Detection of mosaic and population-level structural variants with Sniffles2.

Calling structural variations (SVs) is technically challenging, but using long reads remains the most accurate way to identify complex genomic alterations. Here we present Sniffles2, which improves over current methods by implementing a repeat aware clustering coupled with a fast consensus sequence and coverage-adaptive filtering. Sniffles2 is 11.8 times faster and 29% more accurate than state-of-the-art SV callers across different coverages (5-50×), sequencing technologies (ONT and HiFi) and SV types. Furthermore, Sniffles2 solves the problem of family-level to population-level SV calling to produce fully genotyped VCF files. Across 11 probands, we accurately identified causative SVs around MECP2, including highly complex alleles with three overlapping SVs. Sniffles2 also enables the detection of mosaic SVs in bulk long-read data. As a result, we identified multiple mosaic SVs in brain tissue from a patient with multiple system atrophy. The identified SV showed a remarkable diversity within the cingulate cortex, impacting both genes involved in neuron function and repetitive elements.

Single-cell somatic copy number variants in brain using different amplification methods and reference genomes.

The presence of somatic mutations, including copy number variants (CNVs), in the brain is well recognized. Comprehensive study requires single-cell whole genome amplification, with several methods available, prior to sequencing. We compared PicoPLEX with two recent adaptations of multiple displacement amplification (MDA): primary template-directed amplification (PTA) and droplet MDA, across 93 human brain cortical nuclei. We demonstrated different properties for each, with PTA providing the broadest amplification, PicoPLEX the most even, and distinct chimeric profiles. Furthermore, we performed CNV calling on two brains with multiple system atrophy and one control brain using different reference genomes. We found that 38% of brain cells have at least one Mb-scale CNV, with some supported by bulk sequencing or single-cells from other brain regions. Our study highlights the importance of selecting whole genome amplification method and reference genome for CNV calling, while supporting the existence of somatic CNVs in healthy and diseased human brain.

A new FRDA mouse model [Fxn null:YG8s(GAA) > 800] with more than 800 GAA repeats.

Introduction: Friedreich's ataxia (FRDA) is an inherited recessive neurodegenerative disorder caused by a homozygous guanine-adenine-adenine (GAA) repeat expansion within intron 1 of the FXN gene, which encodes the essential mitochondrial protein frataxin. There is still no effective therapy for FRDA, therefore the development of optimal cell and animal models of the disease is one of the priorities for preclinical therapeutic testing. Methods: We obtained the latest FRDA humanized mouse model that was generated on the basis of our previous YG8sR, by Jackson laboratory [YG8JR, Fxn null:YG8s(GAA) > 800]. We characterized the behavioral, cellular, molecular and epigenetics properties of the YG8JR model, which has the largest GAA repeat sizes compared to all the current FRDA mouse models. Results: We found statistically significant behavioral deficits, together with reduced levels of frataxin mRNA and protein, and aconitase activity in YG8JR mice compared with control Y47JR mice. YG8JR mice exhibit intergenerational GAA repeat instability by the analysis of parent and offspring tissue samples. Somatic GAA repeat instability was also detected in individual brain and cerebellum tissue samples. In addition, increased DNA methylation of CpG U13 was identified in FXN GAA repeat region in the brain, cerebellum, and heart tissues. Furthermore, we show decreased histone H3K9 acetylation and increased H3K9 methylation of YG8JR cerebellum tissues within the FXN gene, upstream and downstream of the GAA repeat region compared to Y47JR controls. Discussion: These studies provide a detailed characterization of the GAA repeat expansion-based YG8JR transgenic mouse models that will help investigations of FRDA disease mechanisms and therapy.

