Rapid IDH1-R132 genotyping panel utilizing locked nucleic acid loop-mediated isothermal amplification.

While the detection of single-nucleotide variants (SNVs) is important for evaluating human health and disease, most genotyping methods require a nucleic acid extraction step and lengthy analytical times. Here, we present a protocol which utilizes the integration of locked nucleic acids (LNAs) into self-annealing loop primers for the allelic discrimination of five isocitrate dehydrogenase 1 R132 (IDH1-R132) variants using loop-mediated isothermal amplification (LAMP). This genotyping panel was initially evaluated using purified synthetic DNA to show proof of specific SNV discrimination. Additional evaluation using glioma tumor lysates with known IDH1-R132 mutational status demonstrated specificity in approximately 35 min without the need for a nucleic acid extraction purification step. This LNA-LAMP-based genotyping assay can detect single base differences in purified nucleic acids or tissue homogenates, including instances where the variant of interest is present in an excess of background wild-type DNA. The pH-based colorimetric indicator of LNA-LAMP facilitates convenient visual interpretation of reactions, and we demonstrate successful translation to an end-point format using absorbance ratio, allowing for an alternative and objective approach for differentiating between positive and negative reactions. Importantly, the LNA-LAMP genotyping panel is highly reproducible, with no false-positive or false-negative results observed.

Mitochondrial aminoacyl-tRNA synthetases trigger unique compensatory mechanisms in neurons.

Mitochondrial aminoacyl-tRNA synthetase (mt-ARS) mutations cause severe, progressive, and often lethal diseases with highly heterogeneous and tissue-specific clinical manifestations. This study investigates the molecular mechanisms triggered by three different mt-ARS defects caused by biallelic mutations in AARS2, EARS2, and RARS2, using an in vitro model of human neuronal cells. We report distinct molecular mechanisms of mitochondrial dysfunction among the mt-ARS defects studied. Our findings highlight the ability of proliferating neuronal progenitor cells (iNPCs) to compensate for mitochondrial translation defects and maintain balanced levels of oxidative phosphorylation (OXPHOS) components, which becomes more challenging in mature neurons. Mutant iNPCs exhibit unique compensatory mechanisms, involving specific branches of the integrated stress response, which may be gene-specific or related to the severity of the mitochondrial translation defect. RNA sequencing revealed distinct transcriptomic profiles showing dysregulation of neuronal differentiation and protein translation. This study provides valuable insights into the tissue-specific compensatory mechanisms potentially underlying the phenotypes of patients with mt-ARS defects. Our novel in vitro model may more accurately represent the neurological presentation of patients and offer an improved platform for future investigations and therapeutic development.

Rapid extraction-free detection of the R132H isocitrate dehydrogenase mutation in glioma using colorimetric peptide nucleic acid-loop mediated isothermal amplification (CPNA-LAMP).

The R132H isocitrate dehydrogenase one (IDH1) mutation is a prognostic biomarker present in a subset of gliomas and is associated with heightened survival when paired with aggressive surgical resection. In this study, we establish proof-of-principle for rapid colorimetric detection of the IDH1-R132H mutation in tumor samples in under 1 hour without the need for a nucleic acid extraction. Colorimetric peptide nucleic acid loop-mediated isothermal amplification (CPNA-LAMP) utilizes 4 conventional LAMP primers, a blocking PNA probe complementary to the wild-type sequence, and a self-annealing loop primer complementary to the single nucleotide variant to only amplify the DNA sequence containing the mutation. This assay was evaluated using IDH1-WT or IDH1-R132H mutant synthetic DNA, wild-type or IDH1-R132H mutant U87MG cell lysates, and tumor lysates from archived patient samples in which the IDH1 status was previously determined using immunohistochemistry (IHC). Reactions were performed using a hot water bath and visually interpreted as positive by a pink-to-yellow color change. Results were subsequently verified using agarose gel electrophoresis. CPNA-LAMP successfully detected the R132H single nucleotide variant, and results from tumor lysates yielded 100% concordance with IHC results, including instances when the single nucleotide variant was limited to a portion of the tumor. Importantly, when testing the tumor lysates, there were no false positive or false negative results.

A translatable RNAi-driven gene therapy silences PMP22/Pmp22 genes and improves neuropathy in CMT1A mice.

Charcot-Marie-Tooth disease type 1A (CMT1A), the most common inherited demyelinating peripheral neuropathy, is caused by PMP22 gene duplication. Overexpression of WT PMP22 in Schwann cells destabilizes the myelin sheath, leading to demyelination and ultimately to secondary axonal loss and disability. No treatments currently exist that modify the disease course. The most direct route to CMT1A therapy will involve reducing PMP22 to normal levels. To accomplish this, we developed a gene therapy strategy to reduce PMP22 using artificial miRNAs targeting human PMP22 and mouse Pmp22 mRNAs. Our lead therapeutic miRNA, miR871, was packaged into an adeno-associated virus 9 (AAV9) vector and delivered by lumbar intrathecal injection into C61-het mice, a model of CMT1A. AAV9-miR871 efficiently transduced Schwann cells in C61-het peripheral nerves and reduced human and mouse PMP22 mRNA and protein levels. Treatment at early and late stages of the disease significantly improved multiple functional outcome measures and nerve conduction velocities. Furthermore, myelin pathology in lumbar roots and femoral motor nerves was ameliorated. The treated mice also showed reductions in circulating biomarkers of CMT1A. Taken together, our data demonstrate that AAV9-miR871-driven silencing of PMP22 rescues a CMT1A model and provides proof of principle for treating CMT1A using a translatable gene therapy approach.

NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.

Molecular markers scalable for clinical use are critical for the development of effective treatments and the design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA, were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141N mouse models as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.

Intracellular Lipid Accumulation and Mitochondrial Dysfunction Accompanies Endoplasmic Reticulum Stress Caused by Loss of the Co-chaperone DNAJC3.

Recessive mutations in DNAJC3, an endoplasmic reticulum (ER)-resident BiP co-chaperone, have been identified in patients with multisystemic neurodegeneration and diabetes mellitus. To further unravel these pathomechanisms, we employed a non-biased proteomic approach and identified dysregulation of several key cellular pathways, suggesting a pathophysiological interplay of perturbed lipid metabolism, mitochondrial bioenergetics, ER-Golgi function, and amyloid-beta processing. Further functional investigations in fibroblasts of patients with DNAJC3 mutations detected cellular accumulation of lipids and an increased sensitivity to cholesterol stress, which led to activation of the unfolded protein response (UPR), alterations of the ER-Golgi machinery, and a defect of amyloid precursor protein. In line with the results of previous studies, we describe here alterations in mitochondrial morphology and function, as a major contributor to the DNAJC3 pathophysiology. Hence, we propose that the loss of DNAJC3 affects lipid/cholesterol homeostasis, leading to UPR activation, ß-amyloid accumulation, and impairment of mitochondrial oxidative phosphorylation.

AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy.

Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X.

Targeted Therapies for Hereditary Peripheral Neuropathies: Systematic Review and Steps Towards a 'treatabolome'.

BACKGROUND: Hereditary peripheral neuropathies are inherited disorders affecting the peripheral nervous system, including Charcot-Marie-Tooth disease, familial amyloid polyneuropathy and hereditary sensory and motor neuropathies. While the molecular basis of hereditary peripheral neuropathies has been extensively researched, interventional trials of pharmacological therapies are lacking. OBJECTIVE: We collated evidence for the effectiveness of pharmacological and gene-based treatments for hereditary peripheral neuropathies. METHODS: We searched several databases for randomised controlled trials (RCT), observational studies and case reports of therapies in hereditary peripheral neuropathies. Two investigators extracted and analysed the data independently, assessing study quality using the Oxford Centre for Evidence Based Medicine 2011 Levels of Evidence in conjunction with the Jadad scale. RESULTS: Of the 2046 studies initially identified, 119 trials met our inclusion criteria, of which only 34 were carried over into our final analysis. Ascorbic acid was shown to have no therapeutic benefit in CMT1A, while a combination of baclofen, naltrexone and sorbitol (PXT3003) demonstrated some efficacy, but phase III data are incomplete. In TTR-related amyloid polyneuropathy tafamidis, patisiran, inotersen and revusiran showed significant benefit in high quality RCTs. Smaller studies showed the efficacy of L-serine for SPTLC1-related hereditary sensory neuropathy, riboflavin for Brown-Vialetto-Van Laere syndrome (SLC52A2/3) and phytanic acid-poor diet in Refsum disease (PHYH). CONCLUSIONS: The 'treatable' variants highlighted in this project will be flagged in the treatabolome database to alert clinicians at the time of the diagnosis and enable timely treatment of patients with hereditary peripheral neuropathies.

Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.

A guide to writing systematic reviews of rare disease treatments to generate FAIR-compliant datasets: building a Treatabolome.

