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
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
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
BACKGROUND: Huntington disease (HD) is a fatal neurodegenerative disorder involving reduced muscle coordination, mental and behavioral changes, and testicular degeneration. In order to further clarify the decreased fertility and penetration ability of the spermatozoa of transgenic HD minipig boars (TgHD), we applied a set of mitochondrial metabolism (MM) parameter measurements to this promising biological material, which can be collected noninvasively in longitudinal studies. OBJECTIVE: We aimed to optimize methods for MM measurements in spermatozoa and to establish possible biomarkers of HD in TgHD spermatozoa expressing the N-terminal part of mutated human huntingtin. METHODS: Semen samples from 12 TgHD and wild-type animals, aged 12-65 months, were obtained repeatedly during the study. Respiration was measured by polarography, MM was assessed by the detection of oxidation of radiolabeled substrates (mitochondrial energy-generating system; MEGS), and the content of the oxidative phosphorylation system subunits was detected by Western blot. Three possibly interfering factors were statistically analyzed: the effect of HD, generation and aging. RESULTS: We found 5 MM parameters which were significantly diminished in TgHD spermatozoa and propose 3 specific MEGS incubations and complex I-dependent respiration as potential biomarkers of HD in TgHD spermatozoa. CONCLUSIONS: Our results suggest a link between the gain of toxic function of mutated huntingtin in TgHD spermatozoa and the observed MM and/or glycolytic impairment. We determined 4 biomarkers useful for HD phenotyping and experimental therapy monitoring studies in TgHD minipigs.
- MeSH
- dýchání MeSH
- geneticky modifikovaná zvířata MeSH
- Huntingtonova nemoc komplikace genetika patologie MeSH
- kyseliny trikarboxylové metabolismus MeSH
- lidé MeSH
- miniaturní prasata MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie metabolismus MeSH
- mutace genetika MeSH
- oxidativní fosforylace MeSH
- prasata MeSH
- protein huntingtin genetika MeSH
- pyruvátdehydrogenasový komplex metabolismus MeSH
- sperma metabolismus MeSH
- spermie metabolismus patologie MeSH
- trinukleotidové repetice genetika MeSH
- věkové faktory MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders currently associated with 27 genes. The most frequent types are caused by expansions in coding CAG repeats. The frequency of SCA subtypes varies among populations. We examined the occurrence of rare SCAs, SCA8, SCA12, SCA17 and dentatorubro-pallidoluysian atrophy (DRPLA), in the Czech population from where the data were missing. We analyzed causal gene expansions in 515 familial and sporadic ataxic patients negatively tested for SCA1-3 and SCA6-7. Pathogenic SCA8 and SCA17 expansions were identified in eight and five patients, respectively. Tay-Sachs disease was later diagnosed in one patient with an SCA8 expansion and the diagnosis of multiple sclerosis (MS) was suspected in two other patients with SCA8 expansions. These findings are probably coincidental, although the participation of SCA8 expansions in the susceptibility to MS and disease progression cannot be fully excluded. None of the patients had pathogenic SCA12 or DRPLA expansions. However, three patients had intermediate SCA12 alleles out of the normal range with 36 and 43 CAGs. Amyotrophic lateral sclerosis (ALS) was probable in the patient with 43 CAGs. This coincidence is remarkable, especially in the context with the recently identified predisposing role of longer SCA2 alleles in ALS. Five families with SCA17 represent a significant portion of ataxic patients and this should be reflected in the diagnostics of SCAs in the Czech population. SCA8 expansions must be considered after careful clinical evaluation.
- MeSH
- amyotrofická laterální skleróza genetika MeSH
- frekvence genu MeSH
- genotyp MeSH
- lidé MeSH
- mutační analýza DNA MeSH
- myoklonické epilepsie progresivní * epidemiologie genetika MeSH
- proteiny nervové tkáně genetika MeSH
- roztroušená skleróza MeSH
- spinocerebelární ataxie * klasifikace epidemiologie genetika MeSH
- trinukleotidové repetice genetika MeSH
- zdraví rodiny MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Huntington's disease (HD) is caused by the expansion of the number of CAG repeats on the chromosome 4p16.3, which results in elongated glutamine tract of huntingtin. The purpose of this work was to examine the interaction between the normal and mutant alleles of this gene and their effect on the clinical onset of HD. We hypothesized that in patients with identical number of CAG repeats within the mutant allele, the age of onset of HD is influenced by the number of CAG repeats within the normal allele. We analyzed the relations between the number of CAG repeats within the normal and mutant alleles, the age at HD onset, and the character of initial symptoms in 468 patients with clinically expressed HD. Although the Cox regression coefficient of 0.15 was significant (P < 0.0001), the regression model explained only 28% of the variance of the age at onset related to the effect of the number of CAG repeats within normal allele. Within the groups of patients with the same number of CAG repeats of the mutant allele, number of CAG repeats of the normal allele was found uncorrelated to the age at onset. Furthermore, when analyzing subgroups of patients with the same allelic composition on both alleles, we failed to observe any correlation with the age at the onset. Our analysis gives no corroboration to the idea of a normal allele having a share in the modification of the age at HD onset. We believe that with the current state of knowledge it is not possible to devise a mathematical model for HD onset prediction because too many entirely unknown modifying factors are still involved.
- MeSH
- dospělí MeSH
- frekvence genu MeSH
- genotyp MeSH
- Huntingtonova nemoc genetika MeSH
- jaderné proteiny genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- logistické modely MeSH
- mladiství MeSH
- mladý dospělý MeSH
- proporcionální rizikové modely MeSH
- proteiny nervové tkáně genetika MeSH
- senioři MeSH
- trinukleotidové repetice genetika MeSH
- věk při počátku nemoci MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Myotonic dystrophy type 1 is caused by the expansion of a CTG repeat in the 3' UTR of the DMPK gene. A length exceeding 50 CTG triplets is pathogenic. Intermediate alleles with 35-49 triplets are not disease-causing but show instability in intergenerational transmissions. We report on the identification of multiple patients with different patterns of CCG and CTC interruptions in the DMPK CTG repeat tract that display unique intergenerational instability. In patients bearing interrupted expanded alleles, the location of the interruptions changed dramatically between generations and the repeats tended to contract. The phenotype for these patients corresponded to the classical form of the disease, but in some cases without muscular dystrophy and possibly with a later onset than expected. Symptomatic patients bearing interrupted intermediate length repeat tracts were also identified, although the role of the interruptions in their phenotype remains unclear. The identification of interruptions in the DMPK repeat has important consequences for molecular genetic testing where they can lead to false negative conclusions.
- MeSH
- delece genu MeSH
- dospělí MeSH
- frekvence genu MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mutační analýza DNA MeSH
- nestabilita genomu MeSH
- protein-serin-threoninkinasy genetika MeSH
- rodina MeSH
- rodokmen MeSH
- sekvence nukleotidů MeSH
- studie případů a kontrol MeSH
- trinukleotidové repetice genetika MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
Huntingtonova choroba (HD) je dědičná neurodegenerativní porucha. Vztah mezi progresí příznaků a počtem CAG opakování u zmutovaného genu IT15 nebyl prozatím z hlediska pohlaví probádán. Cílem bylo zjistit všechny korelace mezi počtem trinukleotidových repeticí CAG u genu IT15 a věkem nástupu HD, symptomy a progresí nemoci, jakož i případné rozdíly mezi výsledky v závislosti na pohlaví. Materiál a metodika: 41 pacientů (23 žen a 18 mužů) s mutací způsobující HD se podrobilo neurologickému hodnocení podle Jednotné hodnotící škály Huntingtonovy choroby (Unified Huntington's Disease Rating Scale, UHDRS). počet Cag repetic na exonu 1 genu IT15 byl stanoven metodou amplifikace DNA pomocí polymerázové řetězové reakce (PCR) a porovnáním výsledného produktu se standardní DNA. Výsledky: Signifikantní korelace mezi výsledky na škále UHDRS, počtem CAG opakování a časem propuknutí choroby byly objeveny pouze u žen. Korelace mezi počtem CAG opakování a věkem nástupu nemoci byla objevena jak u žen, tak u mužů. Závěr: Výsledky naznačují korelaci mezi klinickým stavem pacientek s HD a délkou CAG repetice. To by mohlo souviset s přítomností dalšího faktoru u žen.
- MeSH
- dědičné degenerativní poruchy nervového systému etiologie genetika MeSH
- genetický výzkum MeSH
- Huntingtonova nemoc etiologie genetika MeSH
- klinický obraz nemoci MeSH
- lidé MeSH
- polymerázová řetězová reakce metody využití MeSH
- rozložení podle pohlaví MeSH
- statistika jako téma metody MeSH
- trinukleotidové repetice genetika MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- MeSH
- Alzheimerova nemoc diagnóza etiologie MeSH
- amyotrofická laterální skleróza diagnóza etiologie patologie MeSH
- demence diagnóza etiologie klasifikace MeSH
- lidé MeSH
- nemoci nervového systému diagnóza etiologie patologie MeSH
- neurodegenerativní nemoci diagnóza etiologie patologie MeSH
- Pickova nemoc mozku diagnóza etiologie patologie MeSH
- prionové nemoci diagnóza etiologie klasifikace MeSH
- synukleiny izolace a purifikace metabolismus škodlivé účinky MeSH
- trinukleotidové repetice fyziologie genetika MeSH
- Check Tag
- lidé MeSH
