Generation and Characterization of Tcf4 CTG Trinucleotide Repeat Expansion Knock-in Mouse as a Model for Fuchs Endothelial Corneal Dystrophy
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE283422 | GEO | 2025/06/06
REPOSITORIES: GEO
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Project description:Fuchs endothelial corneal dystrophy (FECD) is a common cause for heritable visual loss in the elderly. Since the first description of an association between FECD and common polymorphisms situated within the transcription factor 4 (TCF4) gene, genetic and molecular studies have implicated an intronic CTG trinucleotide repeat (CTG18.1) expansion as a causal variant in the majority of FECD patients. To date, several non-mutually exclusive mechanisms have been proposed that drive and/or exacerbate the onset of disease. These mechanisms include (i) TCF4 dysregulation; (ii) toxic gain-of-function from TCF4 repeat-containing RNA; (iii) toxic gain-of-function from repeat-associated non-AUG dependent (RAN) translation; and (iv) somatic instability of CTG18.1. However, the relative contribution of these proposed mechanisms in disease pathogenesis is currently unknown. In this review, we summarise research implicating the repeat expansion in disease pathogenesis, define the phenotype-genotype correlations between FECD and CTG18.1 expansion, and provide an update on research tools that are available to study FECD as a trinucleotide repeat expansion disease. Furthermore, ongoing international research efforts to develop novel CTG18.1 expansion-mediated FECD therapeutics are highlighted and we provide a forward-thinking perspective on key unanswered questions that remain in the field.
| S-EPMC7988464 | biostudies-literature
Project description:PurposeTo analyze the expansion of CTG18.1 allele associated with Fuchs' corneal dystrophy (FCD) in our large cohort of late-onset FCD cases.MethodsCTG repeats within the CTG18.1 allele were estimated by short tandem repeat (STR) and triplet primed PCR (TP-PCR) assays in our large cohort of 574 late-onset FCD cases and 354 controls and large multigeneration familial cases. The age versus severity relationships were analyzed in FCD genotypes, namely, nonexpanded (N/N), monoallelic expansion (N/X), and biallelic expansion (X/X) with N ≤ 40 CTG monomers. The threshold for causality conferred by an expansion of CTG18.1 was identified by excluding the population of FCD cases who harbored an allele length equivalent to the maximum CTG monomers observed in the controls.ResultsThe expanded CTG18.1 for (CTG)n>40 showed a strong association (P = 1.56 × 10(-82)) with FCD. Importantly, we delineated the threshold of expansion to 103 CTG repeats above which the allele confers causality in 17.8% of FCD cases. Regression analyses demonstrated a significant correlation between disease severity and age in individuals who harbor either a monoallelic expansion or a biallelic expansion at (CTG) n > 40. These analyses helped predict FCD in two previously unaffected individuals based on their CTG18.1 expansion genotype.ConclusionsA monoallelic expansion of CTG18.1 contributes to increased disease severity and is causal at (CTG)n>103, whereas a biallelic expansion is sufficient to be causal for FCD at (CTG)n>40. This study highlights the largest contributory causal allele for FCD.
| S-EPMC4515948 | biostudies-literature
Project description:Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene.
| S-EPMC6611681 | biostudies-literature
Project description:Fuchs endothelial corneal dystrophy (FECD), the leading indication for corneal transplantation in the U.S., causes loss of corneal endothelial cells (CECs) and corneal edema leading to vision loss. FECD pathogenesis is linked to impaired response to oxidative stress and environmental ultraviolet A (UVA) exposure. Although UVA is known to cause nonapoptotic oxidative cell death resulting from iron-mediated lipid peroxidation, ferroptosis has not been characterized in FECD. We investigated the roles of genetic background and UVA exposure in causing CEC degeneration in FECD. Using ungenotyped FECD patient surgical samples, we found increased levels of cytosolic ferrous iron (Fe2+) and lipid peroxidation in end-stage diseased tissues compared with healthy controls. Using primary and immortalized cell cultures modeling the TCF4 intronic trinucleotide repeat expansion genotype, we found altered gene and protein expression involved in ferroptosis compared to controls including elevated levels of Fe2+, basal lipid peroxidation, and the ferroptosis-specific marker transferrin receptor 1. Increased cytosolic Fe2+ levels were detected after physiologically relevant doses of UVA exposure, indicating a role for ferroptosis in FECD disease progression. Cultured cells were more prone to ferroptosis induced by RSL3 and UVA than controls, indicating ferroptosis susceptibility is increased by both FECD genetic background and UVA. Finally, cell death was preventable after RSL3 induced ferroptosis using solubilized ubiquinol, indicating a role for anti-ferroptosis therapies in FECD. This investigation demonstrates that genetic background and UVA exposure contribute to iron-mediated lipid peroxidation and cell death in FECD, and provides the basis for future investigations of ferroptosis-mediated disease progression in FECD.