BACKGROUND: Rare diseases are individually rare but globally affect around 6% of the population, and in over 70% of cases are genetically determined. Their rarity translates into a delayed diagnosis, with 25% of patients waiting 5 to 30 years for one. It is essential to raise awareness of patients and clinicians of existing gene and variant-specific therapeutics at the time of diagnosis to avoid that treatment delays add up to the diagnostic odyssey of rare diseases' patients and their families. AIMS: This paper aims to provide guidance and give detailed instructions on how to write homogeneous systematic reviews of rare diseases' treatments in a manner that allows the capture of the results in a computer-accessible form. The published results need to comply with the FAIR guiding principles for scientific data management and stewardship to facilitate the extraction of datasets that are easily transposable into machine-actionable information. The ultimate purpose is the creation of a database of rare disease treatments ("Treatabolome") at gene and variant levels as part of the H2020 research project Solve-RD. RESULTS: Each systematic review follows a written protocol to address one or more rare diseases in which the authors are experts. The bibliographic search strategy requires detailed documentation to allow its replication. Data capture forms should be built to facilitate the filling of a data capture spreadsheet and to record the application of the inclusion and exclusion criteria to each search result. A PRISMA flowchart is required to provide an overview of the processes of search and selection of papers. A separate table condenses the data collected during the Systematic Review, appraised according to their level of evidence. CONCLUSIONS: This paper provides a template that includes the instructions for writing FAIR-compliant systematic reviews of rare diseases' treatments that enables the assembly of a Treatabolome database that complement existing diagnostic and management support tools with treatment awareness data.

Multifocal demyelinating motor neuropathy and hamartoma syndrome associated with a de novo PTEN mutation.

OBJECTIVE: To describe a patient with a multifocal demyelinating motor neuropathy with onset in childhood and a mutation in phosphatase and tensin homolog (PTEN), a tumor suppressor gene associated with inherited tumor susceptibility conditions, macrocephaly, autism, ataxia, tremor, and epilepsy. Functional implications of this protein have been investigated in Parkinson and Alzheimer diseases. METHODS: We performed whole-exome sequencing in the patient's genomic DNA validated by Sanger sequencing. Immunoblotting, in vitro enzymatic assay, and label-free shotgun proteomic profiling were performed in the patient's fibroblasts. RESULTS: The predominant clinical presentation of the patient was a childhood onset, asymmetric progressive multifocal motor neuropathy. In addition, he presented with macrocephaly, autism spectrum disorder, and skin hamartomas, considered as clinical criteria for PTEN-related hamartoma tumor syndrome. Extensive tumor screening did not detect any malignancies. We detected a novel de novo heterozygous c.269T>C, p.(Phe90Ser) PTEN variant, which was absent in both parents. The pathogenicity of the variant is supported by altered expression of several PTEN-associated proteins involved in tumorigenesis. Moreover, fibroblasts showed a defect in catalytic activity of PTEN against the secondary substrate, phosphatidylinositol 3,4-trisphosphate. In support of our findings, focal hypermyelination leading to peripheral neuropathy has been reported in PTEN-deficient mice. CONCLUSION: We describe a novel phenotype, PTEN-associated multifocal demyelinating motor neuropathy with a skin hamartoma syndrome. A similar mechanism may potentially underlie other forms of Charcot-Marie-Tooth disease with involvement of the phosphatidylinositol pathway.

PFN2 and GAMT as common molecular determinants of axonal Charcot-Marie-Tooth disease.

BACKGROUND: Charcot-Marie-Tooth type 2 (CMT2) neuropathy is characterised by a vast clinical and genetic heterogeneity complicating its diagnosis and therapeutic intervention. Identification of molecular signatures that are common to multiple CMT2 subtypes can aid in developing therapeutic strategies and measuring disease outcomes. METHODS: A proteomics-based approach was performed on lymphoblasts from CMT2 patients genetically diagnosed with different gene mutations to identify differentially regulated proteins. The candidate proteins were validated through real-time quantitative PCR and western blotting on lymphoblast samples of patients and controls, motor neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) and sciatic nerves of CMT2 mouse models. RESULTS: Proteomic profiling of patient lymphoblasts resulted in the identification of profilin 2 (PFN2) and guanidinoacetate methyltransferase (GAMT) as commonly downregulated proteins in different genotypes compared with healthy controls. This decrease was also observed at the transcriptional level on screening 43 CMT2 patients and 22 controls, respectively. A progressive decrease in PFN2 expression with age was observed in patients, while in healthy controls its expression increased with age. Reduced PFN2 expression was also observed in motor neurons differentiated from CMT2 patient-derived iPSCs and sciatic nerves of CMT2 mice when compared with controls. However, no change in GAMT levels was observed in motor neurons and CMT2 mouse-derived sciatic nerves. CONCLUSIONS: We unveil PFN2 and GAMT as molecular determinants of CMT2 with possible indications of the role of PFN2 in the pathogenesis and disease progression. This is the first study describing biomarkers that can boost the development of therapeutic strategies targeting a wider spectrum of CMT2 patients.

