Project description:Giant Axon Neuropathy (GAN) is a progressive neurodegenerative disease characterised by involvement of peripheral and central nervous system frequently associated with frizzy hair. The phenotype is caused by bi-allelic variants in the GAN gene, leading to loss of functional gigaxonin proteins.. Thus, far the disease cannot be cured, but a first clinical gene therapy trial in the US was currently completed. So far, there has been no systematic reporting of GAN patients in German-speaking countries. Therefore, we conducted a survey using an e-mail list of German-speaking child neurologists of the so-called DACH region reflecting Germany, Austria and Switzerland. Based on their feedback, clinical, genetic and epidemiological data were collected using a standardised data collection form in turn enabling the collection of comprehensive and detailed epidemiological, clinical and genetic information from a total of 15 patients representing one of the largest cohorts systematically described thus far. Average age of patients was 11.7 years at the time of data collection. In line with the frequency of homozygous variants, the majority were of Syrian origin and of consanguineous relationships. In 14 patients, diagnosis was confirmed by molecular genetics, revealing eight different GAN variants, four being reported as pathogenic in literature. 13 patients had a classical GAN phenotype, one was classified as mild whereas another one was too young to draw final clinical conclusions. Proteomics of white blood cells derived from four patients revealed was conducted to obtain unbiased insights into the underlying pathophysiology and revealed dysregulation of 111 proteins implicated in diverse biological processes. Of note diverse of these proteins are known to be crucial for proper synaptic function and transmission and affection of intermediate filament organization and proteolysis is in line with the known functions of gigaxonin. In view of a potential genetic treatment option, these data on the natural course of the disease are important for study design in preparation for gene therapy trials in Europe and moreover provide a catalogue of proteins serving as minimal invasive but cellular biomarkers.

Project description:Myotonic Dystrophy type 2 (DM2) is an autosomal-dominant, multisystemic disease with a core manifestation of proximal muscle weakness, muscle atrophy, myotonia, and myalgia. The disease-causing CCTG tetranucleotide expansion within the CNBP gene on chromosome 3 leads to an RNA-dominated spliceopathy which is currently untreatable. Research exploring the pathophysiological mechanisms in Myotonic Dystrophy Type 1 resulted in new insights into disease mechanisms and identified mitochondrial dysfunction as promising therapeutic target. It remains unclear whether similar mechanisms underlie DM2 and, if so, whether these might also serve as potential therapeutic targets. In this cross-sectional study, we studied DM2 skeletal muscle biopsy specimens on proteomic, molecular, and morphological including ultrastructural levels in two separate patient cohorts consisting of 8 (explorative cohort) and 40 (confirmatory cohort) patients. Seven muscle biopsy specimens obtained from four female and three male DM2 patients underwent proteomic analysis. We performed immunoblotting of respiratory chain complexes, mitochondrial DNA copy number determination and long-range PCR (LR-PCR) to study mitochondrial deletions on six biopsies. Forty-eight biopsy samples were studied by light and electron microscopy. Proteomic analysis revealed a downregulation of essential mitochondrial proteins, namely of subunits of respiratory chain complexes I, III, and IV (e.g. mt-CO1, mt-ND1, mt-CYB, NDUFB6) and associated translation factors (TACO1). Light microscopy showed an age-inappropriate amount of COX-deficient fibers in most biopsy specimens. Electron microscopy revealed widespread ultrastructural mitochondrial abnormalities including dysmorphic mitochondria with paracrystalline inclusions. Immunoblotting and LR-PCR did not reveal significant differences between patients and controls, while mtDNA-copy number measurement revealed a reduction of mtDNA-copy numbers in the patient group compared to controls. This first multi-level study of DM2 unravels thus far undescribed functional and structural mitochondrial abnormalities. While the molecular link between the tetranucleotide expansion, and mitochondrial dysfunction needs to be further elucidated, these findings may provide an additional route for treatment strategies for DM2.

Project description:The elucidation of pathomechanisms leading to the manifestation of neuromuscular diseases (NMDs) represents an important step toward the understanding of the genesis of the respective disease and might help to define starting points for (new) therapeutic intervention concepts. However, these “discovery studies” are often limited by the availability of human biomaterial especially regarding the fact that muscle and nerve biopsies became less common in the diagnostic work-up of patients suffering from NMDs. Moreover, given that results of next-generation-sequencing approaches frequently result in the identification of ambiguous variants, testing of their pathogenicity is crucial but also depending on patient-derived material. To systematically address the question if human skin fibroblasts might serve as a valuable biomaterial for (molecular) studies of NMDs, using proteomic profiling we generated a protein library by decreasing protein complexity via pH8-based sample fractionation: cataloguing of 8280 proteins revealed the expression of a variety of such linked to genetic forms of motor neuron diseases, congenital myasthenic syndromes, neuropathies and muscular disorders. In silico-based pathway analyses revealed expression of a variety of proteins involved in muscle contraction and such decisive for neuronal function and maintenance suggesting the suitability of human skin fibroblasts to study the etiology of NMDs. Based on these findings, next we aimed to further demonstrate the suitability of this in vitro model to study NMDs by a use case: utilizing a data independent acquisition (DIA) approach, the proteomic signature of whole protein extracts of fibroblasts derived from an Allgrove-patient was studied. Paradigmatic dysregulated proteins were confirmed in muscle biopsy of the patient and protein-functions could be linked to neurological symptoms known for this disease. Moreover, LC-MS/MS-based investigation of nuclear protein composition allowed the identification of protein-dysregulations which accord with structural perturbations observed in the muscle biopsy.

			Action	DRS
	20240124_125020_NME_WBC_CRHNE_fullset_2.sne	Other
	Std-QExactive01-06526_NME_WBF_171-23.raw	Raw
	Std-QExactive01-06527_NME_WBF_136-20.raw	Raw
	Std-QExactive01-06528_NME_WBF_57-21.raw	Raw
	Std-QExactive01-06530_NME_WBF_176-23.raw	Raw

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Blood biomarkers in a cohort of patients with CHRNE-associated congenital myasthenic syndrome

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Blood biomarker fingerprints in a cohort of patients with CHRNE-related congenital myasthenic syndrome.

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