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. Author manuscript; available in PMC: 2024 Jun 1.
Published in final edited form as: Exp Eye Res. 2023 May 9;231:109499. doi: 10.1016/j.exer.2023.109499

Dysregulation of DNA repair genes in Fuchs Endothelial Corneal Dystrophy

Shazia Ashraf 1,2,3,*, Neha Deshpande 1,2,3,*, Shivakumar Vasanth 1,2,3, Geetha Melangath 1,2,3, Raymond Jeff Wong 1,2,3, Yan Zhao 2,3, Marianne Price 4, Francis Price Jr 4, Ula V Jurkunas 1,2,3
PMCID: PMC10246500  NIHMSID: NIHMS1900859  PMID: 37169279

Abstract

Fuchs Endothelial Corneal Dystrophy (FECD), a late-onset oxidative stress disorder, is the most common cause of corneal endothelial degeneration and is genetically associated with CTG repeat expansion in TCF4. We previously reported accumulation of nuclear (nDNA) and mitochondrial (mtDNA) damage in FECD. Specifically, mitochondrial DNA damage was a prominent finding in development of disease in the ultraviolet-A (UVA) induced FECD mouse model. We hypothesize that an aberrant DNA repair may contribute to the increased DNA damage seen in FECD. We analyzed differential expression profiles of 84 DNA repair genes by real-time PCR arrays using Human DNA Repair RT-Profiler plates using cDNA extracted from Descemet’s membrane-corneal endothelium (DM-CE) obtained from FECD patients with expanded (>40) or non-expanded (<40) intronic CTG repeats in Transcription Factor 4 (TCF4) gene and from age-matched normal donors. Change in mRNA expression of <0.5- or >2.0-fold in FECD relative to normal was set as cutoff for down- or upregulation. Downregulated mitochondrial genes were further validated using the UVA-based mouse model of FECD. FECD specimens exhibited downregulation of 9 genes and upregulation of 8 genes belonging to the four major DNA repair pathways, namely, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and double strand break (DSB) repair, compared to normal donors. MMR gene MSH2 and BER gene POLB were preferentially upregulated in expanded FECD. BER genes LIG3 and NEIL2, DSB repair genes PARP3 and TOP3A, NER gene XPC, and unclassified pathway gene TREX1, were downregulated in both expanded and non-expanded FECD. MtDNA repair genes, Lig3, Neil2, and Top3a, were also downregulated in the UVA-based mouse model of FECD. Our findings identify impaired DNA repair pathways that may play an important role in DNA damage due to oxidative stress as well as genetic predisposition noted in FECD.

Keywords: Fuchs Endothelial Corneal Dystrophy, TCF4, CTG repeat expansion, oxidative stress, mitochondrial DNA damage, DNA repair

1. Introduction

Fuchs endothelial corneal dystrophy (FECD) is a complex age-related disorder manifesting as progressive loss of corneal endothelial cells due to life-long exposure of the human cornea to various stresses. These include environmental as well as intracellular stresses which cause oxidative stress build-up causing DNA damage and mitochondrial dysfunction (Benischke et al., 2017; Jurkunas et al., 2010) all of which culminates into the pathology of FECD. In FECD, corneal endothelial (CE) cell loss and abnormal extracellular matrix (ECM) deposition (i.e., guttae) lead to corneal edema and blindness (Jurkunas, 2018; Jurkunas et al., 2010). In the United States and Europe, the prevalence of the disease is estimated to be 4–5% among persons over the age of 40 years (Zoega et al., 2006). FECD is a bilateral, genetically heterogeneous disease with most genetic cases associated with an increase in the intronic trinucleotide CTG repeat (>40) in the TCF4 gene (Wieben et al., 2012). Corneal transplantation is the only treatment modality. There are no pharmacologic treatments for FECD, and, thus, there is a significant unmet need to improve the understanding of FECD pathogenesis, as well as to develop therapeutic measures to prevent the need for corneal transplantation.

Human corneal endothelial cells (HCEnC) are terminally differentiated post-mitotic cells that exhibit a high rate of metabolism and mitochondrial activity. HCEnCs are responsible for pumping excess ions and water from the stroma into the anterior chamber. Therefore, HCEnCs inherently exhibit high metabolic levels, intracellular reactive oxygen species (ROS), and DNA damage exacerbated by their lifelong exposure to ultraviolet light. Since these cells possess limited regenerative capacity and are susceptible to oxidized DNA accumulation over time, the DNA repair mechanisms must be efficient in protecting the cells from oxidative stress (Fig. 1A). Our group had reported the involvement of oxidative stress in the pathobiology of FECD, demonstrated by the presence of 8 oxoguanosine, the most common DNA lesions incited by oxidative stressors such as ROS (Jurkunas et al., 2010). Ex vivo FECD specimens harbored extensive 8 oxoguanosine lesions that were localized in the mitochondria (Jurkunas et al., 2010). We further showed by long amplification quantitative PCR that Fuchs specimens harbored significantly greater mtDNA and nDNA damage than age matched healthy corneal endothelia (Jurkunas et al., 2010).

Figure 1. PCR array for DNA repair genes in normal and FECD specimens.

Figure 1.

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(A). The schematic shows the pathogenesis of FECD via the effect of increased ROS in the corneal endothelium. Solid arrow represents pathway that has been elucidated by our laboratory where uncontrolled ROS leads to oxidative stress resulting in mitochondrial fragmentation, nuclear and mitochondrial DNA damage, and apoptosis. The mechanism of the pathology driven by genetic determinants has not been sufficiently deciphered. We hypothesize that oxidative stress disrupts the DNA damage response mechanism, resulting in increased DNA lesions including microsatellite instability such as the expansion of CTG repeats within TCF4. (B) Table representing age and sex status in normal donors and FECD corneal specimens used in this study. (C) Workflow of sample preparation and DNA repair RT-PCR array setup.

Our recently published mouse model of FECD showed that following exposure to an environmental oxidative stressor-UVA light, which represents 95% of the incident solar UV radiations (Zinflou and Rochette, 2017), the corneal endothelia of both male and female mice showed characteristic FECD phenotype noted by the progressive loss of hexagonal morphology and appearance of enlarged cells, correlating to a significant time-dependent decrease in corneal endothelial cell (CEnC) count compared to the unexposed control eye. Furthermore, we showed that UVA-light-induced mtDNA damage occurs early in both males and females, however, it shows a second wave of damage in females at 1 month and stays significantly greater than males up to 3 months post-exposure, which also corresponds to a worse phenotype seen in females (Liu et al., 2020). There are four major DNA pathways responsible for the repair of DNA damage lesions: a) base-excision repair (BER) is involved in the removal of oxidized DNA bases induced by genotoxicants, predominantly ROS; b) nucleotide excision repair (NER) is involved in the repair of bulky lesions generated by chemicals or UV light; c) double-strand breaks (DSB) repair, as the name suggests, repairs double-stranded apurinic gaps in the DNA induced by ionizing radiation or metabolic malfunction and are repaired by either non-homologous DNA end joining or homologous recombination; d) mismatch repair (MMR) is a conserved pathway that repairs base–base mismatches and small insertion or deletions caused by misincorporation errors during DNA replication (Vechtomova et al., 2021).

To identify the cause of increased DNA damage in FECD, we sought to investigate if a deficiency in the DNA repair system fails to repair the ROS-induced DNA damage and may cause the accumulation of toxic DNA lesions in the disease. We hypothesize that the dysregulation of DNA repair mechanisms might contribute to a) a differential expression of a subset of DNA repair genes in FECD with expanded trinucleotide CTG repeats in TCF4; and b) an increased mtDNA damage seen in FECD. To determine the repair genes differentially expressed in FECD compared to normal corneal endothelia, we isolated RNA from DM-CE obtained either from FECD patient specimens post-Descemet’s membrane endothelial keratoplasty (DMEK) or normal cadaveric donor corneas. RNA was used to generate cDNA, which was then subjected to a real-time PCR array of 84 DNA repair genes. We determined the number of CTG repeats in the TCF4 gene in an additional cohort of FECD as either those harboring TCF4 with intronic CTG repeats greater than 40 and identifying as “with expanded CTG repeats” (FECD X) or those harboring TCF4 with intronic CTG repeats less than 40 and identifying as “with non-expanded CTG repeats” (FECD NX). We identified genes that were preferentially upregulated in FECD specimens harboring expanded TCF4 repeats and a sub-set of genes that were downregulated in FECD irrespective of their TCF4 repeats status. Interestingly, 3 mtDNA repair genes that were downregulated in FECD specimens were also found to be depleted in mouse CE after UVA irradiation.

2. Materials and Methods

2.1. Human Tissue Samples

This study was conducted according to the tenets of the Declaration of Helsinki and approved by the Massachusetts Eye and Ear Institutional Review Board. Written and informed consent was obtained from patients undergoing surgical treatment for FECD. Post DMEK, the diseased DM-CE tissue surgically removed from FECD patients was immediately placed in storage medium (Optisol-GS; Bausch & Lomb) at 4 °C, while normal donor corneas were purchased from Eversight eye bank, Illinois. We used the previously published criteria to determine tissue suitability, where average preservation time was between 2.5 and 8 days for normal and FECD specimens (Bitar et al., 2012; Joyce and Zhu, 2004; Jurkunas et al., 2010; Jurkunas et al., 2008; Miyai et al., 2019). RT-PCR array was performed using n=3 normal (age range 68–75 years) and n=6 age-matched FECD specimens. Differentially expressed genes identified from the RT-PCR array were further validated in additional cohorts of age-matched (a) non-stratified FECD (n=5) and normal (n=4), and (b) stratified FECD specimens (n=4 FECD X and n=3 FECD NX) and normal (n=4) samples. All tissues were snap-frozen in liquid nitrogen and stored at −80°C before further processing. For details of human specimens used in the PCR array, refer Fig. 1B and for those used in the validations, refer Supplementary Tables 3 and 4.

2.2. CTG repeat expansion in TCF4 in blood samples from FECD individuals

The number of repeats in CTG18.1 in the TCF4 gene of FECD individuals and normal donor corneas was estimated by short tandem repeat (STR) and triplet-primed PCR (TPPCR) based fragment analysis as previously established(Vasanth et al., 2015) (Fig. 1C). For CTG repeat size estimation, genomic DNA was isolated either from the peripheral blood mononuclear cells (PBMCs) obtained from FECD cases or from half of the normal donor cornea tissue obtained from cadaveric donors. The fragment analysis was performed on data generated by a ABI3730 genetic analyzer in the Genomics Core Facility of Mass Eye and Ear. The literature on FECD suggests that a repeat length of 40 or more CTG repeats is accepted as an expanded allele associated with FECD (Wieben et al., 2012). Accordingly, the FECD patients were stratified as either those with mono- or bi-allelic CTG repeats greater than 40 (expanded) or those with bi-allelic CTG repeats less than 40 (non-expanded) in TCF4. The specimens from FECD patients with expanded CTG repeats were designated as FECD X, while those with non-expanded CTG repeats as FECD NX.