The role of tRNA synthetases in neurological and neuromuscular disorders.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes responsible for charging tRNAs with their cognate amino acids, therefore essential for the first step in protein synthesis. Although the majority of protein synthesis happens in the cytosol, an additional translation apparatus is required to translate the 13 mitochondrial DNA-encoded proteins important for oxidative phosphorylation. Most ARS genes in these cellular compartments are distinct, but two genes are common, encoding aminoacyl-tRNA synthetases of glycine (GARS) and lysine (KARS) in both mitochondria and the cytosol. Mutations in the majority of the 37 nuclear-encoded human ARS genes have been linked to a variety of recessive and dominant tissue-specific disorders. Current data indicate that impaired enzyme function could explain the pathogenicity, however not all pathogenic ARSs mutations result in deficient catalytic function; thus, the consequences of mutations may arise from other molecular mechanisms. The peripheral nerves are frequently affected, as illustrated by the high number of mutations in cytosolic and bifunctional tRNA synthetases causing Charcot-Marie-Tooth disease (CMT). Here we provide insights on the pathomechanisms of CMT-causing tRNA synthetases with specific focus on the two bifunctional tRNA synthetases (GARS, KARS).

Elimination of truncated recombinant protein expressed in Escherichia coli by removing cryptic translation initiation site.

Undesirable truncated recombinant protein products pose a special expression and purification challenge because such products often share similar chromatographic properties as the desired full length protein. We describe here our observation of both full length and a truncated form of a yeast protein (Gcn5) expressed in Escherichia coli, and the reduction or elimination of the truncated form by mutating a cryptic Shine-Dalgarno or START codon within the Gcn5 coding region. Unsuccessful attempts to engineer in a cryptic translation initiation site into other recombinant proteins suggest that cryptic Shine-Dalgarno or START codon sequences are necessary but not sufficient for cryptic translation in E. coli.

Extranucleosomal DNA enhances the activity of the LSD1/CoREST histone demethylase complex.

The promoter regions of active genes in the eukaryotic genome typically contain nucleosomes post-translationally modified with a trimethyl mark on histone H3 lysine 4 (H3K4), while transcriptional enhancers are marked with monomethylated H3K4. The flavin-dependent monoamine oxidase LSD1 (lysine-specific demethylase 1, also known as KDM1) demethylates mono- and dimethylated H3K4 in peptide substrates, but requires the corepressor protein, CoREST, to demethylate nucleosome substrates. The molecular basis for how the LSD1/CoREST complex interacts with its physiological nucleosome substrate remains largely unknown. We examine here the role of extranucleosomal DNA beyond the nucleosome core particle for LSD1/CoREST function. Our studies of LSD1/CoREST's enzyme activity and nucleosome binding show that extranucleosomal DNA dramatically enhances the activity of LSD1/CoREST, and that LSD1/CoREST binds to the nucleosome as a 1:1 complex. Our photocrosslinking experiments further indicate both LSD1 and CoREST subunits are in close contact with DNA around the nucleosome dyad as well as extranucleosomal DNA. Our results suggest that the LSD1/CoREST interacts with extranucleosomal DNA when it productively engages its nucleosome substrate.

RCC1 uses a conformationally diverse loop region to interact with the nucleosome: a model for the RCC1-nucleosome complex.

The binding of RCC1 (regulator of chromosome condensation 1) to chromatin is critical for cellular processes such as mitosis, nucleocytoplasmic transport, and nuclear envelope formation because RCC1 recruits the small GTPase Ran (Ras-related nuclear protein) to chromatin and sets up a Ran-GTP gradient around the chromosomes. However, the molecular mechanism by which RCC1 binds to nucleosomes, the repeating unit of chromatin, is not known. We have used biochemical approaches to test structural models for how the RCC1 beta-propeller protein could bind to the nucleosome. In contrast to the prevailing model, RCC1 does not appear to use the beta-propeller face opposite to its Ran-binding face to interact with nucleosomes. Instead, we find that RCC1 uses a conformationally flexible loop region we have termed the switchback loop in addition to its N-terminal tail to bind to the nucleosome. The juxtaposition of the RCC1 switchback loop to its Ran binding surface suggests a novel mechanism for how nucleosome-bound RCC1 recruits Ran to chromatin. Furthermore, this model accounts for previously unexplained observations for how Ran can interact with the nucleosome both dependent and independent of RCC1 and how binding of the nucleosome can enhance RCC1's Ran nucleotide exchange activity.