HMTase Inhibitors as a Potential Epigenetic-Based Therapeutic Approach for Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disorder caused by a homozygous GAA repeat expansion mutation in intron 1 of the frataxin gene (FXN), which instigates reduced transcription. As a consequence, reduced levels of frataxin protein lead to mitochondrial iron accumulation, oxidative stress, and ultimately cell death; particularly in dorsal root ganglia (DRG) sensory neurons and the dentate nucleus of the cerebellum. In addition to neurological disability, FRDA is associated with cardiomyopathy, diabetes mellitus, and skeletal deformities. Currently there is no effective treatment for FRDA and patients die prematurely. Recent findings suggest that abnormal GAA expansion plays a role in histone modification, subjecting the FXN gene to heterochromatin silencing. Therefore, as an epigenetic-based therapy, we investigated the efficacy and tolerability of two histone methyltransferase (HMTase) inhibitor compounds, BIX0194 (G9a-inhibitor) and GSK126 (EZH2-inhibitor), to specifically target and reduce H3K9me2/3 and H3K27me3 levels, respectively, in FRDA fibroblasts. We show that a combination treatment of BIX0194 and GSK126, significantly increased FXN gene expression levels and reduced the repressive histone marks. However, no increase in frataxin protein levels was observed. Nevertheless, our results are still promising and may encourage to investigate HMTase inhibitors with other synergistic epigenetic-based therapies for further preliminary studies.

The Glutamate Dehydrogenase Pathway and Its Roles in Cell and Tissue Biology in Health and Disease.

Glutamate dehydrogenase (GDH) is a hexameric enzyme that catalyzes the reversible conversion of glutamate to α-ketoglutarate and ammonia while reducing NAD(P)⁺ to NAD(P)H. It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate), lipid biosynthesis (via oxidative generation of citrate), and lactate production. While most mammals possess a single GDH1 protein (hGDH1 in the human) that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. The novel hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1) is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH) in the mitochondria for the biosynthesis of steroidal hormones. Lastly, up-regulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth. In addition, deregulation of hGDH1/2 is implicated in the pathogenesis of several human disorders.

Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence.

Mammalian glutamate dehydrogenase (GDH), a nuclear-encoded enzyme central to cellular metabolism, is among the most abundant mitochondrial proteins (constituting up to 10% of matrix proteins). To attain such high levels, GDH depends on very efficient mitochondrial targeting that, for human isoenzymes hGDH1 and hGDH2, is mediated by an unusually long cleavable presequence (N53). Here, we studied the mitochondrial transport of these proteins using isolated yeast mitochondria and human cell lines. We found that both hGDHs were very rapidly imported and processed in isolated mitochondria, with their presequences (N53) alone being capable of directing non-mitochondrial proteins into mitochondria. These presequences were predicted to form two α helices (α1: N 1-10; α2: N 16-32) separated by loops. Selective deletion of the α1 helix abolished the mitochondrial import of hGDHs. While the α1 helix alone had a very weak hGDH mitochondrial import capacity, it could direct efficiently non-mitochondrial proteins into mitochondria. In contrast, the α2 helix had no autonomous mitochondrial-targeting capacity. A peptide consisting of α1 and α2 helices without intervening sequences had GDH transport efficiency comparable with that of N53. Mutagenesis of the cleavage site blocked the intra-mitochondrial processing of hGDHs, but did not affect their mitochondrial import. Replacement of all three positively charged N-terminal residues (Arg3, Lys7 and Arg13) by Ala abolished import. We conclude that the synergistic interaction of helices α1 and α2 is crucial for the highly efficient import of hGDHs into mitochondria.

Common players in mitochondria biogenesis and neuronal protection against stress-induced apoptosis.

Mitochondria biogenesis is a fundamental process for the organization and normal function of all cells. Since the majority of mitochondrial proteins are synthesized in the cytosol, protein import is the major mechanism for mitochondria biogenesis. We describe the different pathways that ensure correct targeting and intra mitochondrial sorting of mitochondrial proteins. The import process of several proteins of the mitochondrial intermembrane space relies on the Mitochondrial Import and Assembly 40 and Essential for respiration and vegetative growth 1 (Erv1) proteins that together constitute the oxidative folding machinery of the mitochondrial intermembrane space. Recent work has implicated the FAD-oxidase protein Erv1 (ad its human homolog Augmenter of Liver Regeneration) as an anti-apoptotic factor in mammalian cells (including neuronal cells) that undergo Reactive Oxygen Species-triggered apoptosis. The different roles of this protein as a key factor in mitochondria biogenesis, iron-sulfur cluster biogenesis and in neuronal protection against apoptosis are discussed.