2.3. DNA repair real-time PCR array and gene validation in FECD and normal specimens

To determine the repair genes differentially expressed in FECD compared to normal corneal endothelia we isolated RNA from DM-CE obtained either from normal donor corneas or from FECD patients who underwent DMEK using Trizol. Normal or FECD specimens were incubated in 500μl Trizol at room temperature and homogenized by intermittent vortexing for 10 minutes to induce lysis. After a quick spin, 200μl of chloroform was added and vortexed again. The samples were spun at 10,000g for 8 minutes, and the aqueous phase was transferred to a new 1.5ml tube (200–250μl). After adding 250μl of 75% ethanol, samples were mixed by inversion and loaded onto RNeasy Micro prep columns (RNeasy micro kit, #74034, Qiagen). DNase digest was performed for 15 min after RW1 wash. RNA was eluted in 2X 16μl. RNA from 3 normal, 3 FECD X, and 3 FECD NX specimens was used to prepare cDNA which was subjected to a real-time PCR array (RT2 Profiler™ PCR Array Human DNA Repair, GeneGlobe ID - PAHS-042Z, Qiagen) consisting of 84 human DNA repair genes (Fig. 1C; Supplementary Table 1, 2). For cDNA synthesis, 50ng (~15ul elute) of RNA from normal or FECD samples was used with an iScript (Bio-Rad) cDNA synthesis kit, and synthesis was performed per manufacturer protocol. Pre-amplification with DNA repair array primer mix was performed using 12.5μl of Platinum SuperFi 2X master mix, 2.5μl of 0.5X pre-amp TaqMan primer mix for all genes, and 10μl of cDNA. Real-time PCR was performed using 2X Kapa Probe Fast master mix (#KK4703, Roche). GAPDH was used as an internal control for the RT-PCR arrays. Relative gene expression was expressed as mean ± standard error (SE) of 2(−dCT) of FECD normalized to that of normal samples, where 2(−dCT) = fold change of gene expression normalized to GAPDH. A relative gene expression threshold of above 2.0- or below 0.5-fold was set to identify genes that were either up- or downregulated respectively in FECD relative to normal samples. The TaqMan assays used for RT-PCR validation of all differentially expressed human genes in FECD vs normal samples are listed in Supplementary Table 5 (upper panel).

2.4. Animals

C57BL6/N wildtype female mice (8–10 weeks of age) were purchased from Charles River Laboratories (Wilmington, MA) and housed at Schepens Eye Research Institute (SERI), Boston, USA, in a controlled environment with constant temperature, 12-hour light/dark cycle, and food and water available ad libitum. Mice were anesthetized with a combined dose of ketamine (100mg/kg) and xylazine (20 mg/kg) administered intraperitoneally (IP). Animal studies were conducted in accordance with the ARVO Statement for the use of Animals in Ophthalmic and Visual Research as well as the NIH Guide for the Care and Use of Animals. All procedures were performed at SERI with approval from the Institutional Animal Care and Use Committee (IACUC).

2.5. UVA Irradiation of Mouse Cornea

The method and details of in vivo UVA irradiation have been described in our earlier publication (Liu et al., 2020). Briefly, a UVA LED light source (M365LP1; Thorlabs, USA) with an emission peak of 365 nm light, 8 nm bandwidth (FWHM), and irradiance of 398 mW/cm2 was focused down to a 4 mm diameter illumination spot onto the mouse cornea. The time of UVA exposure was adjusted to deliver the appropriate fluence (42 minutes for 1000 J/cm2) measured with a thermal power sensor head (S425C, Thorlabs, USA) and energy meter console (PM100D, Thorlabs). The right eye (OD) was irradiated, while the contralateral eye (OS) served as untreated control and was covered with heat retention drapes (SpaceDrapes, Inc., USA). Mouse eyes were enucleated either at 1 day or 1 month post irradiation in sterile PBS, and DM-CE was isolated, snap-frozen, and used for RT-PCR or western blot analysis.

2.6. In vivo Imaging

Mice were anesthetized as described earlier, and the corneal photographs were taken using a slit lamp biomicroscope attached to a camera (Nikon D100, Tokyo, Japan). The epithelial cell integrity was assessed by observing punctate staining under cobalt blue light using fluorescein (1 mL in 2.5 % PBS; Sigma Aldrich, USA) applied topically onto the lateral conjunctival sac. Anterior segment images were acquired to assess corneal thickness using anterior segment optical coherence tomography (AS-OCT) (Bioptigen Spectral Domain Ophthalmic Imaging System Envisu R2200 with a 12 mm telecentric lens for corneal scans).

For monitoring CE morphology in vivo, mice wrapped in SpaceDrapes were positioned on a secure platform and their corneas were imaged by laser scanning in vivo confocal microscopy using the Heidelberg Retina Tomograph III (HRT) with Rostock Corneal Module (RCM) (Heidelberg Engineering). The laser confocal microscope acquires 2D images representing a coronal cornea section of 400 × 400 μm (160,000 μm2) at selectable corneal depth. Acquired images comprise 384 × 384 pixels with a lateral resolution of 1 μm per pixel. Digital images were stored on a computer workstation at 3 frames per second.

2.7. Mouse DM isolation and real-time PCR in mouse tissue

Mouse eyes were enucleated either at 1 day or 1 month post irradiation in sterile, ice-cold PBS. Under a stereo-zoom microscope (MZ6, Leica), the eyeball was penetrated using a 26-gauge syringe needle to create a slight incision at the corneo-scleral junction. Curved Vannas capsulotomy scissors, 3 3/8” (5677E, Ambler Surgical) were inserted into the incision to snip out the corneal cup from which the lens was removed, and remaining iris and loose tissue was scraped out to visualize the trabecular meshwork (TM). With the corneal endothelium facing up, a superficial pinch was made just below the TM using jeweler’s forceps to grip the DM which was then peeled intact by applying a controlled pull along the edges while rotating the corneal cup in the opposite direction. The DM-CE thus obtained was snap-frozen at −80°C for either RT-PCR or western blot analysis.

To determine the differential expression of mitochondrial DNA repair genes Lig3, Neil2, and Top3a, in UVA-irradiated compared to non-irradiated mouse corneas, we isolated RNA from the DM-CE using RNeasy micro kit (#74034, Qiagen). Two mouse DMs pooled per sample were homogenized in RLT buffer with 10% β-mercaptoethanol using a handheld pestle (#12-141-361, Fisher Scientific) and RNA was isolated as per manufacturer’s protocol. cDNA synthesis, pre-amplification, and real-time PCR was performed as described in section 2.3. The Taqman assays used for RT-PCR validation of three mitochondrial repair genes, lig3, top3a, and neil2 in UVA-irradiated vs non-irradiated mouse corneas are listed in Supplementary Table 5 (lower panel).

2.8. Western blot analysis

Whole cell extracts from DM-CE isolated from at least 3 mice per time-point were taken as described in our earlier publication (Liu et al., 2020; Miyajima et al., 2020). UVA-irradiated CE tissue from one mouse was used per replicate for western blotting. Briefly, proteins were loaded into 4–12% Bis-Tris NuPAGE gels (#NP0336, Thermo Fisher Scientific, USA), run, and transferred to polyvinylidene difluoride membrane (PVDF), blocked with 5% non-fat dry milk, probed with primary antibodies overnight then exposed next day to secondary antibodies for 1-hour and finally probed with Super Signal West Pico or Femto (#34577 or #34096, Thermo Fisher Scientific, USA). Chemiluminescent signal was captured by exposing the blots to an x-ray film and developing it using an automated film processor inside a dark room. Densitometry was performed using Image J software (NIH, USA). The primary antibodies used were LIG3 (1:1000, 611876, BD Biosciences, USA), Neil2 (1:1000, GTX132565, Genetex), Top3a (1:1000, PA5-116710, Life Technologies), β-Actin (1:5000, A1978, Sigma, USA), and gapdh (1:2000, G9545, Sigma, USA) and secondary antibodies were horseradish peroxidase-conjugated mouse anti-rabbit IgG (1:1000, sc-2357) and anti-mouse IgG (1:1000, sc-516102) from Santa Cruz Biotechnology Inc., USA.

2.9. Statistical Analysis

To analyze the raw data from the PCR array, we implemented 84 Mann Whitney rank sum exact tests on 84 genes respectively by comparing 3 values from normal and 6 values from FECD samples. P-values were corrected by using False Discovery Rate (FDR) (Benjamini-Hochberg method) for multiple comparisons and significance level was set to 10%. For validation results, unpaired t test was performed for comparing two groups with equal variance. One-way ANOVAs (Tukey’s multiple comparison tests) were performed for more than two groups using GraphPad Prism 9 (V8, La Jolla, CA). P< 0.05 was considered as statistically significant difference, denoted as * P<0.05, ** P<0.01, *** P < 0.001, and **** P<0.0001.

3. Results

3.1. Eight DNA repair genes are differentially upregulated in FECD compared to normal specimens

To determine the repair genes differentially expressed in FECD compared to normal corneal endothelia, we isolated RNA from DM-CE obtained either from a normal donor cornea or acquired post keratoplasty from FECD patients. cDNA was prepared and subjected to real-time PCR arrays of 84 DNA repair genes (Fig. 1B–C; Supplementary Table 1, 2). We had a sample size of 3 normal and 6 FECD specimens (Fig. 1B). A relative gene expression threshold of greater than 2.0- or less than 0.5-fold was set to identify genes that were either up- or downregulated respectively in FECD relative to normal samples (Table 1, Supplementary Table 1).

Table 1.

Relative expression of DNA repair genes analyzed by real-time PCR based array in normal and FECD specimens.

Gene Symbol Gene name Function Cellular localization Fold change
Upregulated genes (> 2.0 fold)
Normal vs FECD (non-stratified)
CCNH Cyclin H NER nu 2.45
CDK7 Cyclin dependent kinase 7 NER nu 2.375
MRE11A Meiotic recombination 11 homolog A DSB repair mito/nu 2.091
MSH2 MutS Homolog 2, colon cancer, non polyposis type 1 (E.coli) MMR nu 2.501
POLB Polymerase (DNA directed), beta BER mito/nu 2.407
RPA3 Replication potein A3, 14 kDa NER nu 2.791
RAD51C RAD51 homolog C DSB repair nu 2.034
XRCC4 X-ray repair cross-complementing protein 4 BER nu 3.888
Downregulated genes (< 0.5 fold)
Normal vs FECD (non-stratified)
ERCC1 Excision repair cross-complementing rodent repair deficiency, complementation group 1 NER nu 0.208
LIG3 Ligase III, DNA, ATP-dependent BER mito/nu 0.325
NEIL2 Nei Like DNA Glycosylase 2 BER mito/nu 0.3119
PARP3 Poly (ADP-ribose) polymerase family, member 3 DSB Repair nu 0.395
POLL DNA polymerase lambda DSB Repair nu 0.281
TOP3A DNA Topoisomerase III Alpha DSB Repair mito/nu 0.2751
TREX1 Three prime repair exonuclease 1 nu/cyto 0.457
XAB2 XPA binding protein 2 NER nu 0.455
XPC Xeroderma pigmentosum, complementation group C NER nu 0.207
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BER, base excision repair; cyto, cytosolic; DSB, double-strand break; E.coli, Escherichia coli; mito, mitochondrail; MMR, DNA-mismatch repair; NER, nucleotide excision repair; nu, nuclear; vs, versus. P-value < 0.05 is considered statistically significant.

From the PCR array, we identified 8 DNA repair genes, CCNH, CDK7, MRE11A, MSH2, POLB, RAD51C, RPA3, and XRCC4 that were differentially upregulated in FECD specimens not stratified with respect to their TCF4 CTG repeats status (non-stratified FECD), as compared to normal controls (Table 1; Fig. 2A). Out of the 8 DNA repair genes found to be differentially upregulated in non-stratified FECD specimens, DSB repair gene MRE11A and BER gene POLB gene are known to be localized in both the mitochondria and nucleus (Table 1; Fig. 2B). We first validated our findings of upregulated genes from the PCR array in an additional second cohort of non-stratified FECD and normal samples (Supplementary Table 3) using real-time PCR. Out of 8 genes differentially upregulated in FECD in the PCR array, we validated 4 genes, namely, CDK7, MRE11A, MSH2, and POLB to be significantly upregulated in this non-stratified FECD cohort (Fig. 2C, Supplementary Figure 1). Since FECD is known to have a strong association with the presence of trinucleotide CTG repeat expansion in TCF4, we further sought to determine whether CDK7, MRE11A, MSH2, and POLB genes were differentially expressed between FECD with and without CTG repeats in TCF4. In an additional third cohort of FECD, we estimated the number of TCF4 CTG repeats using blood collected from the patients at the time of surgery and classified the specimens as FECD NX and FECD X as defined in methods (Supplementary Table 4). We performed RT-PCR for CDK7, MRE11A, MSH2, and POLB in these stratified FECD specimens and compared their expression levels in each FECD group individually to additional normal samples. Interestingly, we found two mismatch repair genes, MSH2 and BER gene POLB, to be preferentially upregulated in FECD X specimens (Fig. 2D, Supplementary Table 4). MSH2 showed a 3.0-fold increased expression in FECD X (P<0.001) but only 1.7-fold increased expression in FECD NX compared to normal samples (Fig. 2D). POLB showed a 2.1-fold increased expression only in FECD X (P<0.05) but no change in FECD NX compared to normal samples (Fig. 2D). However, CDK7 and MRE11A did not show differential expression in either FECD X or FECD NX compared to normal samples (Supplementary Fig. 2).

Figure 2. DNA repair genes upregulated in FECD compared to normal specimens in an RT-PCR array of 84 DNA repair genes.

Figure 2.

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(A) 8 DNA repair genes showed a significant upregulation represented as relative gene expression >2.0-fold in FECD (non-stratified) compared to normal controls in the RT-PCR array. * indicates corrected p value (q value) <0.1 applying 84 Mann Whitney tests with an FDR of 10% (Supplementary Table 1). (B) Classification of the 8 upregulated genes found in FECD specimens as those involved in base excision repair (BER, β), nucleotide excision repair (NER, η), double-strand break (DSB, δ) repair, DNA mismatch repair (MMR, ϻ) or other DNA repair pathways in accordance with their known cellular localization in mitochondria or nucleus. (C) Validation of the differential expression profiles of 8 genes (from Fig. 2A) in an additional cohort of non-stratified FECD specimen RNAs confirmed significant upregulation of 4 genes: CDK7, MRE11A, MSH2 and POLB. * P<0.05, ** P<0.01, *** P <0.001, and **** P<0.0001 by applying two-tailed unpaired t-test. (D) MSH2, and POLB (but not CDK7 and MRE11A) genes were significantly upregulated only in FECD X, and not in FECD NX compared to normal controls. * P<0.05, and *** P <0.001 by applying one-way ANOVA with Tukey’s Multiple Comparison Test. Relative gene expression was expressed as mean ± SE of 2(−dCT) of FECD and normal samples, where 2(−dCT) = fold change of gene expression normalized to GAPDH.

3.2. Nine DNA repair genes are differentially downregulated in FECD compared to normal specimens

From the PCR array, we identified 9 DNA repair genes, ERCC1, LIG3, NEIL2, PARP3, POLL, TOP3A, TREX1, XPC, and XAB2 that were differentially downregulated in non-stratified FECD specimens as compared to normal controls (Table 1; Fig. 3A). Out of the 9 DNA repair genes found to be differentially downregulated in non-stratified FECD specimens, BER genes, LIG3 and NEIL2, and DSB repair gene TOP3A are known to be localized in both the mitochondria and nucleus (Table 1; Fig. 3B). We first validated our findings of downregulated genes from the PCR array in the additional second cohort of non-stratified FECD and normal samples (Supplementary Table 3) using real-time PCR. Out of 9 genes differentially downregulated in FECD in the PCR array, we validated 6 genes, namely, LIG3, NEIL2, PARP3, TOP3A, TREX1, and XPC to be significantly downregulated in this non-stratified FECD cohort (Fig. 3C, Supplementary Figure 3). To determine whether LIG3, NEIL2, PARP3, TOP3A, TREX1, and XPC were differentially expressed between FECD X and FECD NX, we used an additional third cohort of stratified FECD samples performed RT-PCR and compared their expression levels in each FECD group individually to additional normal samples (Supplementary Table 4). We found that all 6 DNA repair genes LIG3, NEIL2, PARP3, TOP3A, TREX1, and XPC were downregulated to the same extent in both FECD X and FECD NX specimens, and no differences were noted between FECD X and NX. (Fig. 3D).

Figure 3. DNA repair genes downregulated in FECD compared to normal specimens in an RT-PCR array of 84 DNA repair genes.

Figure 3.

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(A) 9 DNA repair genes showed significant downregulation represented as relative gene expression <0.5-fold in FECD (non-stratified) compared to normal controls in the RT-PCR array. * indicates corrected p value (q value) <0.1 applying 84 Mann Whitney tests with an FDR of 10% (Supplementary Table 1). (B) Classification of the 9 downregulated genes found in non-stratified FECD specimens as those involved in base excision repair (BER, β), nucleotide excision repair (NER, η), double-strand break (DSB, δ) repair, DNA mismatch repair (MMR, ϻ) or other DNA repair pathways in accordance with their known cellular localization in the mitochondria or nucleus. (C) Validation of the differential expression profiles of 9 genes (from Fig. 3A) in an additional cohort of non-stratified FECD specimen RNA confirmed significant downregulation of 6 genes: LIG3, NEIL2, TOP3A, PARP3, TREX1, and XPC. * P<0.05, and ** P<0.01 by applying two-tailed unpaired t-test. (D) Validation of the differential expression profiles of 6 downregulated genes (from Fig. 3C) in an additional cohort of non-stratified FECD specimen RNAs confirmed significant downregulation of all 6 genes in both, FECD X and FECD NX compared to normal controls. * P<0.05, *** P <0.001, and **** P<0.0001 by applying one-way ANOVA with Tukey’s Multiple Comparison Test. Relative gene expression was expressed as mean ± SE of 2(−dCT) of FECD and normal samples, where 2(−dCT) = fold change of gene expression normalized to GAPDH.

3.3. Downregulation of mitochondrial DNA repair genes Lig3, Neil2, and Top3a correlates to early mitochondrial DNA damage in UVA mouse model of FECD

Our group established a non-genetic UVA-based mouse model that recapitulates the morphological and molecular changes of FECD and provides the model of late-onset FECD based on the physiologic outcome of CEC susceptibility to oxidative stress(Liu et al., 2020). The irradiation of mouse corneas with UVA (1000 J/cm2) caused enlargement and loss of CECs assessed by Heidelberg Retina Tomography (HRT), increased corneal thickness assessed by AS-OCT, and the formation of guttae-like lesions interspersed around degenerating cells at 1-month post-UVA (Fig. 4A). UVA irradiation also upregulated ROS levels in mouse aqueous humor and caused nuclear and mitochondrial DNA damage, which was also seen in human FECD specimens, ex vivo. Additionally, we have shown that the mitochondrial DNA damage in our UVA mouse model occurs early in both males and females, followed by a second wave of damage occurring at 1-month(Liu et al., 2020). We thus utilized this FECD mouse model further to investigate the expression of downregulated mtDNA repair genes from our DNA repair PCR array, (Fig. 3B), namely, BER genes Lig3 and Neil2, and DSB repair gene Top3a. By employing RT-PCR, we found that these three genes were differentially downregulated in the mouse CE at 1-day and 1-month post-UVA irradiation, where Neil2 was further depleted at 1-month compared to the 1-day time-point (Fig. 4B). Western blot analysis of lysates from mouse CEs at 1-day post UVA showed Lig3 and Neil2, but not Top3a to be significantly downregulated at the protein level (Fig. 4C, Supplementary Fig. 4).

Figure 4. Downregulation of mitochondrial repair genes in a mouse model of FECD based on ultraviolet-A (UVA) light, a physiological stressor of the cornea.

Figure 4.

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(A) Single 1000 J/cm2 UVA irradiation dose causes morphological changes and damage in mouse cornea and mouse corneal endothelial cells (mCEnCs). UVA light constitutes 95% of all incident UV light. Only the right (OD) eye of mice was irradiated with UVA light. Representative AS-OCT images show increased corneal edema and central corneal thickness (CCT) through 1-month post-UVA. Slit lamp (broad beam) imaging of irradiated mouse eyes exhibits changes in corneal opacity. Sodium fluorescein staining shows corneal epithelial defect at 1-day post-UVA. Representative in vivo confocal HRT photographs show progressive loss of mCEnC through 1-month post-UVA. (B) Lig3, Neil2, and Top3a genes involved in mitochondrial DNA repair, are downregulated in mouse CE post 1-day and 1-month UVA irradiation by RT-PCR analysis. Relative gene expression was expressed as mean ± SE of 2(−dCT) of irradiated OD eyes normalized to that of non-exposed control OS eyes, where 2(−dCT) = fold change of gene expression normalized to gapdh. * P<0.05, ** P <0.01, and *** P<0.001 by applying one-way ANOVA with Tukey’s Multiple Comparison Test. (C) Western blot assay showing downregulation of BER proteins DNA lig3 (Lig3) (n=6) and Neil2 (n=4) in UVA-irradiated mouse corneal endothelia at 1-day post exposure of their right eyes to 1000 J/cm2 UVA light, compared to non-exposed control left eyes. Densitometry was analyzed using Image J software and relative protein expression levels were determined by normalizing band intensities to that of β-actin or gapdh. ** or * indicates p <0.01 or <0.05 respectively applying two-tailed unpaired t-test with Welch’s correction.

4. Discussion

Corneal endothelial cells are terminally differentiated cells that depend on DNA repair capacity to maintain the integrity of the transcribed genome and protect against acquired DNA damage that if unrepaired, may lead to the cellular dysfunction and cell death. There are many differences in the DNA repair systems between proliferative and non-proliferative cells such as CE, pointing to yet a lot of unanswered questions of how terminally differentiated cells deal with the acquired damage. In the post-mitotic cells of cornea, a robust resistance to various types of stressors, including UV light, is likely at play via activation of DNA repair systems that are specifically poised to combat oxidative damage. The degeneration of central cells in FECD, consistent with the area of UV light exposure (Liu et al., 2020), points to the concept of dysregulation of repair mechanisms leading to the inability to preserve genome integrity from endogenous and exogenous stressors. In this study, we performed a comprehensive analysis of DNA repair genes by PCR array and detected differential dysregulation of various genes involved in oxidative and UV-induced DNA damage repair in FECD compared to normal corneal endothelium, pointing to a possible incapacity to repair acquired DNA damage in FECD. Herein, we identified 8 differentially upregulated and 9 differentially downregulated genes in FECD specimens, irrespective of TCF4 CTG repeats status, as compared to normal controls; indicating that non-genetic factors such as oxidative stress may significantly affect DNA repair in FECD.

DNA damage response (DDR) is comprised of a diverse network of signal transduction pathways activated by aberrant DNA structures. It results in the initial phosphorylation of the most upstream DDR kinases, such as ATM (ataxia-telangiectasia mutated), ATR (ATM and Rad3-Related), and the subsequent phosphorylation and activation of downstream effectors involved in DNA repair, replication, apoptosis and senescence (Marechal and Zou, 2013; Zhou and Elledge, 2000) (Fig. 1A). UVA induces oxidative DNA lesions and causes single-strand breaks (SSBs) (Cadet et al., 2009; Karran and Brem, 2016; Peak and Peak, 1990) that are repaired by either homologous or non-homologous recombination or generates bulky adducts that undergo NER (Jackson and Bartek, 2009) or BER (Cadet et al., 2009; Karran and Brem, 2016; Peak and Peak, 1990; Rastogi et al., 2010); while catechol estrogens and estrogen-DNA adducts lead to mutagenic apurinic sites, primarily repaired by BER or DSB repair enzymes (Cavalieri et al., 2006; Mailander et al., 2006; Savage et al., 2014; Zhao et al., 2006). BER is the major DNA repair system found in the nucleus (nBER) and mitochondria (mtBER) in post-mitotic cells, the latter being the repair system of oxidatively damaged mtDNA. BER consists of DNA glycosylases, such as OGG1, UNG, MYH, AP endonucleases (APE1), DNA polymerases, and DNA ligases (Maynard et al., 2010).

Mitochondrial dysfunction is a hallmark of the aging process, resulting in increased oxidative stress, and plays a crucial role in the pathogenesis of FECD (Jurkunas, 2018; Jurkunas et al., 2010). Here, we detected the downregulation of 3 mtDNA repair genes comprising 2 key BER [LIG3 (Gao et al., 2011; Synowiec et al., 2015), NEIL2 (Han et al., 2019; Mandal et al., 2012)] and 1 DSB repair [TOP3A (Jiang et al., 2021)] gene. We utilized our non-genetic UVA-based FECD mouse model and demonstrated that these genes are also downregulated in mouse CE after UVA, indicating that lack of mtDNA repair may play a role in generating FECD phenotype in vivo. LIG3, encoding DNA ligase III, is the only DNA ligase operating in mitochondria and is known to be involved in the repair of damaged mtDNA, both at basal levels and under genotoxic stress (Sharma et al., 2014; Synowiec et al., 2015). Consistent with our study, FECD has been previously linked to the polymorphisms rs1052536 and rs3135967 of LIG3 (Synowiec et al., 2015). A previous study has demonstrated that ATM regulates the DNA Lig3 stability and decrease in LIG3 resulted in lower levels of intact mitochondrial DNA (mtDNA) and lower cellular ATP levels that in turn causes cerebellar degeneration (Sharma et al., 2014). Further studies should investigate the role of ATM in maintaining the Lig3 levels and phosphorylation status, as it may be an effector in mtDNA repair in FECD. Deficiency of NEIL2, a DNA glycosylase, has been shown to cause accumulation of oxidative DNA damage in mitochondria which triggers a TP53-mediated intrinsic apoptosis in Xenopus embryos (Han et al., 2019). In a recent study, human topoisomerase, TOP3A, was found to be mutated in Bloom-like syndrome, characterized by growth retardation, sun-sensitive skin, and predisposition to malignancy, and Top3a-deficient cells showed reduced mitochondrial ATP generation and oxygen consumption rates (Jiang et al., 2021).

Furthermore, we identified 3 nDNA repair genes including NER gene, Xeroderma pigmentosum group C (XPC), an exonuclease, three prime repair exonuclease 1 (TREX1), and a DSB gene, Poly ADP-ribose polymerase family member 3 (PARP3) to be downregulated in FECD. XPC, the primary initiating factor in the global genome NER pathway is one of the more common complementation groups associated with Xeroderma pigmentosum (XP), a genetic disease with clinical and cellular hypersensitivity to ultraviolet radiation and defective DNA repair. A systematic medical literature survey of 830 published cases of XP (Kraemer et al., 1994) showed that 41% of the patients had ocular abnormalities that were restricted to tissues exposed to ultraviolet radiation (lid, conjunctiva, and cornea) and included ectropion, corneal opacity leading to blindness, and neoplasms. Expression of XPC was found to be decreased in mouse lungs after chronic cigarette smoke (CS) exposure and its knockdown in CS-exposed human lung epithelial cells decreased their survival due to activation of the intrinsic apoptosis pathway (Sears et al., 2018). TREX1 is a major 3’ DNA exonuclease that degrades extranuclear DNA species in mammalian cells. Deficiency of TREX1 has been shown to trigger ER stress through the accumulation of single-strand DNA and activate unfolded protein response signaling leading to neuronal cell death (Halder et al., 2022). The absence of Parp3, a DSB repair protein, has been shown to provoke Nox4-induced oxidative stress and defective mTorc2 activation leading to inefficient differentiation of post-natal neural stem cells to astrocytes (Rodriguez-Vargas et al., 2020). Interestingly, the 6 downregulated DNA repair genes LIG3, NEIL2, PARP3, TOP3A, TREX1, and XPC were altered in all FECD specimens irrespective of their CTG repeats status in TCF4, suggesting that the alterations may provide a common unifying mechanism of the susceptibility to oxidative stress regardless of FECD genotype.

