BACKGROUND: Genetic factors are involved in the pathogenesis of familial and sporadic amyotrophic lateral sclerosis (ALS) and constitute a link to its association with frontotemporal dementia (FTD). Gene-targeted therapies for some forms of ALS (C9orf72, SOD1) have recently gained momentum. Genetic architecture in Czech ALS patients has not been comprehensively assessed so far. OBJECTIVE: We aimed to deliver pilot data on the genetic landscape of ALS in our country. METHODS: A cohort of patients with ALS (n = 88), recruited from two Czech Neuromuscular Centers, was assessed for hexanucleotide repeat expansion (HRE) in C9orf72 and also for genetic variations in other 36 ALS-linked genes via next-generation sequencing (NGS). Nine patients (10.1%) had a familial ALS. Further, we analyzed two subgroups of sporadic patients - with concomitant FTD (n = 7) and with young-onset of the disease (n = 22). RESULTS: We detected the pathogenic HRE in C9orf72 in 12 patients (13.5%) and three other pathogenic variants in FUS, TARDBP and TBK1, each in one patient. Additional 7 novel and 9 rare known variants with uncertain causal significance have been detected in 15 patients. Three sporadic patients with FTD (42.9%) were harbouring a pathogenic variant (all HRE in C9orf72). Surprisingly, none of the young-onset sporadic patients harboured a pathogenic variant and we detected no pathogenic SOD1 variant in our cohort. CONCLUSION: Our findings resemble those from other European populations, with the highest prevalence of HRE in the C9orf72 gene. Further, our findings suggest a possibility of a missing genetic variability among young-onset patients.

• MeSH
• Amyotrophic Lateral Sclerosis * genetics   MeSH
• DNA-Binding Proteins genetics   MeSH
• Adult MeSH
• DNA Repeat Expansion * MeSH
• Frontotemporal Dementia * genetics   MeSH
• Genetic Predisposition to Disease MeSH
• Cohort Studies MeSH
• Middle Aged MeSH
• Humans MeSH
• C9orf72 Protein * genetics   MeSH
• RNA-Binding Protein FUS genetics   MeSH
• Protein Serine-Threonine Kinases genetics   MeSH
• Aged MeSH
• Age of Onset MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

BACKGROUND: Fuchs endothelial corneal dystrophy (FECD) is the most common repeat-mediated disease in humans. It exclusively affects corneal endothelial cells (CECs), with ≤81% of cases associated with an intronic TCF4 triplet repeat (CTG18.1). Here, we utilise optical genome mapping (OGM) to investigate CTG18.1 tissue-specific instability to gain mechanistic insights. METHODS: We applied OGM to a diverse range of genomic DNAs (gDNAs) from patients with FECD and controls (n = 43); CECs, leukocytes and fibroblasts. A bioinformatics pipeline was developed to robustly interrogate CTG18.1-spanning DNA molecules. All results were compared with conventional polymerase chain reaction-based fragment analysis. FINDINGS: Analysis of bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin. However, clusters of CTG18.1 molecules, encompassing ∼1800-11,900 repeats, were exclusively detected in diseased CECs from expansion-positive cases. Additionally, both progenitor allele size and age were found to influence the level of leukocyte-specific CTG18.1 instability. INTERPRETATION: OGM is a powerful tool for analysing somatic instability of repeat loci and reveals here the extreme levels of CTG18.1 instability occurring within diseased CECs underpinning FECD pathophysiology, opening up new therapeutic avenues for FECD. Furthermore, these findings highlight the broader translational utility of FECD as a model for developing therapeutic strategies for rarer diseases similarly attributed to somatically unstable repeats. FUNDING: UK Research and Innovation, Moorfields Eye Charity, Fight for Sight, Medical Research Council, NIHR BRC at Moorfields Eye Hospital and UCL Institute of Ophthalmology, Grantová Agentura České Republiky, Univerzita Karlova v Praze, the National Brain Appeal's Innovation Fund and Rosetrees Trust.

