Construction of DNA/RNA Triplex Helices Based on GAA/TTC Trinucleotide Repeats

Zhang J, Fakharzadeh A, Pan F, Roland C, Sagui C.; Bio Protoc. 2021 Sep 20;11(18):e4155. doi: 10.21769/BioProtoc.4155. PMID: 34692905; PMCID: PMC8481028. 

Atypical DNA and RNA secondary structures play a crucial role in simple sequence repeat (SSR) diseases, which are associated with a class of neurological and neuromuscular disorders known as "anticipation diseases," where the age of disease onset decreases and the severity of the disease is increased as the intergenerational expansion of the SSR increases. While the mechanisms underlying these diseases are complex and remain elusive, there is a consensus that stable, non-B-DNA atypical secondary structures play an important - if not causative - role. These structures include single-stranded DNA loops and hairpins, G-quartets, Z-DNA, triplex nucleic acid structures, and others. While all of these structures are of interest, structures based on nucleic acid triplexes have recently garnered increased attention as they have been implicated in gene regulation, gene repair, and gene engineering. Our work here focuses on the construction of DNA triplexes and RNA/DNA hybrids formed from GAA/TTC trinucleotide repeats, which underlie Friedreich's ataxia. While there is some software, such as the Discovery Studio Visualizer, that can aid in the initial construction of DNA triple helices, the only option for the triple helix is constrained to be that of an antiparallel pyrimidine for the third strand. In this protocol, we illustrate how to build up more generalized DNA triplexes and DNA/RNA mixed hybrids. We make use of both the Discovery Studio Visualizer and the AMBER simulation package to construct the initial triplexes. Using the steps outlined here, one can - in principle - build up any triple nucleic acid helix with a desired sequence for large-scale molecular dynamics simulation studies.

Longitudinal investigation of brain activation during motor tasks in Friedreich ataxia: 24-month data from IMAGE-FRDA

Shishegar R, Harding IH, Selvadurai LP, Corben LA, Delatycki MB, Egan GF, Georgiou-Karistianis N.; Brain Struct Funct. 2021 Oct 23. doi: 10.1007/s00429-021-02413-6. Epub ahead of print. PMID: 34687355. 

We investigated the hypothesis that progressive functional changes in both the cerebellum and cerebrum are related to longitudinal changes in performance on complex motor tasks in individuals with FRDA. Twenty-two individuals with FRDA and 28 controls participated over 24 months. The longitudinal investigation included finger tapping tasks with different levels of complexity (i.e., visually cued, multi-finger; self-paced, single finger), performed in conjunction with fMRI acquisitions, to interrogate changes in the neurobiology of motor and attentional brain networks including the cerebellum and cerebrum. We demonstrated evidence for significant longitudinal decreased cerebral fMRI activity over time in individuals with FRDA, relative to controls, during an attentionally-demanding motor task (visually cued tapping of multiple fingers) in six cerebral regions: right and left superior frontal gyri, right superior temporal gyrus, right primary somatosensory area, right anterior cingulate cortex, and right medial frontal gyrus. Importantly, longitudinal decreased activity was associated with more severe disease status at baseline, higher GAA1 repeat length and earlier age of onset. These findings suggest a dynamic pattern of neuronal activity in motor, attention and executive control networks over time in individuals with FRDA, which is associated with increased disease severity at baseline, increased GAA1 repeat length and earlier age at onset.

Clinical and Molecular Features of First Mexican Friedreich's Ataxia Patients with Compound Heterozygous FXN Mutations

Boll MC, Gasca-Saldaña D, Mayén-Lobo YG, Dávila-Ortiz de Montellano DJ, Monroy-Jaramillo N.; Neurol India 2021;69:1363-7. DOI: 10.4103/0028-3886.329555

Eighteen patients had a homozygous FXN genotype; whereas five were CH patients with a slow progression and phenotypic variability, including a late-onset case with spastic paraparesis, and a Charcot-Marie-Tooth-like case.

Left atrial appendage thrombosis in a patient with Friedreich Ataxia–related cardiomyopathy, left ventricular systolic dysfunction, and atrial fibrillation

. Michele Russo, Annachiara Nuzzo, Matteo Foschi, Simona Boarin, Stefano Lorenzetti, Corrado Tomasi, Pietro Querzani, Andrea Rubboli. SAGE Open Medical Case Reports. January 2021. doi:10.1177/2050313X211056419 

We present the case of a 45-year-old man with a history of paroxysmal atrial fibrillation and a congestive heart failure, hypertension, age ⩾ 75 years, diabetes mellitus, stroke, vascular disease, age 65–74 years, and female sex (CHA2DS2-VASc) score of only 1 (because of reduced left ventricular ejection fraction) who presented with pneumonia and was also found to have atrial fibrillation with a rapid ventricular response. Despite already being on long-term therapy with a non-vitamin K-antagonist oral anticoagulant, a transesophageal echocardiogram showed a mobile floating thrombus in the left atrial appendage. In accordance with previous necropsy evidence of thrombosis and thromboembolism in Friedreich ataxia subjects who likely have had only non-sex-related CHA2DS2-VASc score ⩽1, this case suggests that the risk of thromboembolism in Friedreich ataxia subjects with atrial fibrillation may not be adequately predicted by the sole CHA2DS2-VASc score.

