALS pathology.ResultsWe demonstrated that S100A4 underlies impaired autophagy and a profibrotic phenotype, which characterize ALS fibroblasts. Indeed, its inhibition reduces inflammatory, autophagic and profibrotic pathways in ALS fibroblasts, and to interfere with different markers known as pathogenic in the disease, such as mTOR, SQSTM1/p62, STAT3, α-SMA and NF-κB. Importantly, niclosamide in vivo treatment of ALS-FUS mice reduces the expression of S100A4, α-SMA and PDGFRβ in the spinal cord, as well as gliosis in central and peripheral nervous tissues, together with axonal impairment and displays beneficial effects on muscle atrophy, by promoting muscle regeneration and reducing fibrosis.ConclusionOur findings show that S100A4 has a role in ALS-related mechanisms, and that drugs such as niclosamide that are able to target inflammatory and fibrotic pathways could represent promising pharmacological tools for ALS.

Download Full-text

Characterization of the human TARDBP gene promoter

Scientific Reports ◽

10.1038/s41598-021-89973-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marco Baralle ◽

Maurizio Romano

Keyword(s):

Gene Promoter ◽

Transcriptional Level ◽

Developmentally Regulated ◽

Cis Acting ◽

Transcript Stability ◽

Intron 1 ◽

Main Component ◽

Als Patients

AbstractThe expression of TDP-43, the main component of neuronal intracellular inclusions across a broad spectrum of ALS and FTD disorders, is developmentally regulated and studies in vivo have shown that TDP-43 overexpression can be toxic, even before observation of pathological aggregates. Starting from these observations, the regulation of its expression at transcriptional level might represent a further key element for the pathogenesis of neurodegenerative diseases. Therefore, we have characterized the human TARDBP promoter, in order to study the transcriptional mechanisms of expression. Mapping of cis-acting elements by luciferase assays in different cell outlined that the activity of the promoter seems to be higher in SH-SY5Y, Neuro2A, and HeLa than in HEK293. In addition, we tested effects of two SNPs found in the promoter region of ALS patients and observed no significant effect on transcription levels in all tested cell lines. Lastly, while TDP-43 overexpression did not affect significantly the activity of its promoter (suggesting that TDP-43 does not influence its own transcription), the presence of the 5′UTR sequence and of intron-1 splicing seem to impact positively on TDP-43 expression without affecting transcript stability. In conclusion, we have identified the region spanning nucleotides 451–230 upstream from the transcription start site as the minimal region with a significant transcription activity. These results lay an important foundation for exploring the regulation of the TARDBP gene transcription by exogenous and endogenous stimuli and the implication of transcriptional mechanisms in the pathogenesis of TDP-43 proteinopathies.

Download Full-text

Analysis of HorseMyostatinGene and Identification of Single Nucleotide Polymorphisms in Breeds of Different Morphological Types

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/542945 ◽

2010 ◽

Vol 2010 ◽

pp. 1-11 ◽

Cited By ~ 19

Author(s):

Stefania Dall'Olio ◽

Luca Fontanesi ◽

Leonardo Nanni Costa ◽

Marco Tassinari ◽

Laura Minieri ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Tata Box ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Analysis Of Molecular Variance ◽

Molecular Variance ◽

Intron 1 ◽

Significant Difference ◽

Intron 2 ◽

Negative Modulator

Myostatin (MSTN) is a negative modulator of muscle mass. We characterized the horse (Equus caballus)MSTNgene and identified and analysed single nucleotide polymorphisms (SNPs) in breeds of different morphological types. Sequencing of coding, untranslated, intronic, and regulatory regions ofMSTNgene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2. The SNPs of the promoter (GQ183900:g.26TC and GQ183900:g.156TC, the latter located within a conserved TATA-box like motif) were screened in 396 horses from 16 breeds. The g.26C and the g.156C alleles presented higher frequency in heavy (brachymorphic type) than in light breeds (dolichomorphic type such as Italian Trotter breed). The significant difference of allele frequencies for the SNPs at the promoter and analysis of molecular variance (AMOVA) on haplotypes indicates that these polymorphisms could be associated with variability of morphology traits in horse breeds.

Download Full-text

A Resource for the Allele-Specific Analysis of DNA Methylation at Multiple Genomically Imprinted Loci in Mice

10.1101/163832 ◽

2017 ◽

Author(s):

Jadiel A. Wasson ◽

Onur Birol ◽

David J. Katz

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Mouse Strains ◽

Nucleotide Polymorphisms ◽

Hybrid Strain ◽

Single Nucleotide ◽

Specific Analysis ◽

Allele Specific ◽

Parent Of Origin ◽

Strain Background

AbstractGenomically imprinted loci are expressed mono-allelically dependent upon the parent of origin. Their regulation not only illuminates how chromatin regulates gene expression but also how chromatin can be reprogrammed every generation. Because of their distinct parent of origin regulation, analysis of imprinted loci can be difficult. Single nucleotide polymorphisms (SNPs) are required to accurately assess these elements allele-specifically. However, publicly available SNP databases lack robust verification, making analysis of imprinting difficult. In addition, the allele-specific imprinting assays that have been developed employ different mouse strains, making it difficult to systemically analyze these loci. Here, we have generated a resource that will allow the allele-specific analysis of many significant imprinted loci in a single hybrid strain of Mus musculus. This resource includes verification of SNPs present within ten of the most widely used imprinting control regions and allele-specific DNA methylation assays for each gene in a C57BL/6J and CAST/EiJ hybrid strain background.

Download Full-text