scholarly journals Comparison of TCF4 repeat expansion length in corneal endothelium and leukocytes of patients with Fuchs endothelial corneal dystrophy

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260837
Author(s):  
Eric D. Wieben ◽  
Ross A. Aleff ◽  
Tommy A. Rinkoski ◽  
Keith H. Baratz ◽  
Shubham Basu ◽  
...  
Keyword(s):  
Corneal Endothelium ◽  
Southern Blotting ◽  
Primary Cultures ◽  
Corneal Dystrophy ◽  
Blood Leukocytes ◽  
Ctg Repeats ◽  
Long Read ◽  
Repeat Expansions ◽  
Transcription Factor 4 ◽  
Endothelial Tissue

Expansion of CTG trinucleotide repeats (TNR) in the transcription factor 4 (TCF4) gene is highly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Due to limitations in the availability of DNA from diseased corneal endothelium, sizing of CTG repeats in FECD patients has typically been determined using DNA samples isolated from peripheral blood leukocytes. However, it is non-feasible to extract enough DNA from surgically isolated FECD corneal endothelial tissue to determine repeat length based on current technology. To circumvent this issue, total RNA was isolated from FECD corneal endothelium and sequenced using long-read sequencing. Southern blotting of DNA samples isolated from primary cultures of corneal endothelium from these same affected individuals was also assessed. Both long read sequencing and Southern blot analysis showed significantly longer CTG TNR expansion (>1000 repeats) in the corneal endothelium from FECD patients than those characterized in leukocytes from the same individuals (<90 repeats). Our findings suggest that the TCF4 CTG repeat expansions in the FECD corneal endothelium are much longer than those found in leukocytes.

scholarly journals Lower Fractions of TCF4 Transcripts Spanning over the CTG18.1 Trinucleotide Repeat in Human Corneal Endothelium

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 2006
Author(s):  
Ida Maria Westin ◽  
Andreas Viberg ◽  
Berit Byström ◽  
Irina Golovleva
Keyword(s):  
Endothelial Cells ◽  
Corneal Endothelium ◽  
Trinucleotide Repeat ◽  
Late Onset ◽  
Corneal Dystrophy ◽  
Digital Pcr ◽  
Corneal Endothelial Cells ◽  
Transcription Start Sites ◽  
Gradual Loss ◽  
Transcription Factor 4

Fuchs’ endothelial corneal dystrophy (FECD) is a bilateral disease of the cornea caused by gradual loss of corneal endothelial cells. Late-onset FECD is strongly associated with the CTG18.1 trinucleotide repeat expansion in the Transcription Factor 4 gene (TCF4), which forms RNA nuclear foci in corneal endothelial cells. To date, 46 RefSeq transcripts of TCF4 are annotated by the National Center of Biotechnology information (NCBI), however the effect of the CTG18.1 expansion on expression of alternative TCF4 transcripts is not completely understood. To investigate this, we used droplet digital PCR for quantification of TCF4 transcripts spanning over the CTG18.1 and transcripts with transcription start sites immediately downstream of the CTG18.1. TCF4 expression was analysed in corneal endothelium and in whole blood of FECD patients with and without CTG18.1 expansion, in non-FECD controls without CTG18.1 expansion, and in five additional control tissues. Subtle changes in transcription levels in groups of TCF4 transcripts were detected. In corneal endothelium, we found a lower fraction of transcripts spanning over the CTG18.1 tract compared to all other tissues investigated.

scholarly journals Impact of TCF4 Repeat Number on Resolution of Corneal Edema after Descemet’s Stripping Only in Fuchs Dystrophy: A Pilot Study

Vision ◽  
2021 ◽  
Vol 5 (4) ◽  
pp. 47
Author(s):  
Natasha Spiteri ◽  
Nino Hirnschall ◽  
Katherine van Bysterveldt ◽  
Alec Lin Hou ◽  
Gregory Moloney ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Corneal Edema ◽  
Corneal Dystrophy ◽  
Partial Least Square ◽  
Least Square ◽  
Partial Least Square Regression ◽  
Ctg Repeats ◽  
Increased Risk ◽  
Transcription Factor 4 ◽  
Allele Repeat