FECD has been associated with trinucleotide CTG repeat expansion beyond 40 repeats in the TCF4 gene and represents the largest contributing locus for FECD cases in the United States (80%); while relatively lower percentages account for this association in non-white populations (China 44%; India 34%; Japan 26%; and Thailand 39%) (Mootha et al., 2015; Okumura et al., 2020; Vasanth et al., 2015; Wieben et al., 2012). Both the autosomal dominant genetics and the knowledge that haploinsufficiency at the TCF4 locus lead to severe congenital diseases [Pitt-Hopkins syndrome (Zweier et al., 2007)] affirms that the pathogenesis of FECD is likely to involve gain-of-function mechanisms (Fautsch et al., 2021). Four mechanisms have been previously proposed to drive and/or exacerbate the onset of TCF4 repeat expansion in FECD, including a) dysregulated TCF4 expression, b) RNA-mediated toxicity leading to dysregulated pre-mRNA splicing events, c) repeat-associated non-AUG (RAN) translation, 4) the instability of CTG repeat length in affected tissue (Fautsch et al., 2021). Additionally, abnormal DNA repair proteins have been directly implicated in few other neurodegenerative repeat expansion diseases (Zhao and Usdin, 2015). Multiple loci in the human genome are prone to expansion or contraction tightly regulated by another DNA repair pathway, MMR, which performs the essential role of correcting DNA lesions, thus maintaining genomic stability. To date, out of 43 genetic diseases caused by gene-specific repeat expansions, 27 of them are affected by aberrant MMR (Zhao and Usdin, 2015). Some of the MMR proteins that have been directly implicated in repeat expansion include MSH2 (Edelbrock et al., 2013; Kovalenko et al., 2012; Manley et al., 1999; Tome et al., 2009), MSH3 (Foiry et al., 2006), MSH6 (Du et al., 2012), MLH1 (Pinto et al., 2013), PMS2 (Gomes-Pereira et al., 2004) and MLH3 (Pinto et al., 2013). In this study, we identified and validated MMR gene, MutS homolog 2 (MSH2) (Edelbrock et al., 2013) and BER gene, Polymerase DNA-directed, beta (POLB) (Kaufman and Van Houten, 2017) to be differentially upregulated in FECD specimens with expanded CTG repeats in TCF4. MSH2 gene has been demonstrated to act as a genetic enhancer in Huntington’s disease, in both of somatic CAG-repeat expansions within HTT gene as well as of CAG-dependent phenotypes in the knock-in mice (Kovalenko et al., 2012). Similarly, another group demonstrated that a functional MSH2 ATPase domain is required for the CTG-CAG repeat somatic instability in Myotonic Dystrophy type 1 (DM1) transgenic mice (Tome et al., 2009). It is also interesting to note that an association between DM1 and FECD has now been described after several members of a cohort of DM1 patients were noted to also have corneal abnormalities consistent with FECD (Gattey et al., 2014). Furthermore, a study provided evidence for a crosstalk between MMR proteins MSH2-MSH3 and BER protein Pol β which promotes trinucleotide repeat expansion during base excision repair (Lai et al., 2016). We postulate that an overdrive of a subset of DNA repair genes, including MSH2 and POLB, may correlate to expansion of trinucleotide repeats within TCF4 in CECs, however, it is also possible that mis-splicing of these DNA repair genes because of RNA toxicity is indeed affecting the efficacy of DNA repair independent of gene expression levels.

One limitation of our study is that normal and FECD specimens had variable preservation times, that could impact the differential gene expression patterns. Although the FECD specimens were stored in the same preservation medium as normal donor corneas, some variability in gene levels may be an outcome of the storage times. Furthermore, studies comprising of a larger sample size of FECD specimens and functional characterization of these differentially expressed genes in disease models as well as correlation with genetic studies should be performed in the future.

In conclusion, we suggest that altered efficiency of oxidative DNA damage repair pathways in FECD, compared to controls may contribute to the development of FECD, especially when oxidative stress is one of the postulated mechanisms of the pathogenesis of the disease. The investigation of the causal mechanism of genomic instability, possibly driven by aberrant DDR, can inform future targeted therapeutic approaches, thus aiding in restoration of dysfunctional DNA repair pathways.

Supplementary Material

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Highlights.

  • DNA repair is impaired in FECD.

  • Base excision repair is the major pathway perturbed in FECD.

  • DNA repair genes MSH2 and POLB are preferentially upregulated in FECD with CTG repeat expansion in TCF4 gene.

  • Mitochondrial DNA repair is deficient and correlates to early DNA damage in the UVA-mouse model of FECD.

Acknowledgements

The work was supported by NIH/NEI R01EY020581 (to U.V.J) and Core Grant P30EY003790 (to Schepens Eye Research Institute).

Abbreviations

DM-CE

Descemet’s membrane-corneal endothelium

CEnCs

Corneal Endothelial Cells

mtDNA

Mitochondrial DNA

nDNA

Nuclear DNA

mtBER

Mitochondrial Base Excision Repair

nMMR

Nuclear Mismatch Repair

Footnotes

Competing interests

The authors declare no competing interest.

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