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
• alely MeSH
• expanze trinukleotidových repetic MeSH
• Fuchsova endoteliální dystrofie * genetika   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mapování chromozomů MeSH
• nestabilita genomu MeSH
• orgánová specificita genetika   MeSH
• senioři MeSH
• transkripční faktor 4 * genetika   metabolismus   MeSH
• trinukleotidové repetice genetika   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články 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
• alternativní sestřih genetika   MeSH
• endoteliální buňky metabolismus   MeSH
• expanze trinukleotidových repetic * genetika   MeSH
• Fuchsova endoteliální dystrofie * genetika   MeSH
• lidé MeSH
• rohovkový endotel metabolismus   patologie   MeSH
• transkripční faktor 4 * genetika   metabolismus   MeSH
• transkriptom genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The aim of this study was to describe the ocular phenotype in a case with Kearns-Sayre syndrome (KSS) spectrum and to determine if corneal endothelial cell dysfunction could be attributed to other known distinct genetic causes. Herein, genomic DNA was extracted from blood and exome sequencing was performed. Non-coding gene regions implicated in corneal endothelial dystrophies were screened by Sanger sequencing. In addition, a repeat expansion situated within an intron of TCF4 (termed CTG18.1) was genotyped using the short tandem repeat assay. The diagnosis of KSS spectrum was based on the presence of ptosis, chronic progressive external ophthalmoplegia, pigmentary retinopathy, hearing loss, and muscle weakness, which were further supported by the detection of ~6.5 kb mtDNA deletion. At the age of 33 years, the proband's best corrected visual acuity was reduced to 0.04 in the right eye and 0.2 in the left eye. Rare ocular findings included marked corneal oedema with central corneal thickness of 824 and 844 μm in the right and left eye, respectively. No pathogenic variants in the genes, which are associated with corneal endothelial dystrophies, were identified. Furthermore, the CTG18.1 genotype was 12/33, which exceeds a previously determined critical threshold for toxic RNA foci appearance in corneal endothelial cells.

• MeSH
• dospělí MeSH
• fenotyp MeSH
• Fuchsova endoteliální dystrofie patologie   MeSH
• genotyp MeSH
• katarakta genetika   MeSH
• Kearnsův-Sayreův syndrom genetika   MeSH
• lidé MeSH
• mitochondriální DNA MeSH
• rohovkový endotel patologie   patofyziologie   MeSH
• sekvenční delece MeSH
• sekvenování exomu MeSH
• transkripční faktor 4 genetika   MeSH
• trinukleotidové repetice * MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disorder categorized into 3 phenotypic variants: infantile, juvenile, and adult. Four recent reports have linked NIID to CGG expansions in the NOTCH2NLC gene in adult NIID (aNIID) and several juvenile patients. Infantile NIID (iNIID) is an extremely rare neuropediatric condition. We present a 7-year-old male patient with severe progressive neurodegenerative disease that included cerebellar symptoms with cerebellar atrophy on brain MRI, psychomotor developmental regression, pseudobulbar syndrome, and polyneuropathy. The diagnosis of iNIID was established through a postmortem neuropathology work-up. We performed long-read sequencing of the critical NOTCH2NLC repeat motif and found no expansion in the patient. We also re-evaluated an antemortem skin biopsy that was collected when the patient was 2 years and 8 months old and did not identify the intranuclear inclusions. In our report, we highlight that the 2 methods (skin biopsy and CGG expansion testing in NOTCH2NLC) used to identify aNIID patients may provide negative results in iNIID patients.

• MeSH
• biopsie MeSH
• dítě MeSH
• intranukleární inkluzní tělíska genetika   patologie   MeSH
• kojenec MeSH
• kůže patologie   MeSH
• lidé MeSH
• mícha patologie   MeSH
• mozek patologie   MeSH
• neurodegenerativní nemoci diagnóza   genetika   patologie   MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• receptor Notch2 genetika   MeSH
• trinukleotidové repetice genetika   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem 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 genetika   MeSH
• exony genetika   MeSH
• expanze trinukleotidových repetic genetika   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• peptidy genetika   MeSH
• protein huntingtin chemie   genetika   MeSH
• protilátky metabolismus   MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• diferenciální diagnóza MeSH
• dospělí MeSH
• expanze trinukleotidových repetic MeSH
• facioskapulohumerální svalová dystrofie genetika   komplikace   MeSH
• fenotyp MeSH
• kosterní svaly patologie   MeSH
• lidé MeSH
• myotonická dystrofie genetika   klasifikace   komplikace   MeSH
• nemoci srdce komplikace   MeSH
• pletencové svalové dystrofie genetika   klasifikace   komplikace   MeSH
• svalová slabost etiologie   MeSH
• svalové dystrofie * diagnóza   etiologie   terapie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• Publikační typ
• přehledy 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 chemie   metabolismus   MeSH
• expanze trinukleotidových repetic * MeSH
• exprese genu MeSH
• Friedreichova ataxie genetika   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• konformace nukleové kyseliny * MeSH
• lidé MeSH
• nádorový supresorový protein p53 chemie   metabolismus   MeSH
• pyrimidiny MeSH
• rekombinantní proteiny MeSH
• trinukleotidové repetice * MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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
• alely MeSH
• CRISPR-Cas systémy genetika   MeSH
• dospělí MeSH
• expanze trinukleotidových repetic genetika   MeSH
• Fuchsova endoteliální dystrofie genetika   patologie   MeSH
• genetická predispozice k nemoci * MeSH
• genotyp MeSH
• introny genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• sekvenční analýza DNA MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• transkripční faktor 4 genetika   MeSH
• trinukleotidové repetice genetika   MeSH
• zobrazení jednotlivé molekuly MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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 genetika   MeSH
• expanze trinukleotidových repetic genetika   MeSH
• genetická terapie metody   MeSH
• genetické vektory genetika   MeSH
• geneticky modifikovaná zvířata MeSH
• Huntingtonova nemoc genetika   metabolismus   terapie   MeSH
• lidé MeSH
• mikro RNA genetika   metabolismus   MeSH
• miniaturní prasata MeSH
• modely nemocí na zvířatech MeSH
• prasata MeSH
• protein huntingtin genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• antisense oligonukleotidy farmakologie   MeSH
• buněčné jádro účinky léků   metabolismus   MeSH
• endoteliální buňky metabolismus   MeSH
• expanze trinukleotidových repetic genetika   MeSH
• Fuchsova endoteliální dystrofie genetika   patologie   MeSH
• genetická predispozice k nemoci * MeSH
• kohortové studie MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• myši inbrední C57BL MeSH
• orgánová specificita MeSH
• posttranskripční úpravy RNA MeSH
• prekurzory RNA genetika   MeSH
• rizikové faktory MeSH
• rohovkový endotel patologie   MeSH
• senioři MeSH
• sestřihové faktory metabolismus   MeSH
• transkripční faktor 4 genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH