Expansion of CTG18.1 Is Associated with Altered mRNA Processing
Transcriptomic analysis of endothelial tissue derived from individuals affected with FECD has previously suggested abnormal regulation of alternative pre-mRNA splicing in CTG18.1 expansion-positive tissue.15, 37 Therefore, we investigated whether signatures of differential splicing were present in cultured primary CECs. Total RNA was isolated from four FECD CTG18.1 expansion-positive and three FECD CTG18.1 expansion-negative CEC lines (CTG18.1 genotype listed in Table S6), in addition to four CEC lines derived from healthy control subjects. RT-PCR analysis was performed to investigate differential splicing of the three most robustly detected aberrant events observed in tissue derived from affected subjects: MBNL1 (MIM: 606516), MBNL2 (MIM: 607327), and NUMA1 (MIM: 164009).37 For each transcript analyzed, significantly different (p < 0.001) patterns of splicing were observed only in the FECD expansion-positive lines compared to FECD expansion-negative and unaffected control lines, supporting the hypothesis that abnormal regulation of mRNA processing is specific to CTG18.1-related pathology (Figure 4).
Figure 4 Altered Pre-mRNA Splicing Events Are Specific to CTG18.1 Expanded Corneal Endothelial Cells (CECs) Derived from FECD-Affected Subjects
(A) Reverse transcriptase (RT)-PCRs reactions are shown for three selected alternative splicing events investigated for the following transcripts: MBNL1, MBNL2, and NUMA1. Samples are grouped in the following categories; controls (lanes 1–4), FECD CTG18.1 expansion positive (lanes 5–8), and FECD CTG18.1 expansion negative (lanes 9–11).
(B) Schematic representations of RT-PCR-generated amplicons are provided for each transcript-specific reaction. Primer locations are denoted with arrows. The respective sizes of all amplified products are given.
(C) Percentage expression of amplicons of interest (A or B) relative to total amplified products, per reaction, are presented as a mean for each respective group (C, E, and NE). Error bars represent ±1 standard deviation. p values were calculated by one-way analysis of variance (ANOVA); ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ns, non-significant.