• MeSH
• Alleles MeSH
• Trinucleotide Repeat Expansion MeSH
• Fuchs' Endothelial Dystrophy * genetics   pathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Chromosome Mapping MeSH
• Genomic Instability MeSH
• Organ Specificity genetics   MeSH
• Aged MeSH
• Transcription Factor 4 * genetics   metabolism   MeSH
• Trinucleotide Repeats genetics   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.

• MeSH
• Alternative Splicing genetics   MeSH
• Endothelial Cells metabolism   MeSH
• Trinucleotide Repeat Expansion * genetics   MeSH
• Fuchs' Endothelial Dystrophy * genetics   MeSH
• Humans MeSH
• Endothelium, Corneal metabolism   pathology   MeSH
• Transcription Factor 4 * genetics   metabolism   MeSH
• Transcriptome genetics   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH

Sandwich ELISA-based methods use Abs that target the expanded polyglutamine (polyQ) tract to quantify mutant huntingtin (mHTT). Using Meso Scale Discovery (MSD) assay, the mHTT signal detected with MW1 Ab correlated with polyQ length and doubled with a difference of only 7 glutamine residues between equivalent amounts of purified mHTTexon1 proteins. Similar polyQ length-dependent effects on MSD signals were confirmed using endogenous full length mHTT from brains of Huntington's disease (HD) knock-in (KI) mice. We used this avidity bias to devise a method to assess average CAG repeat instability at the protein level in a mixed population of HTT proteins present in tissues. Signal detected for average polyQ length quantification at the protein level by our method exhibited a strong correlation with average CAG repeat length at the genomic DNA level determined by PCR method in striatal tissue homogenates from HdhQ140 KI mice and in human HD postmortem cortex. This work establishes that CAG repeat instability in mutant HTT is reflected at the protein level.

• MeSH
• DNA genetics   MeSH
• Exons genetics   MeSH
• Trinucleotide Repeat Expansion genetics   MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic MeSH
• Peptides genetics   MeSH
• Huntingtin Protein chemistry   genetics   MeSH
• Antibodies metabolism   MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Dědičné cerebelární ataxie jsou vzácnou příčinou poruch rovnováhy v dospělosti. V tomto článku shrnujeme základní informace o této heterogenní skupině chorob s důrazem na klinickou diagnostiku a se zvláštním zaměřením na identifikaci léčitelných příčin.

Hereditary cerebellar ataxias are rare causes of adult balance disorders. In this paper, we summarize basic information about this heterogeneous group of diseases, with an emphasis on clinical diagnosis and with a particular focus on identifying treatable causes.

• MeSH
• Ataxia diagnosis   etiology   genetics   therapy   MeSH
• Cerebellar Ataxia * diagnosis   etiology   genetics   therapy   MeSH
• Chronic Disease MeSH
• Heredity MeSH
• DNA Repeat Expansion MeSH
• Friedreich Ataxia diagnosis   etiology   genetics   MeSH
• Genes, X-Linked MeSH
• Humans MeSH
• Metabolic Diseases diagnosis   genetics   MeSH
• Mitochondrial Diseases diagnosis   genetics   MeSH
• Cerebellar Diseases diagnosis   MeSH
• Disease Progression MeSH
• Spinocerebellar Ataxias * diagnosis   etiology   genetics   therapy   MeSH
• Inheritance Patterns MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Check Tag
• Humans MeSH

Svalové dystrofie jsou progresivní hereditární onemocnění, pro které je charakteristické nekolísavé, chronické a (většinou) nebolestivé oslabení svalových skupin a jejich atrofie. Můžou se projevit v dětství nebo v dospělosti. Jde o onemocnění vzácná s překrývajícími se obrazy a podobnými fenotypy a jejich diagnostika je náročná, většinou odkázána na neuromuskulární centra. Některé nejběžnější dystrofie by ale měl poznat každý neurolog. Nejčastější dystrofie dospělého věku jsou dystrofinopatie, myotonické dystrofie, facioscapulohumerální dystrofie, pletencové myopatie. Vzhledem k tomu, že některé dystrofie s charakteristickým fenotypem lze relativně snadno diagnostikovat (facioscapulohumerální, myotonická dystrofie typ 1), je v článku popsána problematika těch nejčastějších svalových dystrofií. Kromě diagnostiky je ovšem důležitá i následná péče – monitorace postižení přidružených systémů (srdce, oči, endokrinní, plíce), genetické poradenství a řešení sociálních problémů spojených s nemocí – to je péče, kterou je schopen nabídnout každý neurolog.

Muscle dystrophies are progressive, hereditary diseases which are characterized by not fluctuating, chronic and (mostly) painless weakness of groups of muscles and their atrophies. They can manifest in childhood or in adult age. These diseases are rare with overlapping clinical pictures and similar phenotypes and their diagnostics is complicated, mostly performed in the neuromuscular centers. The most common muscular dystrophies should be recognized by each neurologist. Dystrophinopathies, myotonic dystrophies, facioscapulohumeral muscular dystrophy and limb girdle dystrophies are the most common dystrophies of adult age. Some dystrophies with characteristic phenotypes can be quite easily diagnosed (facioscapulohumeral muscular dystrophy, myotonic dystrophy type 1), the issue of the most common dystrophies is described in this article. Not only appointment of diagnosis is important, but also – monitoring of disability of associated systems (heart, eyes, endocrine system, lungs), genetic consultations and solutions of social problems associated with the disease – this is a common care of each neurologist.

• MeSH
• Diagnosis, Differential MeSH
• Adult MeSH
• Trinucleotide Repeat Expansion MeSH
• Muscular Dystrophy, Facioscapulohumeral genetics   complications   MeSH
• Phenotype MeSH
• Muscle, Skeletal pathology   MeSH
• Humans MeSH
• Myotonic Dystrophy genetics   classification   complications   MeSH
• Heart Diseases complications   MeSH
• Muscular Dystrophies, Limb-Girdle genetics   classification   complications   MeSH
• Muscle Weakness etiology   MeSH
• Muscular Dystrophies * diagnosis   etiology   therapy   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Publication type
• Review MeSH

Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich's ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich's ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs' non-B DNA structures.

• MeSH
• DNA chemistry   metabolism   MeSH
• Trinucleotide Repeat Expansion * MeSH
• Gene Expression MeSH
• Friedreich Ataxia genetics   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 chemistry   metabolism   MeSH
• Pyrimidines MeSH
• Recombinant Proteins MeSH
• Trinucleotide Repeats * MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.

• MeSH
• Alleles MeSH
• CRISPR-Cas Systems genetics   MeSH
• Adult MeSH
• Trinucleotide Repeat Expansion genetics   MeSH
• Fuchs' Endothelial Dystrophy genetics   pathology   MeSH
• Genetic Predisposition to Disease * MeSH
• Genotype MeSH
• Introns genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Sequence Analysis, DNA MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Transcription Factor 4 genetics   MeSH
• Trinucleotide Repeats genetics   MeSH
• Single Molecule Imaging MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD (tgHD) minipig model. We detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT. The combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic.

• MeSH
• Dependovirus genetics   MeSH
• Trinucleotide Repeat Expansion genetics   MeSH
• Genetic Therapy methods   MeSH
• Genetic Vectors genetics   MeSH
• Animals, Genetically Modified MeSH
• Huntington Disease genetics   metabolism   therapy   MeSH
• Humans MeSH
• MicroRNAs genetics   metabolism   MeSH
• Swine, Miniature MeSH
• Disease Models, Animal MeSH
• Swine MeSH
• Huntingtin Protein genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.

• MeSH
• Oligonucleotides, Antisense pharmacology   MeSH
• Cell Nucleus drug effects   metabolism   MeSH
• Endothelial Cells metabolism   MeSH
• Trinucleotide Repeat Expansion genetics   MeSH
• Fuchs' Endothelial Dystrophy genetics   pathology   MeSH
• Genetic Predisposition to Disease * MeSH
• Cohort Studies MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Organ Specificity MeSH
• RNA Processing, Post-Transcriptional MeSH
• RNA Precursors genetics   MeSH
• Risk Factors MeSH
• Endothelium, Corneal pathology   MeSH
• Aged MeSH
• RNA Splicing Factors metabolism   MeSH
• Transcription Factor 4 genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH