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.

• Klíčová slova
• Fuchs endothelial corneal dystrophy, Optical genome mapping, Somatic mosaicism, Tissue-specific repeat instability, Trinucleotide repeat expansion disease, Triplet repeat expansion-mediated disease,
• 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
• Názvy látek
• TCF4 protein, human MeSH Prohlížeč
• transkripční faktor 4 * MeSH

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.

• Klíčová slova
• Amyotrophic lateral sclerosis, C9orf72 repeat expansion, gene variants, mutation screening, neurogenetics, next-generation sequencing,
• MeSH
• amyotrofická laterální skleróza * genetika   MeSH
• DNA vazebné proteiny genetika   MeSH
• dospělí MeSH
• expanze repetic DNA * MeSH
• frontotemporální demence * genetika   MeSH
• genetická predispozice k nemoci MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• protein C9orf72 * genetika   MeSH
• protein FUS vázající RNA genetika   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• senioři MeSH
• věk při počátku nemoci MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• C9orf72 protein, human MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• FUS protein, human MeSH Prohlížeč
• protein C9orf72 * MeSH
• protein FUS vázající RNA MeSH
• protein-serin-threoninkinasy MeSH
• TARDBP protein, human MeSH Prohlížeč
• TBK1 protein, human MeSH Prohlížeč

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
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• TCF4 protein, human MeSH Prohlížeč
• transkripční faktor 4 * 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
• Názvy látek
• DNA MeSH
• peptidy MeSH
• polyglutamine MeSH Prohlížeč
• protein huntingtin MeSH
• protilátky 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.

• Klíčová slova
• Fuchs endothelial corneal dystrophy, amplification-free sequencing, no-amp targeted sequencing, somatic mosaicism, triplet repeat-mediated 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
• Názvy látek
• TCF4 protein, human MeSH Prohlížeč
• transkripční faktor 4 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.

• Klíčová slova
• DNA hairpin, DNA–protein, frataxin, non-B DNA, p53, trinucleotide repeat,
• 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
• Názvy látek
• DNA MeSH
• nádorový supresorový protein p53 MeSH
• pyrimidiny MeSH
• rekombinantní proteiny MeSH
• TP53 protein, human MeSH Prohlížeč

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.

• Klíčová slova
• AAV, Huntington disease, gene silencing, microRNA, transgenic minipig,
• 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
• Názvy látek
• mikro RNA MeSH
• protein huntingtin 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.

• Klíčová slova
• Fuchs endothelial corneal dystrophy, RNA toxicity, antisense oligonucleotide, corneal dystrophy, non-coding mutations, repeat-expansion, transcription factor 4, triplet repeat-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
• Názvy látek
• antisense oligonukleotidy MeSH
• messenger RNA MeSH
• prekurzory RNA MeSH
• sestřihové faktory MeSH
• TCF4 protein, human MeSH Prohlížeč
• transkripční faktor 4 MeSH

BACKGROUND: The progressive myoclonic epilepsies (PME) are a heterogeneous group of disorders in which a specific diagnosis cannot be made in a subset of patients, despite exhaustive investigation. C9orf72 repeat expansions are emerging as an important causal factor in several adult-onset neurodegenerative disorders, in particular frontotemporal lobar degeneration and amyotrophic lateral sclerosis. An association with PME has not been reported previously. OBJECTIVE: To identify the causative mutation in a Belgian family where the proband had genetically unexplained PME. RESULTS: We report a 33-year old woman who had epilepsy since the age of 15 and then developed progressive cognitive deterioration and multifocal myoclonus at the age of 18. The family history suggested autosomal dominant inheritance of psychiatric disorders, epilepsy, and dementia. Thorough workup for PME including whole exome sequencing did not reveal an underlying cause, but a C9orf72 repeat expansion was found in our patient and affected relatives. Brain biopsy confirmed the presence of characteristic p62-positive neuronal cytoplasmic inclusions. CONCLUSION: C9orf72 mutation analysis should be considered in patients with PME and psychiatric disorders or dementia, even when the onset is in late childhood or adolescence.

• MeSH
• dospělí MeSH
• expanze repetic DNA * MeSH
• fenotyp MeSH
• genetická predispozice k nemoci MeSH
• lidé středního věku MeSH
• lidé MeSH
• mozek patologie   MeSH
• myoklonické epilepsie progresivní genetika   patologie   patofyziologie   psychologie   MeSH
• protein C9orf72 genetika   MeSH
• rodina MeSH
• rodokmen MeSH
• věk při počátku nemoci MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• C9orf72 protein, human MeSH Prohlížeč
• protein C9orf72 MeSH

Retrotransposons comprise a large portion of mammalian genomes. They contribute to structural changes and more importantly to gene regulation. The expansion and diversification of gene families have been implicated as sources of evolutionary novelties. Given the roles retrotransposons play in genomes, their contribution to the evolution of gene families warrants further exploration. In this study, we found a significant association between two major retrotransposon classes, LINEs and LTRs, and lineage-specific gene family expansions in both the human and mouse genomes. The distribution and diversity differ between LINEs and LTRs, suggesting that each has a distinct involvement in gene family expansion. LTRs are associated with open chromatin sites surrounding the gene families, supporting their involvement in gene regulation, whereas LINEs may play a structural role promoting gene duplication. Our findings also suggest that gene family expansions, especially in the mouse genome, undergo two phases. The first phase is characterized by elevated deposition of LTRs and their utilization in reshaping gene regulatory networks. The second phase is characterized by rapid gene family expansion due to continuous accumulation of LINEs and it appears that, in some instances at least, this could become a runaway process. We provide an example in which this has happened and we present a simulation supporting the possibility of the runaway process. Altogether we provide evidence of the contribution of retrotransposons to the expansion and evolution of gene families. Our findings emphasize the putative importance of these elements in diversification and adaptation in the human and mouse lineages.

• Klíčová slova
• LINE, LTR, SINE, gene families, retrotransposons, transposable elements,
• MeSH
• expanze repetic DNA MeSH
• genom lidský * MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• multigenová rodina * MeSH
• myši MeSH
• polymorfismus genetický MeSH
• retroelementy * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• retroelementy * MeSH