C9ORF72 hexanucleotide repeat exerts toxicity in a stable, inducible motor neuronal cell model, which is rescued by partial depletion of Pten.

Amyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease, characterised by progressive failure of the neuromuscular system. A (G4C2)n repeat expansion in C9ORF72 is the most common genetic cause of ALS and frontotemporal dementia (FTD). To date, the balance of evidence indicates that the (G4C2)n repeat causes toxicity and neurodegeneration via a gain-of-toxic function mechanism; either through direct RNA toxicity or through the production of toxic aggregating dipeptide repeat proteins. Here, we have generated a stable and isogenic motor neuronal NSC34 cell model with inducible expression of a (G4C2)102 repeat, to investigate the gain-of-toxic function mechanisms. The expression of the (G4C2)102 repeat produces RNA foci and also undergoes RAN translation. In addition, the expression of the (G4C2)102 repeat shows cellular toxicity. Through comparison of transcriptomic data from the cellular model with laser-captured spinal motor neurons from C9ORF72-ALS cases, we also demonstrate that the PI3K/Akt cell survival signalling pathway is dysregulated in both systems. Furthermore, partial knockdown of Pten rescues the toxicity observed in the NSC34 (G4C2)102 cellular gain-of-toxic function model of C9ORF72-ALS. Our data indicate that PTEN may provide a potential therapeutic target to ameliorate toxic effects of the (G4C2)n repeat

A genome-wide association study of myasthenia gravis

IMPORTANCE: Myasthenia gravis is a chronic, autoimmune, neuromuscular disease characterized by fluctuating weakness of voluntary muscle groups. Although genetic factors are known to play a role in this neuroimmunological condition, the genetic etiology underlying myasthenia gravis is not well understood. OBJECTIVE: To identify genetic variants that alter susceptibility to myasthenia gravis, we performed a genome-wide association study. DESIGN, SETTING, AND PARTICIPANTS: DNA was obtained from 1032 white individuals from North America diagnosed as having acetylcholine receptor antibody–positive myasthenia gravis and 1998 race/ethnicity-matched control individuals from January 2010 to January 2011. These samples were genotyped on Illumina OmniExpress single-nucleotide polymorphism arrays. An independent cohort of 423 Italian cases and 467 Italian control individuals were used for replication. MAIN OUTCOMES AND MEASURES: We calculated P values for association between 8114394 genotyped and imputed variants across the genome and risk for developing myasthenia gravis using logistic regression modeling. A threshold P value of 5.0 × 10(−8) was set for genome-wide significance after Bonferroni correction for multiple testing. RESULTS: In the over all case-control cohort, we identified association signals at CTLA4 (rs231770; P = 3.98 × 10(−8); odds ratio, 1.37; 95% CI, 1.25–1.49), HLA-DQA1 (rs9271871; P = 1.08 × 10(−8); odds ratio, 2.31; 95% CI, 2.02 – 2.60), and TNFRSF11A (rs4263037; P = 1.60 × 10(−9); odds ratio, 1.41; 95% CI, 1.29–1.53). These findings replicated for CTLA4 and HLA-DQA1 in an independent cohort of Italian cases and control individuals. Further analysis revealed distinct, but overlapping, disease-associated loci for early- and late-onset forms of myasthenia gravis. In the late-onset cases, we identified 2 association peaks: one was located in TNFRSF11A (rs4263037; P = 1.32 × 10(−12); odds ratio, 1.56; 95% CI, 1.44–1.68) and the other was detected in the major histocompatibility complex on chromosome 6p21 (HLA-DQA1; rs9271871; P = 7.02 × 10(−18); odds ratio, 4.27; 95% CI, 3.92–4.62). Association within the major histocompatibility complex region was also observed in early-onset cases (HLA-DQA1; rs601006; P = 2.52 × 10(−11); odds ratio, 4.0; 95% CI, 3.57–4.43), although the set of single-nucleotide polymorphisms was different from that implicated among late-onset cases. CONCLUSIONS AND RELEVANCE: Our genetic data provide insights into aberrant cellular mechanisms responsible for this prototypical autoimmune disorder. They also suggest that clinical trials of immunomodulatory drugs related to CTLA4 and that are already Food and Drug Administration approved as therapies for other autoimmune diseases could be considered for patients with refractory disease

A Genome-Wide Association Study of Myasthenia Gravis

Myasthenia gravis is a chronic, autoimmune, neuromuscular disease characterized by fluctuating weakness of voluntary muscle groups. Although genetic factors are known to play a role in this neuroimmunological condition, the genetic etiology underlying myasthenia gravis is not well understood

Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation.Objective: To identify the genetic variants associated with juvenile ALS.Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism.Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members.Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway.Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.</p

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

Algorithms for modeling gene regulation and determining cell type using single-cell molecular profiles

Thesis (Ph.D.)--University of Washington, 2019Single-cell genomic technologies are helping us answer key biological questions that have long remained elusive. How do a single cell and a single genome generate such complex multicellular organisms as humans? More specifically, how do these cells orchestrate specific transcriptional programs depending on their cell type? New technologies like single-cell RNA-seq and single-cell ATAC-seq allow us to examine the transcription and regulation of individual cells as they develop; however, these methods have important limitations. A primary limitation with all single-cell data is data sparsity, which must be overcome computationally to extract useful information from these experiments. In this dissertation, I present two algorithms designed to overcome the sparsity of single-cell data and allow biological discovery. I first introduce Cicero for single-cell chromatin accessibility data, which is both an algorithm that calculates co-accessibility scores to assign distal regulatory elements to genes, and a software system that adapts existing single-cell RNA-seq analysis techniques for use with single-cell chromatin accessibility data. In Chapter 2, I apply Cicero to an in vitro myoblast differentiation assay and find evidence for the use of ”chromatin hubs” during myogenesis. In Chapter 3, I apply Cicero to single-cell ATAC-seq data from mouse bone marrow and recapitulate known patterns of hematopoiesis and known cis-regulation of the b-globin locus. In Chapter 4, I introduce a second algorithm, Garnett, which uses single-cell expression data to train and apply automated cell type classifiers. The accuracy of this technology is demonstrated with data from various single-cell RNA-seq methods and tissue sources. In a final chapter, I reflect on the development of software for biological applications and future directions for this work

Searching for Grendel: origin and global spread of the C9ORF72 repeat expansion.

Recent advances are uncovering more and more of the genetic architecture underlying amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative condition that affects ~6,000 Americans annually. Chief among these was the discovery that a large repeat expansion in the C9ORF72 gene is responsible for an unprecedented portion of familial and sporadic ALS cases. Much has been published on how this expansion disrupts neuronal homeostasis and how gene-based therapy might be an effective treatment in the future. Nevertheless, it is instructive to look back at the origins of this important mutation. In this opinion piece, we attempt to answer three key questions concerning C9ORF72. First, how many times did the expansion occur throughout human history? Second, how old is the expansion? And finally and perhaps most importantly, how did the expansion spread throughout Europe? We speculate that the expansion occurred only once in the past, that this event took place in the Finnish population and that the Vikings and their descendants were responsible for disseminating this mutation throughout the rest of the continent

Identification and annotation of <i>X</i>. <i>tropicalis</i> F-box family of proteins.

<p>Full-length protein sequences of F-box proteins categorized into subgroups were used to construct maximum likelihood phylogenies; <b>A.</b> F-box proteins with WD40 repeats (Fbxw), <b>B.</b> F-box proteins with leucine rich repeats (Fbxl), and <b>C.</b> F-box proteins with other domains (Fbxo). A <i>Xenopus</i> F-box protein (red) is considered homologous if it clusters with mammalian proteins. Green shaded branch shows <i>Xenopus</i> specific Fbxw7-like genes. <i>Xenopus</i> Fbxl sequences in green lack an identifiable LRR domain but are found to be highly associated with Fbxl proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136929#pone.0136929.s001" target="_blank">S1 Fig</a>). Bootstrap values higher than 50 are shown. Arrowheads show <i>X</i>. <i>tropicalis</i> specific F-box proteins.</p

Spatial expression of F-box genes during <i>X</i>. <i>laevis</i> development using <i>in situ</i> hybridizations.

<p>Embryos positions: egg and blastula embryos, lateral view with animal side up; gastrula embryos, lateral view with dorsal up and vegetal to the right; neurula embryos dorso-lateral view with anterior to the right; tailbud embryos, lateral view with anterior to the right. Black arrowheads indicate neural tissue, blue—neural crest, green—pre or somitic mesoderm and red–heart.</p

Domain structures of the <i>Xenopus</i> F-box proteins.

<p>Domains identified by Pfam include F-box motif (F, green), WD40 repeat (WD, gray), leucine-rich repeat (L, orange), F-box-associated domain (FBA), between-ring domain (IBR), Kelch repeat, zf-CXXC zing finger domain (zf-C or zf), Cyclin N and Cyclin C domains, SMI1 KNR4, microtubule interacting and trafficking domain (MIT), zinc finger in N-recognin (Zf-UBR), SPla and the RYanodine Receptor (SPRY), TNF-receptor associated factor (TRAF), Elongin A (E), FIST C domain, Transglutaminase-like superfamily (Transglut), Hemimethylated DNA-binding protein YccV like (YccV), UvrD Helicase (H).</p