Purpose: To investigate whether Fuchs endothelial corneal dystrophy (FECD) genotype, specifically transcription factor 4 (TCF4) CTG triplet repeat “load” predicts time to clearance following Descemet’s Stripping Only (DSO). Methods: This prospective, interventional trial was conducted on consecutive FECD patients undergoing DSO. Genetic analysis using patients’ saliva was performed to assess the extent of CTG expansion using short tandem repeat analysis, corroborated gel electrophoresis and Sanger sequencing. Polymerase chain reaction and bidirectional Sanger sequencing was undertaken. Partial least square regression and logistic regression modelling was used to evaluate the predictive power of TCF4 repeats on corneal clearance. Results: Of 11 eyes of 11 patients, 8 showed complete corneal clearance. For these 8 patients, mean TCF4 allele repeat was 24.8 (SD: 23.7, range: 11–63) and 63.4 (SD: 30.3; range: 11–97), respectively. In total, 9/11 (81.8%) had expanded CTG repeats (>40) in one allele. In cases with an allele repeat ≥80, there was a significantly increased risk of corneal non-clearance (odds ratio 18.2, p = 0.009). Conclusion: Whilst it was not possible to predict time to corneal clearance based on CTG repeats, there is a significant correlation between allele repeats and achievement of corneal clearance.

scholarly journals Accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION

2018 ◽  
Author(s):  
Arne De Roeck ◽  
Wouter De Coster ◽  
Liene Bossaerts ◽  
Rita Cacace ◽  
Tim De Pooter ◽  
...  
Keyword(s):  
Southern Blotting ◽  
Flow Cell ◽  
Current Data ◽  
Whole Genome ◽  
Genome Coverage ◽  
Base Calling ◽  
Length Estimation ◽  
Long Read ◽  
Repeat Expansions

AbstractTandem repeats (TRs) can cause disease through their length, sequence motif interruptions, and nucleotide modifications. For many TRs, however, these features are very difficult - if not impossible - to assess, requiring low-throughput and labor-intensive assays. One example is a VNTR in ABCA7 for which we recently discovered that expanded alleles strongly increase risk of Alzheimer’s disease. Here, we investigated the potential of long-read whole genome sequencing to surmount these challenges, using the high-throughput PromethION platform from Oxford Nanopore Technologies. To overcome the limitations of conventional base calling and alignment, we developed an algorithm to study the TR size and sequence directly on raw PromethION current data.We report the long-read sequencing of multiple human genomes (n = 11) using only a single sequencing run and flow cell per individual. With the use of fresh DNA extractions, DNA shearing to approximately 20kb and size selection, we obtained an average output of 70 gigabases (Gb) per flow cell, corresponding to a 21x genome coverage, and a maximum yield of 98 Gb (30x genome coverage). All ABCA7 VNTR alleles, including expansions up to 10,000 bases, were spanned by long sequencing reads, validated by Southern blotting. Classical approaches of TR length estimation suffered from low accuracy, low precision, DNA strand effects and/or inability to call pathogenic repeat expansions. In contrast, our novel NanoSatellite algorithm, which circumvents base calling by using dynamic time warping on raw PromethION current data, achieved more than 90% accuracy and high precision (5.6% relative standard deviation) of TR length estimation, and detected all clinically relevant repeat expansions. In addition, we identified alternative TR sequence motifs with high consistency, allowing determination of TR sequence and distinction of VNTR alleles with homozygous length.In conclusion, we validated the robustness of single-experiment whole genome long-read sequencing on PromethION, a prerequisite for application of long-read sequencing in the clinic. In addition, we outperformed Southern blotting, enabling improved characterization of the role of expanded ABCA7 VNTR alleles in Alzheimer’s disease, and opening new opportunities for TR research.