| S-EPMC11470242 | biostudies-literature
Project description:PurposeCTG trinucleotide repeat (TNR) expansion is frequently found in transcription factor 4 (TCF4) in Fuchs' endothelial corneal dystrophy (FECD), though the effect of TNR expansion on FECD pathophysiology remains unclear. The purpose of this study was to evaluate the effect of TNR expansion on TCF4 expression in corneal endothelium of patients with FECD.MethodsPeripheral blood DNA and Descemet membrane with corneal endothelium were obtained from 203 German patients with FECD. The CTG TNR repeat length in TCF4 was determined by short tandem repeat (STR) assays and Southern blotting using genomic DNA. Genotyping of rs613872 in TCF4 was performed by PCR. TCF4 mRNA levels in corneal endothelium were evaluated by quantitative PCR using three different probes. Control corneal endothelial samples were obtained from 35 non-FECD subjects.ResultsThe STR assay and Southern blotting showed that 162 of the 203 patients with FECD (80%) harbored CTG trinucleotide repeat lengths larger than 50. Quantitative PCR using all three probes demonstrated that TCF4 mRNA is significantly upregulated in the corneal endothelium of patients with FECD, regardless of the presence of TNR expansion. However, the length of the TNR tended to show a positive correlation with TCF4 expression level. No correlation was shown between the genotype of TCF4 SNP, rs613872, and the level of TCF4 expression.ConclusionsOur findings showed that TCF4 mRNA is upregulated in the corneal endothelium of patients with FECD. Further studies on the effects of TCF4 upregulation on corneal endothelial cell function will aid in understanding the pathophysiology of FECD.
| S-EPMC6392475 | biostudies-literature
Project description:PurposeWe tested the association between two intronic polymorphisms (CTG18.1 and rs613872) in TCF4 and Fuchs' endothelial corneal dystrophy (FECD), and analyzed their segregation patterns in families.MethodsWe recruited 120 unrelated Caucasian subjects with FECD and 100 controls. Available family members of probands were recruited. Genotyping of the single nucleotide polymorphism (SNP) rs613872 was performed using Sanger sequencing or real-time allelic discrimination assay. The trinucleotide repeat polymorphism, CTG18.1, was genotyped using a combination of short tandem repeat assay and triplet repeat primed PCR assay. The cytosine-thymine-guanine (CTG) repeat length of ≥40 was classified as an expanded CTG18.1 allele. Association of the two loci with FECD was evaluated. Segregation in 29 families was examined.ResultsThe two polymorphisms are in linkage disequilibrium (r(2) = 0.65 in cases and 0.31 in controls). Significant associations were found between FECD and rs613872 (P = 3.1 × 10(-17)), expanded CTG18.1 allele (P = 6.5 × 10(-25)), and their haplotypes (P = 5.9 × 10(-19)). The odds ratio (OR) of each copy of the rs613872 G allele for FECD was estimated to be 9.5 (95% confidence interval [CI], 5.1-17.5). The OR of each copy of the CTG18.1 expanded allele was estimated to be 32.3 (95% CI, 13.4-77.6). The expanded CTG 18.1 allele cosegregated with the trait in 52% (15/29) of families with complete penetrance and 10% (3/29) with incomplete penetrance.ConclusionsWe report, to our knowledge, the first independent replication of the expanded CTG 18.1 allele conferring significant risk for FECD (>30-fold increase). The expanded allele cosegregates with the trait with complete penetrance in a majority of families, but we also document cases of incomplete penetrance.
| S-EPMC3880006 | biostudies-literature
Project description:The CTG trinucleotide repeat (TNR) expansion in Transcription factor 4 (TCF4) intron 3 is the main cause of Fuchs' endothelial corneal dystrophy (FECD) and may confer an increased risk of developing bipolar disorder (BD). Usage of alternative 5' exons for transcribing the human TCF4 gene results in numerous TCF4 transcripts which encode for at least 18 N-terminally different protein isoforms that vary in their function and transactivation capability. Here we studied the TCF4 region containing the CTG TNR and characterized the transcription initiation sites of the nearby downstream 5' exons 4a, 4b and 4c. We demonstrate that these exons are linked to alternative promoters and show that the CTG TNR expansion decreases the activity of the nearby downstream TCF4 promoters in primary cultured neurons. We confirm this finding using two RNA sequencing (RNA-seq) datasets of corneal endothelium from FECD patients with expanded CTG TNR in the TCF4 gene. Furthermore, we report an increase in the expression of various other TCF4 transcripts in FECD, possibly indicating a compensatory mechanism. We conclude that the CTG TNR affects TCF4 expression in a transcript-specific manner both in neurons and in the cornea.
