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

Abstract

<jats:title>Abstract</jats:title> <jats:p>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.</jats:p>

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

“…The original RNA-seq study by Chu and others concluded that the TCF4 CTG TNR expansion increases the stability and thus the amount of expanded CTG repeat-including intronic RNAs in the corneal endothelium and causes comprehensive changes in splicing. No alterations in the overall expression of mature TCF4 mRNA was noted 28 . The study by Nikitina et al was a data article and no conclusions were made 27 .…”

“…To describe whether an increased CTG TNR affects the expression of different TCF4 transcripts, we performed a comprehensive analysis of two previously published RNA-seq datasets from the corneal endothelium of FECD patients with an expanded TCF4 CTG TNR and control groups without the repeat expansion 27 , 28 . The 2019 dataset generated by Nikitina et al includes 6 controls and 8 FECD patients with an expanded TCF4 CTG TNR 27 , and the 2020 dataset by Chu et al includes 9 controls and 6 FECD patients with an expanded TCF4 CTG TNR 28 . First, we evaluated how the levels of transcripts beginning from the CTG TNR region change in FECD.…”

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Figure 3 The expression of TCF4 alternative transcripts containing 5′ exons spliced to exon 4 is decreased in the cornea of FECD patients . Two independent FECD RNA-seq datasets—by Nikitina et al 27 (named 2019 in the figure) and Chu et al 28 (named 2020 in the figure)—were used to analyze the expression levels of TCF4 alternative 5′ exons in corneal endothelium. The expression levels of different TCF4 splicing events were quantified using the number of reads crossing the splice junction (shown above the graphs) normalized with the total number of spliced reads and multiplied by million.

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Figure 4 Expression of transcripts encoding TCF4-C isoform are downregulated, whereas transcripts encoding other TCF4 isoforms are upregulated in the cornea of FECD patients . Two independent FECD RNA-seq datasets—by Nikitina et al 27 (named 2019 in the figure) and Chu et al 28 (named 2020 in the figure)—were used to analyze the expression levels of TCF4 exons in corneal endothelium. The expression levels of ( A ) TCF4 isoforms and ( B ) internal exons were quantified using the number of reads ( A ) from transcripts encoding for the respective isoform (shown above the graphs) and ( B ) crossing the indicated splice junction (shown above the graphs).

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“…Detailed analysis of previously published FECD RNA-seq datasets 27 , 28 revealed that the levels of TCF4 transcripts containing alternative 5′ exons 4aI and 4aIII were reduced in the corneal endothelial cells of FECD patients with an expanded CTG TNR. These results support our findings that the TCF4 CTG TNR expansion reduces the activity of proximal downstream promoters linked to these 5′ exons also in human corneal endothelium.…”