U-Net Convolutional Neural Network for Segmenting the Corneal Endothelium in a Mouse Model of Fuchs Endothelial Corneal Dystrophy

Cornea ◽  
2021 ◽  
Vol Publish Ahead of Print ◽  
Author(s):  
Naoki Okumura ◽  
Shohei Yamada ◽  
Takeru Nishikawa ◽  
Kaito Narimoto ◽  
Kengo Okamura ◽  
...  
Keyword(s):  
Neural Network ◽  
Mouse Model ◽  
Convolutional Neural Network ◽  
Corneal Endothelium ◽  
Corneal Dystrophy

scholarly journals Trinucleotide repeat expansion in the transcription factor 4 (TCF4) gene in Thai patients with Fuchs endothelial corneal dystrophy

Eye ◽  
2019 ◽  
Vol 34 (5) ◽  
pp. 880-885 ◽  
Author(s):  
Naoki Okumura ◽  
Vilavun Puangsricharern ◽  
Raina Jindasak ◽  
Noriko Koizumi ◽  
Yuya Komori ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Trinucleotide Repeat ◽  
Corneal Dystrophy ◽  
Repeat Expansion ◽  
Trinucleotide Repeat Expansion ◽  
Transcription Factor 4

scholarly journals Gene expression in the corneal endothelium of Fuchs endothelial corneal dystrophy patients with and without expansion of a trinucleotide repeat in TCF4

PLoS ONE ◽  
2018 ◽  
Vol 13 (7) ◽  
pp. e0200005 ◽  
Author(s):  
Eric D. Wieben ◽  
Ross A. Aleff ◽  
Xiaojia Tang ◽  
Krishna R. Kalari ◽  
Leo J. Maguire ◽  
...  
Keyword(s):  
Gene Expression ◽  
Corneal Endothelium ◽  
Trinucleotide Repeat ◽  
Corneal Dystrophy

scholarly journals The Fuchs corneal dystrophy-associated CTG repeat expansion in the TCF4 gene affects transcription from its alternative promoters

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Alex Sirp ◽  
Kristian Leite ◽  
Jürgen Tuvikene ◽  
Kaja Nurm ◽  
Mari Sepp ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Trinucleotide Repeat ◽  
Corneal Dystrophy ◽  
Protein Isoforms ◽  
Compensatory Mechanism ◽  
Alternative Promoters ◽  
Rna Seq ◽  
Increased Risk ◽  
Specific Manner ◽  
Transcription Factor 4

Abstract 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.

scholarly journals Human-specific tandem repeat expansion and differential gene expression during primate evolution

2019 ◽  
Vol 116 (46) ◽  
pp. 23243-23253 ◽  
Author(s):  
Arvis Sulovari ◽  
Ruiyang Li ◽  
Peter A. Audano ◽  
David Porubsky ◽  
Mitchell R. Vollger ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
Tandem Repeats ◽  
Sequence Data ◽  
Variable Number ◽  
Specific Expression ◽  
Sequence Composition ◽  
Transcription Profiles ◽  
Long Read ◽  
Repeat Expansions ◽  
Human Specific

Short tandem repeats (STRs) and variable number tandem repeats (VNTRs) are important sources of natural and disease-causing variation, yet they have been problematic to resolve in reference genomes and genotype with short-read technology. We created a framework to model the evolution and instability of STRs and VNTRs in apes. We phased and assembled 3 ape genomes (chimpanzee, gorilla, and orangutan) using long-read and 10x Genomics linked-read sequence data for 21,442 human tandem repeats discovered in 6 haplotype-resolved assemblies of Yoruban, Chinese, and Puerto Rican origin. We define a set of 1,584 STRs/VNTRs expanded specifically in humans, including large tandem repeats affecting coding and noncoding portions of genes (e.g., MUC3A, CACNA1C). We show that short interspersed nuclear element–VNTR–Alu (SVA) retrotransposition is the main mechanism for distributing GC-rich human-specific tandem repeat expansions throughout the genome but with a bias against genes. In contrast, we observe that VNTRs not originating from retrotransposons have a propensity to cluster near genes, especially in the subtelomere. Using tissue-specific expression from human and chimpanzee brains, we identify genes where transcript isoform usage differs significantly, likely caused by cryptic splicing variation within VNTRs. Using single-cell expression from cerebral organoids, we observe a strong effect for genes associated with transcription profiles analogous to intermediate progenitor cells. Finally, we compare the sequence composition of some of the largest human-specific repeat expansions and identify 52 STRs/VNTRs with at least 40 uninterrupted pure tracts as candidates for genetically unstable regions associated with disease.

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