| S-EPMC7595208 | biostudies-literature
Project description:PurposeIn the United States, 70% of Fuchs' endothelial corneal dystrophy (FECD) cases are caused by an intronic trinucleotide repeat expansion in the TCF4 gene. CUG repeat RNA transcripts from this expansion accumulate as nuclear foci in the corneal endothelium. In this study, we sought to detect foci in other anterior segment cell types and assess their molecular impact.MethodsWe examined CUG repeat RNA foci appearance, expression of downstream affected genes, gene splicing, and TCF4 RNA expression in corneal endothelium, corneal stromal keratocytes, corneal epithelium, trabecular meshwork cells, and lens epithelium.ResultsCUG repeat RNA foci, the hallmark of FECD in corneal endothelium (found in 84% of endothelial cells), are less detectable in trabecular meshwork cells (41%), much less prevalent in stromal keratocytes (11%) or corneal epithelium (4%), and absent in lens epithelium. With few exceptions including mis-splicing in the trabecular meshwork, differential gene expression and splicing changes associated with the expanded repeat in corneal endothelial cells are not observed in other cell types. Expression of the TCF4 transcripts including full-length isoforms containing the repeat sequence at the 5' end is much higher in the corneal endothelium or trabecular meshwork than in the corneal stroma or corneal epithelium.ConclusionsExpression of the CUG repeat containing TCF4 transcripts is higher in the corneal endothelium, likely contributing to foci formation and the large molecular and pathologic impact on those cells. Further studies are warranted to examine any glaucoma risk and impact of the observed foci in the trabecular meshwork of these patients.
| S-EPMC10204776 | biostudies-literature
Project description:Fuchs endothelial corneal dystrophy (FECD) is a common, familial disease of the corneal endothelium and is the leading indication for corneal transplantation. Variation in the transcription factor 4 (TCF4) gene has been identified as a major contributor to the disease. We tested for an association between an intronic TGC trinucleotide repeat in TCF4 and FECD by determining repeat length in 66 affected participants with severe FECD and 63 participants with normal corneas in a 3-stage discovery/replication/validation study. PCR primers flanking the TGC repeat were used to amplify leukocyte-derived genomic DNA. Repeat length was determined by direct sequencing, short tandem repeat (STR) assay and Southern blotting. Genomic Southern blots were used to evaluate samples for which only a single allele was identified by STR analysis. Compiling data for 3 arms of the study, a TGC repeat length >50 was present in 79% of FECD cases and in 3% of normal controls cases (p<0.001). Among cases, 52 of 66 (79%) subjects had >50 TGC repeats, 13 (20%) had <40 repeats and 1 (2%) had an intermediate repeat length. In comparison, only 2 of 63 (3%) unaffected control subjects had >50 repeats, 60 (95%) had <40 repeats and 1 (2%) had an intermediate repeat length. The repeat length was greater than 1000 in 4 FECD cases. The sensitivity and specificity of >50 TGC repeats identifying FECD in this patient cohort was 79% and 96%, respectively Expanded TGC repeat was more specific for FECD cases than the previously identified, highly associated, single nucleotide polymorphism, rs613872 (specificity = 79%). The TGC trinucleotide repeat expansion in TCF4 is strongly associated with FECD, and a repeat length >50 is highly specific for the disease This association suggests that trinucleotide expansion may play a pathogenic role in the majority of FECD cases and is a predictor of disease risk.
| S-EPMC3504061 | biostudies-literature
Project description:PurposeTo evaluate the association of single nucleotide polymorphisms (SNPs) and the intronic expansion of a trinucleotide repeat (TNR) in the TCF4 gene with Fuchs endothelial corneal dystrophy (FECD) in a Thai population.MethodsIn total, 54 Thai FECD patients and 54 controls were recruited for the study. Five SNPs (rs613872, rs2123392, rs17089887, rs1452787, and rs1348047), previously reported to be associated with FECD, were genotyped by direct sequencing. The repeat length was determined by direct sequencing of PCR-amplified DNA (a short tandem repeat; STR assay) and by triplet repeat primed PCR (TP-PCR).ResultsOnly one of the 54 patients with FECD harboured rs613872 (1.9%). Four SNPs (rs2123392, rs17089887, rs1452787, and rs1348047), which are not rare polymorphisms in the Thai population, were found in approximately half of the patients. Of the 54 patients, 21 (1 homozygous and 20 heterozygous patients; 39%) harboured a TNR ≥ 40, while 33 patients (61%) harboured a TNR < 40.ConclusionsThe association of TNR expansion in TCF4 with FECD is shown for the first time in the Thai population. The intronic TNR expansion identified in various ethnic groups underlines the importance of expansion as a potent pathophysiological cause of FECD.
| S-EPMC7182560 | biostudies-literature