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

ABSTRACTHow genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs’ endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CUG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at 1-2 copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because; 1) Affected tissue is routinely removed during surgery; 2) The expanded CUG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and 3) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cell-specific manner. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in presymptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease.

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

Genes ◽

10.3390/genes12122006 ◽

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.

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Analyzing pre-symptomatic tissue to gain insights into the molecular and mechanistic origins of late-onset degenerative trinucleotide repeat disease

Nucleic Acids Research ◽

10.1093/nar/gkaa422 ◽

2020 ◽

Vol 48 (12) ◽

pp. 6740-6758 ◽

Cited By ~ 1

Author(s):

Yongjun Chu ◽

Jiaxin Hu ◽

Hanquan Liang ◽

Mohammed Kanchwala ◽

Chao Xing ◽

...

Keyword(s):

Gene Expression ◽

Late Stage ◽

Trinucleotide Repeat ◽

Late Onset ◽

Cell Monolayer ◽

Absolute Magnitude ◽

Corneal Dystrophy ◽

Matrix Gene ◽

Affected Tissue ◽

Symptomatic Tissue

Abstract How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs’ endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CTG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at one–two copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because: (i) Affected tissue is routinely removed during surgery; (ii) The expanded CTG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and (iii) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here, we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cells. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in pre-symptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease.

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GAA Trinucleotide Repeat Region Regulates M9/pMGA Gene Expression in Mycoplasma gallisepticum

Infection and Immunity ◽

10.1128/iai.68.2.871-876.2000 ◽

2000 ◽

Vol 68 (2) ◽

pp. 871-876 ◽

Cited By ~ 34

Author(s):

Li Liu ◽

Kevin Dybvig ◽

Victor S. Panangala ◽

Vicky L. van Santen ◽

Christopher T. French

Keyword(s):

Gene Expression ◽

Trinucleotide Repeat ◽

Mycoplasma Gallisepticum ◽

Avian Host ◽

Phase Variable ◽

Repeat Region ◽

Chromosomal Dna ◽

Promoter Regions ◽

Variable Expression ◽

Gaa Repeats

ABSTRACT Mycoplasma gallisepticum, the cause of chronic respiratory infections in the avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) associated with hemagglutination. Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes. The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlation between gene expression and the number of tandem GAA repeat motifs located upstream of the putative promoter. In this study, transposon Tn4001was used as a vector with the Escherichia coli lacZ gene as the reporter system to examine the role of the GAA repeats in M9/pMGA gene expression in M. gallisepticum. A 336-bp M9 gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligated with alacZ gene from E. coli, and inserted into the Tn4001-containing plasmid pISM2062. This construct was transformed into M. gallisepticum PG31. Transformants were filter cloned on agar supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) to monitor lacZ gene expression on the basis of blue/white color selection. Several cycles of filter cloning resulted in cell lineages in which lacZ gene expression alternated between the On and Off states in successive generations of progeny clones. The promoter regions of the M9-lacZ hybrid genes of individual progeny clones were amplified by PCR and sequenced. The only differences between the promoter regions of the blue and white colonies were in the number of GAA repeats. Clones that expressedlacZ had exactly 12 tandem copies of the GAA repeat. Clones that did not express lacZ invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats. Southern analysis of M. gallisepticum chromosomal DNA confirmed that the phase-variable expression of the lacZ reporter gene was not caused by Tn4001 transposition. These data strongly indicate that changes in the length of the GAA repeat region are responsible for regulating M9/pMGA gene expression.

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Trinucleotide repeat expansion length as a predictor of the clinical progression of Fuchs’ Endothelial Corneal Dystrophy

PLoS ONE ◽

10.1371/journal.pone.0210996 ◽

2019 ◽

Vol 14 (1) ◽

pp. e0210996 ◽

Cited By ~ 9

Author(s):

Yu Qiang Soh ◽

Gary Peh Swee Lim ◽

Hla Myint Htoon ◽

Xin Gong ◽

V. Vinod Mootha ◽

...

Keyword(s):

Trinucleotide Repeat ◽

Corneal Dystrophy ◽

Repeat Expansion ◽

Clinical Progression ◽

Trinucleotide Repeat Expansion

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RNA Sequencing of Human Peripheral Blood Cells Indicates Upregulation of Immune-Related Genes in Huntington's Disease

Frontiers in Neurology ◽

10.3389/fneur.2020.573560 ◽

2020 ◽

Vol 11 ◽

Author(s):

Miguel A. Andrade-Navarro ◽

Katja Mühlenberg ◽

Eike J. Spruth ◽

Nancy Mah ◽

Adrián González-López ◽

...

Keyword(s):

Gene Expression ◽

Huntington's Disease ◽

Huntington’S Disease ◽

Peripheral Blood ◽

Blood Cells ◽

Trinucleotide Repeat ◽

Neurodegenerative Disorder ◽

Gene Expression Signature ◽

Interferon Response ◽

Peripheral Blood Cells

Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a trinucleotide repeat expansion in the Huntingtin gene. As disease-modifying therapies for HD are being developed, peripheral blood cells may be used to indicate disease progression and to monitor treatment response. In order to investigate whether gene expression changes can be found in the blood of individuals with HD that distinguish them from healthy controls, we performed transcriptome analysis by next-generation sequencing (RNA-seq). We detected a gene expression signature consistent with dysregulation of immune-related functions and inflammatory response in peripheral blood from HD cases vs. controls, including induction of the interferon response genes, IFITM3, IFI6 and IRF7. Our results suggest that it is possible to detect gene expression changes in blood samples from individuals with HD, which may reflect the immune pathology associated with the disease.

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