Tract-Specific Spinal Cord Diffusion Tensor Imaging in Friedreich's Ataxia

Ana Luisa C.C. Hernandez MD,Thiago J.R. Rezende PhD,Alberto R.M. Martinez MD, PhD,Mariana R. de Brito MD,Marcondes C. França Jr MD, PhD; Mov Disord. doi:10.1002/mds.28841 

DTI uncovered abnormalities in SC WM tracts, which correlated with clinical features in FRDA. CSA and CST FA in C2 correlated best with disease severity, whereas DC FA showed the largest effect size to differentiate patients and healthy controls. SC WM microstructure is a potential neuroimaging biomarker to be explored in the disease.

Targeting 3′ and 5′ untranslated regions with antisense oligonucleotides to stabilize frataxin mRNA and increase protein expression

Yanjie Li, Jixue Li, Jun Wang, David R Lynch, Xiulong Shen, David R. Corey, Darshan Parekh, Balkrishen Bhat, Caroline Woo, Jonathan J Cherry, Jill S Napierala, Marek Napierala, Nucleic Acids Research, 2021;, gkab954, doi:10.1093/nar/gkab954 

Friedreich’s ataxia (FRDA) is a severe multisystem disease caused by transcriptional repression induced by expanded GAA repeats located in intron 1 of the Frataxin (FXN) gene encoding frataxin. FRDA results from decreased levels of frataxin; thus, stabilization of the FXN mRNA already present in patient cells represents an attractive and unexplored therapeutic avenue. In this work, we pursued a novel approach based on oligonucleotide-mediated targeting of FXN mRNA ends to extend its half-life and availability as a template for translation. We demonstrated that oligonucleotides designed to bind to FXN 5′ or 3′ noncoding regions can increase FXN mRNA and protein levels. Simultaneous delivery of oligonucleotides targeting both ends increases efficacy of the treatment. The approach was confirmed in several FRDA fibroblast and induced pluripotent stem cell-derived neuronal progenitor lines. RNA sequencing and single-cell expression analyses confirmed oligonucleotide-mediated FXN mRNA upregulation. Mechanistically, a significant elongation of the FXN mRNA half-life without any changes in chromatin status at the FXN gene was observed upon treatment with end-targeting oligonucleotides, indicating that transcript stabilization is responsible for frataxin upregulation. These results identify a novel approach toward upregulation of steady-state mRNA levels via oligonucleotide-mediated end targeting that may be of significance to any condition resulting from transcription downregulation.

Iron Metabolism in Pancreatic Beta-Cell Function and Dysfunction

Marku, A.; Galli, A.; Marciani, P.; Dule, N.; Perego, C.; Castagna, M.; Cells 2021, 10, 2841. doi:10.3390/cells10112841 

Iron is an essential element involved in a variety of physiological functions. In the pancreatic beta-cells, being part of Fe-S cluster proteins, it is necessary for the correct insulin synthesis and processing. In the mitochondria, as a component of the respiratory chain, it allows the production of ATP and reactive oxygen species (ROS) that trigger beta-cell depolarization and potentiate the calcium-dependent insulin release. Iron cellular content must be finely tuned to ensure the normal supply but also to prevent overloading. Indeed, due to the high reactivity with oxygen and the formation of free radicals, iron excess may cause oxidative damage of cells that are extremely vulnerable to this condition because the normal elevated ROS production and the paucity in antioxidant enzyme activities. The aim of the present review is to provide insights into the mechanisms responsible for iron homeostasis in beta-cells, describing how alteration of these processes has been related to beta-cell damage and failure. Defects in iron-storing or -chaperoning proteins have been detected in diabetic conditions; therefore, the control of iron metabolism in these cells deserves further investigation as a promising target for the development of new disease treatments.

FDA refusal of Stealth Bio drug shows challenges of ultra-rare disease studies

https://medcitynews.com

The FDA refused to review Stealth BioTherapeutics’ Barth syndrome drug, telling the company results in a study of just eight patients are insufficient to support its submission. The impasse highlights the challenges of testing drugs for ultra-rare diseases. Barth is so rare that Stealth is unsure it can recruit patients to run a new study. Stealth said it is evaluating its next steps and expects to provide an update in early November. But Barth is not the only disease at stake. Elamipretide was developed as a way to treat mitochondrial problems in a range of disorders. Cardiomyopathy, the heart muscle weakness that develops in Barth, also develops in two other rare disorders, Duchenne muscular dystrophy and Friedreich’s ataxia. When Stealth reported its second quarter financial results in August, executives said they hope to begin testing elamipretide in Duchenne patients with cardiomyopathy in the first half of next year. An open label Phase 2a study is also expected in Friedreich’s ataxia. Stealth is also evaluating its drug in other diseases.

Mitochondrial Iron-Sulfur Cluster Biogenesis and Neurological Disorders

Arthavan Selvanathan, Bindu Parayil Sankaran; Mitochondrion, 2021, doi:10.1016/j.mito.2021.10.004.

This review focuses on the disorders of ISC biogenesis that have been described in the literature to-date. Key clinical, biochemical and neuroradiological features will be discussed, providing a reference point for clinicians diagnosing and managing these patients. Therapies are mostly supportive at this stage. However, the improved understanding of the pathophysiology of these conditions could pave the way for disease-modifying therapies in the near future.

Variables affecting pricing of orphan drugs: the Italian case

Claudio Jommi, Elisabetta Listorti, Federico Villa, Simone Ghislandi, Armando Genazzani, Agnese Cangini & Francesco Trotta; Orphanet J Rare Dis 16, 439 (2021). doi:10.1186/s13023-021-02022-w

Our results suggest that value arguments and sustainability (dimension of the target population and its impact on budget impact) issues are considered for orphan drugs pricing: the role played by sustainability is systematically supported by our results. A more transparent and reproducible price negotiation process for orphan drugs is needed in Italy.